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Table S2. Summary of annotations of the Actinidia chinensis var. chinensis transcriptome. (DOC 66 kb)

Since 2008 Psa ( Pseudomonas syringae pv. actinidiae) has become a threat for kiwifruit plantation in many Countries. A lot of research and trials have been carried out looking for chemicals able to stop the infection, acting directly on the agent of the disease. This study is targeted on the “environment” corner of the disease triangle, with the aim of creating an overall environment unfavorable to Psa development, considering the effect of change not only on the disease, but also on growing parameters. The objective is achievable growing kiwifruit under green house in different structures. Zespri in conjunction with the University of Bologna built up three years of growing experience under covers, so the present work wants to underline the outcomes of it. It has been proved that growing kiwifruit under cover reduces significantly the symptoms of Psa. This is likely linked to lower bacteria’s population, both epiphytic and endophytic. Beside the effect on bacteria’s population, effect on fruit quality was observed. Earlier fruit set was recorded under the cover due to the warmer spring temperature. It also resulted in bigger fruit size at harvest with less blemish. In fact, plastic covers protect the crop from adverse climate events, such as wind and hail. Generally, under cover kiwifruit needs a limited number of chemical treatments, with benefit on soil compaction (fewer sprays and in better soil condition – no wet soil). The controlled climate condition allowed to operate with no weather constrains: this was particularly helpful at pollination time, but also in a Psa safer environment for summer pruning, harvest and so forth, where wounds could get infected. In the other hand, growing kiwifruit under plastic cover showed some constrains. Primary, building a structure that can resist to climate events, like wind storms or hail and snow precipitation is generally highly expensive. In addition, the plastic covers last few years (5-6 years), or at least the light permeability decreases dramatically, requiring to replace it. This implies frequent substitution of the plastic sheet, with extra charge of cost to add to the balance. Data have been collected about change in amount and quality of the light. Although in Italy it is not a big issue during sunny days, it may become a problem when it is cloudy, causing a decrease of the photosynthesis: this can impact dry matter accumulation, resulting in lower fruit quality. Care must be paid to irrigation and nutrition management, since salinity can easily build up in the root area, due to the quality of irrigation water and/or the fertilizer application. In fact, the complete absence of precipitation avoids regular wash off/dilution of the salinity, accumulating in narrow layer compressed by the irrigation volume applied. In conclusion, this paper as result of ongoing monitoring, highlights all the side effects of plastic covers, both positive and negative, on kiwifruit crop

A forecasting model to predict the kiwifruit bacterial canker infections was developed in New Zealand. In 2012 such forecasting model was intended to be validated in the kiwifruit growing areas of Emilia-Romagna Region. Model, using hourly temperature and leaf wetness data, calculates the bacteria multiplication rates (M). M values were three days accumulated (actual + two preceding days) to provide a daily Risk Index R whenever a rain event (> 1mm) occur during the growing season. Validation was carried over 2012-2014, exposing micro-propagated potted kiwi plantlets under untreated infected plants from march untill november. Every week, after being exposed, 18 Plantlets (9 Hayward and 9 Tomuri) were removed and placed in climate chamber with 24 hours wetness duration. Afterwards, part of them were placed in greenhouse at 15-18°C temperature to check for symptom occurrence, and the remaining analyzed by both pathogen isolation on selective growing media and PCR on isolated bacterial colony. Analysis were carried out as soon as kiwi plantlets were remove from the field (T0) and after 7 days (T7). In 2012 an 2013, the experiment was carried out in two kiwi orchard of the cv Hayward (1 located in the plain and 1 in the hillside), while in 2014 the experiment was carried out on 2 A.deliciosa and 1 A.chinensis orchards. Over the years 2012-2014, symptoms on plantlets were always observed from march to the first week of june. In the same period of time disease symptoms occurred in the unprayed kiwifruit plot, confirming what observed in potted trap plants. However diagnostic analysis showed that bacteria can be detected in potted trap plants throughout the year (particularly in 2013 and 2014), except some sporadic week during summer even without a rain event. Validation experiment using trap plants permitted to effectively validate the forecasting model during springtime (from march to june) showing a robust correspondence between the infection index R and the symptom occurrence. In fact, all the disease symptoms occurred in coincidence of R infection Index higher than 15, value that can be taken as action threshold. At the same time, molecular analysis as well showed a clear relationship between the infection index R and the bacterial inoculum contaminating the trap plants during the growing season.

Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has emerged since 2008 and is the most important disease of kiwifruit, threatening its production in the world. Until now, little is known about the molecular interaction between Psa and kiwifruit plants: to elucidate early molecular events of kiwifruits-Psa interaction, Illumina RNA-seq was conducted at 3 h, 24 h and 48 h after Psa inoculation and mock inoculation, both on acibenzolar-S-methyl (ASM)-pretreated and untreated Actinidia chinensis plants. De novo assembly of kiwifruit transcriptome consists of 39,607 transcripts, about 4500 of them were differentially expressed (DE) in the various situations. During infection process, genes modulation caused by Psa in A. chinensis involve about 3000 transcripts modulated that is mainly evident at 3hpi rather than 24hpi or 48hpi. Only few transcripts were common between the three infections time points (1,5%). Moreover, transcripts modulation in infected ASM-untreated plants were higher than in ASM pre-treated infected plants. To investigate transcriptome expression profile during infection process, both in ASM untreated and ASM pre-treated plants, Hierarchical Cluster Analysis (HCA) were performed to reveal the coordinates change of some important gene functional categories. Moreover, MapMan has been shown to be an effective tool to map transcriptome data, to define functional categories and to perform time course analysis in order to identify differentially-represented functional groups. Regulation overview highlight difference in hormonal balance, transcriptional regulation and signaling. For example, seven major families of TF (AP2/ERF, MYB, Myc-bHLH, GATA, NAC, WRKY and GRAS), reported as linked to plant stress responses such as pathogens, were found DE in response to Psa in ASM pretreated plants with respect to the untreated ones. Different TFs were linked also with signaling and hormonal balance and probably involved in the ability of ASM plants to assemble the right defense response against PSA. On the other side, ASM untreated plants try to mount a response at 3 hpi , but not adequate to counteract the infection. Moreover, the RNA-seq technology has permitted to identify novel differentially expressed transcripts of unknown functions but related with infection and ASM treatment. Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has emerged since 2008 and is the most important disease of kiwifruit, threatening its production in the world. Until now, little is known about the molecular interaction between Psa and kiwifruit plants: to elucidate early molecular events of kiwifruits-Psa interaction, Illumina RNA-seq was conducted at 3 h, 24 h and 48 h after Psa inoculation and mock inoculation, both on acibenzolar-S-methyl (ASM)-pretreated and untreated Actinidia chinensis plants. De novo assembly of kiwifruit transcriptome consists of 39,607 transcripts, about 4500 of them were differentially expressed (DE) in the various situations. During infection process, genes modulation caused by Psa in A. chinensis involve about 3000 transcripts modulated that is mainly evident at 3hpi rather than 24hpi or 48hpi. Only few transcripts were common between the three infections time points (1,5%). Moreover, transcripts modulation in infected ASM-untreated plants were higher than in ASM pre-treated infected plants. To investigate transcriptome expression profile during infection process, both in ASM untreated and ASM pre-treated plants, Hierarchical Cluster Analysis (HCA) were performed to reveal the coordinates change of some important gene functional categories. Moreover, MapMan has been shown to be an effective tool to map transcriptome data, to define functional categories and to perform time course analysis in order to identify differentially-represented functional groups. Regulation overview highlight difference in hormonal balance, transcriptional regulation and signaling. For example, seven major families of TF (AP2/ERF, MYB, Myc-bHLH, GATA, NAC, WRKY and GRAS), reported as linked to plant stress responses such as pathogens, were found DE in response to Psa in ASM pretreated plants with respect to the untreated ones. Different TFs were linked also with signaling and hormonal balance and probably involved in the ability of ASM plants to assemble the right defense response against PSA. On the other side, ASM untreated plants try to mount a response at 3 hpi , but not adequate to counteract the infection. Moreover, the RNA-seq technology has permitted to identify novel differentially expressed transcripts of unknown functions but related with infection and ASM treatment.

