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I
University of Pisa
Symptom observations and multilocus sequence typing
analysis of ‘Candidatus Phytoplasma solani’ strains in
Tuscan vineyards
by
Roberto Pierro
Ph. D. Thesis
Agriculture, Food and Environment
Department of Agriculture, Food and Environment
III
University of Pisa
Symptom observations and multilocus sequence typing
analysis of ‘Candidatus Phytoplasma solani’ strains in
Tuscan vineyards
by
Roberto Pierro
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy in
Agriculture, Food and Environment
Candidate: Roberto Pierro
Supervisor: Dr. Alberto Materazzi
Accepted by the Ph.D School The Coordinator
V A Paola, “… ti serbo fra ciò che il mondo dona di più caro”
VII
Declaration
This thesis is a presentation of my original research work. Wherever contributions of
others are involved, every effort is made to indicate this clearly with due reference to
the literature and acknowledgement of collaborative research and discussions.
This thesis contains no material that has been submitted previously, in whole or in part,
for the award of any other academic degree or diploma.
VIII
Preface
This dissertation is submitted for the degree of Doctor of Philosophy at the University of Pisa. The research described herein was conducted under the supervision of Dr. Alberto Materazzi in the Department of Agriculture, Food and Environment, between November 2015 and February 2019.
Part of this work has been presented in papers published in peer-reviewed journals and proceedings, as follows:
Chapter 2 and 4:
Pierro, R., Passera, A., Panattoni, A., Casati, P., Luvisi, A., Rizzo, D., Bianco, P.A., Quaglino, F., Materazzi, A. (2018). Molecular typing of ‘bois noir’ phytoplasma strains in the Chianti Classico area (Tuscany, central Italy) and their association with symptom severity in Vitis vinifera L. cv. Sangiovese”. Phytopathology 108: 362-373.
Chapter 5:
Pierro, R., Passera, A., Panattoni, A., Rizzo, D., Stefani, L., Bartolini, L., Casati, P., Luvisi, A., Quaglino, F., Materazzi, A. (2018). Prevalence of a ‘Candidatus Phytoplasma solani’ strain, so far associated only with other hosts, in Bois Noir-affected grapevines within Tuscan vineyards. Annals of Applied Biology 173: 202-212;
Pierro, R., Passera, A., Panattoni, A., Casati, P., Luvisi, A., Rizzo, D., Bianco, P.A., Quaglino, F., Materazzi, A. Genetic Variability of “Bois Noir” phytoplasma strains using multilocus sequence typing analysis in Tuscan Vineyards (Central Italy). 5th European Bois Noir Workshop 2018, Ljubljana (SLO), (In press).
1
ABSTRACT
Bois Noir (BN) is a disease of grapevine yellows (GY) complex caused by phytoplasma (Bois Noir phytoplasma, BNp) strains of the species ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) and responsible for serious crop losses in all major vine-growing areas worldwide. BN epidemiological cycle includes several herbaceous plant hosts from which BNp is transmitted by polyphagous insect vectors to grapevine (Vitis vinifera). The great BNp genetic variability and the absence of efficient control strategies represent important factors for its diffusion in different environmental conditions. Currently, BN is the most widespread GY in the world, largely reported in Europe, Southern and Northern America, Asia, Middle East and South Africa. Several studies, based on molecular approach, were carried out in order to improve the knowledge about its genetic structure and dynamics. In the present study, GY symptom observations and Multi Locus Sequence Typing Analysis (MLST) of BNp strains were carried out in a Sangiovese vineyard located in the Chianti Classico area (Tuscany, Central Italy) in order to evaluate symptom intensity and describe the genetic diversity of BNp strains over 3 following growing seasons (2015-2017). Results showed the unique presence of BNp in all symptomatic vines, revealing the highest BN incidence during 2016. Interestingly, statistical analyses carried out between BNp relative quantification and symptom severity classes, revealed a possible high relative concentration of BNp in vines associated with moderate and severe symptoms. Allelic discriminations carried out through TaqMan assay on tuf gene, revealed that BN epidemiological patterns were mainly bindweed-related (Convolvulus arvensis), while vmp1 and stamp nucleotide sequence analyses showed larger variability for stamp gene than vmp1. Moreover, this study identified for the first time BNp strains harboring the sequence variants Vm43
2 (vmp1) and St10 (stamp) (type Vm43/St10), as the prevalent type in the BNp strain population. Further investigations on BNp genetic diversity influencing symptom severity in plant hosts were carried out. In this regard, phylogenetic analyses revealed the possible high virulence of BNp strains grouping in the same vmp1 cluster and mainly associated with vines showing moderate to severe symptoms.
The prevalence of the BNp strain type Vm43/St10, found exclusively in the case study vineyard, have stimulated further genetic studies at Regional level. Analyses carried out on samples collected from the main vine-growing areas in Tuscany confirmed the prevalence of BN strains associated with bindweed-related pathosystem and the large widespread of the type Vm43/St10. Interestingly, such BNp strain type showed a close phylogenetic relationship with ‘Ca. P. solani’ strains, previously found only in
Solanaceae and Plantaginaceae hosts in France and Central Italy, respectively. In
conclusion, it is reasonable to state that BN epidemiology in the Tuscan vineyard agro eco-systems is mainly associated with bindweed, representing one of the most important BNp inoculum source. Moreover, the identification of the BNp strain type Vm43/St10, found exclusively in Tuscany and showing close phylogenetic relationship with phytoplasma strains previously found in weeds in France and Central Italy, could suggest possible recombination or co-evolution events in such area, opening a new scenario in the BNp epidemiological patterns in Tuscany.
3
Acknowledgments
First of all, I would like to express my most sincere gratitude to my Supervisor Dr. Alberto Materazzi, who has been supporting in this challenging journey. Without his knowledge, help and huge patience, this research project would not have been possible. I would especially like to thank Dr. Alessandra Panattoni for her precious helps, remarks and suggestions, aiming to overcome obstacles in all times and situations. Many thanks.
