Diversity of Trichoderma spp. causing Pleurotus green mould diseases in Central Europe

The present study includes the molecular characteristics of Trichoderma pleurotum and Trichoderma pleuroticola isolates collected from green moulded cereal straw substrates at 47 oyster mushroom farms in Poland. The screening of the 80 Trichoderma isolates was performed by morphological observation and by using the multiplex PCR assay. This approach enabled specific detection of 47 strains of T. pleurotum and 2 strains of T. pleuroticola. Initial identifications were confirmed by sequencing the fragment of internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) of the rRNA gene cluster and the fragment including the fourth and fifth introns and the last long exon of the translation–elongation factor 1-alpha (tef1) gene. ITS and tef1 sequence information was also used to establish the intra- and interspecies relationship of T. pleurotum and T. pleuroticola originating from the oyster mushroom farms in Poland and from other countries. Comparative analysis of the ITS sequences showed that all T. pleurotum isolates from Poland represent one haplotype, identical to that of T. pleurotum strains from Hungary and Romania. Sequence analysis of the tef1 locus revealed two haplotypes (“T” and “N”) of Polish T. pleurotum isolates. The “T” type isolates of T. pleurotum were identical to those of strains from Hungary and Romania. The “N” type isolates possessed a unique tef1 allele. Detailed analysis of the ITS and tef1 sequences of two T. pleuroticola isolates showed their identicalness to Italian strain C.P.K. 1540.


Introduction
Pleurotus ostreatus (Jacq.) P. Kumm. is one of the most important commercial crop edible mushrooms in Poland. Together with Italy and Hungary, Poland is the main producer of P. ostreatus in Europe. However, significant disintegration of oyster mushroom production and differences in cultivation conditions affect the appearance of many pests and diseases. In recent years, severe symptoms of green mould have been observed in oyster mushroom farms, resulting in crop losses.
The first reported appearance of green mould on P. ostreatus was in North America (Sharma and Vijay 1996). Serious cases of this disease in commercially grown P. ostreatus were detected thereafter in South Korea (Park et al. 2004a, b), Italy (Woo et al. 2004), Romania (Kredics et al. 2006), Hungary , and most recently in Spain (Gea 2009 (Park et al. 2004a(Park et al. , b, 2006Komon-Zelazowska et al. 2007). Phenotypically, T. pleurotum and T. pleuroticola species are significantly different. T. pleuroticola shows a typical pachybasium-like conidiophore developing in fascicles or pustules which is typical for the Harzianum clade, whereas T. pleurotum is characterised by a gliocladium-like conidiophore morphology (Park et al. 2006;Komon-Zelazowska et al. 2007). In spite of the large phenetic divergence, these species present a very close phylogenetic relationship to the Harzianum clade of Hypocrea/ Trichoderma, which also includes Trichoderma aggressivum Samuels & W. Gams, the causative agent of green mould disease in Agaricus (Park et al. 2004a, b;Hatvani et al. 2007;Komon-Zelazowska et al. 2007).
Trichoderma pleurotum has been found only on cultivated P. ostreatus and its substratum. In contrast, T. pleuroticola has been found both on wild and cultivated P. ostreatus, as well as on the natural and productive substratum of the oyster mushroom (Park et al. 2004a, b;Szekeres et al. 2005;Hatvani et al. 2007;Komon-Zelazowska et al. 2007;Kredics et al. 2009). Additionally, T. pleuroticola has been isolated from soil and wood in Canada, the USA, Europe, Iran, and New Zealand (Park et al. 2004a, b;Szekeres et al. 2005;Komon-Zelazowska et al. 2007).
Until now, it has not been clear which species of Trichoderma is the causative agent of the green mould in oyster mushroom farms of Central Europe. The present study was carried out to confirm the association of the T. pleurotum and T. pleuroticola species with P. ostreatus cultivated in Poland based on morphological and molecular analysis of collected Trichoderma isolates originating from Polish oyster mushroom farms.

Fungal collection
Four T. pleurotum (E135, E136, E138, E139) and five T. pleuroticola strains (E137, M141, M142, M143, M144), used as the reference strains, were kindly supplied by Dr. Monika Komon-Zelazowska, Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Austria. Eighty Trichoderma isolates were collected from green moulded cereal straw substrates at 47 oyster mushroom farms in Poland. The small fragments of cereal straw were taken from substrates used for cultivation of P. ostreatus. Basidiomes were suspended in 10 mL sterile distilled water and 0.2 mL Tween 20 (Sigma), incubated at 25°C for 10 min on a rotary shaker (120 rpm) and diluted 1:10 with sterile distilled water. Inoculation was performed from the suspensions (0.5 mL) onto a potato dextrose agar (PDA, Oxoid) and incubated in darkness at 25°C for 7 days. The resultant fungal colonies were transferred to new plates of PDA and incubated as described above. The strains collected from Polish mushroom farms and investigated in this study are listed in Table 1.

Morphological analysis
Identification was performed by observation of phenotypic characteristics of the colonies and by microscopic studies of the conidia and conidiophores. Colony characteristics were examined from cultures grown in darkness at 25°C for 7 days on PDA. Microscopic observations were made according to Park et al. (2006).

