Isolation and characterization of phages targeting the phytopathogenic bacteria of Actinidia
Abstract
Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker in kiwifruit (Actinidia deliciosa), leading to severe symptoms in plants and consequent considerable production and financial losses. Current methods for controlling this disease rely on the use of copper-based products and, in countries outside the European Union, in the use of antibiotics. These products, besides being considered phytotoxic, can lead to the spread of resistance to both copper and antibiotics. With the increasing need to obtain safe and effective biocontrol strategies against this pathogen, this study focused on the isolation and characterization of (bacterio)phages for the control of Psa. A collection of Psa strains was characterized by PCR and phenotypical analysis, such as Biolog GEN III and phages were isolated from branches, buds, leaves, petals, sepals, and stamens from kiwifruit plants in orchards in the North of Portugal. Phages were isolated using the enrichment procedure with Psa strains CFBP 7286 and P84 as possible hosts, and the lytic spectra of 6 selected phages were tested against the Psa collection. Two phages displayed broader host ranges (between 71% and 84% of efficacy among Psa strains) were selected for further analysis. Phage stability was studied at different temperatures (-20º to 60ºC), pH (1-13) and UV light, and it was observed that the two phages were stable between -20ºC and 50ºC, pH range of 3 to 11 and UV light at 366 nm up to 180 min. The morphological characterization of phages was done by Transmission Electron Microscopy and genome sequencing. In vitro efficacy assays showed that phage 177T was able to reduce the number of CFUs after 4h of inoculation and keeping the bacterial load at low levels for up to 24h post-infection with MOI=1. Phage VC3 was able to decrease the OD600 through 24h. In a preliminary test, the infection of Psa and phage in kiwi plants was examined, and the evolution of symptoms after 12 days of inoculation with just Psa was observed. One phage has been sequenced and confirmed to be lysogenic, prophages might be employed to combat illnesses caused by harmful bacteria. In this study was possible to highlight the potential of phages as promising biocontrol agents against Psa, that could be used in the future to replace copper compounds and antibiotics.
Full text
Rute Lúcia Loureiro Rego Isolation and characterization of phages targeting the phytopathogenic bacteria of Dezembro 2021 UMinho | 2021 Rute Rego Isolation and characterization of phages targeting the phytopathogenic bacteria of Universidade do Minho Escola de Ciências Actinidia Actinidia
Rute Lúcia Loureiro Rego Isolation and characterization of phages targeting the phytopathogenic bacteria of Actinidia Master Thesis Plant Molecular Biology, Biotechnology and Bioentrepreneurship Work performed under the guidance of Doctor Francisca Rodrigues Reis Doctor Diana Priscila Penso Pires Universidade do Minho Escola de Ciências Dezembro de 2021 Actinidia
ii DIREITOS DE AUTOR E CONDICES DE UTILIZACO DO TRABALHO POR TERCEIROS Este um trabalho acadmico que pode ser utilizado por terceiros desde que respeitadas as regras e boas praticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licenca abaixo indicada. Caso o utilizador necessite de permisso para poder fazer um uso do trabalho em condices no previstas no licenciamento indicado, dever contactar o autor, atraves do RepositoriUM da Universidade do Minho. License granted to users of this work Atribuição-Não Comercial-Sem Derivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
iii Acknowledgments I'd want to express my gratitude to Dr. Francisca Reis for accepting me as my advisor and for always being able to assist and support me, as well as her research group, from which I had extremely encouraging feedback. To Dr. Priscila Pires for her guidance at the Biological Engineering Center, for her attention and help during this year. I owe you a big thank you for this journey that was undoubtedly very enriching precisely because of the way you guide me, always available and concerned about my evolution during this year. I must thank Dr. Joana Azeredo, for her kindness in accepting me in her laboratory and in her team this year. I also thank the L-Phage team, for the companionship, for being willing to help whenever necessary. To Dr. Luísa Moura from the Escola Superior Agrária of the Polytechnic Institute of Viana do Castelo (ESA-IPVC), for encouraging me to pursue my academic career, for trusting me for new challenges and for being available to help me whenever necessary. Thank you to the whole GesPsaKiwi team for their assistance, curiosity, and confidence in me to carry out this research. To KiwiGreenSun, SA for providing the material needed for this study. I have to thank my friends Kelly and Liliana, who are also like me, finishing this chapter, thanks for the encouragement and the intensive days of writing together to move together to the next stage of our lives. I also have to thank Ana, for her help in the laboratory work, for listening to my uncertainties and giving me strength, she was without a doubt my everyday companion. Finally, I must thank my family for the unconditional support they have given me this year. To my parents, for the strength they gave me to continue day after day to fight for my goals. To my siblings for all the trust they placed in me in believing that I could go further, even when I doubted my abilities. This work was funded by the European Regional Development Fund (FEDER), through the Regional Operational Program North 2020, within the scope of the project "GesPSA KiwiFerramenta Operacional para gestão sustentável do cancro bacteriano (Psa) da ActinídeaNORTE-01-0247-FEDER-033647.
iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. University of Minho, 29 of December 2021 --------------------------------------------------------------------------- Rute Lúcia Loureiro Rego
v Abstract Isolation and characterization of phages targerting the phytopathogenic bacteria of Actinidia Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of bacterial canker in kiwifruit ( Actinidia deliciosa ), leading to severe symptoms in plants and consequent considerable production and financial losses. Current methods for controlling this disease rely on the use of copper-based products and, in countries outside the European Union, in the use of antibiotics. These products, besides being considered phytotoxic, can lead to the spread of resistance to both copper and antibiotics. With the increasing need to obtain safe and effective biocontrol strategies against this pathogen, this study focused on the isolation and characterization of (bacterio)phages for the control of Psa. A collection of Psa strains was characterized by PCR and phenotypical analysis, such as Biolog GEN III and phages were isolated from branches, buds, leaves, petals, sepals, and stamens from kiwifruit plants in orchards in the North of Portugal. Phages were isolated using the enrichment procedure with Psa strains CFBP 7286 and P84 as possible hosts, and the lytic spectra of 6 selected phages were tested against the Psa collection. Two phages displayed broader host ranges (between 71% and 84% of efficacy among Psa strains) were selected for further analysis. Phage stability was studied at different temperatures (-20º to 60ºC), pH (1-13) and UV light, and it was observed that the two phages were stable between -20ºC and 50ºC, pH range of 3 to 11 and UV light at 366 nm up to 180 min. The morphological characterization of phages was done by Transmission Electron Microscopy and genome sequencing. In vitro efficacy assays showed that phage 177T was able to reduce the number of CFUs after 4h of inoculation and keeping the bacterial load at low levels for up to 24h post-infection with MOI=1. Phage VC3 was able to decrease the OD600 through 24h. In a preliminary test, the infection of Psa and phage in kiwi plants was examined, and the evolution of symptoms after 12 days of inoculation with just Psa was observed. One phage has been sequenced and confirmed to be lysogenic, prophages might be employed to combat illnesses caused by harmful bacteria. In this study was possible to highlight the potential of phages as promising biocontrol agents against Psa, that could be used in the future to replace copper compounds and antibiotics. Keywords: Actinidia deliciosa ; bacterial canker; bacteriophages; kiwifruit; Pseudomonas syringae pv. actinidia (Psa)
vi Resumo Isolamento e caracterização de fagos contra bactérias fitopatogénicas na Actinidia Pseudomonas syringae pv. actinidiae (Psa) é o agente da doença do cancro bacteriano da Actinidea ( Actinidia deliciosa ), e é responsável por perdas consideráveis produtivas e financeiras, fazendo com que as plantas sofram sintomas severos. Os métodos atuais de controlo da doença contam com o uso de produtos à base de cobre e, em países fora da União Europeia, com o uso de antibióticos. Esses produtos, além de serem considerados fitotóxicos, podem levar ao aparecimento da resistência tanto ao cobre quanto aos antibióticos. Com a necessidade cada vez maior de se obterem estratégias biológicas, seguras e eficazes contra esse patogénio, este estudo abordou o isolamento e a caracterização de bacteriófagos para o controlo de Psa. Uma colecção de estirpes de Psa foi caracterizada por métodos moleculares e análises fenotípicas, como Biolog GEN III. Os fagos foram isolados de ramos, botões, folhas, pétalas, sépalas e estames de plantas de kiwi em pomares no Norte de Portugal usando o procedimento de enriquecimento com as estirpes Psa CFBP 7286 e P84 como possíveis hospedeiros, e o espectro lítico de 6 fagos selecionados foram testados contra a coleção de Psa. Dois fagos exibiram eficácia entre estirpes de maneira mais ampla (entre 71% e 84% de eficácia entre as estirpes de Psa) e foram selecionados para determinar análises posteriores. A estabilidade dos fagos foi estudada em diferentes temperaturas (-20º a 60ºC), pH (1-13) e luz UV, e foi observado que os dois fagos eram estáveis entre -20ºC e 50ºC, de pH de 3 a 11 e luz UV a 366 nm até 180 min. A caracterização morfológica foi feita por Microscopia Eletrônica de Transmissão e procedeu-se à sequenciação do genoma. Ensaios de eficácia in vitro mostraram que o fago 177T foi capaz de reduzir o número de UFC após 4h de inoculação e manter a carga bacteriana em níveis baixos por até 24h pósinfecção com MOI = 1. O fago VC3 foi capaz de diminuir a OD600 ao longo de 24h. Num teste preliminar, foi examinada a infeção de Psa e fago em plantas de kiwi, e foi observada a evolução dos sintomas após 12 dias de inoculação apenas com Psa. Neste estudo apenas um fago foi sequenciado e confirmado como lisogénico, os profagos podem ser benéficos para combater doenças prejudiciais causadas por bactérias patogénicas. Neste estudo foi possível destacar o potencial dos fagos como agentes de biocontrolo promissores contra Psa, que podem ser usados no futuro para substituir compostos de cobre e antibióticos Palavras-chave: Actinidia deliciosa ; cancro bacteriano; bacteriófagos; kiwi; Pseudomonas syringae pv. actinidia (Psa)
