Hodnocení citlivosti vybraných peckovin k izolátům fytoplazmy ESFY s využitím kvantifikace FLO pomocí metody qPCR

Ing. Tomáš Kiss

Doctoral thesis

Evaluation of the sensitivity of selected stone fruit species to phytoplasma ESFY isolates using phytoplasma quantification by qPCR method.

Abstract:

Úlohou dizertačnej práce bolo porovnať dostupné metódy kvantifikácie DNA fytoplaziem pomocou real-time PCR. Ďalej boli molekulárne charakterizované izoláty fytoplazmy ‘Candidatus Phytoplasma prunorum’ v Českej republike a nakoniec bola definovaná citlivosť a náchylnosť podpníkov a odrôd z rodu Prunus k infekcii fytoplazmou ‘Ca. P. prunorum’. Pre porovnanie real-time PCR protokolov detekujúcich fytoplazmy zo skupiny 16SrX boli vybrané protokoly využívajúce interkalačné farbivá i hydrolyzačné TaqMan sondy. Protokoly boli testované na skupine DNA vzoriek získaných z prirodzene infikovaných stromov. Všetky testované protokoly potvrdili svoju špecifitu, no líšili sa hlavne v citlivosti detekcie. Ako najmenej citlivá bola vyhodnotená štandardná detekčná metóda pomocou nested PCR, od ktorej boli protokoly na báze interkalačných farbív 10krát citlivejšie. Nakoniec protokoly na báze hydrolyzačných TaqMan sond boli 10krát citlivejšie než tie na báze interkalačných farbív a boli schopné detekovať i jednotky kópií cieľového fragmentu DNA v reakcii. Úlohou druhej časti bolo molekulárne charakterizovať genotypy ‘Ca. P. prunorum’ v izolátoch zozbieraných z niekoľkých lokalít južnej Moravy a vyhodnotiť ich vzťah so symptomatickým prejavom rastlín. Izoláty boli charakterizované pomocou analýzy štyroch génov (aceF, imp, pnp a secY). Výsledky potvrdili existenciu koinfekcie viacerými genotypmi ‘Ca. P. prunorum’. Najzastúpenejším genotypom génu aceF bol A6, imp I4, secY S2 a pnp P2. Výsledky tiež preukázali, že všetky stromy s nízkou intenzitou symptómov alebo bez symptómov boli infikované genotypom ‘Ca. P. prunorum’ A6, konkrétne haplotypmi A6-P2-I10-S2 a A6-P2-I4-S2. Pre hodnotenie citlivosti genotypov rodu Prunus na fytoplazmu ‘Ca. P. prunorum’ boli použité kombinácie 3 podpníkov (St. Julien A, M-VA-1 a GF-305) a 2 marhuľových odrôd (Hargrand a Poyer), kde každá odroda bola infikovaná iným haplotypom fytoplazmy. Z testovaných podpníkov bol podpník St. Julien A vyhodnotený na napadnutie ‘Ca. P. prunorum’ ako najmenej náchylný a tolerantný, zatiaľ čo podpník M-VA-1 bol náchylnejší a citlivejší a podpník GF-305 bol rovnako náchylný ako M-VA-1 no z testovaných podpníkov bol na ‘Ca. P. prunorum’ najcitlivejší. Výsledky tiež indikovali, že samotný podpník St. Julien A ovplyvňuje i koncentráciu ‘Ca. P. prunorum’ v pletivách na ňu naočkovaných odrôd.

Abstract:

The task of the dissertation thesis was to compare selected real-time PCR protocols for quantification of phytoplasmas. Furthermore, the ‘Candidatus Phytoplasma prunorum’ isolates collected in the Czech Republic were molecularly characterised and finally the sensitivity and susceptibility of rootstocks and varieties of the genus Prunus to infection by ‘Ca. P. prunorum’ was defined. Protocols using both intercalating dyes and hydrolysis TaqMan probes were selected for comparison of real-time PCR protocols detecting phytoplasmas of the 16SrX group. The protocols were tested on a set of DNA samples obtained from naturally infected trees. All tested protocols confirmed their specificities, however they differed in detection sensitivities. The standard nested PCR detection method was the least sensitive, from which the protocols using intercalation dye were 10 times more sensitive. Finally, protocols using hydrolysis TaqMan probes were 10 times more sensitive than those based on intercalating dyes and were able to detect units of copies of the target DNA in the reaction. The task of the second part was to molecularly characterise genotypes of ‘Ca. P. prunorum’ in isolates collected from several places in South Moravia and to evaluate their relationship with the symptomatic manifestation of the plants. Isolates were characterized by analysis of four genes (aceF, imp, pnp and secY). The results confirmed the existence of coinfections of several ‘Ca. P. prunorum’ genotypes in one plant. The most common genotype of the aceF gene was A6, imp I4, secY S2 and pnp P2. The results also showed that all trees with low or no symptom manifestation were infected with the A6 genotype, in particular haplotypes A6-P2-I10-S2 and A6-P2-I4-S2. To evaluate the susceptibility of Prunus genotypes to ‘Ca. P. prunorum’, combinations of 3 rootstocks (St. Julien A, M-VA-1 and GF-305) and 2 apricot varieties (Hargrand and Poyer) were used, where each variety was infected with a different phytoplasma haplotype. Of the tested rootstocks, the rootstock St. Julien A was evaluated as the least susceptible and tolerant to ‘Ca. P. prunorum’, while the M-VA-1 rootstock was more susceptible and sensitive and the GF-305 rootstock was as susceptible as M-VA-1 but the most sensitive of the tested rootstocks. The results also indicated that the St. Julien A also affects the concentration of ‘Ca. P. prunorum’ in the tissues of grafted varieties.

Keywords

‘Candidatus Phytoplasma prunorum’, real-time PCR, Prunus, molekulárna charakterizácia, zvinutka listov, chloróza listov

Keywords

‘Candidatus Phytoplasma prunorum’, Prunus, molecular characterisation, leafroll, leaf chlorosis, real-time PCR

Language used: Czech

Date on which the thesis was submitted / produced: 19. 7. 2021

Thesis defence

• Supervisor: Ing. Tomáš Nečas, Ph.D.
• Reader: Jaroslav Salava, Ph.D., doc. Ing., Jana Fránová, externi

Mendel University in Brno

Faculty of Horticulture

Doctoral programme / field:
Horticultural Engineering / Horticulture