I show my gratitude to Dr. Fabio Quaglino, for giving me friendly and useful advices about the research studies conducted in the last three years.
I am also very grateful to Dr. Andrea Luvisi, who was encouraging and extremely helpful when I needed.
Furthermore, I would like to thank Dr. Domenico Rizzo, for giving me continuous support in these years and the President of TOS.CO.VIT., Dr. Roberto Bandinelli, for his huge wisdom.
My thesis has benefited from valuable comments by Prof. Piero Attilio Bianco and Prof. Gianfranco Romanazzi, which definitely improved this work. I also thank them both. Last but not least, I would like to thank my friends for their generosity and my family for their unlimited love.
5
TABLES OF CONTENTS
CHAPTER 1 1
GENERAL INTRODUCTION AND THESIS OUTLINE 1
1.1. Overview on phytoplasma 2
1.2 Symptomatology and economic impact 4
1.3 Phytoplasma genome 6 1.4 Grapevine Yellows 7 1.5 Bois Noir 9 1.6 Control strategies 12 1.7 Thesis outline 12 1.8 References: 13 CHAPTER 2 19
SYMPTOM OBSERVATIONS IN THE CASE STUDY VINEYARD 19
ABSTRACT 20
2.1 INTRODUCTION 20
2.2 MATERIALS AND METHODS 22
2.2.1 Vineyard description 22 2.2.2 Symptom observations 22 2.2.3 Sampling 23 2.3 RESULTS 24 2.4 DISCUSSION 25 2.5 References 26 CHAPTER 3 28
BOIS NOIR IDENTIFICATION 28
ABSTRACT 29
3.1 INTRODUCTION 29
6
3.2.1 DNA extraction 30
3.2.2 GY phytoplasma detection and relative quantification 31
3.3 RESULTS 32
3.4 DISCUSSION 36
3.5 References 37
CHAPTER 4 39
BOIS NOIR PHYTOPLASMA CHARACTERIZATION IN THE CASE STUDY VINEYARD BY MULTI LOCUS SEQUENCE TYPING (MLST) AND
PHYLOGENETIC ANALYSES 39
ABSTRACT 40
4.1 INTRODUCTION 40
4.2 MATERIALS AND METHODS 42
4.2.1 Bois Noir phytoplasma characterization using tuf gene 42 4.2.2 Bois Noir phytoplasma characterization using vmp1 gene 42 4.2.3 Bois Noir phytoplasma characterization using stamp gene 43 4.2.4 Phylogenetic analysis, selective pressure and association of Bois Noir phytoplasma strains with
symptom severity 44
4.3 RESULTS 45
4.3.1 Bois Noir phytoplasma characterization using tuf gene 45 4.3.2 Bois Noir phytoplasma characterization using vmp1 gene 45 4.3.3 Bois Noir phytoplasma characterization using stamp gene 47 4.3.4 Phylogenetic analysis, selective pressure and association with symptom severity 49
4.4 DISCUSSION 59
4.5 References 61
CHAPTER 5 65
GENETIC DIVERSITY OF BOIS NOIR PHYTOPLASMA IN TUSCANY 65
ABSTRACT 66
5.1 INTRODUCTION 66
5.2 MATERIALS AND METHODS 67
5.2.1 Field observations 67
5.2.2 DNA extraction and phytoplasma detection 68
5.2.3 Genetic characterization of Bois Noir phytoplasma using tuf, vmp1 and stamp genes 68 5.2.4 Phylogenetic analyses and selective pressure on Bois Noir phytoplasma population 69
7
5.3.1 Bois Noir phytoplasma detection 70
5.3.2 Bois Noir phytoplasma characterization 70
5.3.3 Phylogenetic analysis and selective pressure on Bois Noir phytoplasma population 72
5.4 DISCUSSION 80
5.5 References 80
CHAPTER 6 83
CONCLUSION 83
6.1 OVERALL DUSCUSSION AND CONCLUSIONS 84
6.2 References 87
AUTHOR’S CV AND PUBLICATIONS 89
1
Chapter 1
2 1.1. Overview on phytoplasma
Numerous yellows-type diseases of plants, initially related to viral agents, were subsequently associated with prokaryote organisms, morphologically resembling mycoplasmas (mycoplasma-like organisms: MLOs) (Doi et al., 1967). Further studies based on nucleotide sequence analyses of the 16S rDNA allowed the MLOs assignment to a distinct monophyletic taxon in the Mollicutes class, order of the Acholeplasmatales (Lee et al., 2000) and provisionally into genus ‘Candidatus Phytoplasma’, that is assigned to a family of incertae sedis (Firrao et al., 2004; Bertaccini et al., 2014). Currently, Restriction Fragment Length Polymorphism (RFLP) analysis of 16S rDNA fragments amplified from known phytoplasmas allowed their attribution to 33 distinct taxonomic ribosomal groups, divided in several subgroups (Bertaccini et al., 2014).
Phytoplasmas are polymorphic cell wall-less bacteria with variable sizes from 200 to 800 nm (Fig. 1.1). They are intracellular parasites introduced by insect vectors during feeding activity into plant sieve tube elements, from which they move through the plants systemically. In details, phytoplasmas traverse the intestinal tract wall of infected insect vector, multiply in the hemolymph, reaching the salivary glands where they multiply further. Subsequently, when the infected insect feeds on a new host plant, phytoplasmas are generally transmitted in a persistent manner into the phloem tissue along with salivary fluids, allowing spread disease (Moriwaki et al., 2003; Kube et al., 2012).
The phloem-feeding insects transmitting phytoplasmas belong to the families
Cicadellidae, Cixidae, Psyllidae, Delphacidae and Derbidae (Weintraub and Beanland,
2006), therefore their host range is strongly dependent upon feeding preferences of their insect vectors (Alma et al., 1997). Their research proved transovarial phytoplasma transmissions in insects (Alma et al., 1997), while other studies showed the possibility
3 of seed transmission into commercial seedlings of alfalfa, tomato, corn and winter oil seed rape (Khan et al., 2002; Botti and Bertaccini, 2006; Calari et al., 2011; Chung and Jeong, 2014). Further evidences also showed that phytoplasmas can be spread via vegetative propagation such as grafting, cuttings, and other methods using infected plant tissues (Botti and Bertaccini, 2006).
