Wang et al. Plant Methods (2019) 15:145 https://doi.org/10.1186/s13007-019-0530-9 Plant Methods RESEARCH Open Access A quick and efficient hydroponic potato infection method for evaluating potato resistance and Ralstonia solanacearum virulence Huijuan Wang1†, Jinxue Hu1†, Yao Lu1†, Mancang Zhang1†, Ning Qin1, Ruize Zhang1, Yizhe He1, Dongdong Wang1, Yue Chen1, Cuizhu Zhao1, Núria S. Coll2, Marc Valls2,3, Qin Chen4* and Haibin Lu1* Abstract Background: Potato, the third most important crop worldwide, plays a critical role in human food security. Brown rot, one of the most destructive potato diseases caused by Ralstonia solanacearum, results in huge economic losses every year. A quick, stable, low cost and high throughout method is required to meet the demands of identification of germplasm resistance to bacterial wilt in potato breeding programs. Results: Here we present a novel R. solanacearum hydroponic infection assay on potato plants grown in vitro. Through testing wilt symptom appearance and bacterial colonization in aerial part of plants, we found that the opti- mum conditions for in vitro potato infection were using an ­OD600 0.01 bacterial solution suspended with tap water for infection, broken potato roots and an open container. Infection using R. solanacearum strains with differential degree of aggressivity demonstrated that this infection system is equally efficient as soil-drench inoculation for assessment of R. solanacearum virulence on potato. A small-scale assessment of 32 potato germplasms identified three varieties highly resistant to the pathogen, which indicates this infection system is a useful method for high-throughout screen- ing of potato germplasm for resistance. Furthermore, we demonstrate the utility of a strain carrying luminescence to easily quantify bacterial colonization and the detection of latent infections in hydroponic conditions, which can be efficiently used in potato breeding programs. Conclusions: We have established a quick and efficient in vitro potato infection system, which may facilitate breed- ing for new potato cultivars with high resistance to R. solanacearum. Keywords: Ralstonia solanacearum, Potato, In vitro infection, Brown rot, Bacterial wilt Background production [1]. This soil-borne bacterium enters root Ralstonia solanacearum is the causal agent of bacte- through wounds or natural openings and multiplies in rial wilt (also known as brown rot on potato), one of the the vascular tissues, which results in xylem dysfunction most destructive plant diseases on many crops in tropical and ultimately kills the plant host [2, 3]. The bacterium and subtropical areas, which leads to huge losses in food is also able to survive for years in soil and water retaining the ability to invade host plants [4]. *Correspondence: chenpeter2289@nwsuaf.edu.cn; luhaibin011@hotmail. R. solanacearum is a heterogeneous species com- com posed of many genetic groups referred to as the R. sola- † Huijuan Wang, Jinxue Hu, Yao Lu and Mancang Zhang contributed nacearum species complex (RSSC) [2, 3]. Phylogenetic equally to this work 1 College of Agronomy and State Key Laboratory of Crop Stress Biology analyses of several conserved genes revealed that the for Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China RSSC is divided into four phylotypes: phylotype I isolates 4 State Key Laboratory of Crop Stress Biology for Arid Areas, College are mainly from Asia, phylotypes II and III are, respec- of Food Science and Engineering, Northwest A & F University, Yangling 712100, Shaanxi, China tively, composed of American and African strains and Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Plant Methods (2019) 15:145 Page 2 of 11 phylotype IV strains originate from Indonesia, Japan, R. solanacearum-potato interactions. Using this new Australia and the Philippines [5, 6]. method, we successfully characterized the virulence of Besides its worldwide geographic distribution, R. sola- several R. solanacearum strains on potato and screened nacearum possesses an extraordinarily broad host range, for potato varieties showing resistance to R. solan- causing disease on more than 200 plant species from 50 acearum. We also simplified the screening process using different botanical families [3]. The pathogen not only a luminescent pathogen that can be tracked in vivo in infects solanaceous crops such as tomato, eggplant, pea- infected plants. This new method will promote the study nut, pepper and potato, but also other plants from both of potato resistance to R. solanacearum and provide the dicot and monocot families, and new hosts are being insights for investigating other root pathogens of potato discovered continuously [7]. Due to its wide