Pseudomonas syringae pv. actinidiae (Psa),the causative agent of bacterial canker of kiwi, was firstly isolated in 1992, but the second outbreak dating back to 2008 is now in charge of a large pandemic that has led to major economic losses worldwide. Despite the high economic impact of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit industry, and the many research efforts, the molecular mechanisms involved in pathogenesis are still not fully characterized. In particular, the precise mechanism of recognition/communication between Psa and its host plant has not been yet elucidated. Our findings shows that plant crude extracts or xylem juices of Actinidia deliciosa and A. chinensis were able to increase Psa virulent in comparison with artificial growing media such as Luria Broth or Nutrient agar. The study of gene expression in response to plant-based or artificial growing medium was performed by QPCR and showed a differential expression of many virulence related genes. Also genes related with biofilm formation, motility and inter cell signaling (e.g. quorum sensing in the broad sense) were differencially expressed according to the type of growing media. Interestringly, many of the studied gene expression responded differentially depending both to plant extracts and Psa cell density. The molecular data were corroborated by phenological studies aiming to test the effect of plant based medium on biofilm formation, motility and symptom development in microcuttings of Actinidia spp. The effect of other plant extracts such as tobacco, tomato, tea, hazelnut and A. arguta were also studied to determine whether the activation of genes and phenotypes observed is related to a non specific plant molecules. To determine which plant molecules are specifically recognized by PSA, the putative bacterial receptors have been cloned, expressed heterologously, purified and immobilized in chromatographic columns in order to capture their ligands present in those plant extracts specifically activating Psa genes. The presented study demonstrates the existence of a not yet identified, host-specific group of molecules able to active the pathogenesis machinery of Psa. Moreover these molecules could led to different physiological and molecular behavior depending on Psa population density. Finally, it was also demonstrated that these molecules were differentially present in leaves and xylem sap collected by different Actinidia species.

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker kiwifruit. This disease spread very rapidly, also due to the circulation of infected plant material, till becoming pandemic. In order to prevent local spread and future introduction into Psa-free area, the early diagnosis of asymptomatic material is essential. Moreover, Psa symptoms in open filed are represented, from spring to autumns, mainly by leaf spots which may be caused also by other pathogens (i.e. P. syringae pv. syrinagae). Thus a fast, reliable, easy to use an inexpensive diagnostic method may represent a key tool screen nursery material or to tailor control treatments, orchard management inputs. Due to its plasticity, cost effective, rapidity and sensitivity, loop-mediated isothermal amplification (LAMP) assay is an ideal approach to develop such diagnostic tool. LAMP primers were designed on the AvrPT01 protein, a virulence factor specific of Psa and tested for specificity with genomic DNA from different Pseudomonas syringae pathovars (tomato, tobacco, glycinea, syringae and theae), other bacteria commonly found on Actinidia species (Pseudomonas viridflava, Pantoea agglomerans, Pantoea vagans, Paenibacillus spp, Pseudomonas fluorescens) and other bacteria not related to kiwifruit (Erwinia amylovora, Escherichia coli). Positive diagnosis, which is indicated in a clear color change or the reaction medium, was found only on Psa pathovars 1, 2 and 3. The method was also tested using crude extracts from infected Actinidia deliciosa and Actinidia chinensis plants. The sensibility of Lamp-based methodology here described was 1000 CFU/ml, comparable to other molecular tools commonly used for Psa identification such as end-point PCR and QPCR

