1. IntroductionKiwifruit has a good reputation and is generally known as “the king of fruits” due to its flavor and nutritional properties, such as its high vitamin C content [1]. Currently, the global production of kiwifruit is about 1.5–1.6 million tons/year. China, Chile, Italy, Iran, and New Zealand are the main kiwifruit-producing countries, representing more than 90% of the world’s total kiwifruit production [2]. However, the bacterial canker has been considered the most devastating disease in kiwifruit production, caused by Pseudomonas syringae pv. actinidiae (Psa). Severe losses caused by this disease have been reported in several kiwifruit-growing countries [3,4,5]. Psa infection can cause various symptoms and, in severe cases, plant death, which significantly reduces the yield and quality of kiwifruit [6,7,8,9].Psa was first isolated in Japan in 1984 [10]. It was subsequently reported worldwide in countries such as Korea, China, Italy, Portugal, Spain, France, Turkey, South America, New Zealand, Slovenia, Greece, Georgia, Switzerland, Chile, and Australia [3,9,11,12]. The severe outbreak of this disease may be partially attributed to the clonal propagation of kiwifruit plants, which makes the pathogen spread quickly via seedlings [13]. Notably, other reasons for its widespread include its high phenotypic and genetic diversity among the Psa isolates as well as the emergence of new virulent strains [14,15,16,17]. Indeed, Psa isolates have been classified into six biovars, designated biovars 1–6, based on virulence and biochemical characteristics [18,19,20]. Among them, biovar 4 strains were transferred to the new pathovar actinidifoliorum due to its lower aggressiveness, which is substantially different from the other Psa biovars [21]. Furthermore, Psa strains belonging to biovar 3 have been found to be involved in the global pandemic of kiwifruit cankers [22,23].Psa has caused and is still causing severe worldwide economic losses [3,4,5,6,7,8]. For example, the land value of orchards growing the popular kiwifruit variety Hort16A has depreciated from 300,000 to 46,000 USD per hectare, resulting in enormous damage to the New Zealand economy [8]. Indeed, New Zealand exported the highest dollar value worth (USD 2 billion, accounting for 50.9% of total kiwifruit exports) of kiwifruit in 2021 (https://www.worldstopexports.com/kiwifruit-exports-by-country/ (accessed on 1 November 2022)). Meanwhile, Psa infection could be effectively alleviated by the characterization of the pathogen and the development of a sensitive detection method [24,25,26,27,28]. Furthermore, copper bactericide and streptomycin have been widely applied to reduce the damage of this disease. However, their effectiveness has been restricted due to the emergence of antibiotic resistance. Additionally, antibiotic residues in leaves and fruits, environmental pollution, and changes in soil bacterial communities have limited their application [29,30,31]. Due to these concerns and restrictions to its effectiveness, the use of streptomycin to control plant diseases has been banned in some European countries, such as Italy and Portugal [2]. Some other methods, including physical, agricultural, and biological, have also been used to control Psa [13,32,33,34]. For example, several antagonistic bacteria and their metabolites have exhibited promising in vitro antibacterial activity against Psa; however, their efficacy in controlling Psa infection was less consistent under field conditions [35,36,37,38]. Interestingly, there was an upsurge in interest in using phage therapy to control plant bacterial diseases [39,40]. Several phage products, such as AgriPhage™ and EcoShield™, have already been developed and commercialized in the market [1]. Therefore, phages are regarded as eco-friendly alternatives for controlling Psa infection in kiwifruit plants due to their environmental safety, high specificity for host bacteria, non-toxicity to plants and beneficial microflora, and ability to kill antibiotic-resistant bacteria [41].

This review summarizes the recent advances in the use of phages to control bacterial canker disease in kiwifruit, together with the potential challenges of phage therapy and its prospects.