vii Index Acknowledgments ................................................................................................ iii Abstract ................................................................................................................ v Resumo ............................................................................................................... vi Index .................................................................................................................. vii List of abbreviations ............................................................................................. ix List of figures ........................................................................................................ x List of tables ....................................................................................................... xiii 1. Introduction ....................................................................................................... 1 1.1. Actinidia cultivation and kiwifruit production ................................................ 1 1.2. Overview of Pseudomonas syringae pv. actinidiae ....................................... 2 1.2.1. Morphology and Taxonomy ...................................................................... 2 1.2.2. Propagation and Symptoms .................................................................... 3 1.2.3. Current methods for Psa control ................................................................ 5 1.3. Biocontrol of phytopathogenic bacteria on Actinidia using bacteriophages.... 7 1.3.1. Bacteriophages - definition, classification and life cycles ........................... 7 1.3.2. The use of phages for plant disease control ............................................. 9 1.3.3. The use of phages on actinidia plants .................................................... 11 1.4. Aims ........................................................................................................ 13 2. Material and Methods ...................................................................................... 14 2.1. Isolation and Characterization of Pseudomonas syringae pv. actinidiae ...... 14 2.1.1. Pathogen Isolation ................................................................................ 14 2.1.2. Morphological characterization .............................................................. 14 2.1.3. Molecular characterization..................................................................... 14 2.1.4. Phenotypic characterization ................................................................... 17 2.1.5. Pseudomonas syringae pv. actinidiae Growth Curve ............................... 17 2.2. Isolation and Characterization of phages targeting Psa .............................. 18 2.2.1. Phage extraction ................................................................................... 18 2.2.2. Phage isolation, propagation and titration .............................................. 18 2.2.3. Host Range ........................................................................................... 20 2.2.4. Phage growth parameters ..................................................................... 20 2.2.5. Phage Stability ...................................................................................... 21
1 1. Introduction 1.1. Actinidia cultivation and kiwifruit production The genus Actinidia, belongs to the Actinidiacea family, it has 76 species described, and 125 taxa worldwide (Zehra et al ., 2020). The most popular species are Actinidia deliciosa (green kiwifruit) and Actinidia chinensis (yellow kiwifruit) (Guroo et al ., 2017). Although A. chinensis has a sweeter and aromatic fruit, its shelf life is very reduced, limiting its commercialization. Unlike A. deliciosa and its predominant cultivar, A. deliciosa “Hayward” has a bigger fruit and is more suitable for production, having longer shelf life (Drzewiecki et al ., 2016; Guroo et al ., 2017). Besides A. deliciosa and A. chinensis, other species are included in this genus: A. Melanandra (red kiwifruit), A. arguta (baby kiwifruit), A. purpurea (purple kiwifruit), A. kolomikta (artic kiwifruit), A. polygama (silver vine) and A. eriantha (velvet vine) (Guroo et al ., 2017). Actinidia species was first described in 1750 as ornamental plants found in China. Although seeds of A. chinensis were sent to Europe, it was only in 1904 when A. deliciosa was inserted in New Zealand that the great exploitation of the potential of the kiwifruit started (Zhen et al ., 2004; Zehra et al ., 2020). Kiwifruit industry has a strong economic impact with an annual production reaching 4.3 million tons worldwide in 2019 (IndexBox, 2020). The biggest Kiwifruit producers are China, Italy and New Zealand, and this massive production leads to a retail market value of ten billion euros(Donati et al ., 2020). Only in New Zealand the production of kiwi led to an annual revenue of one billion $NZ (Cameron and Sarojini, 2014). In Portugal, Actinidia was introduced later, in 1973, but it was after 1980 that the interest in kiwi production increased, due to its good productivity at that time (Franco, 2008). Kiwi production can reach 32 thousand tons and, in 2019 alone, there was a 126% increase in the installation of kiwi orchards (INE, 2020). Over time, the installation of Actinidia orchards has spread throughout the country, but its largest production is found in the regions Entre Douro e Minho and Beira Litoral (Moura et al ., 2015). Although there is a large production of kiwi, this cultivation, like many others, suffers very serious diseases caused by phytopathogenic bacteria. One of the most serious problems is the bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae , which is considered
2 the most significant limiting factor in kiwifruit production that leads to tremendous economic losses (Morán, 2018). 1.2. Overview of Pseudomonas syringae pv. actinidiae 1.2.1. Morphology and Taxonomy Pseudomonas syringae pv. actinidiae (PSA) is an gram-negative bacteria with a white circular and convex colony (Figure 1) (Takikawa et al ., 1989). Since PSA is considered the biggest threat to the production of kiwi worldwide, it has been included on the A2 list of European Plant Protection Organization as a quarantine strain( Abelleira et al ., 2014; EPPO 2021). Psa is divided by 6 different populations (also known as biovars) that are capable to infect Actinidia with distinct levels of virulence, which are denominated by Psa biovar 1, 2, 3, 4 ,5 and 6. Psa biovar 1 was the first strain to be described in Japan in 1984 causing massive economic losses in the kiwifruit industry. Later, in 1992, it was also detected in Italy causing less damage when compared to the outbreak in Japan due to climatic factors. These strains have in their genome phaseolotoxin, which is a toxin responsible for the halo symptoms on the leaves. Psa biovar 2 was only found in South Korea, causing major economic losses after infection of A. deliciosa and A. chinensis . This one doesn’t produce phaseolotoxin but instead has coronatine in the genome (Cameron and Sarojini, 2014; Figueira et al ., 2020). Coronatine is known to be a Figure 1.1Colonies of Pseudomonas syringae pv. actinidiae in King’s B medium.
3 toxin that enhances bacterial growth and develop the disease symptoms, which can be produced by several Pseudomonas syringae strains (Zheng et al ., 2012). Psa biovar 3 is the most aggressive population of them all and, unlike Psa Biovar 1 and 2, this one does not present phaseolotoxin or coronatine (Wicaksono et al ., 2018). It can be very destructive and cause severe economic losses and is responsible for the outbreaks in Italy, France, Spain, Portugal, New Zealand, Chile, China, Korea and Japan. Psa3 is also responsible for the bacterial canker in Actinidia (Abelleira et al ., 2014; He et al. , 2019; Vaz et al ., 2018). Psa biovar 4 was found in New Zealand and Australia (Cameron and Sarojini, 2014); this population was unable to cause infections and is considered low virulent, causing only leaf spots and no damage to kiwifruit, after comparing different characteristics on phylogenetic and phenotypic analysis it was decided to rename this population to Pseudomonas syringae pv. actinidifoliorum (Psaf) (Abelleira et al ., 2015; Cunty et al ., 2014; Morán, 2018). Psa biovar 5 and biovar6 were both found in Japan in limited areas (Morán et al ., 2018). It was verified that biovar 5 is more related to biovar 2 than the other populations of Psa, but biovar 5 does not produce both coronatine and phaseolotoxin(Figueira et al ., 2020; Fujikawa & Sawada, 2016) Biovar 6 produces both toxins and is more related to biovar 1 and 3(Sawada et al ., 2016). 1.2.2. Propagation and Symptoms Psa is common in the surface of plants, mostly in leaves, buds, twigs and flowers (Figueira et al ., 2020). Psa infection occurs with temperatures ranging between 12ºC and 18ºC, being the autumn and spring seasons two moments of high risk for plant infection and bacterial dissemination (Vanneste et al ., 2011). There are several sources of plant infection by Psa, as this population also resides outside Actinidia without causing any symptoms. The sources of infection of Psa can be over short distances, when infected plants interact in an orchard of healthy plants. Also, the use of pruning material or other material used on infected plants is also a source of infection and helps spreading the bacteria. Climatic conditions such as rain, frost and wind can also contribute to the spread of the bacteria among plants: rain and frost help Psa to enter tissues and the wind can take infected pollen to healthy plants within a short distance (Vanneste, 2013; Vanneste et al ., 2011) The disease symptoms also can appear during the spring, on leaves, flowers and branches (Figure 1.2) (Abelleira et al ., 2015). Although with less favorable temperatures, in winter, with frost, Psa
4 symptoms can be observed. As the disease aggravates it mixes with the dark coloured bark tissue producing a red rusty exudate (Vanneste et al ., 2011). Figure 1.2Life cycle of Pseudomonas syringae pv. actinidiae . In each season it is described the major symptoms (inside the circle), and the justification of why these symptoms occur (outside the circle). Adapted from Vanneste et al ., (2011). The symptoms of Psa are identified by brown necrotic spots on the leaves, surrounded, or not, by a yellow halo, necrosis and abortion of flower buds and red or white exudates in the trunks and branches (Figure 1.3) (Butler et al ., 2013; Vanneste, 2013). When the bacteria begin to proliferate throughout the plant, the symptoms are severe: wilt on the branches, leaf fall and dehydration of the fruits, which end up losing their commercial value, and finally, Psa can lead to the death of the plant or death of the entire orchard (Carvalho et al ., 2017). To infect the internal tissues of the plant, Psa needs to go through the epiphytic phase to the endophytic phase. The bacteria enter naturally through the opening of the stomata or also through lesions in the trichomes, leaf abscission and fresh cuts. In the winter, with the lesions caused by frost, the entry of the bacteria into the plant is facilitated and later on can cause its multiplication (Figueira et al ., 2020; Spinelli et al ., 2011).Trichomes on kiwi are able to create a favorable environment for the growth of bacteria due to their retention of humidity and to its hairy •Infection takes place during leaf fall and harvest •Multiplication of Psa inside the plant tissues •Plant reacts to presence of bacteria •Multiplication of Psa outside the plant •Occasional infection leading to leaf spot •Production of inoculum •New Infections SPRING Leaf Spots Wilting of Shoots SUMMER Shoots tips die back FALL WINTER White exudate Red exudate
5 structures, also protecting the bacteria from adverse conditions (Figueira et al ., 2020; Spinelli et al ., 2011). Once in the endophytic phase, the bacteria can migrate between the leaves, shoots and twigs through the apoplast and then through xylemic vessels (Donati et al ., 2020; Figueira et al ., 2020). Psa can invade the majority of the tissues of the plant and can create an systemic infection when the bacteria migrates to the xylem (Spinelli et al ., 2011). 1.2.3. Current methods for Psa control Currently, there is no product or solution that eliminates Psa completely, but there are different means of combat that aim to control the effects of the bacteria on the plants (Carvalho et al ., 2017). To arrest the spread of Psa among plants in an orchard, the implementation of good plant cultivation practices is essential such as the disinfection of cut material used in infected plants and the elimination and destruction of the infected organs (DGAV, 2013). In countries of the European Union, it is mandatory that all the transited plants have a phytosanitary passport to prevent the B) C) A) Figure 1.3Symptoms of Pseudomonas syringae pv. actinidiae . A) Brown spots surrounded by a yellow halo; B) Red exudate on infected trunk; C) Abortion of flower buds. (Kiwi GreenSun, SA, 2020).
