Loop-mediated isothermal amplification (LAMP) assay for rapid detection of Phoma macdonaldii, the causal agent of sunflower black stem

Phoma macdonaldii, the causal agent of sunflower black stem, severely affects sunflower yield and quality. A rapid and sensitive detection method is necessary for diagnosis of this disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the pathogen from diseased sunflower tissues. The LAMP primers were designed targeting the rDNA region of the fungus. The reaction condition was optimized to 60°C water baths for 45 min. The detection limit of the LAMP assay was 100 fg DNA or 10 conidia/g seeds. The LAMP assay was validated by detecting P. macdonaldii from infected sunflower tissue samples including leaves, stems and seeds, and applied to seed samples randomly collected from sunflower fields. This LAMP assay will be useful to estimate disease prevalence and implement sustainable management of sunflower black stem.

Mycoses as causes of biotic stress in sunflower production

Osjetljivost suncokreta na biotski stres uvelike određuje vrijednosti agronomskih svojstava. Uzročnici biotskog stresa mogu biti različiti, a u svijetu pa tako i u Hrvatskoj se po svome utjecaju izdvajaju bolesti. Uslijed pojave različitih biljnih bolesti, u pojedinim godinama prinosi zrna i ulja suncokreta mogu biti reducirani i preko 50%. Najčešći uzročnici bolesti suncokreta su gljive, a prema stupnju štete koje prave ističu se Sclerotinia sclerotiorum, Diaporthe/Phomopsis helianthi i Plasmopara halstedii te u nešto manjoj mjeri Alternaria helianthi, Phoma macdonaldii, Puccinia helianthi, Verticillium spp., Botrytis cinerea, Macrophomina phaseolina i Erysiphe cichoracearum. Kreiranje otpornih/tolerantnih linija iz divljih formi suncokreta, a zatim transfer poželjnih gena u komercijalne linije i hibride je najčešći oblik povećanja otpornosti/tolerantnosti linija i hibrida suncokreta na biotski stres. U ovom postupku značajnu ulogu ima primjena molekularnih markera. Pored toga važan je stalni monitoring, ali i pravovremena primjena agrotehničkih, kemijskih i bioloških mjera.

Occurrence of black stem on Helianthus annuus caused by Phoma macdonaldii and resistant evaluation of different sunflower varieties

The first reported case of Phoma macdonaldii Boerema that caused sunflower black stem disease in China was from Xinjiang Uygur Autonomous Region in 2008. Black spindle lesions were observed on sunflower stems in the fields of Inner Mongolia region in China. The causal agents were isolated from the infected stems. Cultures on potato dextrose agar (PDA) plates were whitish with black particles underneath. Dark and pear-like pycnidia with yellowish or dirty whitish exudates were also observed on the surface of the cultures. The cultures produce primarily non-septate conidia. The cultures could infect sunflowers stems by artificial inoculation and developed symptoms consistent with those in the field. Morphological characteristics and molecular identification indicated the isolated causal agents were P. macdonaldii. Phylogenetic analysis based on ITS1, 5.8 s RNA and ITS2 region confirmed the identification. Disease resistance assessments were performed on twenty sunflower varieties and the results suggested that the resistance of seven sunflower varieties to P. macdonaldii was above the average; SC89 and Longkuiza1 were susceptible to P. macdonaldii.

Secondary screening of strains of antagonists to a Phoma pathogen on sunflower

There are presented data of the first stage of the secondary screening of antagonist strains from a collection of the biological methods laboratory of V.S. Pustovoit All-Russian Research Institute of Oil Crops to the aggressive isolate of Phoma macdonaldii Boerema infecting sunflower. We used methods of ‘agar blocks’ and ‘perforated Petri dishes’. As a result of a screening of 55 antagonist strains to Phoma macdonaldii pathogen we selected 17 promising strains-producers of microbiological preparations having biological efficiency exceeding 40.0 %. In control variant without seed treatment sunflower seedlings infection was up to 76.2 %, and maximal biological efficiency of laboratory samples from fungi-producers were determined for a strain M-24 Penicillium sp. (73.8 %); from bacteria of a genus Bacillus – for a strain D-10 Bacillus sp. (74.2%); from bacteria of a genus Pseudomonas – for a strain Sgc-1 Pseudomonas sp. (73.8 %). Maximal colonizing activity were noted for fungi-producers M-24 Penicillium sp. (85.7 %) and Xk-1 Ch. olivaceum (71.4 %), as well as for bacteria of a genus Bacillus – 5B-1 B. subtilis (71.4 %) and a genus Pseudomonas – Oif 2-1 Pseudomonas sp. and 14-3 P. chlororaphis (71.4 %) against Phoma infection in control of 70.0 %.