Recovery of Vitis vinifera L. cv. ‘Kékfrankos’ from ‘bois noir’ disease

Investigation of diseases caused by phytoplasmas, a group of cell-wall-less gram-positive bacteria has received significant attention in plant pathology. Grapevine is a host of two, genetically distinct phytoplasmas: Line Flavescence dorée (FD) phytoplasma associated to ‘flavescence dorée’ and ‘Candidatus Phytoplasma solani’ responsible for ‘bois noir’ (BN) disease. In the current study, we focused on BN diseased grapevines (Vitis vinifera L. cv. ‘Kékfrankos’), measured their photosynthetic performance and leaf hydrogen peroxide (H2O2) concentration. The latter is generally considered as a key molecule in the process of ‘recovery’ which is a spontaneous and unpredictable long-term remission of disease symptoms. This phenomenon also occurred during the time of our experiment. Infection resulted in reduced gas exchange performance and maximum quantum efficiency of PSII with an increased regulated non-photochemical quenching of PSII and H2O2 concentration. Changes in gas exchange seem to be a systemic response, while reduced photochemistry is a local response to ‘Ca. P. solani’ infection. H2O2 accumulation in BN phytoplasma infected plants, unlike in FD disease, was found to be a typical response to the appearance of a biotic stressor.

Phytoplasmas are pleomorphic, cell-wall-less prokaryotes with a cell size under 1 μm in diameter. Their genome is extremely small among bacteria (0.6-1.6 Mbp) and lacking many pathways synthesizing important metabolites (Kube et al. 2012), which makes them obligate parasites of their plant hosts and insect vectors (Bertaccini and Duduk 2009). They are grouped in the genus 'Candidatus Phytoplasma' and further divided in subgroups based upon their gene sequence coding 16S rRNA. The two most important ones infecting European grapevines are from distinct subgroups. Flavescence dorée (FD) phytoplasmas are part of subgroups 16SrV-C and 16SrV-D and are associated to 'flavescence dorée' disease, while 'Candidatus Phytoplasma solani' belongs to subgroup 16SrXII-A and is associated with 'bois noir' (BN) disease (Lee et al. 2000). As these phytoplasmas induce identical symptoms, they cannot be differentiated by visual inspection. Shoots become partially non-lignified and have shorter internodes. Leaves roll downwards and change their colour during vegetation: white varieties become yellow to golden, red varieties become reddish to purple. Flowers and berries wither and may also die (Kölber 2011), resulting in great economic loss. Despite the alterations in essential functions, the infection may disappear spontaneously. This phenomenon is called 'recovery' and it has been highly investigated but mostly in connection with FD disease (Musetti et al. 2007;Gambino et al. 2013). Different models predicting infection and recovery fluencies were suggested in the past few years (Panassiti et al. 2015(Panassiti et al. , 2017Rotter et al. 2018;Tomkins et al. 2018). In the case of FD and other phytoplasma-diseases in different cultivated plants the key element of recovery is hydrogen peroxide (H 2 O 2 ) accumulation which serves as a signal to induce defense responses in plants, such as the Systemic Acquired Resistance (SAR) (Musetti et al. 2004(Musetti et al. , 2005(Musetti et al. , 2007Gambino et al. 2013). In our study, H 2 O 2 concentration and photosynthetic activity of BN phytoplasma infected grapevine (Vitis vinifera L. cv. 'Kékfrankos') leaves were measured in midsummer, when interfering effects of seasonal physiological changes do not occur.