The quorum sensing (QS) is a complex bacterial intra and inter communication system which is regulated by signalling molecules produced by cell density dependent mechanisms. The QS may regulate different phenotypes such as virulence, motility and biofilm production. The aim of the research was to investigate the existence of a QS system in Pseudomonas syringae pv. actinidiae (Psa). Psa lacks of the canonical QS system based on signal-synthase gene. However, at least three signal receptors (PsaR1, PsaR2, PsaR3) were recently identified. The presented study was conducted in order to clarify the effects of different cell densities on the intraspecific communication in Psa cultures. Therefore, interspecific interaction between Psa and other bacteria associated with the plant host (i.e. in the phyllosphere) or known to have an active QS system were also investigated. A bioinformatic research was conducted for the identification of genes putatively involved in Psa QS system. About 30 genes were selected due to their involvement in influencing specific phenotypes such as virulence, density perception, biofilm formation and motility. The regulation of these genes was analysed by QPCR in Psa samples derived from different culture conditions (i.e. density; co-cultures). Therefore, after a nucleotide sequences blast analysis of several bacterial signal synthase promoters that respond to all Psa signal receptors, homology with the promoter region in front of a transcriptional regulator was found. We suppose that this regulator may be involved in the QS signal transduction. Motility and biofilm formation crucial phenotypes in the early stages of host colonization, are under control of QS circuitries. The experiments conducted demonstrated Psa ability of producing biofilms in LB liquid media in a population density manner. Psa motility resulted particularly enhanced on QS-bacteria supernatants (such as Pseudomonas fluorescens, or an overproducing QS-signals strain of Pseudomonas putida). Quantitative expression of QS-related genes was analysed by QPCR. The expression fold change significantly varied in Psa populations depending on the cell density (from 105 up to 108 cfu/ml). Therefore, the gene expression profiles also changed when Psa cultures were grown in bacterial co-cultured media, remarking the importance of interspecific communication on Psa QS-system and potentially on Psa virulence. In conclusion, from our preliminary studies we observed that QS-phenotypes seem to be influenced not only by the cell density, but also by the microbial community that may share with Psa the same environment. For now, these findings are leading to the hypothesis that Psa has evolved to eavesdrop signals coming from the environment to regulate the complexity of its QS mechanisms. Presumably only by considering a multiple model system, where Psa interacts both with the plant host and other bacterial species, the functionality of Psa QS-system will be fully elucidated.

The bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae, is considered one of the most severe diseases affecting several cultivated Actinidia species, including A. chinensis and A. deliciosa. Kiwifruits have been always considered fruit with an high intrinsic quality due to the strong nutraceutical value, but also to the absence of contaminants such as pesticide residues. With the emergence of this devastating disease, the use of pesticides rapidly increased and therefore the kiwifruit quality can be maintained only by ensuring agricultural practices that reduce the need of toxic xenobiotic compounds. The aim of this study was to provide an in-depth understating of the influence of agricultural practices on disease development and spread and to harmonise the agricultural practices with the current disease control inputs. Therefore, the role of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning on the incidence and epidemiology of PSA were examined. Nitrogen fertilization had a direct effect on the pathogen's endophytic growth. Furthermore, the depletion of some micronutrients, such as iron, increased the disease. The water stress consequent to a reduced irrigation resulted in higher symptoms. Concerning pruning, the open cuts remains a possible entry point for more than 30 days. In addition, the pruning performed in late season resulted more risk for infection. To test curative pruning, preliminary data were collected on the migration rate of Psa inside the different plant organs. Among the different training system evaluated in this study, the gender double curtain the allowed a more efficient penetration of the phytosanitary treatments that may increase the disease control. However, no differences on disease incidence were observed. Concerning the use of bioregulators, synthetic gibberellin, such as forchlorfenuron, reduced in controlled condition both the disease incidence and severity. Synthetic auxins, on the other hand, showed a detrimental effect with higher symptomatology. In field conditions, none of the used bioregulators showed any effect on the disease incidence and development. Several species were tested for their susceptibility to Psa in order to develop a tolerant rootstock. Arguta spp. resulted the most tolerant to the disease and further experiments are need to test its possible exploitation as a commercial rootstock. Finally, the a preliminary guideline harmonizing the best cultural practices with the most widely used control methods (i.e resistance inducers and copper) is provided.