DNA isolation and amplification
Mycelium for DNA extraction was obtained as described previously (Błaszczyk et al. 2011). Isolation of total DNA was performed using the CTAB method (Doohan et al. 1998).
The ITS1 and ITS2 region of the rDNA gene cluster was amplified using primers ITS4 and ITS5 (White et al. 1990). A fragment of 1.2-kb tef1 gene was amplified using primers Ef728M (Carbone and Kohn 1999) and TEF1LLErev (Jaklitsch et al. 2005) as well as the set of primers (FPforw1, FPrev1, PSrev1) designed for the rapid detection of T. pleurotum and T. pleuroticola (Kredics et al. 2009).

DNA sequencing and comparative analyses
The 0.4-kb ITS and 1.2-kb tef1 amplicon purification steps and sequencing were carried out as described previously (Chełkowski et al. 2003;Błaszczyk et al. 2011). Sequences were edited and assembled using Chromas v. 1.43 (Applied Biosystems). The sequences were identified Table 1 The list of strains collected from oyster mushroom farms in Poland and identified on the basis of multiplex PCR and ITS and tef1 sequence analysis
The comparative analyses were based on the ITS and tef1 sequences of the 49 T. pleurotum/T. pleuroticola isolates obtained in the present study and 9 reference strains, as well as on the sequences of 21 other T. pleurotum/T. pleuroticola strains, deposited in NCBI GeneBank (www.ncbi.nlm.nih. gov, Table 2). The sequences of 8 T. pleurotum, and 13 T. pleuroticola strains, sourced from Hungary, Italy, Romania, Canada, USA, Netherlands, and Colombia, were used in order to determine the relationship of these strains and the isolates originating from Poland. ClustalW (Thompson et al. 1994) was used to align the sequences.

Identification of T. pleurotum and T. pleuroticola isolates
Preliminary identifications of the 80 Trichoderma isolates collected from the 47 oyster mushroom farms in Poland and  The initial identifications of 2 T. pleuroticola and 47 T. pleurotum isolates collected from Poland as well as 9 reference Trichoderma strains were confirmed by sequencing two different phylogenetic markers: the fragment of the ITS1-5.8S-ITS2 rRNA region and the fragment of the tef1 gene (Table 1). The sequence analyses were also used to identify the remaining Trichoderma isolates collected from oyster mushroom farms in Poland. These isolates were identified as Trichoderma harzianum Rifai (17 isolates) and Trichoderma atroviride P. Karst (14 isolates) ( Table 1).
Comparison of ITS and tef1 sequences of T. pleurotum and T. pleuroticola isolates The comparative analyses were based on the ITS and tef1 sequences of the T. pleurotum and T. pleuroticola strains both obtained in this study and published previously by Hatvani et al. (2007), Komon-Zelazowska et al. (2007), and Kredics et al. (2009).
DNA sequence alignment showed that the ITS allele detected in 47 T. pleurotum isolates from Poland was identical to that of T. pleurotum strains from Hungary (C.P.K. 2113, C.P.K. 2096, C.P.K. 2097, C.P.K. 2100, C.P.K. 2116, C.P.K. 2117) and Romania (C.P.K. 2814) but differed by one single nucleotide polymorphism (SNP) from the Italian strain C.P.K. 1532. Similarly, 2 T. pleuroticola isolates from Poland and 11 strains from: Canada (DAOM 175924), USA (DAOM 22996), Italy (C.P.K. 1540), Romania (C.P.K. 2816, C.P.K. 2817), Hungary (C.P.K. 2104, C.P.K. 3266), Netherlands (G.J.S. 95-81), and Colombia (T 1295) possessed an identical allele in the ITS locus, while their ITS1 sequences were different by one SNP from the sequences of Italian strain C.P.K. 1550 and Hungarian strain C.P.K. 3193. Single nucleotide polymorphism (A/C transversion) was also observed between ITS alleles of T. pleurotum and T. pleuroticola isolates used in the present study. The intra-and interspecies variability in the ITS sequences, deriving from single nucleotide indel or transition (A-C), is given in Fig. 1. As shown in Fig. 2, T. pleurotum and T. pleuroticola were clearly divergent in the tef1 analysis. Their tef1 sequences were separated by several indel and nucleotide substitutions. The set of 47 T. pleurotum isolates originating from Poland were found to be polymorphic and represented two tef1 alleles ("T" type and "N" type), distinguishable based on one single nucleotide insertion/deletion (Fig. 2, Table 1). Nineteen Polish isolates of T. pleurotum possess the tef1 allele ("T" type) identical to three isolates from Hungary (C.P.K. 2113, C.P.K. 2116, C.P.K. 2117) and Romania (C.P.K. 2814), but different from the alleles represented by Hungarian strain C.P.K. 2096, C.P.K. 2097, and C.P.K. 2100, and Italian strain C.P.K. 1532. The "N" type of the tef1 allele, found in the remaining T. pleurotum isolates from Poland, has one position (indel or transition A/G) that differs from the allele type of five strains from Hungary (C.P.K. 2113, C.P.K. 2116, C.P.K. 2117, C.P.K. 2110) and Romania (C.P.K. 2814), two positions (indel and transition A/G) that differ from the allele type of two Hungarian strains C.P.K. 2096 and C.P.K. 2097, and several positions that differ from the allele type of Italian strain C.P.K. 1532. The tef1 sequences of two T. pleuroticola isolates from Poland were identical to that of T. pleuroticola strains DAOM 175924 from Canada, DAOM 229916 from the USA, and C.P.K. 1540 and C.P.K. 1544 from Italy, but different by four A/G and T/C transitions from the sequences of C.P.K. 3266, C.P.K. 3193, C.P.K. 2816, C.P.K. 2817, and T 1295 strains. More polymorphism was detected between the tef1 sequences of Polish T. pleuroticola isolates and that of C.P.K. 2104, C.P.K. 1550, and C.P.K. 1551 strains.