Phytoplasma are associated with diseases in several hundreds of woody and herbaceous plant species (wild and cultivated) all over the world, including many economically important crops where they cause heavy economic losses due to the reduction of yields, fruit quality and plant vitality (Maixner, 2011). The wide diffusion of phytoplasmas is possible thanks to the high pathogen adaptability that allows them to survive as parasites in plant phloem and insect hemolymph modulating their gene expression (Mayer et al., 2008). Interestingly, studies conducted by Oshima et al. (2001), revealed that approximately 33% of the gene expression changes during host switching between plant and insect. Gene changes might involve transporters, secreted proteins, and metabolic enzymes in host-specific way.
4
Figure 1.1. Cross sections of sieve tubes showing phytoplasmas with different sizes and shapes
(Electron micrographs from Bertaccini et al., 2014).
1.2 Symptomatology and economic impact
Plants affected by phytoplasma-related diseases generally exhibit a range of symptoms including leaf yellowing, virescence and/or phyllody, sterility of flowers, proliferation of buds resulting in a witches’ broom behavior, abnormal internodes elongation, generalized stunting green flower (Fig. 2.1) and many other non-specific symptoms resulting from plant stresses (Bertaccini, 2007). Some evidences could explain such symptom expressions. For example, leaf yellowing is caused by the inhibition of photosystem II, causing alteration in carbohydrate synthesis and transportation, together with inhibition of chlorophyll and carotenoids, as described by Bertamini and Nedunchezhian (2001). Witches broom behavior is mainly caused by the loss of apical dominance with an uncontrolled production of axillary shoots and length reductions, while virescence and green flowers are mainly caused by the loss of pigment in the petal cells (Lee et al., 2000).
5 Phytoplasmas are associated with diseases in several hundreds of plant species, including many relevant vegetable and fruit crops and represent the main limiting factor all over the world (Bertaccini and Duduk, 2009). For example, Grapevine Yellows (GY) are largely widespread in Europe, Australia, North and South America, South Africa and Asia; Potato Witches’ Broom (PWB) and Maize Bushy Stunt (MBS) cause severe yield losses in economically important crops, as potato and corn in Central and South America; Rice Yellow Dwarf (RYD) severely affects rice crops in South-Eastern Asia. Moreover, Pear Decline (PD), Apple Proliferation (AP) and other fruit declines represent serious issues in several countries across Europe (Bertaccini and Duduk, 2009).
Figure 2.1. Symptoms associate to phytoplasma diseases in different plant hosts: (A) tomato infected
with stolbur phytoplasma; (B) comparison between healthy (left) and phytoplasma-infected hydrangea (right) flower; (C) virescent and malformed fruits in strawberry; (D) European Stone Fruit Yellows in plum; (E) Apple Proliferation (form Bertaccini et al., 2009; Oshima et al., 2013; Bertaccini et al., 2014).
6 1.3 Phytoplasma genome
Generally, phytoplasma genomes consist of one circular double-stranded DNA chromosome with many gene duplication and redundancy (Oshima et al., 2013). Several plasmids with different sizes were also found in all members of the Aster Yellows (AY) phytoplasma group (16SrI), “stolbur” group (16SrXII) and in some members of the X-disease (16SrIII) and Clover Proliferation (16SrVI) groups (Bertaccini, 2015).
Phytoplasma chromosome is the smallest genome among bacteria: sizes range from 680 to 1600 kb (Bertaccini, 2015). Sequence analysis of 16S rDNA and other housekeeping genes have also shown a low G+C content (21-28%), suggesting their close similarity with mycoplasmas and endosymbiotic bacteria (Wernegreen, 2002; Gasparich et al., 2004).
Phytoplasmas have small genomes since the selection process could have caused the loss of some genes involved in biosynthetic pathways and host responses, such as Microbe/Pathogen-Associated Molecular patterns (MAMPs or PAMPs) (Jones and Dangl, 2006). As a consequence, phytoplasmas became directly dependent on metabolic compounds from their host cells (Bai et al., 2006), preserving the genes for cellular functions, such as DNA replication, transcription, translation, and protein translocation (Kakizawa et al., 2001; Jung et al., 2003). Phytoplasma genome also contains transposon genes, insertion sequences and many genes encoding transporter systems, responsible for their great genetic variability that allowed adaptations in different environments and led to the emergence of distinct phytoplasma lineages (Wei et al., 2008).
Genetic studies identified genes clustered in Sequence-Variable Mosaics (SVMs). SVMs are repetitive and targeted insertions of ancient phage genomes, probably
7 acquired by horizontal transfer (Jomantiene and Davis, 2006). SVMs encode different putative membrane-targeted proteins, potentially involved in the plant-host interactions and putative virulence factors. They represent “platforms for genome plasticity”, allowing specific acquisition of new genes and targeting of mobile genetic elements into phytoplasma chromosome (Bai et al., 2006; Jomantiene and Davis, 2006; Davis et al., 2007; Wei et al., 2008).
Currently, the whole genome sequences have been completed for some phytoplasma strains (Oshima et al., 2004; Bai et al., 2006; Kube et al., 2008; Tran-Nguyen et al., 2008), allowing studies focused on different genetic approaches.
From a holistic point of view, the unique genetic and biological features of phytoplasmas make them interesting microorganisms and the development of media supporting phytoplasma growth (Contaldo et al., 2013; Contaldo et al., 2016) could help further research in physiology and genetics.
1.4 Grapevine Yellows
Grapevine Yellows (GY) represent one of the most important threat for viticulture all over the world. GY are a complex of phytoplasma-associated diseases causing undistinguishable symptoms in grapevine, but differing in their etiology and epidemiology (Belli et al., 2010).