3. Genome Analysis of Psa PhagesThe genomes of phages consist of either single- or double-stranded (ds) DNA or RNA, which can be classified as either lytic or temperate according to their life cycle. All phages of the class Caudoviricetes have a genome with ds DNA. As shown in Table 3, the genome of Psa phages could be either linear or circular. The genome size of Psa phages ranged from 40,472 bp to 305,260 bp, with the G+C content ranging from 43.1% to 60.44%. Estimates of the genome size using pulsed field gel electrophoresis (PFGE) indicated that the genome size of Psa173 was about 110 kb, the genome size of ΦPsa17 was about 30 kb, and the genome sizes of the other 21 phages were about 95 kb [49]. Interestingly, preliminary sequence data indicated that the size of the ΦPsa21 genome was about 300 kb, which is greater than that of other Psa phages. The phages with genome sizes larger than 200 kb were designated as Jumbo phages, evolutionarily divergent from phages with smaller genomes. Indeed, Jumbo phages have larger capsids and more genes than smaller phages. This genome size enables them to be less dependent on the replication mechanisms of their hosts. Interestingly, Wojtus et al. [58] recently found that the transcription of a Jumbo phage happens independently of the host bacteria by encoding their own RNA polymerases.Psa phage φPsa17 has been identified as a member of the T7-like virus genus (now named as Podovirus) based on a combination of genomic and proteomic assays as well as cryo-EM morphological observation [49,59]. Interestingly, a genomic analysis suggested that all Psa phages from Chile are closely related and similar to T7-like phages, having high similarity with other Psa phages from different countries, such as phiPSA2 (φPSA2) from Italy, phage PPPL-1 from Korea, and phiPSA17 from New Zealand. [48]. Therefore, it can be inferred that there is a global distribution of Psa phages, which is consistent with the pandemic of Psa biovar 3 at a global scale. Furthermore, the PFGE data correlated well with the genomic assembly, indicating that PFGE could be regarded as a useful tool to help and confirm the assembly of phage genomes [49].Following the genome-sequencing results, the Psa phages from Northern Italy could be divided into four groups based on the similarity of their sequences. In accordance with the phylogenetic analysis, psageA1 and psageB1 are considered two newly defined species of phages infecting Psa in the class Caudoviricetes [52]. Furthermore, phage PHB09 has been regarded as a novel genus in the class Caudoviricete based on phylogenetic analysis of the complete genome sequence and amino acid sequences of the conserved proteins [42]. To investigate more subtle differences in the DNA sequences of these phages, restriction fragment length polymorphism (RFLP) analysis was also carried out using restriction enzyme digestion. Based on the sizes of these bands, the Psa phages exhibited different size patterns. Indeed, the genomic DNAs of most phages were digested with restriction enzymes, such as NheI, EcoRI, and SphI, while some phages could be digested with HhaI and MspI. However, all of the tested phages could not be digested by the following enzymes: MnlI, NcoI, EcoRV, Sau3AI, SphI, RsaI, StuI, XhoI, DraI, Acc65I, HinfI, KpnI, and Tsp45I. These results indicated high diversity among the Psa phages collected from kiwifruit orchards [48,50].To investigate genetic diversity, we downloaded all Psa phage genomes available in the NCBI database. As shown in Table 4, these Psa phages originating from different countries differed in genome length, GC content, gene numbers, and classification. Interestingly, all Psa phages from Chile are podoviruses, while the Psa phages from China, New Zealand, and Italy are more variable. Furthermore, the genetic relationship of the Psa phages originating from different countries was revealed using the phylogenetic tree of the Psa phages, which was constructed based on the genome sequences available in the NCBI database using maximum composite likelihood (Figure 1). The result revealed that the Italian and Chinese strains exhibited greater diversity compared to the Chilean and New Zealand strains, which is generally consistent with the result of Table 4. However, in the same family, a very high similarity of genome sequences was observed among the Psa phages from different countries, which may be because they have the same origin. The wide distribution of phages in the same family may be mainly due to the international trade of kiwifruit seedlings. In addition, phylogenetic analysis of phages was also carried out in some studies based on large subunit terminases, which exhibited a similar result to that of genomes [60]. 4. Infection Mechanism of PhagesThe infection of host bacteria by a lytic phage involves a series of processes, which includes the attachment of phages to the host cells, injection of the DNA into the host cells, and self-replication in the host cells, leading to the death of host bacterial cells. The lytic activity of phages toward host bacteria has been, at least partially, attributed to endolysin and holin, which have been regarded as two lytic enzymes of phages [43,44,60]. Indeed, holins and endolysins have been widely reported to be able to damage the inner cell membrane and the peptidoglycan layer, respectively. For instance, it has been reported that phage endolysin LysPN09, produced by phage PN09, can cause the lysis of bacterial cells by effectively degrading the murein sacculus, which is the primary structural component of the cell wall in bacteria [47]. On the other hand, the function of endolysin is dependent on holin, which helps to transport the muramidases of the phage to the murein sacculus by perforating the inner cell membrane and determining the exact time point for the lysis of bacterial cells.Recently, increased attention has been paid to finding new phage endolysins and their potential in agriculture as novel antibacterial agents [43,44,60]. Many studies have reported that Gram-positive bacteria are more sensitive to phage endolysins than Gram-negative bacteria. This may be because the peptidoglycan layers of Gram-negative bacteria cannot be degraded by endolysin due to the outer membrane layer. However, the lysis of phage endolysin on Gram-negative bacteria could be facilitated by EDTA, which has usually been used as an outer-membrane permeabilizer. For example, when