geographi- under gnotobiotic conditions. cal distribution, broad host range, long persistence in soil and highly aggressivity on plants, the bacterium was Results ranked by scholars as the second most important bacte- Development of an in vitro potato infection system for R. rial plant pathogen [1]. solanacearum Potato is currently the third most important staple Aiming at the quick identification of potato resistance food crop for human direct consumption just after rice to R. solanacearum, we designed a method for infection and wheat and it ranks first in energy and protein pro- in vitro Fig. 1a). A ­107–108 colony forming units (cfu)/ duction per unit of water [8, 9]. In protein produced per ml R. solanacearum suspension is used to infect plant acre land, potato ranks second to soybean. Importantly, hosts such as Arabidopsis, tomato and potato [13, 20, 22]. potato is rich in microelements and vitamins essential Thus, we grew potato plants hydroponically in MS liquid for the human diet, such as vitamin C, potassium and in medium for two weeks, injured the roots and transferred fiber [8]. Brown rot caused by R. solanacearum is one of them to the same medium containing 1 × 108 cfu/ml of the most notorious potato diseases, estimated to result R. solanacearum strain GMI1000, belonging to the phy- each year in 1 billion US$ economic losses worldwide lotype I. The leaves of the infected plants started wilting [1]. Breeding new potato cultivars with resistance to at 4 days post inoculation (dpi). At 5 dpi, almost all plants brown rot is essential for integrated management of this exhibited severe wilting symptoms, while the leaves of disease. To this end, the development of procedures to plants mock-treated with water kept green and healthy facilitate the screening for resistance in germplasm from (Fig. 1b). This suggested that in vitro infection could be wild potato collections, progenies from crosses between employed to test the virulence of R. solanacearum in potato species and potato transgenic lines will help potato. potato breeding programs. Wilt symptoms appear when water transport in the Soil-drench and stem-puncture inoculation are the xylem is blocked by lipopolysaccharides produced by R. two methods most widely used to study plant resistance solanacearum [2]. We thus hypothesized that increased to R. solanacearum [10–13]. However, adult full-size evapotranspiration by opening the growth container’s plants are needed in these procedures with the ensu- lids could accelerate plant wilting. To test this possibil- ing use of space, energy and time. Furthermore, since R. ity, plant inoculations were carried out in parallel under solanacearum is a soil-borne root pathogen, stem pen- opened and closed lid conditions. Although the same etration bypasses potential root resistance mechanisms. number of wilted plants was observed in both condi- The disadvantage of soil-drench inoculation is that the tions, plants in the open jars exhibited more severe wilt- opacity of soil hinders the direct investigation of root ing symptoms than those in the close jars (Fig. 2a). We responses to the pathogen. Vass et al. inoculated tomato further measured R. solanacearum growth in the aerial plants grown in hydroponic conditions to study R. sola- part of potato plants. The amount of bacteria in plants nacearum root colonization [14]. More recently, in vitro in the open and close jars was comparable (Fig. 2b), sug- pathogenicity assays have been successfully established gesting the air exchange does not affect bacterial growth, on tomato [15], Arabidopsis [16, 17], petunia [18] and but enhances wilting. In nature, R. solanacearum enters M. truncatula [19]. However, miniaturized in vitro infec- root at the emergence sites of lateral roots or root tips tion assays have not been set up to screen for resistance [2]. To test whether this natural infection also worked in potato to the pathogen. We previously generated con- in our in vitro infection system, 2-week-old plants with- stitutively luminescent R. solanacearum reporter strains, out injury were directly infected with the 1 × ­108 cfu/ a tool that we have used to characterize the colonization ml R. solanacearum solution. Even at 9 dpi, no potato and the defense responses of potato breeding lines [20, plant exhibited wilting symptoms in these conditions 21]. Here we have established a faster, highly efficient, (Fig. 3a). To confirm successful colonization in plants, low-cost potato hydroponic infection method to study we measured bacterial loads in the aerial plant tissues. Wang et al. Plant Methods (2019) 15:145 Page 3 of 11 Fig. 1 R. solanacearum inoculation causes wilting of potato grown under hydroponic conditions. