Pathogens identification and resistance evaluation on bacterial canker in Actinidia arguta germplasm

Pathogen isolation and identification were performed on Actinidia arguta ‘Longcheng No. 2’ occurring bacterial canker from Liaoning Province, China. The pathogenic bacteria were identified as Pseudomonas syringae pv. actinidiae (Psa) by the analysis of morphology,16S rRNA and gyrB sequence, which were identified as Psa biovar 2 by Psa-specific primer sequence analysis. The pathogenicity tests were carried out with the isolate ‘R12’ and type strain ‘M228’ (biovar 3) as a control; the results showed that the phloem of green stems in A. arguta ‘Kuilv’ could be infect rapidly by R12, and milky mucus flowed from wounds, then the phloem turned black-brown, but it had strong resistance to Psa M228. In order to evaluate the resistance on Psa R12, 54 A. arguta germplasm resources were infected by artificial inoculation of stems, with A. deliciosa cv. ‘Hongyang’ and A. chinensis cv. ‘Xuxiang’, as control plant material, and their resistance levels were classified according to the disease index. The 54 tested materials exhibited differences in resistance to Psa R12, but no immune materials were found. In general, the germplasms were divided into five disease resistance categories, including 2 accessions with high resistance ‘Jianfengkuilv’ and ‘TL20013’, accounted for 3.70% of all the inoculated accessions; there were 11 resistant accessions, 15 tolerant accessions, 21 susceptible accessions, 5 highly susceptible accessions among them, accounted for 20.37%, 27.78%, 38.89% and 9.26%, respectively. In this study, the screening of disease-resistant germplasms could lay a foundation for further research on gene mapping, resistance mechanisms and breeding-resistant varieties of A. arguta to Psa.


Introduction
Actinidia arguta (Sieb.et Zucc.) is an important deciduous fruit that belongs to Actinidia in Actinidiaceae, and is also known as hardy kiwifruit, kiwiberry, baby kiwi or mini kiwi. Although the fruit of A. arguta is smaller in size than A. deliciosa 'Hayward', its fruit is smooth, edible and rich in nutrients such as vitamin C, phenolics, carotenoids and various minerals (Nishiyama et al. 2004;Okamoto and Goto 2005;Fisk et al. 2006;Latocha 2007;Latocha and Krupa 2008;Latocha et al. 2010). A. arguta has been widely planted as a promising species worldwide, especially in cold areas, because of its high cold hardiness (up to −30 °C in midwinter) and relatively short vegetation period (150 days) (Latocha 2008;Piotr et al. 2011). In recent years, many commercial orchards of A. arguta have been established in China, New Zealand, the USA, Japan, Chile and some European countries (Okamoto and Goto 2005;Williams et al. 2003).
With the continuous development of kiwifruit industry, related diseases began to occur. Among them, the bacterial canker is a common and devastating disease of kiwifruit, and its causal agent is Pseudomonas syringae pv. actinidiae (Psa). The pathogen was first discovered in the orchards of A. chinensis from California, USA (Opgenorth et al. 1983) and Shizuoka Prefecture, Japan (Serizawa et al. 1989), and subsequently also reported in Korea (Koh and Lee 1992;Koh et al. 1994), Italy (Scortichini 1994), Portugal (Balestra 1 3 et al. 2010) and New Zealand (Everett et al. 2011). In China, bacterial canker was first discovered in 1985. Then, large outbreaks occurred in areas of Sichuan, Shanxi, Anhui and others within a short period, resulting in numerous plant deaths and serious economic losses (Cheng et al. 1995;Liang et al. 2000). Psa can infect many species of Actinidia, among which A. chinensis and A. deliciosa are very susceptible, while A. arguta and A. pubescens are highly resistant to the pathogen (Song et al. 2020;Zhang et al. 2014;Shi et al. 2014). To date, Psa infection in A. arguta plants has only been reported in Japan (Ushiyama et al. 1992;McCann et al. 2013McCann et al. , 2017, New Zealand (Vanneste et al. 2014) and South Korea ) accompanied by mild symptoms, such as brown spot with yalo on leaves, which did not lead to serious losses. However, we found that the plants of A. arguta 'Longcheng No. 2' were infected with bacterial canker in Liaoning Dandong of China (N 40°13′; E 124°30′), and spread rapidly with adjacent vines. Its infection speed is no less than that of Psa in A. chinensis. This is contrary to the previous studies that A. arguta is resistant to bacterial canker. Although bacterial canker has only affected few areas at present (only three gardens), it has potential to become the deadliest disease of A. arguta, causing great harm to the industry.
In this study, pathogens of bacterial canker in A. arguta Longcheng No. 2 plants were firstly identified by morphological identification, 16S rRNA and gyrB gene sequence analysis. Then, the species of the pathogen was further identified by specific primers, including Psa F1/R2, Psa F3/R4, Tac F/R, PsaJ-F/R and PsaK-F/R. Finally, the resistance of 54 A. arguta germplasm resources to Psa was evaluated by artificial inoculation on stems in vitro. The above related studies could provide a foundation for resistant breeding to bacterial canker in A. arguta.