combined with EDTA, the endolysin LysPN09 of phage PN09 was able to effectively infect all of the 29 tested Psa strains and exhibited strong activity on the Psa cells so that the outer membrane was permeabilized with good thermal and pH stability. On the other hand, some other mechanisms have been proposed for phage lysis [47]. For example, Ni et al. [46] recently found that the biofilm formation of Psa strains was effectively inhibited by the suspensions of either a single phage or a phage cocktail. The biofilm-removal mechanism is mainly attributed to the ability of phages to produce specific enzymes, which drives them to actively disturb and reduce the formation of the host’s bacterial biofilm. Furthermore, lytic phages can stimulate their host bacteria to produce more EPS-degrading enzymes, facilitating the penetration and movement of phages through the host’s bacterial biofilm. Subsequently, phages first penetrate into host bacterial cells via the biofilm, then proliferate within their host’s bacterial cells, and finally eliminate host bacteria via lytic activity. 5. Tolerance to Environmental StressesTo effectively control the bacterial canker disease, it is necessary to check the activity of phages under various natural environments in kiwifruit-growing orchards, which is a key factor for effectively controlling phage therapy. Generally, Psa phages are negatively affected by various environmental conditions such as high temperatures, extreme pH, and ultraviolet (UV) radiation [45,47,50,51]. Although phages could still serve as a biocontrol agent of bacterial cankers, their population reduction by these factors has limited their efficiency and application in the field [41,61,62,63,64]. The hypothesized mechanisms of these abiotic factors have included that Psa phages are inactivated by damage to their structural elements and/or the promotion of DNA structural changes, which results in a decline in phage titers in the phyllosphere [2]. Notably, the reduction in phage activity depends on the specific strain of the phage, in which some were more tolerant than others [48]. Therefore, the utmost attention should be paid to the environmental adaptability factors of phages during their selection for the biocontrol of kiwifruit bacterial canker disease in the field.Among these environmental factors, temperature and pH have been reported to play an important role in the survival and stability of phages by influencing the attachment, penetration, intracellular replication, and amplification of particles within host bacterial cells [2]. When the temperature is low, only some phages can inject their genetic materials into host bacterial cells, while when the temperature is high, the capsid proteins can be degraded, resulting in a longer phage latency period [42,56]. Furthermore, the extreme pH values prevented the attachment of phages to receptor sites of host bacterial cells by interfering with either the lysozyme enzyme or with other capsid proteins of phages [42]. In general, it has been reported that the optimum pH value for the lytic activity of most Psa phages and their proteins is between 6 and 8 [2,41,42,46,47,48,49,50,53,56]. However, as shown in Table 5, several Psa phages have been documented to tolerate a wide range of pH values, ranging from 2.0 to 12.0.UV irradiation has been widely considered the most crucial factor for the reduction and loss of phage activity in the natural environment by affecting the longevity of phages in the plant phyllosphere [44,61,62,63,64]. Indeed, UV radiation can directly damage free viruses by degrading the proteins of free phage particles, changing the nucleic acid structure, and reducing phage infectivity [2]. In particular, the irreversible effect induced by shorter wavelengths was found on the genomic material, which resulted in both the modification of viral proteins and the formation of lethal photoproducts [2]. Compared to RNA phages, DNA phages are generally more sensitive to UV radiation due to the formation of lethal photoproducts such as thymine dimers induced by UV radiation, while dsDNA or dsRNA phages exhibited greater resistance to UV radiation than ssDNA or ssRNA phages [2]. However, the sensitivity of phage particles to UV radiation can be overcome by using different measures, such as high-titer phages in the morning or at night when radiation is limited [42]. 8. Conclusions and Perspectives

Due to the extremely high abundance of phages in the natural ecology, phages have received increasing research interest as an alternative and eco-friendly approach for controlling Psa infection in kiwifruit orchards. The main limitations of the application of phage therapy in agriculture are their high sensitivity to UV radiation and the emergence of bacterial resistance to phages. However, this can be overcome by using phage formulation via micro- and/or nanocarriers, phage cocktails, or preadapted phages, as well as the adjustment of application timing to avoid UV radiation exposure. Interestingly, compared to phage-susceptible bacteria, phage-resistant bacteria grow slower and are less virulent to host plants. Additional strategies to increase the efficiency of phage therapy and prevent the development of bacterial resistance include the application of phages in combination with currently available biological, physical, and chemical treatments. Other strategies are using mutant phages obtained from the wild-type and isolating new or modified phages that exhibit strong lytic activity against the resistant bacteria. Furthermore, the survival of phages in the orchards could be increased using protective formulations and effective inoculation methods. In addition, the government and organizations should develop relevant legislation to guarantee the phages’ large-scale production and safe use. Although several studies have revealed that phages can be successfully used to control bacterial cankers in kiwifruit plants, more in vivo field experiments should be carried out to elucidate the ecological and evolutionary effect of phage therapy in the rhizosphere and phyllosphere. Additionally, a good knowledge of microbial interaction between the phage and host bacteria is necessary to produce an effective phage cocktail, which will be very helpful for applying Psa phages to kiwifruit orchards. In conclusion, phages have great potential to control bacterial cankers either alone or together with other control methods.