Discussion
The present study states the association of T. pleurotum and T. pleuroticola with Pleurotus green mould in Polish mushroom farms. T. pleurotum was also the most common species collected from Hungarian oyster mushroom farms (Komon-Zelazowska et al. 2007). The predominance of T. pleurotum species in samples originating from Polish and Hungarian Pleurotus farms may be due to the use of similar technologies in the production of cereal straw substratum for mushroom cultivation. These technologies are different from the methods used in Italy (probably adverse for the T. pleurotum infection), where T. pleuroticola was the major contaminant of Pleurotus substratum (Komon-Zelazowska et al. 2007).
Other species isolated from green moulded substrata for Pleurotus cultivation in Poland were: T. harzianum and T. atroviride. The presence of these species in the cultivation of P. ostreatus was also noted by Hatvani et al. (2007). Additionally, Hatvani et al. (2007) found individual isolates of Trichoderma longibrachiatum Rifai, Trichoderma ghanense Yoshim. Doi, Y. Abe & Sugiy, and Trichoderma asperellum Samuels, Lieckf. & Nirenberg. Five of these seven species, namely T. pleuroticola, T. harzianum, T. atroviride, T. longibrachiatum, and T. asperellum, were isolated from the substrate and the basidiomes of wild-grown P. ostreatus in Hungary. T. pleurotum was not found in these samples.
The preliminary identification of the collected Trichoderma isolates was based on phenetic observations and multiplex PCR assay. DNA markers used in the present work and  Fig. 1 The intra-and interspecies variability in the ITS sequences of selected T. pleurotum and T. pleuroticola isolates from oyster mushroom farms in Poland and strains deposited in NCBI GeneBank (Tables 1 and 2). The nucleotide polymorphism for T. pleurotum and T. pleuroticola strains are enclosed. Single nucleotide polymorphism (A/C transversion) between ITS alleles of T. pleurotum and T. pleuroticola is shown by the arrow specific for T. pleurotum and T. pleuroticola were recently described by Kredics et al. (2009). These authors (Kredics et al. 2009) demonstrated that T. pleurotum and T. pleuroticola can be distinguished from each other, as well as from other fungal species, using three oligonucleotide primers: FPforw1, FPrev1, and PSrev1, based on tef1 sequences. The present paper validates the specificity and the usefulness of the multiplex PCR assay developed by Kredics et al. (2009). As shown here, the PCR markers enabled the rapid screening of 80 Trichoderma isolates and specific detection of T. pleurotum and T. pleuroticola, collected from green moulded substrata for Pleurotus cultivation.
The ITS and tef1 sequence information was used to establish the intra-and interspecies relationship of T. pleurotum and T. pleuroticola originating from the oyster mushroom farms in Poland and those from other countries. The comparative analysis of the ITS sequences showed that all T. pleurotum isolates from Poland represent one haplotype, identical to that of T. pleurotum strains C.P.K. 2113, C.P.K. 2096, C.P.K. 2097, C.P.K. 2100, C.P.K. 2116, C.P.K. 2117 from Hungary and C.P.K. 2814 from Romania, but different from Italian strain C.P.K. 1532. However, the sequence analysis of the tef1 locus revealed two haplotypes of Polish T. pleurotum isolates-"T" type and "N" type. The "T" type isolates of T. pleurotum have identical tef1 allele to that of strains C.P.K. 2113, C.P.K. 2116, C.P.K. 2117 from Hungary and C.P.K. 2814 from Romania, whereas the "N" type isolates are unique at the tef1 locus. As observed in the present study, the distribution of "T" type and "N" type isolates in Poland is not correlated with the location of the mushroom farms from which they originated (Table 1). According to a previous study (Komon-Zelazowska et al. 2007), the source of T. pleurotum infection is the substratum for mushroom cultivation. Thus, the composition of two T. pleurotum haplotypes most likely depends on the manufacturer (source) of the cereal straw substratum used for the mushroom cultivation. The trading (import-export) of the Pleurotus substratum among European countries could also explain the identicalness of the "T" type isolates to the   Fig. 2 The intra-and interspecies variability in the tef1 sequences of selected T. pleurotum and T. pleuroticola isolates from oyster mushroom farms in Poland and strains deposited in NCBI GeneBank (Tables 1 and 2).