FUNGI AND LICHENS IN THE BALTICS AND BEYOND XVIII ...

DIRECT AMPLIFICATION OF FUNGAL DNA FROM VARIOUS SAMPLES
A. KAČERGIUS
Institute of Botany, Nature Research Center, Žaliųjų ežerų Str. 49, LT-08406, Vilnius,
Lithuania
E-mail: audrius.kacergius@botanika.lt
Thermo Scientific Phire Plant Direct PCR Kit enables DNA amplification directly
from various plant tissues, including fungal samples: mycorrhiza, diseased plant parts and
axenic cultures. Such procedure saves both time and costs. The simple protocol for target
fungal DNA amplification without primary DNA purification is presented. The Phire Plant
Direct PCR Kit contains reagents and tools for two alternative methods: direct and dilution
protocols. Dilution protocol is also suitable for storing the DNA samples for short periods at
+4°C, and for long-time storage at -20 °C. The kit employs Phire Hot Start II DNA
Polymerase, a specially engineered enzyme that exhibits extremely high resistance to many
PCR inhibitors found in plants and fungi. We applied the method for mycorrhizal fungi,
amplifying part of rRNR gene, as well as phytopathogenic fungi from axenic cultures and
directly from diseased plants. Suitable amplicons were sequenced after conventional
EXO/SAP purification, manually editing ambiguous readings, and queried in sequence
database at NCBI using blastn algorithm. These protocols were employed for identification of
mycorrhizae in the project “Changes in biotic and abiotic ecosystem components induced by
an invasive species: case study of the great cormorant”, funded by a grant (No. LEK-23/2010)
from the Research Council of Lithuania.
PHYLOGENETIC ANALYSIS OF Phomopsis ISOLATES FROM DIFFERENT
HOST PLANTS
A. KAČERGIUS 1 , R. RODEVA 2 , J. GABLER 3 , Z. STOYANOVA 2
1 Institute of Botany, Nature Research Center, Žaliųjų Ežerų Str. 49, LT-08406, Vilnius,
Lithuania
2 Institute of Plant Physiology and Genetics of BAS, 1113 Sofia, Bulgaria
3
Institute for Epidemiology and Pathogen Diagnostics of the Julius Kühn-Institute (JKI) –
Federal Research Centre of Cultivated Plants, D-06484 Quedlinburg, Germany
E-mails: audrius.kacergius@botanika.lt, r.rodeva@abv.bg, jutta.gabler@jki.bund.de
Phomopsis is a large genus of plant-inhabiting Coelomycetes fungi distributed
worldwide. Phomopsis is the only known anamorph of Diaporthe. Fungi in the genus
Phomopsis and its sexual state Diaporthe cause serious diseases on a wide variety of hosts.
Lately new emerging plant diseases caused by Phomopsis spp. have been established on
various hosts in Bulgaria, Germany and Lithuania. So far their identification to the species
level was unsatisfying because of the insufficient morphological and cultural characteristics.
Phylogenetic analysis was performed on 76 isolates of Phomopsis using sequences from the
Internal Transcribed Spacers 1 and 2, including 5.8S region of the nuclear ribosomal DNA. In
this study we used NCBI database, CLUSTALW and DNASTAR Lasergene 8.1.4 softwares.
The results from phylogenetic trees indicated, that some non-identified to species level
isolates had very close relationships to Diaporthe angelicae (97–99 %) and to Phomopsis
diachenii (98–99 %). Bootstrap analysis showed strongly defined clade (99 %) of Phomopsis
isolates from Lactuca sativa. The results of present study confirm previous observations that