IDENTIFICATION OF SUNFLOWER PATHOGENIC FUNGUS PLENODOMUS LINDQUISTII USING PCR WITH SPECIES-SPECIFIC OLIGONUCLEOTIDE PRIMERS

Plenodomus lindquistii causes Phoma black stem of sunflower which is the most common stem disease of this crop in Russia. The diagnostics of both field specimens and pure cultures of P. lindquistii is troublesome. Molecular methods involving the use of the PCR are rapid diagnostic express tests that can precisely identify and detect fungal species. The aim of this study was to develop species-specific oligonucleotide primers for selective amplification of P. lindquistii DNA. The primers LepliF2/LepliR2 were designed on the basis of ITS region analysis and showed stable amplification of the target fungus DNA with no cross-reaction with other fungal species. The primers are recommended for express detection of the causative agent of Phoma black stem of sunflower. This is the first PCR assay that could be used to rapidly reveal and identify this pathogen.

The diagnostics of P. lindquistii under the field conditions is rather difficult because Phoma black stem can be confused with Phomopsis stem canker (causal agents are Diaporthe spp.). Identification of P. lindquistii isolates is usually based on morphological criteria of asexual structures: pycnidia and conidia, but it is often unreliable due to substantial morphological similarity of many related phoma-like species. Correct identification of P. lindquistii in pure culture is laborious, time consuming, and requires special conditions and different culture media.
Molecular methods based on PCR are rapid diagnostic express tests that can contribute to detection and precise identification of fungal species in vitro. Nuclear rDNAs particularly in the internal transcribed spacer (ITS) regions are good targets for phylogenetic analysis in fungi (Bruns et al. 1991). It was demonstrated that oligonucleotide specific primers targeting the ITS region selectively detect many agriculturally important fungi including sunflower pathogen Macrophomina phaseolina (Babu et al., 2007) and some Phoma-like fungi, e.g. P. lingam and P. biglobosus (Mahuku et al., 1996).
Currently there are no molecular techniques based on PCR for correct identification of P. lindquistii -the causal agent of Phoma black stem of sunflower. The aim of this study was to develop specific oligonucleotide primers and to subsequently evaluate their efficiency and specificity for identification and detection of P. lindquistii.

Materials and Methods
Fungal isolates. As a result of the extensive studies of fungal biodiversity on sunflower carried out in 2015-2019 in different geographical locations in Russia 177 P. lindquistii isolates were collected by authors from the surface of sterilized stems exhibiting typical symptoms of Phoma black stem. All isolates were stored in the collection of pure cultures of the All-Russian Institute of Plant Protection (VIZR, St. Petersburg).
DNA extraction, PCR and sequencing. Mycelium was obtained from cultures, incubated on potato sugar agar (PSA) and macerated with 0.3 mm glass sand on a MM400 mixer mill (Retsch, Germany). Genomic DNA was then extracted according to a standard CTAB/chloroform method (Doyle, Doyle, 1990).
Four isolates, i.e. one from Lipetsk region (MF Ha15-001) and three from Krasnodar territory (MF Ha16-001, MF Ha16-004, and MF Ha16-005), were selected for sequencing of ITS region. The primers ITS1 and ITS4 (White et al., 1990) were used to amplify the ITS region. The amplification reactions had a total reaction volume of 25 μl which was composed of dNTPs (200 μМ), each of the forward ITS1 and reverse ITS4 primers (0.5 μМ), Taq DNA-polymerase (5 U/μl), 10× PCR buffer with Mg2+ and NH 4 + ions and total genomic DNA (approx. 1 ng). The PCR conditions were as follows: predenaturation of DNA at 95 °C for 5 min; 35 cycles of denaturation at 92 °C for 50 s, annealing at 55 °C, 40 s, and elongation at 72 °C for 75 s; followed by a final elongation step for 5 min at 72 °C.
Amplicons were purified according to the standard method with a DNA-binding silica matrix (Boyle, Lew, 1995). Visualization and concentration measurements of the purified PCR products were implemented by electrophoresis in 1 % agarose gel stained with ethidium bromide and MassRuler 1000 bp as a marker of concentration.
Amplicons were sequenced by Sanger's method (1977) on ABIPrism 3500 (Applied Biosystems -Hitachi, Japan), with the Big Dye Terminator v3.1 Cycle Sequencing Kit (ABI, Foster City, USA), according to the manufacturer's instructions. All sequences were deposited in the GenBank with the following accession numbers: MK495985, MK495986, MK495987, and MK495988.
Development of specific oligonucleotide primers. Four sequences obtained during this study, reference sequence of the ex-type culture of P. lindquistii CBS 381.67 and sequences of other fungi were aligned using the ClustalX 1.8 (Thompson et al., 1997). The regions, which were conserved among the isolates and specific for P. lindquistii, were selected to design species specific oligonucleotide primers. Three pairs of primers were designed using Primer3plus online software with default options. The parameters such as percentage of G+C content and absence of self-complementarity were analyzed by Primer3plus. Sequences, annealing temperature and size of product are listed in the Table. The theoretical specificity of the primers set was checked with the sequences from the other fungi in GenBank by the BLASTn analysis.
The PCR conditions were as follows: predenaturation of DNA at 94 °C for 2 min; 30 cycles of denaturation at 92 °C for 50 s, annealing at according temperature (Table) for 30 s, and elongation at 72 °C for 75 s; followed by a final elongation step for 5 min at 72 °C. Specificity evaluation of oligonucleotide primers was carried out by PCR with DNA of eight P. lindquistii isolates (MF Ha16-001 -MF Ha16-008) as positive amplification control. As negative amplification control we used the DNA of the next 16 isolates representing various groups of fungi, including both ascomycetes and basidiomycetes, i.e.

Results and Discussion
The primers LepliF/LepliR failed to amplify ITS region of eight tested P. lindquistii isolates. Whereas primers LepliF2/ LepliR2 and LepliF3/Lepli3R yielded single amplified product each of 250 and 180 bp respectively (Fig. 1). However, amplification with the primers LepliF3/LepliR3 generated the target product for six isolates out of eight. Amplification with primers LepliF2/LepliR2 was successful for all DNA samples (Fig. 1).