Seasonal colonisation of apple trees by ‘Candidatus Phytoplasma mali’ revealed by a new quantitative TaqMan real-time PCR approach

Abstract

Apple proliferation (AP), caused by 'Candidatus Phytoplasma mali', is an economically important disease affecting many apple-growing areas in Europe. A new TaqMan real-time PCR assay was established for absolute quantification of 'Ca. P. mali' by using a single-copy gene of the host plant as a reference, which is amplified with the pathogen DNA in a single-tube reaction. Normalised estimates of phytoplasma concentration are ultimately expressed as the number of phytoplasma cells per host plant cell. The assay was used to monitor the 'Ca. P. mali' titre over the course of two growing seasons in roots and branches of symptomatic and asymptomatic but AP-positive apple trees. All 252 root samples from symptomatic and asymptomatic trees tested positive, with an average number of 59.8±5.68 (standard error) and 55.1±9.83 'Ca. P. mali' per host cell, respectively. From the 378 shoot samples analysed, 81% of the symptomatic and only 20% of the asymptomatic samples were AP-positive with an average number of 9.4±1.04 and 0.7±0.13 'Ca. P. mali' per host cell, respectively. This strengthens evidence that not the pathogen occurrence alone but the presence of a certain quantity of 'Ca. P. mali' in the aerial tree sections is involved in symptom expression. In addition, pronounced seasonality of the phytoplasma concentration was found, not only in branches, but also for the first time in roots of symptomatic and asymptomatic apple trees. Highest phytoplasma levels in roots were detected from December to May.

Resistance Inducers and Plant Growth Regulators Show Only Limited and Transient Effects on Infection Rates, Growth Rates and Symptom Expression of Apple Trees Infected With ‘candidatus Phytoplasma mali’

“…DNA isolates were normalized to 10 ng μl -1 and a standard curve-based real-time PCR procedure was used for quantitative analysis of 'Ca. P. mali' as described in Baric et al (2011).…”

“…The underlying mechanism is poorly understood, but several studies indicate that recovered plants have reduced levels of reactive oxygen species (ROS) and scavenging enzymes, particularly ascorbate peroxidases (APX) and catalases (CAT). Several studies have reported that recovered trees were colonized by the phytoplasma in the roots at a similar level as symptomatic plants, whereas the shoots were either phytoplasma-free (Carraro et al 2004) or colonized by lower phytoplasma titres (Baric et al 2011).…”

Die Landwirtschaftliche Forschung im Obstbau Südtirols

“…Der erreger, 'Candidatus Phytoplasma mali', ein zellwandloses bakterium, ist nicht kultivierbar und damit für die erforschung schwer erfassbar. Zudem ist der erreger nicht gleichmäßig in der Pflanze verteilt: er kommt in einem befallenen baum auf Dauer nur in den Wurzeln vor, während er die oberirdischen Pflanzenteile nur saisonal besiedelt (Schaper und Seemüller 1982;baric et al 2011b). auf grund von unsachgemäßer Probennahme unterliegt es einem bestimmten Zufall, ob man den erreger nachweisen kann oder nicht.…”

Amarelo da ameixeira: Modelo de colonização do hospedeiro por um fitoplasma associado à doença

“…`Candidatus Phytoplasma mali´, agente causal da proliferação de ramos em macieira, também teve sua concentração estimada, apresentando valores variáveis de 732 a 2,5 x 10 5 (BARIC et al, 2011). e máxima de 30ºC.…”

“…Outros estudos também têm comprovado a eficiência dessa nova técnica para fins de diagnose e investigações sobre a distribuição sazonal de fitoplasmas em hospedeiros portadores de relevantes doenças, como a proliferação de ramos da macieira, doença da raiz em coqueiro, "Bois noir" em videira, declínio da pereira e o amarelo das fruteiras de caroço (TORRES et al, 2005;ALDAGHI et al, 2007;HREN et al, 2007;MANIMEKALAI et al, 2011;BARIC et al, 2011;BARIC, 2012). A técnica de PCR quantitativo também vem sendo utilizada para melhor entendimento das relações patógeno-vetor.…”