6 entry and spread of Psa (Commission Implementing Decision No. 2012/756 / EU, of 5 December) (Borg, 2012). Copper-based chemicals (copper hydroxide and copper sulfate) are commonly used products to fight bacterial canker in kiwifruits; however, these treatments should be avoided due to its phytotoxicity, due to the decrease of soil diversity, and also due to the risk of spreading resistance of Psa to these compounds. In Asian countries and in New Zealand, antibiotic products (like streptomycin) are also used to fight this bacterial disease; however, according with the 2004/129/CE Decision of the European Commission on the 91/414/CEE Directive, the use of antibiotics as phytopharmaceuticals are not allowed in the European Union (Bryne, 2003; Bukman, 1991; Cameron & Sarojini, 2014; Carvalho et al ., 2017; Mariz-Ponte et al ., 2021). Both of the chemical options mentioned above have shown phytotoxicity problems, and can cause resistance in bacteria and leave residues on fruits (Donati et al. , 2014). In addition, streptomycin can lead to chlorosis and “cupping” on leaves, while copper can damage stalks and leaves (Cameron and Sarojini, 2014). Besides copper and streptomycin, others compounds were studied to realize the ability to control Psa,over the last years several alternatives of biological control have been studied as substitutes for chemical products. One of these studies was with terpenes, two monoterpenes, geraniol and citronellol were able to create an inhibitory effect on in vitro assays against Psa. These monoterpenes disturb the lipid membrane, changing its permeability leading to cell death (Cameron & Sarojini, 2014; Ferrante & Scortichini, 2010). Chitosan, that is obtained from shrimp shells was also tested against Psa and has the advantage of being both biocompatible and biodegradable (Donati et al ., 2014). Chitosan interacts with the negative charged membranes of the pathogen that leads to the leakage of the intracellular content and it can also inhibit the microbial growth (Cameron & Sarojini, 2014). Even though chitosan can decrease the epiphytic inoculum loads, once the pathogen enters the plant tissue, it is no longer effective (Donati et al ., 2014). Bacillus subtilis is one of the most used biologic alternative, Bacillus subtilis has the advantage to create antibiosis by competing with the pathogenic bacteria by killing it or by reducing its growth and, can also induce systemic resistance. It is used in Gray Mold, provoked by Botrytis cinerea , Bacterial spot ( Xanthomonas spp.), Pseudomonas syringae pv. tomato , among others bacterial or fungal strains(Carvalho et al ., 2017; Stewart et al ., 2011). Essential oils like clove bud ( Syzigium aromaaticum ), thyme ( Thymus vulgaris ) and oregano ( Origanum vulgare ) were also tested for their antimicrobial activity and production of radical reactions when interacting with lipids
7 of the cell membrane, causing cell death (Pucci et al ., 2018; Song et al ., 2016; Vavala et al ., 2016). Recently, antimicrobial peptides (AMPs) began to be studied for its protective power of the host in attacks against the pathogen and for its low toxicity (Mariz-Ponte et al ., 2021). AMPs are constituted with less than 50 amino acids, are amphipathic and have a cationic charge that become a great binding site with the plasmatic membrane of Psa that has anionic groups. This creates a displacement of the metal ions, damaging the outer membrane and creating access to the inter membrane. This process allows the leakage of intercellular material and can provoke disintegration of the membrane and lysis (Cameron & Sarojini, 2014). The study of plant-induced resistance was also deepened, Acibenzolar-S-methyl (ASM) was authorized as a biological product due to the emergency to control the bacterial canker caused by Psa in Italy (Bion® or Actigard®)(Pucci et al ., 2018). ASM belongs to the benzothiadiazole chemical group and operates as a functional analogue of salicylic acid leading to an increase at the resistance to pathogens and an upregulation of pathogenesis-related genes (Cameron & Sarojini, 2014; Jong et al ., 2019). Nevertheless, resistant inducers can be effective in controlled conditions but the host response in the field could be variable and also lead to a decrease of fruit quality and production(Reglinski et al ., 2013). Lastly, a great interest in the study of bacteriophages has arisen as a biological control against Psa, bacteriophages are viruses that have the advantage of being able to control bacterial infections, without affecting the normal microbiota (Pinheiro et al ., 2020) and also has a high specificity of the host with strong lytic activity (Ni et al ., 2020). 1.3. Biocontrol of phytopathogenic bacteria on Actinidia using bacteriophages 1.3.1. Bacteriophages - definition, classification and life cycles Bacteriophages (also known as phages) are viruses that specifically infect, and in many cases, kill bacteria without affecting the host’s microbiota (Chan et al ., 2013; Frampton et al ., 2012). They were described in 1915 and also in 1917 by Twort and d'Hérelle, respectively, and soon after started being used as therapeutic agents against bacterial diseases (Terms, 2011; Twort, 1915). Although the early promising results and effective treatments, the use of phages has drastically decreased with the introduction of antibiotics. However, the extensive use of antibiotics over time led to the spread of bacterial resistance throughout the world and so, these compounds became increasingly ineffective. This fact has triggered the interest on phages that reemerged as
8 a promising and attractive alternative to antibiotics and other chemicals to combat bacterial pathogens (Sieiro et al ., 2020). As phages are considered the most abundant entities on Earth, they can be found in many different environments were their bacterial hosts are present ( Clokie et al. , 2011). Most of the phages belong to the Caudovirales order, which is characterized by grouping tailed phages. Recently it was added new families to this order but the three largest families are Myoviridae that represents phages with contractile tails, Siphoviridae, representing phages with long, noncontractile tails and Podoviridae that englobes phages with short, noncontractile tails (Maniloff & Ackermann, 1998; Turner et al. , 2021; Yu et al ., 2016) . The structure of a phage is composed of a head with a nucleic acid genome enveloped by a capsid, consisting on a single or double–stranded DNA or RNA. According to their life cycle, phages can be classified as virulent or temperate (Sieiro et al ., 2020; Yu et al. , 2016). Virulent phages have a lytic life cycle that starts when the phage binds to specific receptors found in the bacterial cell surface and injects its DNA into the cell. Inside the cell, the phage DNA is replicated, new progeny phages are assembled and then released due to bacterial cell lysis. These phages are able to start another round of infection (self-replicating capability) by targeting other pathogenic bacteria found in the same location. In the lysogenic life cycle, after injection of the genetic material, the phage genome is inserted into the host's chromosome and replicates with cell division (Figure 4). This phage is referred to as a prophage. Therefore, temperate phages do not immediately cause Figure 1.4Ilustration of a tailed phage, from the Caudovirales order (Doss et al ., 2017)
9 bacterial cell lysis as it only occurs when there is an environmental change or when there are signs of stress (Doss et al ., 2017). Figure 1.5Lytic and Lysogenic cycle of bacteriophages (Batinovic et al ., 2019) 1.3.2. The use of phages for plant disease control The first study developed on isolation of phages in plants was performed Mallmann & Hemstreet, (1924), the authors were able to inhibit the development of Xanthomonas campestris pv. campestris by filtering decaying cabbages. As mentioned above, the use of antibiotics replaced as well, in plants, the interest of using phages against pathogenic bacteria. Producers mostly started to use streptomycin and also copper-based products to inhibit the development of bacteria. However, over time, the use of these products led to bacterial resistance. Copper-based products can cause phytotoxicity when accumulated in the soil and, can be also toxic for plants and humans. These products can as well, affect the fruit itself by leaving toxic residues in them. Antibiotics on the other hand, can harm both pathogenic and beneficial bacteria for the plant (Sieiro et al ., 2020), thus being necessary to find different approaches. Bacteriophages can be a biological alternative to chemical products, with the benefit of controlling bacterial diseases that have no effective biological control method, since phages have the advantage of having no negative effect against animal or plant cells (Chan et al ., 2013; Gill & Hyman, 2010; Yu et al ., 2016). The ease of phage isolation, efficiency against biofilms and the capacity of self-replication are some of the advantages of using bacteriophages as a control method, as well as being environment friendly and non-toxic to eukaryotes (Sieiro et al ., 2020).
