PCR-Diagnostic of Septoria nodorum Berk. Aggresivenes in Wheat Tissue - Biotechnology of Fungal Genes - page 289

To obtain culture medium, fungal mycelium of different strains was
grown for 14 days in Czapek liquid medium containing 1×10 -2 % M H 2 PO 4 ,
2×10 -2 % NaNO 3 , 5×10 -3 % MgSO 4 , 5×10 -3 % KCl, 1×10 -6 % FeSO 4 . 0.5% glucose
was used as a source of carbohydrates during cultivation of fungi in the
culture medium.
The enzyme activity of CDA of S. nodorum was determined in culture
liquid using titration method on multimeter «Expert-1234» (Econix-Expert,
Russia) and chitin. Titrant NaOH was gradually added to the test portion
with chitin. After each addition of titrant the readings were recorded.
Titration curve of chitin has strongly pronounced S-shaped type. Control
chitin with certain degree of acetylation of 85% (ZAO “Sonat”, Russia)
was used.
DNA from mycelium and infected plants was isolated by phenol-
detergent method (Graham 1978). Isolation and purifi cation of RNA were
carried out by P. Chomczynski and N. Sacchi (1987) method.
For PCR we used primers to gene variable fragment CDA of fungus
S. nodorum (table 1). They were selected using «Primer Select» software
(DNAStar) on the basis of nucleotide sequences of the CDA gene of
fungi Mucor rouxii, Saccharomyces cerevisiae, Colletotrichum lindemutianum,
Blumeria graminis, Cryptococcus neoformans, Candida albicans, from a database
GeneBank ( http://www.ncbi.nlm.nih.gov/entrez) . The size of the amplicon
was presumably ~ 387 bp (Figure 1).

Table 1. The chitin deacetylase (CDA), internal transcribed spacer (ITS) and β -tubulin (Tub)
primers sequences of S. nodorum.

Gene Primer sequences (5'-3') Ref.

CDA For CGTGGCGCTCTCGATGGTGACT

http://www.ncbi.
nlm.nih.gov/entrez

Rev GTGGGCAAGGACAATGGGGTGAC

ITS For TCCTCCGCTTATTGATATGC

White et al. 1990

Rev GGAAGTAAAAGTCGTAACAAGG

Tu b For TGGTATGGGTACGCTTTTGATCTC

Fraaije et al. 1999

Rev GTAGCGACCGTTGCGGAAGTCAGA

To confi rm that amplicon is a fragment of CDA gene, its sequencing
was carried out with sequencer ABI Prism 310 Genetic analyser (ABI,
USA). After DNA sequencing of amplicons it was found that a nucleotide
sequence coincided with the data of the corresponding gene fragment CDA
SN15 strain of the fungus from the genebank at 99% (Figure 1). Moreover,
we found individual replacement in the nucleotide sequence of the DNA
fragment of the studied strains 4VD, 6VD, 9MN and Bas1. The data obtained