GY phytoplasmas have been characterized and assigned to different groups: Aster Yellows (16SrI), Peanut Witches’ Broom (16SrII), X-disease (16SrIII), Elm Yellows (16SrV), Clover proliferation (16SrVI), Ash Yellows (16SrVII) and Stolbur (16SrXII) phytoplasma groups (Bianco et al., 1993; Davis et al., 1993; Maixner et al., 1995; Bertaccini et al., 2014; Zambon et al., 2018).
8 “Flavescence Dorée” (FD), associated with Elm Yellows group (16SrV) phytoplasmas (Martini et al., 1999), and “Bois Noir” (BN), associated with Stolbur group (16SrXII) phytoplasmas (Quaglino et al., 2013), are surely the most important diseases occurring in all major vine-growing areas worldwide (Bertaccini et al., 1995; Choueiri et al., 2002; Botti and Bertaccini, 2006; Gajardo et al., 2009; Duduk et al., 2010; Salem et al., 2013; Ertunc et al., 2015; Mirchenari et al., 2015).
FD was identified for the first time in France in 1957 (Caudwell, 1957) and it is currently a quarantine pathogen in the main vine-growing areas of Europe.
BN was also reported in France for the first time (Caudwell, 1961) and it is currently widespread in the Euro-Mediterranean countries (Belli et al., 2010), South America (Chile) (Gajardo et al., 2009), North America (Canada) (Rott et al., 2007), Asia (China, Middle East) and South Africa (Choueiri et al., 2002; Duduk et al., 2010; Salem et al., 2013; Mirchenari et al., 2015) (Fig. 3.1). BN is associated with ‘Candidatus Phytoplasma solani’ and -related strains (BNp), classified in the rDNA RFLP 16SrXII-A subgroup. BNp is mainly transmitted to grapevine (Vitis vinifera) by the polyphagous leafhopper Hyalesthes obsoletus, living preferentially and completing its biological cycle on nettle (Urtica dioica), bindweed (Convolvulus arvensis), mugworth (Artemisia
vulgaris) and chaste tree (Vitex agnus-castus) (Alma et al., 1988; Maixner, 1994;
Langer and Maixner, 2004; Sharon et al., 2005).
Other GY diseases were reported in several European countries (Maixner et al., 1995), in the USA (Davis et al., 1998), in Asian countries, such as China (Cai et al., 2016), India (Tripathi et al., 2017; Yadav et al., 2016), Korea (Chung et al., 2005), Japan (Takinami et al., 2013), Thailand (Sarindu et al., 1993), and Australia (Davis et
9 Outbreaks of GY epidemics represent a risk in the vineyard agro-eco systems all over the world with negative economic impacts. Monitoring and control strategies against GY are essential in order to prevent epidemic phytoplasma diffusion, both in historical vine-growing areas in Europe and in more recent devoted vine areas in the USA and Asia.
Figure 3.1. Bois Noir reports (in red) in Vitis vinifera all over the world.
1.5 Bois Noir
In V. vinifera BN induces typical GY symptoms including: vine decline, desiccation of inflorescences, berry shrivel, reduction of growth, irregular ripening of wood, leaf rolling with reddening or yellowing on red and white cultivars, respectively (Belli et al., 2010) (Fig. 4.1).
BNp strains belong to the species ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), subgroup 16SrXII-A (Quaglino et al., 2013).
10 Due to the erratic feeds of H. obsoletus on V. vinifera, infected grapevines are considered a “dead-end host” of BNp.
BN spreads also in areas where H. obsoletus does not occur (Belli et al., 2010; Maixner et al. 2011), and recent studies proved that Reptalus panzeri acts as vector of BNp to grapevine in Serbian and French vineyards (Cvrkovic et al., 2014). In addition, further studies identified several herbaceous hosts directly involved in BN diffusion (Berger et al., 2009; Cvrković et al., 2014; Landi et al., 2015; Marchi et al., 2015; Mori
et al., 2015; Oliveri et al., 2015; Chuche et al., 2016; Kosovac et al., 2016), indicating
that ‘Ca. P. solani’ has a great intra-species variability that allowed its adaptation in different ecosystems. BNp genetic diversity could be a consequence of selective process, mainly influenced both by BNp interactions with plant hosts and insect vectors and by geographic conditions in different climatic areas (Quaglino et al., 2017). In order to investigate BNp genetic diversity in relation with its plant hosts and insect vectors in different geographic areas, several studies were carried out by nucleotide sequence analyses of BNp and ‘Ca. P. solani’ -related strains on molecular markers associated with specific biological features. The most used markers are the genes tuf, encoding the translation elongation factor Tu (Schneider et al., 1997), secY, encoding a translocation protein (Fialovà et al., 2009), vmp1 and stamp, encoding membrane proteins, presumably involved in the interaction with its hosts (Cimerman et al., 2009; Fabre et
11
Figure 4.1. Grapevine Yellows symptoms observed in grapevine on two cultivars (Sangiovese and
Chardonnay): (A-B-C) leaf reddening on Sangiovese; (D) berry shrivel on Chardonnay; (E-F) leaf rolling and yellowing on Chardonnay; (G) irregular ripening on Sangiovese wood.
12 1.6 Control strategies
Pesticides against insect vectors and/or removal of infected plants could be used for to control the outbreaks of phytoplasma disease epidemics.
Other methodologies also consider the use of antibiotics or other chemicals (Bertaccini, 2007), although they are expensive and prohibited in several countries. Moreover, methodologies based on the use of antibiotics could cause development of resistance mechanisms.
Currently, results in limiting phytoplasma diffusion are not very effective and outbreaks are increasing year by year all over the world. On the one hand, the use of pesticides against insect vectors is an effective strategy for the control of the insect vector population, obtaining decreased levels of phytoplasma transmission to healthy plants. On the other hand, the extensive use of pesticides has negative environmental impacts (Firrao et al., 2007). The removal of infected plants from the agriculture eco-systems could be a suitable method for reducing phytoplasma disease spread by monophagous vectors (Bertaccini and Duduk, 2009).