10 Through the years, a lot of studies have been taking place to characterize phages against plant diseases caused by phytopathogenic bacteria. Studies in fruit trees, citrus, tomato and potato are the most common performed with phages (Balogh et al ., 2010), that is the case of Xanthomonas axonopodis pv. citri , causal agent of asiatic citrus canker, phages were tested as a biocontrol for citrus canker and citrus bacterial spot and it was observed 59% of reduction in disease severity by using a phage cocktail (Balogh et al ., 2008). The control of bacterial spot on tomato, caused by Xanthomonas campestris pv. vesicatoria was also studied with phages to stop spot disease on tomato and pepper, in this study, besides decreasing the disease severity, there was an increase on total weight of the fruit by using phages (Flaherty et al. , 2000). Still within the Xanthomonas genus, the prevention of the disease of bacterial blight of granium, provoked by Xanthomonas campestris pv. pelargonii was also studied by using h-mutant phages that were able to reduce up to 75% of incidence (Flaherty et al. , 2001). Erwinia amylovora , the causal agent of fire blight that appears mostly in fruit trees, phages were able to reduced infection by 84% to 96% infection in studies with pear blossom by using the bacteria Pantoea agglomeran s as a carrier (Boulé et al ., 2011). In the case of Dickeya solani, the bacteria that causes soft rot and blackleg diseases on potato, in vitro assays demonstrated that phages stopped completely the growth of this pathogenic bacteria (Czajkowski et al ., 2014). Bacterial wilt, caused by Ralstonia solanacearum was inhibit by using phage and also application of phage with a surfactant (Silwet L-77) (Young et al ., 2012). Pectobacterium carotovorum that causes soft rot in several crops like potato and tomato, was reduced when treated with phage PP1 (Lim et al ., 2013). In addition to phage-only treatments, the application of phage with integration of products already used for Psa control was also studied (Balogh et al ., 2010). Application of phages with acibenzolar-S-methyl was shown to suppress hypersensitive reaction on bacterial spot disease in tomato incited by Xanthomonas campestris pv. vesicatoria (Obradovic et al ., 2005) and also reduce of leaf blight of onion, caused by Xanthomonas axonopodis pv. allii (Lang et al ., 2007) . At the time, several phage biocontrol products are already available (Buttimer et al ., 2017). In the United States there are several products registered and approved by United States Environmental Protection Agency produced by OmniLytics Inc., such as AgriPhage, created to control tomato and pepper bacterial spot caused by Xanthomonas campestris pv. vesicatoria and also control Pseudomonas syringae pv. tomato (EPA, 2005), Agriphage – Fire Blight, to control Erwinia amylovora on apple and pear trees (EPA, 2018), Agriphage – CMM, to control tomato bacterial canker caused by Clavibacter michiganensis pv. michiganensis (OmniLytics Inc., 2019)
17 was carried out at 100 V for 1h30 min. The results were checked using a transilluminator under UV light, and images saved in digital format for further analysis. 2.1.4. Phenotypic characterization For the phenotypic characterization of bacteria, BIOLOG GEN III Microplates (MicroplateTM, Biolog) were used. Twenty portuguese isolates previously identified as Psa by duplex-PCR were used. Additionally, CFBP 7286, an Italian Psa biovar 3 reference strain, as well as P84, a Portuguese Psa biovar 3 strain previously referred in prior publications (Garcia et al. , 2018; Moura et al ., 2015), the pathotype stain of Psa CFBP 4909T (Japanese, biovar 1), and Pseudomonas syringae pv. actinidifoliorum CFBP 7812 (reclassification of Psa biovar 4) were also included in this analysis. Suspensions of pure cultures each isolate were prepared using the fluid IF-A (Biolog), the concentration were standardized uising a Biolog Turbidimeter and 100 μl of the bacterial suspension was dispensed in each well of the Biolog GEN III microplate. The plates were incubated at 28ºC and the optical density (590 nm) were read after 24, 48 and 72 hours, using the Biolog GEN III Micro Station™ ID System and the software MicroLogTM. Additionally, the optical density was read in a ASYS UVM 340 microplate reader (Hitech GmbH, Austria). The results of physiologic and biochemical characteristics of bacterial strains obtained with Biolog GEN III microplates, were analyzed by PAST 3 software (Øyvind Hammer, Natural History Museum, University of Oslo) and were compared using the correlation coefficient and the unweighted pair-group method using arithmetic averages (UPGMA) (Unweigted Pair Group Method using arithmetic Averages) (Sneath and Sokal, 1973). 2.1.5. Pseudomonas syringae pv. actinidiae Growth Curve The different phases of the bacterial growth were assessed in vitro using one of the Portuguese Psa strains, named P84. This strain was grown overnight, and then the culture was diluted with a dilution factor (DF) of 1:50 by adding 500 µL of the overnight grown culture on 25 mL of Tryptic Soy Broth (TSB) and incubated at 28ºC under agitation (120 rpm). The OD was measured at 600 nm every hour since time zero until 9 h of incubation, and then it was measured
18 after 24 h to 26 h. Simultaneously, 50 µL of the bacterial suspension was taken in every time point and serial dilutions (1:10) were made in NaCl 0,9%. After, 10 µL of each dilution was placed in a petri dish with Tryptic Soy Agar (TSB + 1.2% of agar) and allowed to run down the plate to create a drop effect in order to allow the quantification of colony forming units (CFUs) after overnight incubation at 28ºC for 48h. These data allowed the construction of a calibration curve OD vs. CFUs. 2.2. Isolation and Characterization of phages targeting Psa 2.2.1. Phage extraction Leaves, flowers, flowers buds, stems, weed, and soil samples from kiwifruit orchards were used for phage extraction. Each sample was placed in stomacher bags with 30 mL of distilled sterile water. The samples were homogenized in the Seward Stomacher 400 for 120 seconds and the mixture was filtered through 0.22 µm nylon membrane filter and collected into 15 mL falcon tubes. In leaf samples, only one leaf was added to each stomacher bag; flowers and buds were added 3 per bag. For soil samples, 10 g of soil was weighed and placed in a stomacher bag and. After homogenization, the samples were centrifuged at 5,000 rpm for 10 minutes, filtered through 0.22 µm nylon membrane filter and collected into 15 mL falcon tubes. For an initial screening of the presence of phages in the homogenized samples, a double layer of soft TSA (TSB + 0.6% (w/v) of agar) was mixed with 1 mL of filtrate and 300 µL of Psa bacterial suspensions (P84 and CFBP 7286) in TSA petri dishes. The plates were incubated at 28ºC for 24h. 2.2.2. Phage isolation, propagation and titration After checking the presence of phage plaques on the extracts, phages were isolated using the enrichment procedure with Psa strains CFBP 7286 and P84 as possible hosts. For this, 6 mL of each extract collected from kiwi leaves, flowers, buds, trunks, soil and weeds were mixed with 15 mL of double concentrated TSB and also 25 µL of both CFBP 7286 and P84 cultures. The cultures were incubated at 28ºC, 120 rpm for 24h and then centrifuged (9,000 × g, 4ºC for 10 min), filtered and the spot test was performed by placing a 10 µL drop on a bacterial lawn (previously prepared by mixing 100 µL of bacterial suspension with 3-5 mL of TSB top-agar (TSB
19 + 0.6% (w/v) of agar) in a TSA plate). The plates were incubated at 28ºC for 24h and after, the presence of lysis zones was analyzed. If an inhibition halo was observed in the plates, individual phage plaques were then isolated and purified using sterile toothpicks and papers until single plaque morphology was observed (Figure 2.1.) (Azeredo et al ., 2014). After isolation, phage production was performed according to previously described protocols with minor modifications (Azeredo et al ., 2014). Briefly, for each phage, a well isolated phage plaque was picked with a toothpick to TSA plates containing the lawns of the host P84 strain and then spread throughout the plates with paper strips. The petri dishes were incubated at 28ºC for 24h and, after incubation, 2.5 mL of Saline Magnesium Buffer (SM Buffer) (5,8 g/L NaCl, 2 g/L MgSO4, 50 mL/L 1 M Tris-HCl pH 7.5) were added to each petri dish and incubated at 4ºC with agitation (80 rpm) for 5-6h. A) B) C) Figure 2.1– A) First test for phage observation in plant tissue extracts; B) Spot test after enrichment procedure; C) Phage isolation technique to obtain phage plaque purification.
20 After, the SM Buffer and top agar were collected, centrifuged (9000 x g, 4ºC for 10 min) and the supernatant was recovered and filtered (0.22 µm). The phage suspension was stored at 4ºC until further use. The phage titration was performed using the double agar overlay technique. Briefly, serial dilutions of phage stock solutions were made in SM Buffer. Subsequently, 10 µL of each phage dilution was placed in a TSA plate containing the host bacterial lawn to create a drop effect. The plates were incubated at 23ºC for 24h and the number of PFUs were counted. To calculate the phage titre, the following equation was used: Equation 2.1.- Bacteriophage titer Bacteriophage titer (PFU per mL) =Nr.of bacteriophage plaques formed × Diluition factor Volume of bacteriophage sample (mL) 2.2.3. Host Range The host range of the isolated phages was evaluated using a collection of 41 Psa strains previously isolated and identified. One drop (10 µL) of each phage was spotted on the lawns of the different bacterial isolates. The petri dishes were incubated at room temperature (23ºC) for 24h and the presence of an inhibition halo for each phage was analyzed. Later, 10 µL of decimal serial dilutions of each phage were tested on the same bacterial lawns, with phage titers ranging from 103 to 106 PFU/mL, in order to assess if the lysis observed could be caused by Lysis from without (LFW). This observation was done after overnight incubation of the plates at 23ºC. The isolated phages were morphologically characterized by Transmission Electron Microscopy (TEM) using a procedure previously described (Melo et al. , 2014; Pires et al ., 2021). 2.2.4. Phage growth parameters The phage growth parameters were evaluated through one-step growth curves (OSGC) (Di Lallo et al ., 2014; Liu et al. , 2021; Ni et al. , 2021). The host strain P84 biovar 3 was grown in TSB until the cells reached the exponential phase (OD600 = 0.3 to 0.5). Then, the bacterial suspension
21 was centrifuged (7,000 x g, 4 ºC, 5min), the pellet was resuspended in 5 mL of fresh media and 5 mL of phage were added to obtain a multiplicity of infection (MOI) of 0.01. The culture was incubated with agitation for 5 minutes to allow the phages to adsorb to the host cells and after, it was centrifuged (7,000 x g, 4 ºC, 5min), the supernatant was discarded and the pellet was resuspended with 10 mL of fresh TSB medium. One sample was immediately taken (t0) and the culture was then incubated at 26ºC with agitation of 120 rpm. Samples were taken at 20 minutes intervals until 180 minutes, and serial dilutions were performed to enumerate the number of PFUs in each time point. 2.2.5. Phage Stability The thermal stability of the phages was assessed by incubating 108 PFU/mL of each phage at different temperatures: -20ºC, 4 ºC (control), 28 ºC, 37 ºC, 50 ºC and 60ºC for 24 and 48 hours. Similarly, the stability of phages to pH was evaluated using a universal buffer (150 mM of KCl, 10mM of KH2PO4 and 10mM of C6H5Na3O7) with different pH values: 1, 3, 5, 7 (control), 9, 11 and 13. Phages (108 PFU/mL) were incubated in each pH at 4ºC for 24 and 48 hours. All tests were performed in triplicate. To study the phages’ stability to UV light, a protocol by Yu et al . (2016) was followed with slightly modifications. Phage aliquots with 108 PFU/mL in SM Buffer were incubated into a 96 well microtiter plate and placed over a 366 nm UV light. Samples were taken every 15 minutes for 3 hours. After incubation under the different conditions described above, serial dilutions of phages were made and the PFUs were evaluated. All the assays were carried on two and three independent assays performed in duplicate.
22 2.2.6. Phage DNA extraction For phage DNA extraction, 1 mL of phage lysate was transferred to a 2 mL eppendorf tube and then added 12.5 µL of 1M MgCl2 and mixed gently. Then, 1 µL of DNAse (10 mg/mL) and 1 µL of RNAse A (100 mg/mL) was added to the lysate, vortexed and incubated at room temperature for 1 hour. After incubation, of the following solutions were sequentially added: 40 µL of 0,5M of EDTA, 5 µL of Proteinase K (10 mg/mL) and 50 µL of 10% SDS; then, the mixture was vigorously vortexed and incubated overnight at 55ºC. After overnight incubation, 500 µL of the mixture were transferred into two 1.5 mL microcentrifuge tubes and 500 µL of phenol were added. This mixture was centrifuged (13,000× g, 10 min at room temperature) and then, the aqueous phase was transferred to a new tube and added 250 µL of chloroform and 250 µL of phenol followed again by centrifugation (13,000× g, 10 min at room temperature). The aqueous phase was extracted again and 500 µL of chloroform were added and centrifuged with the same conditions mentioned previously. The top aqueous layer above the white interphase was recovered and the DNA was precipitated by adding 1mL of absolute ethanol and 50 µL of 3M sodium acetate solution. The samples were placed on ice for 30 minutes to help with precipitation. After, the samples were centrifuged (15 minutes at 14,000 × g at 4ºC) and the pellets were washed with 500 µL of 70% (v/v) of ethanol. After a new centrifugation (14,000 × g, 4ºC, 5 min) the ethanol was removed and the pellets were air dried for approximately 2 hours. After, the pellets were dissolved in 5 µL of sterile dH2O and the DNA samples were stored at -20ºC until further analysis. To verify if the phage DNA extraction was successful, the DNA concentration was measured using a NanoDrop and then the samples were loaded through an agarose gel (1% (w/v)) electrophoresis with a sample mix of 1.5 µL of DNA, 5 µL of H2O and 2 µL of Loading Dye. In the agarose gel it was also added 5 µL of 1kb marker (Grisp).
