Mitochondrial plasmids in Trichoderma strains associated with green

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					Mitochondrial plasmids in Trichoderma strains associated with green mould disease of commercially grown mushrooms

                                       Zsuzsanna Antal1, János Varga2, László Kredics1, Lóránt Hatvani2, András Szekeres2, László Manczinger2
                                                                        Csaba Vágvölgyi2, Elisabeth Nagy1,3
                         1Microbiological    Research Group, Hungarian Academy of Sciences and University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary
                                                 2Department of Microbiology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary
                                            3Institute of Clinical Microbiology, University of Szeged, Somogyi Béla tér 1, H-6725 Szeged, Hungary

                                                                                E-mail: zs.antal@bio.u-szeged.hu


INTRODUCTION

      Trichoderma species are common fungi found in many cultivated and natural soils, and used as biological control agents of fungus-associated plant diseases. However, green
mould epidemics on commercially grown mushrooms caused by Trichoderma species spread in the last two decades both in Europe and North America.
      Double-stranded DNA plasmids were found in the mitochondria of numerous filamentous fungi. The plasmid carrying fungi generally do not show any symptoms, but special
phenotypes are associated with their presence in certain cases. In the present study we investigated the presence of extrachromosomal dsDNA molecules in Trichoderma isolates
derived from Hungarian Agaricus compost and Pleurotus substrate samples as well as from the CBS culture collection




MATERIALS AND METHODS                                                                                Hybridization experiments carried out using the 5.0 kb fragment as probe
                                                                                               revealed that these fragments exhibit sequence homology with each other. The
       Trichoderma strains were isolated from Agaricus compost and Pleurotus substrate         DNA samples derived from the isolated mitochondria of the strain also
derived from three Hungarian mushroom farms. The Trichoderma isolates were identified          contained the fragments, indicating the mitochondrial localization of the plasmid
by sequence analysis of the ITS and tef regions.
                                                                                               molecules. Similar sized band exhibiting sequence homology with the labeled
      Trichoderma aggressivum f. aggressivum (CBS 450.95, CBS 100527) strains derived
                                                                                               5.0 kb fragment were observed in the undigested total DNA preparations of
form the CBS culture collection.
      The total DNA content of the strains was extracted from the dried mycelia by Gentra      strains T. aggressivum f. aggressivum strain CBS 100527, T. aggressivum f. sp.
Systems Puregene DNA Purification Kit. The double-stranded DNA nature of the bands             europaeum strain B1, and Trichoderma sp. DAOM 175924 C15 as well (Fig. 2).
was checked by S1 nuclease and RNase treatments. Following agarose gel electrophoresis,        It was pointed out in the case of Neurospora and Fusarium that mitochondrial
the size of the plasmid was determined with the "GelBase/GelBlot Pro" Gel Analysis             plasmids seem to be highly mobile and their horizontal transfer occurs
Software from Ultra Violet Product Ltd, using a HindIII-digested λ molecular size marker.      frequently in nature. A similar mechanism could have caused the widespread
The DNA samples were blotted onto Hybond N+ membrane and hybridizations were                   appearance of these plasmids in these green mould associated Trichoderma
performed according to the protocol of the DIG DNA Labeling and Detection Kit (Roche).         strains.
                                                                                                1.   2.   3.   4.

                                                                                                                     Fig. 2. Blot of a gel hybridized with the 5.0 kb fragment as
                                                                                                                     probe
                                                                                                                     1. T. aggressivum f. aggressivum strain CBS 450.95
 RESULTS AND DISCUSSION
                                                                                                                     2. T. aggressivum f. aggressivum strain CBS 100527
                                                                                                                     3. T. aggressivum f. sp. europaeum strain B1
                                                                                                                     4. Trichoderma sp. DAOM 175924 C15
       In addition to a large DNA band, further bands both in the low-molecular-
 weight region and in the very-high-molecular-weight region (>23 kb) were
 observed in the undigested total DNA preparation of T. aggressivum f.
 aggressivum strain CBS 450.95 (Fig 1.). These fragments were resistant to S1
 nuclease and RNase treatment, indicating their double-stranded DNA nature.                   Fig. 3. A gel and its blot hybridized with the 5.0 kb fragment as probe
                                                                                              1. The total DNA of T. aggressivum f. aggressivum strain CBS 450.95
                                                                                                 digested with EcoRI
                                                                                              2. Undigested total DNA of strain CBS 450.95                 1. 2. M           1. 2.
                                                                            M
                                                                                              M: pUC-mix molecular size marker                                     - 19 .3
                                                                                                   Although plasmid ladders derived from                           - 7.7
                                                                                             circular mitochondrial plasmids have already been                     - 5.5
      Fig. 1. The undigested total DNA of T. aggressivum f.                                  reported in some Trichoderma isolates, the
                                                                                - 23.1                                                                             - 4.2
      aggressivum strain CBS 450.95.                                            - 9.4        complete sequence of only a single 2.6 kb plasmid
                                                                                - 6.6                                                                              - 3.3
      M: HindIII-digested λ molecular size marker                               - 4.4
                                                                                             detected in T. harzianum strain T95 is known.
                                                                                                                                                                   - 2.7
                                                                                             Therefore we started to clone and sequence the                        - 2.3
                                                                                             plasmid of the T. aggressivum f. aggressivum strain
                                                                                - 2.3                                                                              - 1.9
                                                                                - 2.0        CBS 450.95. The EcoRI restriction enzyme cut the
                                                                                             plasmid molecules into three pieces (Fig. 3)
                                                                                             suggesting the size of the plasmid around 10 kbp.
                                                                                             The sequence of the smallest fragment was determined, and the sequence analysis
                                                                                             revealed that it has no sequence homology neither at DNA nor at aminoacid level
                                                                                             with any mitochondrial plasmid or other DNA sequences accessible in the Genbank
                                                                                             database.
                                                                                                   Further studies are in progress to determine the sequence of the whole plasmid
Acknowledgements                                                                             and to clarify whether these plasmids have any effect on the virulence of the
This work was supported by the Hungarian Government with the grant OM-00083/2004.            harboring strains.