The identification of resistant varieties or transgenic plants producing antibodies could help contrast phytoplasma diffusion. In this regard, some interesting results were discovered (Chen and Chen, 1998; Le Gall et al., 1998; Albertazzi et al., 2009).
1.7 Thesis outline
The present research, carried out over three following years (2015-2017) in a case study vineyard located in the Chianti Classico area, was mainly focused on: (i) the evaluation of the symptom severity and evolution of GY symptomatic vines (Chapter 2); (ii) specific detection of BNp, FDp and AYp, determining the relative quantification of
13 BNp in BN-infected vines showing different symptom intensity (Chapter 3); (iii) analyses of BNp genetic diversity using the MLST approach (Chapter 4).
Based on the results obtained in the case study vineyard further genetic studies were carried out in 17 vineyards located in the main vine-growing areas of Tuscany (Chapter 5), extending the investigation of BNp genetic diversity at Regional level.
Finally, a general conclusion based on the main results obtained in this PhD research is provided (Chapter 6).
1.8 References:
Albertazzi, G., Milc, J., Caffagni, A., Francia, E., Roncaglia, E., Ferrari, F., Tagliafico, E., Stefani, E., Pecchioni, N. (2009). Gene expression in grapevine cultivars in response to Bois Noir phytoplasma infection. Plant Science, 176(6): 792-804.
Alma, A., Arnò, C., Arzone, A., Vidano, C. (1988). New biological reports on Auchenorrhyncha in vineyards. Proceedings, 6th Auchenorrhyncha Meeting, Turin, 509-516.
Alma, A., Bosco, D., Danielli, A., Bertaccini, A., Vibio, M., Arzone, A. (1997) Identification of phytoplasmas in eggs, nymphs and adults of Scaphoideus titanus Ball reared on healthy plants. Insect Molecular Biology, 6: 115-121.
Bai, X., Zhang, J., Ewing, A., Miller, S.A., Radek, A.J., Shevchenko, D.V., Tsukerman, K., Walunas, T., Lapidus, A., Campbell, J.W. and Hogenhout, S.A. (2006). Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. Journal of Bacteriology, 188: 3682-3696.
Belli, G., Bianco, P. A., Conti, M. (2010). Grapevine yellows in Italy: past, present and future. Journal of Plant Pathology, 92: 303-326.
Berger, J., Dalla, Via J., Baric, S. (2009). Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir. European Journal of Plant Pathology, 124:521-526.
Bertaccini, A., Vibio, M., Stefani, E. (1995). Detection and molecular characterization of phytoplasmas infecting grapevine in Liguria (Italy). Phytopathologia Mediterranea, 34: 137-141.
Bertaccini, A. (2007). Phytoplasmas: diversity, taxonomy, and epidemiology. Frontieres in Bioscience, 12: 673-689.
Bertaccini, A., Duduk, B. (2009). Phytoplasma and phytoplasma diseases: a review of recent research. Phytopathologia Mediterranea, 48: 355-378.
14
Bertaccini, A., Duduk, B., Paltrinier, S., Contaldo, N. (2014). Phytoplasmas and phytoplasma diseases: a severe threat to agriculture. American Journal of Plant Sciences, 5: 1763-1788.
Bertaccini, A. (2015). Phytoplasma research between past and future: what directions? Indian Journals 5: S1-S4.
Bertamini, M., Nedunchezhian, N. (2001). Effect of phytoplasma, stolbur-subgroup (Bois noir-BN) of photosynthetic pigments, saccarides, ribulose-1,5-bisphosphate carboxylase, nitrate and nitrite reductases and photosynthetic activities in field-grow grapevine (Vitis vinifera L. cv Chardonnay) leaves. Photosynthetica, 39: 119-122.
Bianco, P.A., Davis, R.E., Prince, J.P., Lee, I.M., Gundersen, D.E., Fortusini, A., Belli, G. (1993). Double and single infections by aster yellows and elm yellows MLOs in grapevines with symptoms characteristic of flavescence doree. Rivista di Patologia Vegetale, 3(3): 69-82.
Botti, S., Bertaccini, A. (2006). Phytoplasma infection trough seed transmission: further observations. 16th Congress IOM, 9-14 July, Cambridge, UK, 76: 113.
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Chapter 2
20 ABSTRACT
Symptom observations were carried out in a case study vineyard located in the Chianti Classico area (Tuscany, Central Italy) over 3 following growing seasons (2015-2017). In each year, GY symptomatic vines were visually assessed according to the symptom severity classes (0-3). Results showed that GY diffusion more than doubled from the first year to the second one. Subsequently, it slightly decreased during 2017. Data also revealed that symptom severity in symptomatic vines is variable though the years and the higher phytoplasma transmission rates represented the main driving force operating in the GY diffusion during 2016. Conversely, the lower GY incidence observed in 2017 could be explained by a stronger influence of abiotic/biotic parameters in phytoplasma spreading.
2.1 INTRODUCTION
Symptom intensity in grapevine is related to several factors. One of the most important aspects influencing symptom severity is strictly related to the erratic phytoplasma localization and its concentration in plant tissues (Riedle-Bauer et al., 2010; Landi and Romanazzi, 2011).
Other studies showed that symptom intensity also depends on weather conditions (Hren et al., 2009; Landi and Romanazzi, 2011; Murolo et al., 2014). In particular, symptom expression is influenced by cold temperature and the amount of rain during the summer, with relative changes in gene expression in plants. Down-regulation of several photosynthetic genes, up-regulation of several genes encoding for the
21 pathogenesis-related protein (PR protein) and changes in gene expression involved in carbohydrate metabolism were reported in the above mentioned studies.
Symptom intensity also depends on the cultivar susceptibility to phytoplasma strains and on the variable range of strain virulence, as reported by Quaglino et al. (2016).
GY symptomatic plants may have a spontaneous remission of disease symptoms. Over the years, symptoms can disappear temporarily or disappear completely (recovery phenomenon) and ‘recovered’ plants may remain asymptomatic if not exposed to infective vectors again (Margaria et al., 2014). Currently, little is known about the mechanisms involved in the recovery phenomenon and plant-pathogen interactions. Some studies have reported changes in carbohydrate metabolism, polyphenol production, sugar and amino acid transportation in grapevine and periwinkle (Catharanthus roseus) as a consequence of recovery phenomenon (Constable et al., 2003; Hren et al., 2009).