23 2.2.7. Genome alignment and annotation The phages genomic DNA was sequenced in an external laboratory. The obtained data was assembled and annotated using Geneious Prime software, myRAST, and online platforms, such as Phobius for transmembrane domains detection, SignalP 4.1 server for signal peptides, Promoter 2.0 Prediction Server, Arnold - FInding terminators for detection of Rho-Independent Terminators and Aragorn for tRNA detection. BLASTn was used to compare the genome to the NCBI database. Manual annotation was performed by using BLASTp to compare Geneious Prime predicted ORFs to the GenBank protein database. 2.3. In vitro phage infection assays 2.3.1. In vitro infection assays in culture medium To assess the in vitro antibacterial activity of phages, the host strain P84 was grown in 25 mL of TSB for 24h at 26ºC with agitation (120 rpm). Bacterial culture was adjusted to 0.4 (OD600) with fresh medium and the bacterial suspension was divided in volumes of 15 mL in two 50 mL flasks. One of the cultures was infected with phage at a MOI = 1 and the other was used as a control (no phage). Both cultures were then incubated again at 26ºC and 120 rpm. Samples were taken in duplicate after 0, 2, 4, 6, 24, 27, 30 and 48h of incubation for OD measurements and quantification of PFUs and CFUs, through the drop technique already described (2.2.2. Phage isolation, propagation and titration). PFU’s were observed after 24h incubation at 23ºC and CFUs were counted after 48h incubation. 2.4. In vivo phage infection assays 2.4.1. In vivo infection assays in Actinidea leaf discs In vivo assays using leaf discs obtained from healthy kiwifruit plants ( Actinidea deliciosa “Hayward”) were performed according to a protocol described by Flores et al . (2020) with slightly alterations. This assay was performed in 3 different groups: two groups infected with (Psa strain
24 P84 + phage 177T; P84 + phage VC3). Leaf discs only infected with Psa were used as a control group. Each group correspond to four leaf discs on a humidity chamber (Figure 2.2). Kiwi leaf discs with 2 cm diameter were disinfected with sodium hypochlorite 1% (v/v) and washed two times with sterile distilled water. Then, the four leaf discs were placed on sterile humidity chambers, which are formed by cotton wrapped with filter paper, soaked in 25 mL of sterile distilled water in glass petri dishes. Each leaf disc was inoculated with three drops of 10 µL of Psa P84 (108 CFU/mL). Each phage was added to its correspondent group, two hours after inoculation with Psa, with a MOI =1. All humidity chambers were incubated at 20ºC with relative humidity at 70% and with a photoperiod of 14h. One disk leaf inoculated was taken at the different time points: 0, 3, 24 and 48h post-infection. To quantify the bacterial and phage loads, leaf discs were homogenized with 1 mL of SM Buffer, followed by serial dilutions and plated on TSA medium (for CFU counts) or on bacterial lawns formed on TSA medium (for PFU counts). All the assays were carried out in biological triplicate. Figure 2.2Humid chambers. A) Humid chamber with leaf discs inoculated with strain P84 at 108 CFU/mL. B) Humid chamber of all groups in triplicate. 2.5. Phage infection assays on plants Preliminary plant infection assays were carried out in 2-year-old healthy kiwi plants (Flores et al ., 2020). Micro-lesions were created (Song et al ., 2021) and then, one of the leaves was inoculated with 2mL of P84 Psa suspension (108 CFU/mL) with a syringe on the adaxial and abaxial part. The leaf was covered with a plastic bag to retain the humidity and induce the proliferation of the bacteria. On another leaf, the same bacterial suspension and also a phage solution with phage A) B)
25 177T a MOI=1 was inoculated on both adaxial and abaxial part of the leaf, which was also covered with a plastic bag. Plants stayed at a photoperiod chamber with controlled temperature of 20ºC and relative humidity of 70%. Observations of both leaves’ evolution were performed every week. 2.6. Statistical analysis GraphPad Prism 9 was used for statistical analysis, which included a two-way repeated measures analysis of variance (ANOVA) with Bonferroni post hoc testing. The data is provided as a mean with a standard deviation. For P-values less than 0.05, differences between samples were considered statistically significant.
26 3. Results & Discussion 3.1. Isolation of Pseudomonas syringae pv. actinidiae After collecting a diverse number of samples, a total number of 238 bacterial isolates were obtained, and it was possible to isolate 55 Pseudomonas syringae pv. actinidiea (Psa) strains from stems, leaves, sepals, petals and flower buds of kiwifruit plants during 2019, 2020 and 2021. Most of the bacterial strains were isolated from an orchard in Briteiros, Guimarães, but some isolates were also collected from orchards in Braga, Famalicão and Póvoa de Lanhoso (Annex A). Besides the morphology of the bacterial isolates, all the Gram-negative and oxidase-negative strains were tested by Duplex-PCR to obtain its identification. 3.1.1. Molecular identification and characterization The molecular identification of the bacterial isolates was performed using the two sets of primers: KN-F, KN-R designed by Koh and Nou (2002) and AvrDdpx-F, AvrDdpx-R that were previously described by Gallelli et al . (2011). In the presence of Psa, these primers will lead to the amplification of two DNA fragments of 492 bp and 226 bp, allowing a fast identification of the isolates obtained in the orchard as Psa (Figure 3.1). Figure 3.1Example of a result obtained from Duplex-PCR using a few Psa strains with primers KN-F/R and AvrdDpx - F/R. M: Molecular marker (100bp plus DNA Ladder, BIORON), (-): negative control (Milli-Q water + PCR mix), 1 – 14: bacterial isolates collected from kiwi leaves from 492 bp 226 bp
33 Figure 3.6Calibration curve of CFU in function of OD (600nm) for P84 Psa strain 3.2. Phage isolation and characterization 3.2.1. Isolation of phages After the enrichment procedure using the extracts collected from different tissues of the kiwifruit orchards, it was possible to isolate 6 phages targeting Psa host P84 from leaves, stems and trunks from the Guimarães and Póvoa de Lanhoso orchards. Bacteriophages were identified as 8, 10, 18, 66, VC3 and 177T as mentioned in Table 3.2. Table 3.2Summary of the phages isolated in this study Plant Material Orchard Date 8 Leaf Guimarães 25/07/2019 10 Flower 18 Leaf Póvoa de Lanhoso 33 Leaf Guimarães 66 VC1 Branches Guimarães 19/02/2021 VC2 VC3 VC6 173T Trunk Guimarães 13/03/2021 175T 177T
34 The isolates used in this study were extracted from flowers, leaves, canes and trunk. In addition, extracts obtained from in soil and water samples collected from Psa-infected orchards were also tested for phage isolation, but in the case of this study, there was no growth of phage plaques. The size of the phage plaques tends to be small with an average of 2mm. Phage plaques are quite round, isolated and clear as is possible to see in figure 3.7. According to the literature, the phages studied to fight Psa are mostly extracted from soil (Lim et al ., 2013; Liu et al. , 2021; Park et al ., 2018; Yu et al ., 2016), water from lagoons and streams (Flores et al ., 2020; Ni et al ., 2020; Yin et al ., 2018), wastewater (Frampton et al ., 2014; Martino et al ., 2021) and kiwi leaves (Di Lallo et al. , 2014). The phage plaques studied so far have different sizes. Usually all have clear plaques but the size of the plaques can be quite different. In the case of Flores et al ., (2020), all selected phages produced large, clear and lytic plaques, but occasionally smaller phage plaques could also be observed. Lim et al ., (2013) also describes that the isolated phage under study had large plaques and Martino et al ., (2021) phage plaques, clear with 5 mm in diameter. On the contrary, Liu et al . (2021) presented that the studied phage would have approximately 2mm in diameter. To prove the variation in size between phage plaques, Di Lallo et al ., (2014) demonstrates that one of their phages forms clear plaques with a diameter between 0.5 to 2 mm, and another phage also has clear plaques of larger size, with a diameter of 3 to 7mm. These studies prove the heterogeneity and size diversity among the isolated phages, regardless of their location of extraction. Figure 3.7Phage 8 isolated on host P84
35 3.2.2. Host Range The host range of the isolated phages was evaluated against a collection of 41 Psa strains isolated in this study, as well the reference Psa strains CFBP7286 and P84, Pseudomonas viridiflava (strain CFBP2353) and Pseudomonas syringae pv. syringae (Pss 10604) (Table 3.3) Table 3.3Host range of the isolated phages against Psa strain PHAGES STRAIN 8 10 18 66 VC3 177T CFBP7286 + + + + + + CFBP2353 - - - - - - Pss 10604 - - - - - - P84 + + + + + + 113S - - - - - - 117P H - H H + + 106S H H H - - 111F - - - - + + 104S - - - - - - 115F H H H H H - 112F H - H - - - 107F H H H H + + 113F H H H H + + 120F H H H H + + 119F H H H H + + 108F H H H H + + 388S - - - - H - 102F H H H H + + 123S - - - - - - 121F - - - - + + 112B + + + + + + 23 - - - - H - 29 - - - - + + 6 - - - - + + 4 - - H - + + 3 H - H - + + 1 - - - - + + 22 - - - - + + 7 - - - - + + 28 + + + + + + 5 - - - - + + 17 - - - - + +