Plant materials
The soft green stems with semi-lignification of A. arguta cv. 'Kuilv', A. deliciosa cv. 'Hongyang' and A. chinensis cv. 'Xuxiang', which were cut during the growing season and uniform thickness, were used as experimental materials in the pathogenicity tests of new isolates. The resistance of Xuxiang, Hongyang and 54 A. arguta germplasm resources to the new strain Psa 'R12' were evaluated by pathogenicity testing with healthy dormant woody stems collected from the A. arguta germplasm resource nursery (N 44°00′; E 126°01′) locating in Institute of Special Animals and Plants Sciences of CAAS, Changchun. Among 54 A. arguta germplasm resources, 1 cultivar was from Russia, 12 were Chinese cultivars, and the remaining 41 were wild resources collected from three provinces (Heilongjiang, Jilin and Liaoning) in the northeast of China.

Isolation, purification and morphological characteristics of pathogenic bacteria
Stems were collected from the diseased plants (Fig. 2a). According to the method of Fang (1998), the pathogenic bacteria were isolated and cultured with NA medium at 24 °C. Then, single colonies were inoculated onto new plates for purification and cultured for 72 h at 24 °C in the dark. The morphology and growth of colonies were observed with a microscope, and the morphology of pathogens were observed under electron microscope, with the type strain Psa 'M228' (CP032631, biovar 3) as a control strain.

Molecular identification
Pathogenic bacteria were inoculated in buffered peptone water (BP) (Gao et al. 2016) liquid medium and then incubated for 18 h on a shaking table at 24 °C and 180 r·min −1 . After centrifugation at 1200 rpm for 2 min, the bacterial pellet was collected. Finally, pathogen DNA was extracted by the Tianjin Lanrui Biotechnology DNA Extraction Kit. The pathogen gene sequences were obtained by PCR amplification using primers 16S rRNA and gyrB gene (Yamamoto and Harayama 1995) respectively, and the sequences were deposited to GenBank (16S rRNA: OP218747-OP218754; gyrB: OP598821-OP598822,OP586787). PCR products were detected by agarose gel electrophoresis, tapped and recovered, TA ligated and transformed, and then sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequences were compared with those of standard strains in the GenBank database by BLAST to determine their taxonomic status. For further genome-level analysis, specific sequences were obtained by PCR amplification with the extracted DNA as a template and Psa-specific primers PsaF1/R2, PsaF3/R4 (Rees-George et al. 2010), Tac-F/R (Koh et al. 2014), PsaJ-F/R and PsaK-F/R . The PCR of gyrB gene was performed by an initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 62 °C for 1 min, extension at 72 °C for 2 min and final elongation at 72 °C for 8 min. The other PCR was performed by an initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing for 30 s, extension at 72 °C for 30 s and final elongation at 72 °C for 5 min. The nucleotide sequences, annealing temperatures and amplicon sizes of the primers used in this experiment are shown in Table 1.