Studies focused on the endophytic bacterial community of grapevine leaves identified differences in microbial community among recovered, healthy and diseased plants, suggesting the possible role of endophytes in protecting plants from re-infections (Bulgari et al., 2012; Bulgari et al., 2014).
Generally, GY symptom in grapevine is clearly visible in September and disease severity for each vine is assessed using empirical scales based on symptom intensity observed on leaf, shoots, inflorescences and berries (Riedle-Bauer et al., 2010; Murolo
et al., 2014; Quaglino et al., 2016).
The study was aimed at evaluating the incidence of GY symptoms and its evolution over 3 following years (2015-2017) in a case study vineyard in the Chianti Classico area.
22 2.2 MATERIALS AND METHODS
2.2.1 Vineyard description
The study was conducted over three following years (2015, 2016 and 2017) in a selected area within a vineyard including 735 V. vinifera cv. Sangiovese, I-SS F9 A5 48. Selected area is localized in a traditional grapevine-growing area of Chianti Classico (Greve in Chianti, Firenze district - 43° 33’ 21’’ N, 11° 18’ 8’’ E; 460 m a.s.l.), in Tuscany (central Italy). Vineyard planted in 1997 with a planting density 2.3 m between the rows and 0.8 m along the row, was bordered by forests and conducted according to organic management. Vines were trained as cordon.
2.2.2 Symptom observations
Over three following years (2015, 2016 and 2017), each vine was visually assessed for the presence of GY disease in September of each year. Symptom severity was classified and sorted according to the GY symptomatic scale from 0 to 3 according to Riedle-Bauer and colleagues (2010), partially modified as follow:
i. class 0: asymptomatic plants;
ii. class 1: one shoot with mild leaf symptoms; iii. class 2: 2-3 shoots with leaf symptoms;
23 The overall disease severity (S) was evaluated using the formula proposed by Murolo and Romanazzi in 2015:
S = ∑ (c · f) / n
where:
S is disease severity; c is symptom severity class;
f is the frequency of the symptom severity class;
n is the number of symptomatic plants.
Chi square test was adopted to estimate statistically significant differences in symptom severity among the years.
2.2.3 Sampling
During field surveys conducted for each year, ten leaves from each GY symptomatic plants and ten from randomly selected symptomless plants were mapped and sampled for the subsequent phytoplasma detection and characterization analyses. Fresh central midribs were dissected and stored at -20°C until total nucleic acids (TNA) extraction.
V. vinifera cv. Sangiovese, obtained from the screenhouse of the Department of
Agriculture, Food and Environment (DAFE, University of Pisa, Italy), was used as healthy control plants, while V. vinifera plants previously found infected by Bois Noir phytoplasma (BNp) (subgroup 16SrXII-A), Flavescence Dorée phytoplasmas (FDp)
24 (subgroups 16SrV-C or -D) and Aster Yellows phytoplasmas (AYp) (subgroups 16SrI-B or -C), were used as infected reference control (IC).
2.3 RESULTS
During the field surveys conducted over the three following years, all symptom severity classes were observed.
In details, in 2015, 24 out of 735 (3.3%) grapevine plants showed typical GY symptoms. The most observed symptom severity class belonged to the class 2 (37.5% of the symptomatic vines) followed by the class 3 (33.3%) and the class 1 (29.2%) (Fig. 2.1).
In 2016, 53 out of 734 (7.2%) grapevine plants showed typical GY symptoms: (i) 33 plants out of 53 exhibited GY symptoms for the first time; (ii) 20 plants out of 53 showed symptoms in both years; (iii) 3 plants, showing symptoms in 2015, were symptomless in 2016. Moreover, one plant, showing symptoms in 2015, was eradicated after death. The most observed symptom severity class belonged to the class 3 (47.2% of the symptomatic vines) followed by the symptom severity class 1 and 2, which were equally abundant (26.4% each) (Fig. 2.1).
In 2017, 42 out of 732 (5.7%) grapevines showed typical GY symptoms: (i) 12 plants exhibited GY symptoms for the first time; (ii) 15 plants showed symptoms over the three years; (iii) 13 showed symptoms in 2016 and 2017; (iv) 2 showed symptoms in 2015 and 2017; (v) 23 showing symptoms in 2016, were symptomless in 2017. Moreover, 2 plants, showing symptoms in the previous years, were eradicated after death. The most observed symptom severity belonged to the classes 1 and 2 (38.1% for both), followed by the class 3 (23.8%) (Fig. 2.1).
25 Overall disease severity (S) was determined as 2.04 in 2015, 2.20 in 2016 and 1.46 in 2017. GY incidence was referred to the number of symptomatic plants on the total plants (Fig. 2.1).
Statistical analysis, carried out using Chi square test, revealed significant differences in the symptom severities for each year (p = 0.004).
Figure 2.1. Symptom severity classes (%) observed in the case study vineyard over 3 following growing
seasons (2015-2017). S: Overall Disease Severity; In: GY incidence
2.4 DISCUSSION
Symptom observations, carried out in September in the case study vineyard, showed clear GY symptoms and different symptom severities over the years (2015-2017).
In each year, all symptom severity classes were observed and the BN incidence ranged from 3.3 to 7.2%. In particular, filed surveys revealed that BN disease incidence
26 more than doubled from 2015 to 2016 (3.3 to 7.2%). Differently, it slightly decreased during 2017 (7.2 to 5.7%).
