BseGI Restriction of the Polymerase Chain Reaction Amplicon Th444 Is Required to Distinguish Biotypes of Trichoderma aggressivum Causing Serious Losses in Mushroom (Agaricus bisporus) Production

in HortScience

Green mold is a serious disease of the cultivated mushroom causing losses in production of economical importance. In the present study, digestion of a Th444 amplicon with endonuclease BseGI was useful to discriminate Trichoderma aggressivum f. aggressivum (T.a.f.a) from the T. aggressivum f. europeanum (T.a.f.e.). The informative restriction fragments of 260 and 300 bp were revealed in the corresponding reference strains T.a.f.a. and T.a.f.e. The 300-bp marker was found in all 28 Polish mushroom isolates tested.

Abstract

Green mold is a serious disease of the cultivated mushroom causing losses in production of economical importance. In the present study, digestion of a Th444 amplicon with endonuclease BseGI was useful to discriminate Trichoderma aggressivum f. aggressivum (T.a.f.a) from the T. aggressivum f. europeanum (T.a.f.e.). The informative restriction fragments of 260 and 300 bp were revealed in the corresponding reference strains T.a.f.a. and T.a.f.e. The 300-bp marker was found in all 28 Polish mushroom isolates tested.

Trichoderma aggressivum f. aggressivum (T.a.f.a.) and T. aggressivum f. europeanum (T.a.f.e.) cause green mold disease in the cultivated mushroom (Agaricus bisporus) in North America and Europe, respectively. T.a.f.a. and T.a.f.e. were previously reported as T. harzianum biotypes Th4 and Th2 (Samuels et al., 2002). The disease is characterized by a rapid infestation of the compost by the pathogen and inhibition of A. bisporus fructification, resulting in serious yield losses worldwide in mushroom production (Ospino-Giraldo et al., 1999). Various molecular approaches were successfully used to characterize these isolates and distinguish them from non-aggressive Th1 (T. harzianum) and Th3 (T. atroviride) biotypes (Miyazaki et al., 2009; Ospino-Giraldo et al., 1999; Samuels et al., 2002). Based on Th4 genomic DNA, the polymerase chain reaction (PCR) marker of 444 bp was developed, which was amplified form both aggressive biotypes (Chen et al., 1999). To distinguish T.a.f.a. and T.a.f.e., the random amplified polymorphic DNA technique was successfully used (Chen et al., 1999; Szczech et al., 2008). However, this technique often suffers from poor reproducibility. Hatvani et al. (2007) used restriction fragment length polymorphism of mitochondrial DNA to distinguish Trichoderma strains. We report a simple, reliable approach to recognize T.a.f.a. and T.a.f.e. by endonuclease restriction of the amplicon Th444. Six isolates, CBS 466.94 (Th 1), CBS 693.94 (Th 3), CBS 100.528 (T.a.f.a.), CBS 450.95 (T.a.f.a.), CBS 689.96 (T.a.f.e.), and CBS 100.526 (T.a.f.e.), were obtained from the Centraalbureau voor Schimmelcultures (CBS) collection (Utrecht, The Netherlands). Twenty-eight Polish mushroom isolates of T.a.f.e. were received from the Research Institute of Vegetable Crops collection, Skierniewice, Poland (Table 1). Morphological and molecular studies of the T.a.f.e isolates are described in Szczech et al. (2008).

Table 1.

Polish T.a.f.e isolates used in this study.

Table 1.

DNA was extracted from mycelium grown on malt extract agar according to Aljanabi and Martinez (1997). The PCR marker Th444 was visible as a strong band on ethidium bromide-stained agarose gels both in T.a.f.a. and T.a.f.e. and was not generated in T. harzianum biotypes Th1 and Th3 (data not shown), because it has been reported by Chen et al. (1999). Polymorphism in T.a.f.a. and T.a.f.e. was revealed by digestion of Th444 with the restriction enzyme BseGI (Fermentas, UAB, Lithuania). The specific restriction fragment of ≈260 bp was informative for the T.a.f.a. reference isolates CBS 100.528 and CBS 450.95 (Fig. 1, Lanes 1 and 17, respectively), whereas the restriction product of 300 bp was visible in the reference isolates T.a.f.e. CBS 689.96 and CBS 100.526 (Fig. 1, Lanes 2 and 18, respectively) and all 28 T.a.f.e. Polish isolates (Fig. 1, Lanes 3 to 16 and 19 to 32). To our knowledge, it is the first example of the reproducible molecular markers that can considerably simplify detection of aggressive biotypes of Trichoderma in compost used in mushroom production.

Fig. 1.
Fig. 1.

Restriction patterns of the polymerase chain reaction marker Th444 amplified in the Trichoderma aggressivum reference strains and Polish isolates digested with BseGI. Lanes 1 and 17: amplicons obtained in T.a.f.a. strains CBS 100.528 and CBS 450.95, respectively; Lanes 2 and 18: amplicons obtained in T.a.f.e. strains CBS 689.96 and CBS 100.526, respectively. Lanes 3 to 16 and 19 to 32, Polish isolates: 3- SP.22.07, 4-11.02, 5-28.05, 6-7.06, 7-16.11.2A, 8-13.02.B, 9-29.05.1B, 10-11.04, 11-PGU12, 12-2-P-9.08-4, 13-PGU25, 14-30.01, 15-11.03, 16-27.12.2, 19-19.09.E, 20-11.08.C, 21-2.02.A, 22-5-0-7.07-9, 23-4-0-9.08-1, 24-3-0-9.08-1, 25-14.12.2C, 26-T4/3.30.03, 27-3-9.08-4, 28-5-0P-7.07-4, 29-1-0P-9.08-3, 30-5.11, 31-5/3.30.03, 32-2-0P-9.08-1. M contains the 100-bp DNA ladder as a molecular size marker.