Pathogenicity tests of pathogenic bacteria
According to the method of Hoyte et al. (2015), pathogenicity tests of new strain Psa R12 were performed on the soft green stems in vitro from healthy A. arguta cv. Kuilv, A. deliciosa cv. Hongyang and A. chinensis cv. Xuxiang, with Psa M228, as a control. The soft green stems (15 cm long) in vitro were sterilized and then wounded, penetrating into the xylem, in the middle part of the stems with a sterile knife, and 20 μL of bacterial suspension (10 8 CFU·mL −1 ) of Psa R12 and M228 was added to the wound respectively. Similarly, controls were inoculated using sterile bacteriological saline (0.85% NaCl). The upper and lower incisions of the stems were wrapped with absorbent cotton containing sterile water to keep wet, and the water has been sterilized by high temperature. Sterile water was sprayed into the plastic boxes in which the plant materials were held once a day to maintain humid conditions. Stems were placed in a Petri dish with moist filter paper and kept at 18 °C in the dark for 21 days. Finally, the pathogenesis of the materials was observed and recorded. Each treatment was conducted with 5 stems and 3 replicates. Finally, bacterial colonies reisolated from the diseased spots of Kuilv and Hongyang green stems inoculated with Psa R12 were identified by PCR tests using specific primers Psa F1⁄R2 and Psa F3⁄R4.

Evaluation on disease resistance of A. arguta germplasm resources
According to the method of Hoyte et al. (2015), the resistance was evaluated by artificial inoculation in vitro in 54 A. arguta germplasm resources, with Hongyang and Xuxiang as control plant materials. The dormant woody stems of Hongyang, Xuxiang and 54 A. arguta germplasm resources were artificially inoculated with Psa R12 in 2019 and 2020. The dormant woody stems (15 cm long) in vitro were sterilized and then wounded, penetrating into the xylem, in the middle part of the stems with a sterile knife, and 20 μL of bacterial suspension (10 8 CFU·mL −1 ) of Psa R12 was added to the wound. The upper and lower incisions of the stems were wrapped with sterile absorbent cotton. Sterile water was sprayed into the plastic boxes in which the plant materials were held once a day to maintain humid conditions. Stems were placed in a Petri dish with moist filter paper and kept at 18 °C in the dark for 21 days. Finally, the disease incidence was determined, and the resistance level was divided into 7 grades based on the standards shown in Table 2 and symptoms shown in Fig. 1.

Statistical analysis
All disease severity index data were analysed by SAS PROC ANOVA using Duncan's multiple range tests at p ≤ 0.05 (SAS Version 9.2).

Description of disease symptoms
Symptoms caused by Psa were first observed on A. arguta in Dandong, Liaoning Province, China, in June 2019. In 2020, similar diseases occurred in more than three Longcheng No.
2 plantings, but disease development was terminated due to eradication of the infected vines and subsequent periodic spraying with the proper bactericides in 2021. The disease mainly harmed the stems of plants, and reddish-brown mucus overflowed from the wounds and buds of the diseased stems (Fig. 2a, b). The diseased leaves had obvious polygonal brown spots surrounded by yellow halos (Fig. 2c). Finally, the twigs became wilted and scorched. When the stems were placed in a Petri dish with moist filter paper for 24 h, reddish-brown bacterial oozing was observed (Fig. 2d).

Morphological characteristics of colonies and pathogens
Eight representative round and convex colonies were isolated from the infected tissues of the vines. The colonies were white-creamy or light yellow, round, semiraised and smooth, with neat edges (R12 is shown as Fig. 3a) on NA medium as well as the type strain M228 (Fig. 3b). After culture for 72 h, the colony diameter was only 1-2 mm, Fig. 1 Grading standards for bacterial canker of A. arguta. The resistance level was divided into 7 grades based on the standards reflecting a very slow growth rate. Electron microscopy showed that both R12 (Fig. 3c) and M228 (Fig. 3d) were rod-shaped, with blunt round ends and flagella. R12 was 2.3-3.9 μm long and 0.6-1.1 μm wide, with a length/width of 3.68. M228 was 2.6-4.9 μm long and 0.7-1.5 μm wide, with a length/width of 3.60.