The study was conducted over a 2-year period, 2014-2015, in a commercial vineyard located near Villány (Villány Wine District, South-Hungary; latitude 45°51′ N, longitude 18°26′E; elevation 120 m asl). Twenty-sixyear-old vines of V. vinifera L. cv. 'Kékfrankos' (syn. 'Blaufr nkisch') were studied under non-irrigated conditions. 'Kékfrankos' vines were grafted on a commonly used rootstock variety 'TK5BB' (V. berlandieri x V. riparia). The soil was a Ramann-type brown forest soil mixed with clay. Vines were grown with 3 × 1 m vine spacing with North-South row direction. Vines were cordon-trained and hand-pruned in umbrella system (vertically shoot-positioned trellis). The site is situated within the Praeillyricum (plant geographical district), which on average receives 680 mm of precipitation per year, 2010 h of sunshine annually and with an annual mean temperature of 10.8°C (Dövényi 2010 'Ca. P. solani' infection was confirmed by our laboratory in 2014 (data not shown). For the infection test, whole leaves were frozen in liquid nitrogen and ground with a mortar and pestle. A purification step was carried out (Xu et al. 2004) followed by a standard CTAB based total DNA extraction protocol. Multiplex nested PCR was performed to detect the phytoplasmas associated to FD or BN, respectively FD phytoplasma or 'Ca. P. solani', following the protocol suggested by the European and Mediterranean Plant Protection Organisation (EPPO 2007). Amplicons were visualised with 1.2% agarose gel electrophoresis stained with ECO Safe Nucleic Acid Staining Solution 20,000x (Avegene Life Science, Taipei, Taiwan) fluorescent dye. Plants were retested for infection in July and September 2015 ( Fig. 1A and B). FD phytoplasma was not detected in any of the examined grapevines. Although 'Ca. P. solani' was detected only in one plant in July (despite the rolling and discoloration symptoms) but its presence was verified in three grapevines in September, proving that DNA based methods for phytoplasma detection are more reliable from samples collected in autumn. Some of the examined grapevines went through remission and the pathogen was not detected from leaf samples collected in 2015 ( Fig. 1.).
Physiological measurements were performed in July 2015 (Table 1). Photosynthesis was assessed by measuring gas exchange of leaves (same ones were used for pathogen detection) with an LCA-4 type open system IRGA (ADC LCA-4 Bioscientific Ltd., Hoddesdon, UK). Photosynthetic rate (A, μmol CO 2 m −2 s −1 ), transpiration rate (E, mmol H 2 O m −2 s −1 ), stomatal conductance to H 2 O (g s , mol H 2 O m −2 s −1 ), intercellular CO 2 concentration (C i , μmol CO 2 mol −1 ) was measured and water use efficiency (WUE, μmol CO 2 / mmol H 2 O) was calculated. Three records (three technical repetitions) were taken from one leaf and means were used in statistical analyses. Then five 0.8 cm diameter discs were cut from the leaves (including veins), placed in a falcon tube filled with 6% TCA (trichloroacetic acid) and kept on ice during transportation to the laboratory to measure H 2 O 2 concentrations. The assay was performed as described earlier (Mátai and Hideg 2017). Leaves from the next node showing the same symptoms were removed from plants, stored in water during transportation from field to laboratory and kept in darkness for 45 min before chlorophyll fluorescence measurements with the MAXI-version of the Imaging-PAM (Heinz Walz GmbH, Effeltrich, Germany). Maximum (Fv/fm) and effective (Y(II)) PSII quantum yields were calculated according to Genty et al. 1989. Yields of regulated (Y(NPQ)) and non-regulated (Y(NO)) nonphotochemical quenching were determined according to Klughammer and Schreiber 2008. Differences between healthy, recovered and infected group of leaves were assessed with two-sample Student's t-tests for either equal or unequal variances, depending on results of F-tests using MS Excel Analysis ToolPak (Version 2007, Microsoft Corporation, Redmond, WA, USA). Significantly different (p < 0.05) means are marked with different letters in Table 1.
To our knowledge, effect of 'Ca. P. solani' infection on gas exchange performance of grapevines cv. 'Kékfrankos' was examined for the first time in this study. As it was expected from the visual symptoms and the economic impact of the disease, infected plants had impaired CO 2 assimilation (79% of healthy plants), transpiration (86% of healthy plants), stomatal conductance (61% of healthy plants) and intercellular CO 2 concentration (90% of healthy plants). In contradiction with the results of a previous study examining V. vinifera L. cv. 'Chardonnay' (Endeshaw et al. 2012) water use efficiency was unaffected which may indicate that 'Kékfrankos' is possibly a less sensitive cultivar to 'Ca. P. solani' infection. Another study with BN phytoplasma infected V. vinifera L. cv. 'Chardonnay' plants reported that infection decreases the maximum quantum efficiency of PSII (Bertamini et al. 2002). However, these authors found a more intense effect than in the  O m −2 s −1 ), intercellular CO 2 concentration (C i , μmol CO 2 mol −1 ) water use efficiency (WUE, μmol CO 2 / mmol H 2 O), maximum yield of PSII (Fv/fm), effective yield of PSII (Y(II)), regulated non-photochemical quenching (Y(NPQ)), non-regulated non-photochemical quenching (Y(NO)), hydrogen peroxide content (H 2 O 2 , nmol g fw −1 ). Different letters represent significant (p < 0.05, two-sample Student's t test) difference between means of groups case of our study (92% of healthy plants). This also may suggest that the examined cultivar is different in susceptibility or it could be due to the different sampling time. Moreover, we further analysed the light acclimated photochemical and non-photochemical yields of examined grapevines, that are missing from the literature, and found a significant increase in Y(NPQ) (105% of healthy plants). Accumulation of H 2 O 2 in infected leaves (196% of healthy plants) is a typical biotic stress response described in many papers (e.g. Apel and Hirt 2004;Foyer and Noctor 2005). Recovered plants showed the same or even more decreased gas exchange characteristics as the BN phytoplasma infected plants (A, 78% and 79%, respectively; E, 76% and 86%, respectively; g s , 58% and 61%, respectively; C i , 91% and 90%, respectively) but the same photochemistry (Fv/fm, 100%; Y(NO), 107%) and H 2 O 2 concentration (94%) as the healthy ones. This shows that recovery is not equal to the total disappearance of physiological changes induced by phytoplasma infection but the maintenance of systemic responses.
In conclusion, effects of 'Ca. P. solani' infection on gas exchange performance, photochemical processes and leaf H 2 O 2 content of V. vinifera L. cv. 'Kékfrankos' plants were examined first time in this study. The presence of the pathogen resulted in decreased photosynthesis and elevated H 2 O 2 concentration which is a typical biotic stress response. Among these, only low gas exchange parameters were maintained after the plants went through recovery. H 2 O 2 levels in the examined recovered plants were the same as in the healthy ones.
In the literature, one may find that plants recovered from other phytoplasma diseases had much higher leaf H 2 O 2 concentration than healthy plants (Musetti et al. 2005). In other case the level of H 2 O 2 in the leaves rather correlated with the severity of the symptoms (Pitino et al. 2017). Our findings are more concordant with the latter observations. However, our results are based on a small number of cases. Therefore, further research is necessary on a bigger group of plants to verify these results and to understand the mechanisms of recovery in 'Kékfrankos' grapevines.
Acknowledgements Open access funding provided by University of Pécs (PTE). We thank Ferenc Ugor oenologist and owner of the vineyard for his permission to collect samples and perform field measurements. We thank Prof. Ernő Szegedi for providing FD and BN positive control DNA samples and Prof. Éva Hideg for her help with PAM measurements.
Author contributions AM and GJ conceived and designed the study. AM and PT collected samples and performed measurements. AM carried out data analysis. AM wrote the manuscript with the participation of PT and GJ.
Funding information The project has been supported by the European Union, co-financed by the European Social Fund Grant no.: EFOP-3.6.1.-16-2016-00004 entitled by Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs; and by the Higher Education Institutional Excellence Programme of the Ministry for Innovation and Technology in Hungary, according to its decision TUDFO/47138/ 2019-ITM within the framework of 'Innovation for sustainable and healthy living and environment' thematic programme of the University of Pécs.

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