Citation: HortScience horts 45, 12; 10.21273/HORTSCI.45.12.1910

Literature Cited

  • AljanabiS.M.MartinezI.1997Universal and rapid salt—extraction of high quality genomic DNA for PCR-based techniquesNucleic Acids Res.2546924693

    • Search Google Scholar
    • Export Citation
  • ChenX.RomaineC.P.Ospina-GiraldoD.RoyseD.J.1999A polymerase chain reaction based test for the identification of Trichoderma harzianum biotypes 2 and 4 responsible for the worldwide green mold epidemic in cultivated Agaricus bisporusAppl. Microbiol. Biotechnol.52246250

    • Search Google Scholar
    • Export Citation
  • HatvaniL.AntalZ.ManczingerL.SzekeresA.DruzhininaI.S.KubicekC.P.NagyA.NagyE.VágvölgyiC.KredicsL.2007Green mold diseases of Agaricus and Pleurotus spp. are caused by related but phylogenetically different Trichoderma speciesPhytopathology97532537

    • Search Google Scholar
    • Export Citation
  • MiyazakiK.TsuchiyaY.OkudaT.2009Specific PCR assays for the detection of Trichoderma harzianum causing green mold disease during mushroom cultivationMycoscience509499

    • Search Google Scholar
    • Export Citation
  • Ospino-GiraldoD.RoyseD.ChenX.RomaineC.P.1999Molecular phylogenetic analysis of biological control strains of Trichoderma harzianum and other biotypes of Trichoderma spp. associated with mushroom green moldPhytopathology89308313

    • Search Google Scholar
    • Export Citation
  • SamuelsG.J.DoddS.L.GamsW.CastelburyL.A.PetriniO.2002Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporusMycologia94146170

    • Search Google Scholar
    • Export Citation
  • SzczechM.StaniaszekM.HabdasH.UlińskiZ.SzymańskiJ.2008Trichoderma spp.—The cause of green mold on Polish mushroom farmsVegetable Crops Research Bulletin69105114

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

To whom reprint requests should be addressed; e-mail mstan@inwarz.skierniewice.pl.

  • View in gallery

    Restriction patterns of the polymerase chain reaction marker Th444 amplified in the Trichoderma aggressivum reference strains and Polish isolates digested with BseGI. Lanes 1 and 17: amplicons obtained in T.a.f.a. strains CBS 100.528 and CBS 450.95, respectively; Lanes 2 and 18: amplicons obtained in T.a.f.e. strains CBS 689.96 and CBS 100.526, respectively. Lanes 3 to 16 and 19 to 32, Polish isolates: 3- SP.22.07, 4-11.02, 5-28.05, 6-7.06, 7-16.11.2A, 8-13.02.B, 9-29.05.1B, 10-11.04, 11-PGU12, 12-2-P-9.08-4, 13-PGU25, 14-30.01, 15-11.03, 16-27.12.2, 19-19.09.E, 20-11.08.C, 21-2.02.A, 22-5-0-7.07-9, 23-4-0-9.08-1, 24-3-0-9.08-1, 25-14.12.2C, 26-T4/3.30.03, 27-3-9.08-4, 28-5-0P-7.07-4, 29-1-0P-9.08-3, 30-5.11, 31-5/3.30.03, 32-2-0P-9.08-1. M contains the 100-bp DNA ladder as a molecular size marker.

  • AljanabiS.M.MartinezI.1997Universal and rapid salt—extraction of high quality genomic DNA for PCR-based techniquesNucleic Acids Res.2546924693

    • Search Google Scholar
    • Export Citation
  • ChenX.RomaineC.P.Ospina-GiraldoD.RoyseD.J.1999A polymerase chain reaction based test for the identification of Trichoderma harzianum biotypes 2 and 4 responsible for the worldwide green mold epidemic in cultivated Agaricus bisporusAppl. Microbiol. Biotechnol.52246250

    • Search Google Scholar
    • Export Citation
  • HatvaniL.AntalZ.ManczingerL.SzekeresA.DruzhininaI.S.KubicekC.P.NagyA.NagyE.VágvölgyiC.KredicsL.2007Green mold diseases of Agaricus and Pleurotus spp. are caused by related but phylogenetically different Trichoderma speciesPhytopathology97532537

    • Search Google Scholar
    • Export Citation
  • MiyazakiK.TsuchiyaY.OkudaT.2009Specific PCR assays for the detection of Trichoderma harzianum causing green mold disease during mushroom cultivationMycoscience509499

    • Search Google Scholar
    • Export Citation
  • Ospino-GiraldoD.RoyseD.ChenX.RomaineC.P.1999Molecular phylogenetic analysis of biological control strains of Trichoderma harzianum and other biotypes of Trichoderma spp. associated with mushroom green moldPhytopathology89308313

    • Search Google Scholar
    • Export Citation
  • SamuelsG.J.DoddS.L.GamsW.CastelburyL.A.PetriniO.2002Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporusMycologia94146170

    • Search Google Scholar
    • Export Citation
  • SzczechM.StaniaszekM.HabdasH.UlińskiZ.SzymańskiJ.2008Trichoderma spp.—The cause of green mold on Polish mushroom farmsVegetable Crops Research Bulletin69105114

    • Search Google Scholar
    • Export Citation
All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 61 31 0
PDF Downloads 34 23 3