36 Table 3.3Host range of the isolated phages against Psa strain (continuation) +: Lysis; -: No Lysis; H: Hazy halo NT: Not tested The lytic spectra results presented in Table 3.3 revealed that phages 8, 10, 18, and 66 had a narrow host range, being only able to infect 47.05%, 38.23%, 50% and 38.23% of the strains tested, respectively. Furthermore, the phage plaques formed by these phages were turbid or very turbid on the strains examined. Phages VC3 and 177T were particularly effective against the majority of the Psa strains tested, infecting 84.44% and 71.11%, respectively. Phages were also tested on different strains that also appear on kiwi fruit orchards, which is the case of Pseudomonas viridiflava (CFBP2353) and Pseudomonas syringae pv. syringae (Pss 10604), but no phage plaques were obtained for both strains. These results showed that the isolated phages are specific for Psa strains, a result also reported by Ni et al ., (2021). These authors tested the specificity of the PN09, a lytic phage, against 29 Psa strains and 5 distinct strains ( Vibrio parahaemolyticus , Salmonella derby , Staphylococcus aureus , Pseudomonas aeruginosa , and two Escherichia coli strains). The phage was able to cause lysis in all Psa strains, but not in the other five strains mentioned before. However, other authors (Frampton et al ., 2014 and Yin et al ., 2018 and Yu et al ., 2016) described phages that were not only effective against Psa biovar 3 strains but also against other Psa biovars, like P. syringae pv. tabaci , P. syringae pv. tomato and P. syringae pv. phaseolicola . In the host range experiment conducted by Liu et al ., (2021), the phage PHB09 27 - - - - + + 19 + + + + + + 110B - - - - + + VC2 NT NT NT NT H - VC3 NT NT NT NT H - VC4 NT NT NT NT H - VC5 NT NT NT NT - - 124F NT NT NT NT + + 117S NT NT NT NT + + 115S NT NT NT NT + + 101S NT NT NT NT + + 388E NT NT NT NT + + 104B NT NT NT NT + + 21 NT NT NT NT + + % INFECTION 47.05 38.23 50.00 38.23 84.44 71.11
37 exhibited a narrow host range, with a limited capacity to infect Psa and an inability to infect other Pseudomonas sp. strains ( P. orzyhabitans , P. fluorescens , P. putida , P. gessardii , P. fragi , Enterobacter hormaechei , E. cloacae and E. coli ). Based on the results obtained from lytic spectra, phages VC3 and 177T were selected for further studies. 3.2.3. Phage Morphological characterization Transmission Electron Microscopy (TEM) was used to examine phage morphology, and it was observed that phages VC3 and 177T are members of the Caudovirales order, belonging to the Siphoviridae and Myoviridae families, respectively (Figure 3.8), according to the classification system of the International Committee on Taxonomy of Viruses (Adams et al ., 2016). Most of the phages isolated and identified to control Psa belongs to the Caudovirales order, the Myoviridae family represents for 52.1% of the phages identified and tested to control Psa, while the Podoviridae family represents for 41.7% and the Siphoviridae family represents for 6.3%(Pereira et al ., 2021). Siphoviridae phages have lengthy flexible tails, while Myoviridae phages have doublelayered contractile tails (Yu et al ., 2016) . Figure 3.8TEM visualization of phages’ morphology; A) Phage VC3 from Siphoviridae family with a noncontractile tail (100 nm scale); B) Phage 177T from Myoviridae family with a contractile tail (100 nm scale). A) B)
38 3.2.4. Phage Stability The stability of phages to particular environmental conditions like as temperature, pH, and UV radiation is crucial for their practical application as biocontrol agents for plant diseases. As a result, the stability of the two selected phages was measured by counting the number of PFU’s following treatment at various temperatures and pH levels, as well as under UV light exposure. The stability of phages at different temperatures is critical for research with applications that are also affected by environmental factors, as it affects their capacity to adhere, penetrate, and grow within the bacterial host. Only a few phages inject their genome into host cells at temperatures below the optimum, limiting the number of phages participating in amplification. High temperatures cause the latent stage to last longer. Furthermore, high temperatures have the potential to destroy the proteins that make up the capsid (Pereira et al ., 2021). In the case of kiwi plants, excessively cold temperatures and frost cause harm to the plants as well as an increase in the spread of Psa as a result of the damage. The stability of the phages at various temperatures was tested and the results are displayed in figure 3.9. Figure 3.9– VC3 and 177T phage stability at different temperatures (-20ºC, 4ºC (control) 28ºC, 37ºC, 50ºC and 60ºC) during 24 hours. Error bars represent standard deviation for three independent assays performed in duplicate. Statistical analysis was performed by the two-wayANOVA. ** and **** indicates p≤0.01 and p≤0.0001, respectively.
39 Phages were tested at various temperatures (-20, 4, 28, 37, 50 and 60ºC) to better understand their stability at severe conditions compared to the standard incubation temperature (4ºC). As observed in Figure 3.9, phage 177T was stable between -20ºC and 37ºC and it was completely inactivated in temperatures above 50 ºC. Phage VC3 was also stable between -20ºC and 37ºC with no statistic differences among these conditions. Contrary to phage 177T, after 24h incubation of phage VC3 at 50ºC, the majority of the phages remained active although the phage titer dropped 5 log10 PFU/mL comparatively with the control (4ºC) ( p <0.05), while at 60ºC the phage titer decreased 3 log10 PFU/mL ( p <0.05). On studies performed by Di Lallo et al ., (2014), Yu et al ., (2016) and Park et al ., (2018), phages were stable at 40ºC after one hour of incubation and phage titer started to decrease when temperatures reached 50ºC and phages were inactive at 60ºC. Phages isolated by Ni et al ., 2021 were stable at temperatures between 25 and 35ºC and also decreased when temperatures were above 45 and 55ºC, and were also inactive at 60ºC. On assays by Flores et al ., (2020), phages were tested at the temperatures of 4, 18 and 37ºC, and at this last temperature it already shown reduction of titer. Yin et al ., (2018) identified phages that were all ineffective between the temperatures of 25 and 60ºC. According to Liu et al ., (2021), phage PHB09 remained stable for twelve hours at temperatures ranging from 4 to 37ºC before starting to lose titer. The phage titer dropped after twenty-four hours at temperatures ranging from 37 to 50ºC. As the average temperature for kiwi fruit growth is 20ºC and usually do not reach temperatures above 50ºC, the two selected phages are both stable at these cultivation conditions and there’s no risk of phage inactivation. The acidity of the environment is also an important aspect to consider as it influences phage stability, which may ultimately impact phage attachment, infectivity, intracellular replication, and multiplication. Because of their protein nature, phage lifespan reduces slowly as the environment becomes more acidic, inducing irreversible coagulation and precipitation(Pereira et al ., 2021). Since phages would be in contact with different environmental conditions when placed in kiwi orchards, it is also critical to address phages’ stability to varied pH's, especially because phages can be trapped in biofilms, reversibly adsorbed to soil particles, and inactivated by the soil’s low pH (Doffkay et al ., 2015). The results obtained are shown in Figure 3.10.
40 Figure 3.10VC3 and 177T phage stability at different pH (1, 3, 5, 7(control), 9, 11, 13), for 24h. Error bars represent standard deviation for three independent assays performed in duplicate. Statistical differences were studied with the two-way ANOVA (p<0.05). The profile of both phages to the different pH’s were quite similar. Both phages were particularly sensitive at pH 1 and pH 13 as no phage plaques could be detected after incubation at 4ºC for 24 hours at these pH's. In the case of phage 177T, although its concentration decreased on pH 3 and pH 11, no statistic differences were found ( p> 0.05) comparatively with the control (pH=7). For pH’s between pH 5 and pH 9, the concentration of this phage remained stable. On the other hand, the titre of phage VC3 was only lower at pH 3 and was stable between pH 5 and pH 11 with no significant differences were observed (p>0.05). Other studies regarding the stability of the phage at various pH levels are consistent with the results obtained in this study. For instance, Yu et al ., (2016), Park et al ., (2018) and Liu et al ., (2021) obtained phages with stability between pH 3 and pH 11; however, considerable decreases at pH 3 and pH 11 were seen by Liu et al ., (2021). Ni et al ., (2021) had great stability between pH 6 and pH 9, decreased at pH 2 and inactivated at pH 1, the phage concentration was relatively high at pH 12. In the study by Yin et al ., (2018) the phages were stable between pH2 and pH 12. Flores et al ., (2020) only studied stability at pH 4, 5 and 6 where all phages are stable except for one which had little stability at all three pH’s. Due to the field conditions not being found pH 1 or 13, in which the phages are shown to be inactive, and therefore, in the pH conditions to which they will be exposed when applied, the phages can be stable.
41 The phage's ability to survive in the phyllosphere is influenced by UV light. UV light (100– 400 nm) is biologically detrimental because it causes protein breakdown in free phage particles and changes the nucleic acid structure, lowering the phage's infectivity (Pereira et al ., 2021).The durability of phages against UV rays, in addition to temperature and pH is critical for this study since the major goal is to apply the phages to plants that are exposed to UV rays outside in orchards. As illustrated in Figure 3.11, the stability of these two phages to UV radiation at 366 nm was tested. Figure 3.11VC3 and 177T phage stability at UV light (366nm) at different time points (0 (control), 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 and 180 minutes). Error bars represent standard deviation for two independent assays performed in duplicate. Statistical differences were studied with the two-way ANOVA (p<0.05). According to the results shown in the figure 3.11, both phages were stable to UV-A rays (366nm) during the 180 minutes of the study and no significant differences were observed (p>0.05). In studies by Yu et al ., (2016) and Park et al ., (2018), both tested their phages in UV-A (365 nm) and UV-B (306 nm) light and both had similar results. Yu et al ., (2016) observed phage stability up to 60 minutes of study at 365 nm wavelengths, while at 306 nm wavelength all phages had a 50% reduction in efficacy. Park et al ., (2018) also determined that the PPPL-1 phage was stable in UV-A light but its stability rapidly declined in UV-B light. When tested with UV radiation at 365 nm, Liu et al ., (2021) reported that phage PHB09 remained stable for up to 60 minutes.