The micro-climatic conditions inside orchards and the vine canopy are critical factors in determining the local severity and extent of the bacterial canker of kiwifruit ( Pseudomonas syringae pv. actinidiae -Psa). Among these conditions, the effect of light quality and intensity has received, so far, little attention. Light intensity and quality are crucial factor influencing plant development, but they may also affect movement, survival and virulence of Psa. The use of plastic covers is an emerging practice showing some effect in reducing Psa incidence and severity. The effect of plastic tunnel is probably related to both a modification of the microclimatic conditions (i.e. a reduction in leaf weatness) and to an increased permanence of control treatments on the leaves. Our study aimed to evaluate the effect of plastic covers on Psa symptom development and on the micro-climatic conditions in the orchard. In this study the use of permanently closed tunnel and seasonal closed tunnel were tested in two infected commercial A. chinensis orchards. The use of permanently closed tunnel showed a positive result in the control of the disease by the reduction of the leaf wetness. Finally, since commercial plastic tunnel might modify light intensity and composition, trials were performed in laboratory conditions to evaluate the effect of light on the bacterium pathogenicity and plant defenses. Concerning Psa virulence, phenotypes and the expression of genes related to motility, biofilm formation and virulence were considered. To evaluate the influence on plant resistance, callose formation, expression of defense related genes and susceptibility were considered.

DNA-based protocols are the golden standard for the diagnosis of infected plant material. Nevertheless, these methods are time-consuming and require trained personnel, with an efficacy depending on the sampling procedure. Given that plant responses induced by pathogens also include changes in emission of volatiles, the electronic-nose may represent a sensitive, accurate and operator-friendly alternative for rapid and reliable screening of asymptomatic plant material. In comparison with DNA-based diagnosis, which is hampered by the sampling procedure of the plant material, volatiles-based diagnosis can be performed on the whole plant. In the present study, the analysis of volatile compounds is used for the discrimination of two apple diseases: fire blight (Erwinia amylovora) and blossom blight (Pseudomonas syringae pv. syringae) on propagation material. The profile of volatiles emission was initially performed by gas chromatography-mass spectroscopy (GC-MS), proton-transfer time of flight mass spectroscopy (PTR-TOF-MS) to identify possible marker compounds. In addition, two different electronic noses, EOS507 (Sacmi Scrl, Imola, Italy) and PEN3 (Airsense Analytics GmbH, Schwering, Germany), both based on metal oxide semiconductors, were used for diagnosis. After the method validation on in vitro micropropagated plants, a sampling protocol was developed to scale up the diagnosis to real nursery conditions on cold-stored, asymptomatic dormant scions. Electronic-nose can be successfully used in practical conditions, such as nurseries or customs, to screen large quantity of asymptomatic plant material in order precisely steer molecular diagnosis

Le condizioni di campo più predisponenti e le interazioni tra ospite e patogeno: numerosi i possibili siti di infezione (ferite, stomi, lenticelle, fiori) e i rischi derivati dalla normale gestione agronomica. L'agevole migrazione del batterio all'interno della pianta causa con facilità infezioni sistemiche.

Pseudomonas syrinage pv actinidiae (Psa) infetta, pur se con gravità della sintomatologia differente, tutte le specie del genere Actinidia comprese l'A. deliciosa e l'A. chinensis, che sono quelle maggiormente coltivate. Nonostante il forte impatto economico, a tutt'oggi, le conoscenze scientifiche riguardanti questa patologia sono ancora incomplete. Al fine di sviluppare efficaci e sostenibili strategie di intervento per ridurre i danni causati dalla Psa è cruciale conoscere la biologia del patogeno, i meccanismi di patogenesi, l'epidemiologia della malattia e le risposte della pianta all'infezione. Lo scopo di questo lavoro è riesaminare e presentare in maniera organica le conoscenze attuali sull’epidemiologia del patogeno e in particolare su: - come il patogeno penetri all’interno della pianta e quale sia l’importanza relativa dei diversi siti di infezione - come il patogeno colonizzi i diversi tessuti della pianta - come il patogeno migri all’interno della pianta - come il patogeno si diffonda da una pianta all’altra - come e per quanto tempo il patogeno sopravviva nell’ambiente - quale sia l’importanza delle tecniche agronomiche e delle attività umane nella diffusione della malattia