Sequence analysis results of pathogenic bacteria
In 8 isolated strains, a 1368-bp fragment was amplified using the 16S rRNA primer set 16F27/1492R, while a 984bp fragment was amplified using gyrB gene. The 16S rRNA and gyrB gene phylogenetic tree was constructed by comparison with published 16S rRNA and gyrB gene sequences of the relative bacterial species using the neighbour-joining method in MEGA 6.0 (Fig. 4). Combined with the results of morphological identification, the pathogen was identified as P. syringae pv. actinidiae (Psa).
There are differences in genetic structure of Psa prevalent in different regions or different periods. On the basis of physiological, biochemical, and genomic evidence, Psa can be divided into five separate clades (now popularly referred to as biovars 1-5) (McCann et al. 2013;Chapman et al. 2012;Fujikawa and Sawada 2016). To further determine the clade, 5 pairs of specific primers were used to conduct PCR amplification on the 8 isolated strains and M228 (biovar 3). The amplified products were analysed by agarose gel electrophoresis. The expected fragments of 280 bp with primer pairs Psa F1⁄R2 and 175 bp with primer pairs Psa F3⁄R4 were amplified from all nine strains (Fig. 5a, b lanes 1 to 9), indicating that they corresponded to Psa. Specific primers PsaJ-F/R, PsaK-F/R and Tac F/R were used to distinguish biovars 1, 2 and 3, respectively. Primers PsaJ-F/R produced a 481-bp region with genomic DNA of biovar 1 strains, while primers PsaK F/R amplified a 413-bp region present only in the genome of biovar 2 strains, and primers Tac F/R amplified a 545 bp region present only in the genome of biovar 3 strains. No signal was obtained from these 9 bacterial strains when using the primers PsaJ F/R (Fig. 5c). The 8 isolated strains produced a 413-bp amplicon (Fig. 5d lanes 2 to 9), while M228 did not show amplification of this DNA segment with the primer pair PsaK-F/R (Fig. 5d lanes 1). In contrast, the expected fragments of 545 bp were amplified from M228 ( Fig. 5e lane 1), but the 8 new strains did not produce any amplicons (Fig. 5e lanes 2 to  9) with the primer pair Tac F/R. Based on these results, it is suggested that these 8 strains belong to Psa biovar 2.

Pathogenicity tests
Pathogenicity tests of the isolate Psa R12 were conducted on the soft green stems of Kuilv, Hongyang and Xuxiang in vitro, with Psa M228 as a control. After inoculation with R12 for 21 days, obvious brown or black-brown diseased spots could be observed at the inoculation sites of Kuilv stems (Fig. 6a), Milky white mucus overflowed from the wounds, and the phloem turned black-brown or black, which was more serious than Hongyang (Fig. 6b) and Xuxiang (Fig. 6c). Inoculated with Psa M228, the incidence of Hongyang (Fig. 6e) was more serious than that of Xuxiang (Fig. 6f), while that of Kuilv was less serious (Fig. 6d). No disease symptoms developed on the control stems. These symptoms were similar to those caused by natural infections. Bacterial colonies reisolated from the diseased spots of Kuilv and Hongyang green stems inoculated with Psa R12 were identified as Psa by PCR tests using specific primers Psa F1⁄R2 and Psa F3⁄R4, fulfilling Koch's postulates.