42 In another approach, Flores et al ., (2020) directly tested the phages to solar radiation in 30and 60-minute trials. The results were maintained in the two periods of study, which determines a good stability of the phages to solar radiation. 3.2.5. One Step Growth Curve One-step growth curves are used to determine phage growth parameters such as the latency period and the burst size (Clokie et al ., 2018). Whenever the bacteria is destroyed, it is done by bursting. Phage corpuscles proliferate continuously within the bacterial cell and are always released when the cell ruptures. However, the time required for fixation, the time required for bacterium rupture, and the number of young phage corpuscles developing within the bacterium to be liberated with its rupture all differ in each case, depending on a variety of conditions that differ from one experiment to the next (Hyman & Abedon, 2009). These parameters are represented in Figure 3.12. Figure 3.12One-Step Growth Curve of 177T and VC3 phages In the diagram above, phage 177T has an 80-minute latency time, a rise period of 60 minutes and a burst size of 14, while phage VC3 it was not possible to obtain a typical OSGC curve and it could be necessary to make some optimizations to the protocol used, such as carrying out
49 When comparing results with other studies carried out in vitro , Di Lallo et al ., (2014) describes the “Time of Death” as the ability of phages to manage to kill the bacteria by their adsorption rate and by the possible reduction in the OD of the bacterial population. In the case of Di Lallo et al ., (2014), with an absorbance reading of 600nm at an MOI=0.01 the ΦPSA2 phage determined a kill time of 155 minutes. Because the OD of the bacterial culture never went below its original value, it was impossible to calculate the "time of death" using the ΦPSA1 phage. In this 24-hour investigation, ΦPSA2 significantly reduced bacterial culture turbidity compared to the control, but the turbidity of the ΦPSA1 phage only marginally decreased, with no major changes from the control. In the study developed by Yu et al ., (2016), the phages used (MOI=0.01) for in vitro infection had some differences between them. KhuΦ38 demonstrated a late lytic activity compared to KHUΦ59 and KHUΦ74 phages. Lytic activity increased during the first 24 hours after infection but then decreased slightly. KHUΦ34 and KHUΦ44 phages only showed lytic activity after 24 hours of infection. However, the lytic activity of KHUΦ34, KHUΦ38 and KHUΦ 44 phages after 80 hours of treatment was quite similar. The PPPL-1 phage, studied by Park et al ., 2018 with MOI=0.01 resulted in a bacterial density gradually reduced to an OD of 0.1 for 12 hours and then slowly increased until the 80h of the study. Phages tested by Ni et al ., (2020) were evaluated with MOI's of 0.1, 1, 10 and 100 and the results showed that phage PN05 inhibited Psa for 12 hours with MOI of 0.1, 1 and 10 and no growth was observed until at 22h of rehearsal where the OD increased from that moment on. With MOI 100, the phage completely inhibited the bacteria up to 24 hours and then some bacterial growth was observed. In the case of PN09 phage, it inhibited bacterial density for 18 hours at an MOI of 0.1, 1 and 10, no bacterial growth was observed until 26 hours of testing and from 26 hours onwards the OD600 began to increase slightly, already with MOI =100 the phage inhibited bacterial growth up to 26 hours of assay and soon thereafter had a regrowth after this incubation time. Flores et al ., (2020) also tested phages with MOI's of 0.1, 1 and 10 and in all of them, the results showed that the CHF1 phage was the most efficient in causing lysis in the bacterial culture only after two hours of infection, while the CHF19 phage required 6 hours to clear the Psa culture. Pinheiro et al ., (2020) studied the potential application of phage ϕ6, to control Psa in vitro. The study of this phage was tested with two Psa strains, CRA-FRU 12.54 and CRA-FRU 14.10 (two Psa strains biovar 3). The inactivation of Psa was verified in vitro, using liquid culture medium and
50 in vitro assays, phage ϕ6 was effective against both strains (maximum reduction of 2.2 and 1.9 log CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). Recently, Liu et al ., (2021) tested the phage PHB09 MOI=0.001 and demonstrated that the bacterial density treated with this phage gradually decreased to an OD600 of 0.29 at 12 hours of assay and then slowly increased until 48 hours of incubation. In general, after analyzing the data of this study and, compared to the data obtained by other authors described above, it is possible to verify that even with different multiplicities of infection (MOI), there is no variation between the phages in terms of performance against the decrease in bacterial growth of Psa. It is also possible to understand that most of the phages mentioned here can obtain a growth inhibition for at least 12 hours, and even with some growth up to 24, 26 or 48 hours, it is possible to see that the phages have good lytic activity to be considered for a biological control of Psa. 3.2. Infection assays in vivo 3.2.1. Infection assays in vivo in kiwi leaves discs Given the potential for these phages to be used as biocontrol agents against Psa, it is important to evaluate their performance under laboratory conditions with kiwifruit leaf samples. Leaf discs (2 cm diameter) were used in in vivo tests to assess the effect of phages in the Psa load as observed on Figure 3.17. Phages were individually applied, two hours post Psa infection of the leaf discs. CFUs at various time points were analyzed and no significant differences were observed from the control. Furthermore, phages concentration is decreasing over time and should happen the opposite , the phage could be replicating in the presence of the bacteria and increasing the concentration. The phage could be integrating into the bacteria's genome Phage VC3 showed significant differences between 3h and 48h (p≤0.1). The bacterial density concentration reduced slightly after infection of the VC3 phage, however the greatest outcome was observed 3 hours after application of the phage with a concentration of 7×106 CFU/mL, compared to the control with only the Psa strain (1×107 CFU/mL). During the rest of the assay VC3 maintained the bacterial load slightly lower than the control ([P84+VC3] T48= 4.67×106 CFU/mL; [P84] T48= 5.67×106CFU/mL). There was no
51 observation of significant differences between the control and the leaf disks inoculated with P84 and each phage (p<0.05). The method used might possibly be an influence in the results obtained, as homogeneity of the material for phage plaque quantification proved difficult to achieve. In the future, it would be ideal to repeat the test with different extraction techniques as well as MOI 0.1 and 10 to analyze if there are any differences between these results. Relating the colony quantification data and plaque quantification, it is possible to observe that the data are coherent, where there is an increase in phage plaques (PFU/mL) there is a response of bacterial density reduction (CFU/mL). Figure 3.17– Efficacy of 177T and VC3 phages to control Psa in vitro. Bacterial Psa load present in leaf discs inoculated with Psa (dark grey), Psa + VC3 (light grey) and Psa + 177T (black) after 48 hours. Striped bars represent the phage concentration in leaf discs inoculated with the Psa + each phage. Error bars represent standard deviation for two independent assays performed in duplicate. Statistical differences were studied with the two-way ANOVA (p<0.05). * represents significant differences (p≤0.1) The methodology used in this assay was similar to that one tested by Flores et al., 2020. Their results indicate that a cocktail of their isolated phages CHF1, CHF7, CHF19 and CHF21
52 managed to reduce the bacterial density of Psa below the limit of detection (20 CFU /mL) after 3 hours of infection, and, Psa remained undetectable up to 24 hours of study. During the same period, the bacterial load of Psa from the leaf discs not treated with phages obtained concentrations between of 105 and 107 CFU/mL. Bacteriophages were detected throughout the experiment, demonstrating an increase in their concentration at 3 hours, due to Psa replication. The same procedure was used by Liu et al ., (2021) in a 72-hour experiment with the PHB09 phage with MOI 1, and it was determined that this phage can lower the bacterial load on kiwi leaves between 24 and 72 hours of infection. Throughout the experiment, phage was detected, with a rise in concentration after 12 hours. Pinheiro et al ., (2020) also developed a similar methodology, using kiwi leaves, cut squares (3cm x 3cm), decontaminated with a 3% H2O2 solution (v/v), washed and placed to sterilize in UV light. After sterilization, 100 µL of Psa (1x105 CFU/mL) and the same volume of phage (1x106 PFU/mL) were added. Samples were incubated in small petri dishes, placed inside larger petri dishes that contained PBS to prevent dehydration. At each sampling time, the sample was removed and dried, added to PBS and placed in an orbital incubator for 30 minutes. Finally, serial dilutions were made to quantify Psa and phage. The results obtained with this method demonstrated an increase in Psa in the control groups with strains Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10 of 2.2 log and 2.9 log CFU/mL, respectively. Strains CRA-FRU 12.54 and Psa CRA-FRU 14.10 with added phage ϕ 6 obtained a reduction of 0.4 and 0.6 log CFU/mL after 12 hours, respectively, and after 24 hours there was inactivation of 1.1 and 1.8 log CFU/ mL, respectively. Regarding the phage quantification, the phage concentration with strain Psa CRA-FRU 12.54 increased by 1.4 log PFU/mL and with strain Psa CRA-FRU 14.10 it increased to 2.8 log PFU/mL. However, these results are lower that the in vitro assay performed by the authors, suggesting that in the ex vivo experiments, the target bacteria may be incorporated inside the leaves' intricate matrix, insulating them from phage particles and preventing phage reproduction. Through these results obtained by Flores et al ., (2020) and by Liu et al ., (2021), it is possible to observe that although the use of only one phage is able to reduce the bacterial load of Psa, the use of a phage cocktail may have the best result. The phage cocktail also has the advantage that the phages involved can complement each other and increase the possibility to create a larger host range.
53 3.3. Ex vivo Infection assays in kiwi plants The preliminary tests in-plant were carried out on a two-year-old, healthy Actinidia deliciosa “Hayward”. Microlesions were made to aid the bacteria's entrance into the leaf, both on the leaf infected with just Psa and on the leaf treated with a combination of Psa and the phage 177T. The leaves were observed for a total of 34 days and it was possible to detect that in the leaf infected with only Psa, the differences and the appearance of necrosis by the leaf were notorious. In the case of the leaf inoculated with Psa and phage, the leaf remained without symptoms of Psa (Figure 3.18.). These preliminary results obtained by visual observation indicate that it is possible to determine that phage 177T will be able to fight Psa infections in the leaves of kiwifruit plants. Psa 177T Psa(P84) nfection day 12 dpi 34 dpi 16 dpi Figure 3.18– Efficacy of 177T phage in a two-year-old Actinidia deliciosa “Hayward” plant. Visual pattern Psa symptoms during 12-, 16and 34-days post infection (dpi). First row, inoculation with Psa, strain P84. Second row, inoculation of Psa and 177T phage.
54 To confirm these observations, it would be necessary, in future trials, to quantify the bacteria and phage population, to reach better conclusions. Also in the future, the installation of a larger trial and the spraying of Psa solutions and a combination of Psa and phage may lead to better conclusions and confirm the results obtained in this first trial, which aimed the observation of Psa symptoms. The results already obtained in in plant trials by other authors who tested combinations of several phages are indicators of the great interest in using phages for biological control of Psa, so it will be necessary to continue with the studies now initiated in this work. Flores et al ., (2020), inoculated healthy 2-year plants grown in a greenhouse at 20°C with a regular photoperiod with a bacterial suspension (106 CFU/mL) by spraying the suspension directly onto the plant's leaf surface. The quantity of Psa was determined after 24 hours. The author found that the phage cocktail reduced the bacterial load on the leaves by more than 75% when compared to untreated plants that were solely infected with Psa after 24 hours. Figure 3.19– Closer look of the differences of leaf infected with Psa (top row) and leaf infected with Psa and 177T phage (bottom row).
55 In the case of the study by Song et al ., (2021) it was used a different method to test phage PPPL-1 on leaves. It was used a silicon brusher to create microlesions and then spread the phage on both sides of the leaf (108 pfu/mL). Psa suspension (OD600=108 CFU/mL) was added after 2 hours. The PPPL-1 control was compared to two additional phages, KHUϕ34 and KHUϕ38, which were examined separately and in a cocktail of the three phages with a final concentration of 108. In comparison to the untreated leaves, the administration of the PPPL-1 phage effectively protected the treated leaves with Psa on the reduction in evident symptomatic spots after 14 days after inoculation. In planta, solo treatment of KHUo34 and KHUo38 phages had less impact than PPPL1, but a phage cocktail with three phages had equivalent efficacy to PPPL-1 phage alone.