L’attività di ricerca svolta nei tre anni di dottorato si è concentrata sullo studio delle Proteine da Shock Termico (Heat Shock Proteins, HSP) vegetali e sul loro potenziale utilizzo in vaccinologia. Le HSP comprendono diverse famiglie di proteine, fra cui quella delle HSP70 che, oltre alle funzioni di chaperons molecolari, sono state dimostrate avere la capacità di attivare il sistema immunitario. Tale funzione è da ascrivere non alle HSP tal quali, ma al loro essere associate a peptidi derivanti dal catabolismo proteico cellulare. L’obiettivo del progetto è stato quello di valutare se i complessi HSP70-peptide estratti da piante che esprimono una proteina eterologa di interesse vaccinale siano in grado di attivare una risposta immunitaria specifica contro peptidi derivanti dalla proteina eterologa, al pari di corrispondenti complessi di origine animale. Al fine di raggiungere questo obiettivo è stato seguito un approccio sperimentale multidisciplinare basato su: esperimenti di bioinformatica (per mettere a punto un modello tridimensionale della HSP70 vegetale che consentisse di verificare il grado di omologia esistente tra HSP animali e vegetali), esperimenti di fisiologia vegetale (per definire le condizioni in cui è massima l’espressione delle HSP70 nei tessuti vegetali), esperimenti di biochimica (per mettere a punto il protocollo di purificazione dei complessi HSP70-peptidi da foglie di Nicotiana benthamiana), esperimenti di espressione transiente in tessuti vegetali (per ottenere mediante agroinfiltrazione l’espressione in foglie di N. benthamiana della Nucleoproteina del virus A dell’Influenza) e esperimenti di immunologia (per verificare l’efficacia dei complessi HSP-peptide estratti da tessuti vegetali che esprimono la proteina NP di indurre, in appropriati modelli murini, l’attivazione di risposte immuni specifiche per l’antigene). I risultati ottenuti sono incoraggianti perché sembrano confermare l’avvenuta associazione di peptidi derivanti dalla proteina NP alle HSP70 vegetali. Nonostante ulteriori esperimenti siano necessari per rendere il dato immunologico statisticamente valido, questa caratteristica delle HSP70 vegetali, insieme al fatto che la somministrazione di tali complessi non richiede l’impiego di sostanze adiuvanti, aprono prospettive nuove e di grande rilievo nel campo della ricerca vaccinologica, gettando nuova luce sul ruolo delle piante biofabbrica nell’ambito della ricerca biofarmaceutica. The three years PhD activity focused on plant Heat Shock Proteins (HSP) and their potential application vaccinology. HSPs include many proteins family, between them the HSP70 one. Besides their historical functions as molecular chaperons, was demonstrated that HSP70 can interact and active the mammalian immune system. This feature was not due to HSP70 alone, but to their association with peptides deriving from cell protein catabolism. The main goal of the project was evaluating if HSP70-complexes derived from plants ex pressing an antigenic protein, were able to interact with mammalian immune system and trigger a specific response against the antigen, as was demonstrated for mammalian HSP70-pepide complexes. In order to get these result was followed an experimental approach based on: bioinformatic experiments (in order to obtaining tridimensional model plant HSP70 which can be verify the homology with plant and mammalian HSP0), physiology studies (in order to identify the conditions in which the HSP70 expression in plant tissues was the most high), biochemistry experiments (In order to set up a purification protocol to obtaining HSP70-peptides complexes from N. benthamiana leaves) transient expression experiments in plant tissues (in order to obtain Influenza A Virus Nucleoprotein expression by agroinfiltration) and immunological experiments (in order to verify the potency of the HSP70- peptide complexes purified from plant expressing NP protein to elicit a specific immune response in appropriate murine model). The achieved results are encourageing, they seem to confirm the association between NP deriving peptides and plant HSP70. Despite that, other experiments are necessary in order to give strength to the immunological result. These feature of plant derived HSP70 with the fact that their immunization doesn’t need any adjuvants, open brand new perspectives in vaccinology research field, giving new interest to plant in the pharmaceutical research. Dottorato di ricerca in Biotecnologie vegetali