Considering that (i) phytoplasmas in the agro eco-systems are transmitted from vine/weeds to vine, mediated by different insect vectors (Belli et al., 2010; Bertaccini et
al., 2014), (ii) vines not showing any longer symptoms for 2 following years can be
considered recovered (Maixner, 2001; Mori et al., 2015; Murolo et al., 2014) and (iii) vineyard management, clonal grapevine cultivar and sampling time were fixed parameters in the case study vineyard, it is reasonable to state that the high phytoplasma transmission frequency in the vineyard is one of the main driving forces playing a role in GY diffusion from 2015 to 2016. Conversely, the lower GY diffusion observed in 2017 could be partially influenced by (i) abiotic parameters, mainly including temperature, rains and relative humidity, as reported in several studies investigating phytoplasma disease incidence and severity in vineyards according to weather conditions (Albertazzi et al., 2009; Hren et al., 2009; Murolo et al., 2014) or (ii) biotic factors, including the possible role of endophytic bacterial community in phenotype expression during the growing seasons, as previously reported (Bulgari et al., 2012, 2014). Differences in symptom intensity over the years were statistically significant, reinforcing the possible role of parameters described above.
2.5 References
Albertazzi, G., Milc, J., Caffagni, A., Francia, E., Roncaglia, E., Ferrari, F., Tagliafico, E., Stefani, E., Pecchioni, N. (2009). Gene expression in grapevine cultivars in response to Bois Noir phytoplasma infection. Plant Science, 176 (6): 792-804.
Belli, G., Bianco, P. A., Conti, M. (2010). Grapevine yellows in Italy: past, present and future. Journal of Plant Pathology, 92: 303-326.
Bertaccini, A., Duduk, B., Paltrinier, S., Contaldo, N. (2014). Phytoplasmas and Phytoplasma Diseases: A Severe Threat to Agriculture. American Journal of Plant Sciences, 5: 1763-1788.
27
Bulgari, D., Bozkurt, A.I., Casati, P., Caglayan, K., Quaglino, F., Bianco, P.A. (2012). Endophytic bacterial community living in roots of healthy and 'Candidatus Phytoplasma mali'-infected apple (Malus domestica, Borkh.) trees. International Journal of General and Molecular Microbiology, 102(4): 677-687.
Bulgari, D., Casati, P., Quaglino, F., Bianco, P.A. (2014). Endophytic bacterial community of grapevine leaves influenced by sampling date and phytoplasma infection process. BMC Microbiology, 14(1): 198. Constable, F.E., Gibb, K.S., Symon, R.H. (2003). Seasonal distribution of phytoplasmas in Australian
grapevines. Plant Pathology, 52: 267-276.
Hren, M., Nikolić, P., Rotter, A., Blejec, A., Terrier, N., Ravnikar, M., Dermastia, M., Gruden, K. (2009). ‘Bois Noir’ phytoplasma induces significant reprogramming of the leaf transcriptome in the field grown grapevine. BMC Genomics, 10: 460.
Landi, L., Romanazzi, G. (2011). Seasonal Variation of Defense-Related Gene Expression in Leaves from Bois noir Affected and Recovered Grapevines. Journal of Agricultural and Food Chemistry, 59: 6628-6637.
Maixner, M. (2011). Recent advances in Bois noir research. Petria, 21: 95-108.
Margaria, P., Ferrandini, A., Caciagli, P., Kedrina, O., Schubert, A, Palmano, S. (2014). Metabolic and transcript analysis of the flavonoid pathway in diseased and recovered Nebbiolo and Barbera grapevines (Vitis vinifera L.) following infection by Flavescence Dorée phytoplasma. Plant, Cell and Environment, 37: 2183-2200.
Mori, N., Quaglino, F., Tessari, F., Pozzebon, A., Bulgari, D., Casati, P., Bianco, P.A. (2015). Investigation on ‘bois noir’ epidemiology in north-eastern Italian vineyards through a multidisciplinary approach. Annals of Applied Biology, 166: 75-89.
Murolo, S., Mancini, V., Romanazzi, G. (2014). Spatial and temporal stolbur population structure in a cv. Chardonnay vineyard according to vmp1 gene characterization. Plant Pathology, 63: 700-707.
Murolo, S., Romanazzi, G. (2015). In-vineyard population structure of 'Candidatus Phytoplasma solani' using multilocus sequence typing analysis. Infection Genetics and Evolution, 31: 221-230.
Quaglino, F., Maghradze, D., Casati, P., Chkhaidze, N., Lobjanidze, M., Ravasio, A., Passera, A., Venturini, G., Failla, O., Bianco, P.A. (2016). Identification and characterization of new ‘Candidatus Phytoplasma solani’ strains associated with Bois Noir disease in Vitis vinifera L. cultivars showing a range of symptoms severity in Georgia, the Caucasus region. Plant Disease, 100: 904-915.
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28
Chapter 3
29
ABSTRACT
The only way to identify the causal agent of GY is via molecular detection. In the case study vineyard, TaqMan assay using specific primer pairs for the 16S rDNA amplification of BNp, FDp and AYp was carried out. Results detected the exclusively presence of BNp in all symptomatic grapevines over the 3 following growing seasons. Interestingly, statistically significant differences were identified in BNp relative concentration associated with grapevines showing different symptom intensity.
3.1 INTRODUCTION
On the basis of GY symptom observations it is not possible to attribute a specific etiological agent to the disease (Belli et al., 2010).
The earliest phytoplasma identification methods relied on electron microscopy of phloem sections. In 1990s, more sensitive diagnostic tools, based on Polymerase Chain Reaction (PCR) followed by Restriction Fragments Length Polymorphism (RFLP) were developed for phytoplasma identification (Bertaccini and Duduk, 2009).
The primary requirement to perform an accurate phytoplasma detection by PCR is the good quality of DNA, however some issues must be taken into account: (i) the amount of phytoplasma DNA is less than 1% out of the total DNA extracted from plant tissues (Bertaccini and Duduk, 2009), (ii) phytoplasma concentration is highly variable in the same symptomatic plant (Angelini et al., 2007) and (iii) grapevine plant tissues contain significant concentration of PCR inhibitors (Boudon- Padieu et al., 2003).