Evaluation of resistance of A. arguta germplasm resources to Psa
The dormant stems of 54 A. arguta germplasm resources, Hongyang and Xuxiang were inoculated with Psa R12 by artificial wounding. After inoculation for 10 days, milky white mucus could be observed from some germplasms. After inoculation for 21 days, milky white, milky yellow or rust-red mucus overflowed conspicuously from the wounds of stems (Fig. 7a-d). Moreover, the mucus colour varied among germplasms. The phloem and xylem also turned brown or dark brown (Fig. 7e, f).
The 54 A. arguta germplasm resources showed significant differences in their degree of resistance to Psa R12 (Table 3), and there were no immune materials. Germplasm resources with a disease index below 30% accounted for 24.07%, indicating very high resistance to Psa R12. Twenty-five accessions, accounting for 55.56%, had a disease index greater than 50%. According to the 2-year comprehensive disease index, the 54 A. arguta germplasm resources were divided into five disease-resistance grades. Among them, 2 accessions, 'Jianfengkuilv' and 'TL20013', were highly resistant with a disease index less than 10%, accounting for 3.70% of all inoculated accessions; 11 accessions, 'Jiayuan 5', were resistant, accounting for 20.37%; 15 tolerant accessions accounted for 27.78%; 21 accessions were susceptible, accounting Fig. 3 The colonies of Psa 'R12' were white-creamy or light yellow, round, semiraised and smooth, with neat edges on NA medium (a) as well as the type strain 'M228'(b). Electron microscopy showed that there was no difference in morphology between R12 (c) and M228 (d), and they were rod-shaped, with blunt round ends and flagella with a length of 0.6-3.9 μm, 0.7-4.9 μm respectively Fig. 4 Phylogenetic analysis of the new isolated strains and the relative bacterial species published in GenBank based on sequences of 16S rRNA (a) and gyrB gene (b). Confidence values on the stems were obtained for 1,000 bootstrap replicates with Mega version 6.0 software for 38.89%; and 5 accessions were highly susceptible, accounting for 9.26%. Hongyang, which is extremely susceptible to Psa biovar 3, showed disease resistance, and Xuxiang, which has strong resistance to Psa biovar 3, showed high resistance to Psa R12.
Pearson's correlation analysis was carried out on the 2-year disease index results of the tested germplasms, and the results showed that there was a very significant positive correlation between the 2-year disease index (p < 0.0001, r = 0.83957). The results showed that the evaluation method was stable, reliable and accurate and could be used to identify the canker resistance of A. arguta.