56 4. Conclusions and future perspectives The main goal of this study was to isolate, characterize and understand the potential of novel phages against phytopathogenic bacteria present on Actinidia , based on preliminary results of this study, it was possible to determine that phages can be a good alternative to antibiotics and also to copper-based products to fight bacterial cancer in kiwi fruit, caused by Psa, and also the disease that causes great economic and productive losses. The phages isolated from composed material of kiwi fruit (VC3) and also from kiwi tree (extract from the trunk – 177T) belong to the Caudovirales order, from Siphoviridae and Myoviridae families, respectively. The stability testes revealed that the isolated phages can be stable under conditions present in kiwi orchards as they were capable of retaining their efficiency at temperatures ranging from -20°C to 37°C, pH ranging from 3 to 11, and UV radiation resistance. According to the in vitro infection assays performed on suspended cultures, both VC3 and 177T phages were able to reduce the bacterial loads after 4 hours of infection, as measured by the optical density along the time and also observed by a decrease in the quantification of cells and the rise in phage plaques. Although in vivo assays on infected discs did not reveal promsing results as no differences were found between phage infected samples and the control, the preliminary ex vivo assays in plants demonstrated visual effects after phage application directly on the leaf. In the future, significantly more ex vivo experiments, large-scale measurement of colonies and phage plaques, and monitoring of symptoms across different parameters would be required. It would also be important to study the combination of multiple phages in a cocktail to understand if the efficacy in biocontrol would increase comparatively with single phage application. Despite the fact that just one phage has been sequenced and confirmed to be lysogenic, prophages might be employed to combat illnesses caused by harmful bacteria. The experiments would be more accurate if the phages were virulent (lytic); consequently, future research should focus on converting these lysogenic phages by genetic modification of genomes, in which the integrase gene, responsible for integration in the host genome, could be removed. Prophages can bestow the capacity to produce poisons, resist antibiotics, boost virulence, and reject subsequent phage infections by modifying the host phenotype, a process known as lysogenic conversion.
57 5. Bibliographic References Abelleira, A., Ares, A., Aguín, O., Picoaga, A., López, M. M. & Mansilla, P. (2014). Current situation and characterization of Pseudomonas syringae pv. actinidiae on kiwifruit in Galicia (northwest Spain). Plant Pathology , 63 (3), 691–699. https://doi.org/10.1111/ppa.12125 Abelleira, A., Yebra, A., Casal, O. & Vázquez, P. (2015). Método de deteção de Pseudomonas syringae pv. actinidiae (Psa) em ramos assintomáticos de actinídea. Revista de Ciencias Agrarias , 38 (2), 206–212. https://doi.org/10.19084/rca.16916 Adams, M. J., Lefkowitz, E. J., King, A. M. Q., Harrach, B., Harrison, R. L., Knowles, N. J., Kropinski, A. M., Krupovic, M., Kuhn, J. H., Mushegian, A. R., Nibert, M., Sabanadzovic, S., Sanfaçon, H., Siddell, S. G., Simmonds, P., Varsani, A., Zerbini, F. M., Gorbalenya, A. E. & Davison, A. J. (2016). Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016). Archives of Virology , 161 (10), 2921–2949. https://doi.org/10.1007/s00705-016-2977-6 Azeredo, J., Sillankorva, S. & Pires, D. P. (2014). Pseudomonas Bacteriophage Isolation and Production. In Methods in molecular biology (Clifton, N.J.) (Vol. 1149, p. v). https://doi.org/10.1007/978-1-4939-0473-0 Balogh, B., Canteros, B. I., Stall, R. E. & Jones, J. B. (2008). Control of citrus canker and citrus bacterial spot with bacteriophages. Plant Disease , 92 (7), 1048–1052. https://doi.org/10.1094/PDIS-92-7-1048 Balogh, B., Jones, J., Iriarte, F. & Momol, M. (2010). Phage Therapy for Plant Disease Control. Current Pharmaceutical Biotechnology , 11 (1), 48–57. https://doi.org/10.2174/138920110790725302 Batinovic, S., Wassef, F., Knowler, S. A., Rice, D. T. F., Stanton, C. R., Rose, J., Tucci, J., Nittami, T., Vinh, A., Drummond, G. R., Sobey, C. G., Chan, H. T., Seviour, R. J., Petrovski, S. & Franks, A. E. (2019). Bacteriophages in natural and artificial environments. Pathogens , 8 (3), 1–19. https://doi.org/10.3390/pathogens8030100 Bilung, L. M., Pui, C. F., Su’Ut, L. & Apun, K. (2018). Evaluation of BOX-PCR and ERIC-PCR as Molecular Typing Tools for Pathogenic Leptospira. Disease Markers , 2018 . https://doi.org/10.1155/2018/1351634 Biolog. (2008). GEN MicroPlate. Cabot Blvd. , 1–8. Borg, T. (2012). COMMISSION IMPLEMENTING DECISION of 5 December 2012 as regards measures to prevent the introduction into and the spread within the Union of Pseudomonas syringae pv. actinidiae Takikawa, Serizawa, Ichikawa, Tsuyumu & Goto. Official Journal of the European Union , 335 (2), 49–54. Boul, J., Sholberg, P. L., Lehman, S. M., O’Gorman, D. T. & Svircev, A. M. (2011). Isolation and characterization of eight bacteriophages infecting Erwinia amylovora and their potential as biological control agents in British Columbia, Canada. Canadian Journal of Plant Pathology , 33 (3), 308–317. https://doi.org/10.1080/07060661.2011.588250 Bryne, D. (2003). Council and Parliament prohibit antibiotics as growth promoters: Commissioner Byrne welcomes adoption of Regulation on feed additives. Ip/03/1058 , July , 2–4.
58 http://europa.eu/rapid/press-release_IP-031058_en.htm?localen%0Ahttp://europa.eu/rapid/press-release_IP-031058_en.htm?locale=en Bukman, P. (1991). Directiva do Conselho de 15 de Julho de 1991 relativa à colocação de produtos fitofarmacêuticos no mercado (91/414/EEC). Jornal Oficial Das Comunidades Européias , 230 , 40–52. Butler, M. I., Stockwell, P. A., Black, M. A., Day, R. C., Lamont, I. L. & Poulter, R. T. M. (2013). Pseudomonas syringae pv. actinidiae from Recent Outbreaks of Kiwifruit Bacterial Canker Belong to Different Clones That Originated in China. PLoS ONE , 8 (2). https://doi.org/10.1371/journal.pone.0057464 Buttimer, C., McAuliffe, O., Ross, R. P., Hill, C., O’Mahony, J. & Coffey, A. (2017). Bacteriophages and bacterial plant diseases. Frontiers in Microbiology , 8 (JAN), 1–15. https://doi.org/10.3389/fmicb.2017.00034 Cameron, A. & Sarojini, V. (2014). Pseudomonas syringae pv. actinidiae: Chemical control, resistance mechanisms and possible alternatives. Plant Pathology , 63 (1), 1–11. https://doi.org/10.1111/ppa.12066 Carvalho, T. P., Nave, A., Rodrigues, S. & Costa, C. A. (2017). Racionalização da luta química no controlo de Pseudomonas syringae pv. actinidea (Psa) na região da Bairrada. Revista de Ciências Agrárias , 40 (SP), S103–S110. https://doi.org/10.19084/rca16168 Chan, B. K., Abedon, S. T. & Loc-Carrillo, C. (2013). Phage cocktails and the future of phage therapy. Future Microbiology , 8 (6), 769–783. https://doi.org/10.2217/fmb.13.47 Clokie, M. R. J., Millard, A. D., Letarov, A. V. & Heaphy, S. (2011). Phages in nature. Bacteriophage , 1 (1), 31–45. https://doi.org/10.4161/bact.1.1.14942 Clokie, M. R., Kropinski, A. M. & Lavigne, R. (2018). Bacteriophages, Methods and Protocols Volume III. In Methods in Molecular Biology (Vol. 1681). Cunty, A., Poliakoff, F., Rivoal, C., Cesbron, S., Fischer-Le Saux, M., Lemaire, C., Jacques, M. A., Manceau, C. & Vanneste, J. L. (2014). Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov. Plant Pathology , 64 (3), 582–596. https://doi.org/10.1111/ppa.12297 Czajkowski, R., Ozymko, Z. & Lojkowska, E. (2014). Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 ('D. solani’). Plant Pathology , 63 (4), 758–772. https://doi.org/10.1111/ppa.12157 DGAV. (2013). Plano de ação nacional para o controlo da flavescência dourada da videira . Di Lallo, G., Evangelisti, M., Mancuso, F., Ferrante, P., Marcelletti, S., Tinari, A., Superti, F., Migliore, L., D’Addabbo, P., Frezza, D., Scortichini, M. & Thaller, M. C. (2014). solation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker. Journal of Basic Microbiology , 54 (11), 1210–1221. https://doi.org/10.1002/jobm.201300951 Doffkay, Z., Dömötör, D., Kovács, T. & Rákhely, G. (2015). 59291. Bacteriophage Therapy against Plant, Animal and Human Pathogens , 59 (Twort 1915), 291–302.
65 6. Annex Annex A Isolates Plant Structure Orchard Collection Date 102F Leaf Guimarães 24/05/2019 107F 108F 111F 112F 113F 115F 119F 120F 121F 124F 117P Petal Guimarães 24/05/2019 101S Sepal Guimarães 24/05/2019 104S 106S 111S 113S 115S 117S 120S 123S 388S 388E Stamen Guimarães 24/05/2019 103B Buds Guimarães 24/05/2019 104B 108B 110B 112B 1 Leaf Cabeceiras de Basto 20/07/2019 6 7 17 Braga 19 21 22 23 Póvoa de Lanhoso 27 Cabeceiras de Basto 28 Póvoa de Lanhoso 29 3 Stem Cabeceiras de Basto 20/07/2019 4 5 Stalk Cabeceiras de Basto 20/07/2019 43 Leaf Guimarães 26/06/2020 48 50 52 132 20/10/2020 135 6.2. Leaf Famalicão 13/11/2020 11.1 V2 Branches Guimarães 09/12/2020 V5 VC2 Table 6.1 – All verified Psa isolates are included in the table below
66 Annex B Figure 6.1 - Biolog Gen III MicroPlate layout
67 Annex C Table 6.2– Summary of sequenced phages targeting Psa