Different nested PCR protocols, amplifying universal and group-specific phytoplasma 16S rDNA gene, followed by RFLP analysis of the amplicons using appropriate restriction enzymes, were developed for the classification in subgroups
30 (Deng and Huruki, 1991; Schaff et al., 1992; Lee et al., 1994; Gibbs et al., 1995; Schneider et al., 1995; Gundersen and Lee, 1996; Daire et al., 1997; Lee et al., 1998; Martini et al., 1999; Boudon Padieu et al., 2003; Clair et al., 2003; Duduk et al., 2004). In order to reduce labor-intensive steps and get results fast, in the last decades Real-Time PCR protocols for the detection and quantification of phytoplasma associated with several plant diseases were developed. Real-Time PCR system with SYBR® Green I technology (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was developed by Galetto et al. (2005) on phytoplasma-infected grapevine and insects. Specific primer pairs and TaqMan probes were designed on the 16S rDNA phytoplasma sequences of FD, BN and AY by Angelini et al. (2007), Hren et al., (2007) and Pellettier and Foissac (2009), obtaining solid results with regards to sensitivity and specificity, when compared to nested PCR methods.
The aim of this study was to identify BNp in GY symptomatic V. vinifera plants and determine the association between symptom severity classes and BNp relative concentration, in the case study vineyard.
3.2 MATERIAL AND METHODS
3.2.1 DNA extraction
DNA was extracted with 2% cetyltrimethylammonium bromide (CTAB) based buffer from leaf veins according to the protocol described by Li et al. (2008), with some modifications: briefly, leaf veins (1 g) were homogenized in plastic bags (Bioreba, Switzerland) with 7 ml of 2% CTAB buffer using Homex 6 (Bioreba, Switzerland), then the homogenate was incubated at 65°C for 15 minutes. DNA was extracted by one
31 volume of chloroform:iso-amylalcohol (24:1) and precipitated with one volume of isopropanol. Pellets were washed with 70% ethanol, air-dried, suspended in 80 µl of deionized water and stored at -20°C until use.
3.2.2 GY phytoplasma detection and relative quantification
TaqMan assay for the specific detection of phytoplasma associated with BN, FD and AY through the amplification of 16S ribosomal DNA was carried out using the Rotor-Gene Q (Qiagen, Germany), following PCR protocols and reaction conditions according to Angelini et al. (2007). The grapevine chloroplast chaperonin 21 gene was used as endogenous control, while the total nucleic acids extracted from healthy control (HC) and positive control (IC) plants were used as negative and positive controls, respectively. Threshold cycles (Ct) < 37 were associated with the presence of GY phytoplasmas (Angelini et al., 2007; Mori et al., 2015) and the template used in the assay was a 1:10 dilution of the DNA extracted from the samples.
For each sample, the relative quantification of phytoplasmas was carried out according to the previous studies conducted by Baric (2012) and Minguzzi et al. (2016) using the following formula:
ΔCt = Ctp - Ctg
Where:
ΔCt is the normalized value;
32
Ctg is the Ct obtained from amplification of the endogenous control used in the
reaction (grapevine chaperonin gene).
ΔCt values were compared through one-way ANOVA, followed by Tukey's Exact Test, performed in SPSS statistical package for Windows, v. 24.0 (IBM Corporation, Armonk, NY) to determine if the symptom severity classes, assessed during field observations, were associated with different phytoplasma relative abundance in the plant hosts.
3.3 RESULTS
Over the three following years, specific TaqMan assay for the BNp, FDp and AYp detection was carried out for each symptomatic grapevine and grapevines no longer showing symptoms during the years. BNp was detected in all symptomatic plants in 2015, in 45 out of 53 symptomatic plants in 2016 and in 41 out of 43 symptomatic plants in 2017. BNp was never detected in symptomless plants and FDp and AYp were never identified neither in symptomatic nor symptomless plants.
Over the three years, Ct values obtained by the TaqMan detection assays varied from 28 to 35 among samples, while Ct values obtained for the endogenous control (chaperonin gene) varied from 15 to 25; ΔCt values were from 3 to 11 (Tab. 3.1; Tab. 3.2). Assay consistency was confirmed by no amplification signals from HC and reaction mixtures devoid of TNA, while ICs of BNp, FDp and AYp gave the expected amplification signals. In average, Ct values were equal to 30 for BNp and 28 for FDp and AYp, respectively.
33 One-Way ANOVA, using Tukey test, revealed statistically significant differences in ΔCt values associated with grapevines showing symptom severity class 1, 2, and 3 (p = 0.007) (Fig. 3.1).
Figure 3.1. Relative abundance of Bois Noir phytoplasma (ΔCt) associated with grapevines showing
different symptom severity classes in the case study vineyard.
34
Table 3.1. Symptom severity classes observed in the case study vineyard and relative phytoplasma
abundance (ΔCt) over 3 following years.
2015 2016 2017 Plant
Symptom 16S rDNA Symptom 16S rDNA Symptom 16S rDNA severity ΔCt severity ΔCt severity ΔCt
San1 3 7 3 6.08 1 7.01 San2 3 7.5 3 6.54 1 8.06 San3 1 4.5 0 - 0 - San4 1 10 2 6.95 2 5.45 San5 2 6.5 3 6.69 1 8.13 San6 2 6.5 2 7.53 1 9.65 San7 1 9 1 5.83 1 2.67 San8 3 5.5 3 7.17 2 3.89 San9 3 8 Eradicated - Eradicated - San10 2 8.5 3 7.38 2 6.89 San11 2 7 3 4.97 3 9.08 San12 1 6 0 - 2 2.88 San13 1 10.5 1 2.12 0 - San14 3 7 2 8.07 0 - San15 3 8.5 1 10.41 1 9.87 San16 3 7 3 6.27 3 4.66 San17 2 8 1 7.68 1 5.66 San18 1 11 0 - 2 5.78 San19 1 8 2 1.04 1 8.55 San20 2 6 3 7.66 Eradicated - San21 3 6.5 3 7.18 0 - San22 2 6.5 1 6.73 3 6.03 San23 2 7 3 5.01 0 - San24 2 6.5 2 5.64 2 6.99
