ANALYSIS OF ‘CANDIDATUS PHYTOPLASMA PRUNORUM’ TITER IN THE TISSUES OF

APRICOT TREES (PRUNUS ARMENIACA L.) THROUGHOUT THE YEAR.

GABRIELA TREMPETIC1, TOMAS KISS*1, TOMAS NECAS1
1 Department of Fruit Science, Faculty of Horticulture in Lednice, Mendel University in Brno, Valtická 337, 691 44 Lednice, Czech Republic
*coresponding author e-mail

1. INTRODUCTION ‘Candidatus Phytoplasma prunorum’, the causal agent of European stone fruit yellows (ESFY) is one of the most detrimental
pathogens of stone fruit trees (genus Prunus) in Europe (Seemuller and Schneider, 2004). It is an unculturable gram-positive cell-wall deficient
bacteria harboring phloem tissues (IRPCM, 2004). One of the most affected species is apricot (P. armeniaca), where susceptible cultivars may render
unproductive 8 to 10 years after planting (Marcone et al., 2010). Detection efficiency of ‘Ca. P. prunorum’ during the year is not the same (Jarausch
et al., 1999), which suggests uneven phytoplasma distribution in plant tissues throughout the year. Real-time PCR enables absolute but also relative
quantification of phytoplasma in plant tissues during the year. The work was aimed to monitor ‘Ca. P. prunorum’ titer during the year and analyse the
relationship of phenological phases and weather conditions with the quantity of ‘Ca. P. prunorum’.

2. MATERIALS AND METHODS

Three different apricot genotypes (H980/74; H990/177 and
H990/95) with 3 ‘Ca. P. prunorum’ positive trees per each genotype
were used in the study. Sampling was performed once a month. From
Jan to May one-year-old shoots and from Jun to Dec annual shoots
were sampled. DNA extraction from phloem tissue was performed
according to modified protocol by Maixner et al. (1995). The real-time
PCR protocol by Christensen et al. (2004) was used for phytoplasmal
(16 S gene) and plant (18 S gene) DNA detection. Real-time PCR was
performed in Biorad CFX 96 (Biorad, CA, USA) detection system and
the results were analysed in Biorad CFX Maestro software (Biorad, CA,
USA). For relative quantification the ddct method (Livak and
Schmittgen, 2001) with the 18 S gene as a reference was used. For the
absolute quantification, the standard curve was used for calculation of
phytoplasmal cells per gram of phloem tissues.
Phenophases and growth patterns were checked regularly during
the year and meteorological data were recorded on automatic
meteorological weather station situated in the orchard.
For correlation analyses software Statistica 14 (Tibco, CA, USA) was
used.

Figure 1. Absolute (left) and relative (right) quantity of ‘Ca. P. prunorum’ in three apricot
hybrids during the year.

Table 1. Correlation coefficients between different quantification methods and

quantifications and meteorological data.

* refers to significant correlation (p˂ 0,05) according to Spearman's correlation method.

Figure 2. Meteorological data and phenophases in 2022.

3. RESULTS AND DISCUSSION

Phytoplasma titer was stable in one-year-old shoots from the beginning of the year with a slight drop in May (Fig.1). Phytoplasma titer was increasing in annual
shoots from June until September and remained stable or slightly decreased towards the end of the year. Correlation between relative and absolute quantification
was high or very high (Tab.1). Slight differences were observed between both quantification methods, especially in H990/177 and H980/74 where the drop of ‘Ca. P.
prunorum’ in May is more obvious in relative quantities (Fig.1).
From meteorological data, the average day temperature (ADT) gave the best correlation with phytoplasma titers (Tab. 1). ADT corresponded better to relative
quantities (moderate negative correlation) than to absolute quantities, where apart from moderate negative correlation, no correlation was observed at H 990/95.
Although low to moderate negative correlations were observed between other meteorological data and quantification methods, they were not consistent in all
hybrids and are not suitable for the estimation of phytoplasma fluctuation in plant tissues.
A significant drop in ‘Ca. P. prunorum’ quantity was observed between May and June in all three hybrids (Fig. 1). This drop happened around June fruit drop (Fig.
2), which is connected to a change in phytohormone and carbohydrate concentrations in plant tissues. These changes could affect the phytoplasma content in the
tissues. However, it could be also caused by the transition of sampling sources (one-year-old shoots to annual), but, the phytoplasma content started to decrease in
one-year-old shoots already during the first growth period (Apr-May). And, on the contrary, the phytoplasma content in annual shoots was gradually increasing
during the second growth period and slightly decreasing after leaf senescence (Oct-Dec), which can be connected to the translocation of carbohydrates to the trunk
and roots.

4. CONCLUSION
Correlation between absolute and relative quantification of ‘Ca. P. prunorum’ is high
or very high. From meteorological data, the average day temperature had the best
correlation with relative ‘Ca. P. prunorum’ quantities (moderate negative correlation).
Phytoplasma titer in plant tissues is affected by phenophases, especially during the June
fruit drop. Phytoplasma content also decreased during the first growth period and
increased during the second growth period.
6. ACKNOWLEDGEMENT
Plant materials were financial supported by the Ministry of Agriculture of the Czech Republic, subsidy programme No. 6.2.10/MZE-
62216/2022-13113 “National Program of Conservation and Utilization of Plant Genetic Resources and Agrobiodiversity,” This
research used the infrastructure acquired by project CZ.02.1.01/0.0/0.0/ 16_017/0002334 Research Infrastructure for Young
Scientists, which is co-financed by the Operational Program of Research, Development and Education.
5. REFERENCES
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