Discussion
The pathogen of bacterial canker in kiwifruit is Pseudomonas syringae pv. actinidiae (Psa), which can quickly infect kiwifruit plants and cause outbreaks in a short time period. It is considered a fatal threat to the kiwifruit industry worldwide. At present, it had a serious impact on the kiwifruit industry in Europe, Asia, New Zealand, Chile and other regions (Balestra et al. 2010;Abelleira et al. 2011;Everett et al. 2011;Vanneste et al. 2011;Koh et al. 2012;Zhao et al. 2013;Sawada et al. 2015).
Although many species of Actinidia can be infected by Psa, there are great differences in disease resistance among the species. The disease sensitivity of A. chinensis has been found in Italy, New Zealand and South Korea. According to the species of Actinidia, the disease incidence of A. chinensis cv. 'Hort16A' was significantly higher than that of A. deliciosa cv. Hayward. And the disease incidence of A. deliciosa cv. Hayward, 'Yate' and 'Qinmei' decreased in turn. In China, Shen et al. (2008) and Gao et al. (2011) also found that the incidence in Hongyang was the highest and that in Xuxiang was the lowest. The similar result was found in New Zealand that the susceptibility of Hongyang was the highest, while the susceptibility levels of Hort16A, 'Gold 3', 'Gold 9' and Hayward gradually decreased. After the screening of disease-resistant species, a number of diseaseresistant varieties were obtained by crossing these diseaseresistant varieties of A. deliciosa with A. chinensis. However, it is rare for A. arguta plants to be infected by Psa, and was only reported in Japan (Ushiyama et al. 1992;McCann et al. 2013McCann et al. , 2017, New Zealand (Vanneste et al. 2014) and South Korea  with mild symptoms and no serious losses. Psa was isolated and reported to cause disease symptoms on A. arguta for the first time in China; its symptoms are similar to those of kiwifruit canker (Koh et al. 2010;Scortichini et al. 2012;Everett et al. 2011) and include reddish-brown mucus overflowing from the wounds and buds of diseased stems and obvious polygonal brown spots surrounded by yellow halos on diseased leaves.
Many studies have shown that there are differences in the genetic structure of Psa prevalent in different regions and even in different periods. At least five clades of Psa are distributed in different regions of the world (McCann et al. 2013;Chapman et al. 2012;Fujikawa and Sawada 2016), and they also show certain differences at the physiological, biochemical and genetic levels. Biovar 1 comprises strains isolated from Japan in 1984 and Italy in 1992, which contain an argK-tox gene cluster (Chapman et al. 2012). The occurrence of this biovar is high in Japan but is only sporadic in Italy. Biovar 2 is represented by strains isolated from South Korea in 1988, and it can produce coronatine (Shim et al. 2003), but the argK-tox gene cluster is deleted in this group. Biovar 3 was discovered in Italy in 2008, has high virulence and is the causative agent of the current outbreaks of kiwifruit canker disease in Europe, New Zealand, Chile and Asia (Chapman et al. 2012;Ferrante and Scortichini 2010;Marcelletti et al. 2011). Recently, biovar 4 was identified as the de novo pathovar P. syringae pv. actinidifoliorum (Pfm)  Table 1; Lane 1: Pseudomonas syringae pv. actinidiae M228; lanes 2-9: isolated strains (Cunty et al. 2015), and biovar 5 is currently only found in a localized area of Japan (Fujikawa and Sawada 2016). Based on morphological methods and 16S rRNA and gyrB gene amplification fragment alignment, the isolated strains were identified as P. syringae. The isolated strains were further characterised as Psa biovar 2 by amplified specific primer Fig. 6 After inoculation with R12 for 21 days, obvious brown or black-brown diseased spots could be observed at the inoculation sites of 'Kuilv' stems (a), which was more serious than 'Hongyang' (b) and 'Xuxiang' (c). After inoculation with M228, the incidence of Kuilv was mild, while the incidence of Hongyang was extremely severe (e), significantly more serious than that of Xuxiang (f) Fig. 7 Milky white, milky yellow, pink or rust-red mucus overflowed (a-d) from the inoculation wound of dormant stems, and the phloem turned brown or dark brown by 21 days after inoculation (e, f)   (Koh et al. 1994(Koh et al. , 2010. Field screening is the most intuitive method for the determination of disease resistance, but this method is easily affected by various factors, such as climate and the environment, causing the easy spread of pathogenic bacteria and inconsistent identification results. Many studies have shown that artificial inoculation in vitro is an effective method to evaluate the resistance to Psa of kiwifruit varieties (Hoyte et al. 2015;Zhang et al. 2014;Shi et al. 2014); the results of lesion length bioassays correlate positively with actual differences in Psa resistance observed in the field and are used by the New Zealand kiwifruit industry to screen for Psa resistance in their breeding programs Hoyte et al. 2015;Nardozza et al. 2015). In this experiment, inoculation of dormant stems in vitro was used to evaluate the disease resistance through 2 consecutive years. And it presented a highly positive correlation between the 2-year disease indexes, which further suggested that the evaluation method was stable, reliable and accurate. The evaluation results showed that milky mucus overflowed in some germplasms 10 days after inoculation, and all materials were infected with bacterial canker 21 days after inoculation. The severity of symptoms of many germplasms was no milder than that of Psa on Actinidia chinensis according to Song et al. (2020). However, as mentioned above, many previous studies showed that A. arguta has strong resistance to Psa Song et al. 2020;Shi et al. 2014), and in our previous studies (Wen et al. 2020), it was also found that A. arguta germplasm resources showed strong resistance to biovar 3 M228. In this experiment, A. arguta was generally susceptible to Psa R12, which belongs to biovar 2, while Hongyang was extremely susceptible to biovar 3, and Xuxiang, which was highly resistant, showed resistance or high resistance to this strain, indicating that there are differences in the pathogenicity of different groups of Psa to different kiwifruit species. These differences could be evidence for the physiological race differentiation observed in Psa. According to their disease index values, 54 germplasm resources of A. arguta were divided into five levels of disease resistance, and the resistant or highly resistant resources only accounted for 24.07% of the total resources. This pathogen has strong pathogenicity to A. arguta and poses a potential threat to this industry. In this study, the screening of germplasms resistance lays a foundation for further research on gene mapping, resistance mechanisms and resistance breeding for A. arguta.
Funding This research was supported by the Central Public-interest Scientific Institution Basal Research Fund (#1610342020011) and the Project of Science and Technology Department of Changchun City (#21ZGN09).

Data availability
The sequences data generated in this study were deposited to GenBank and freely available to any researcher.

Declarations
Ethics approval The authors declare that the research complies with ethical standards.

Competing interests The authors declare no competing interests.
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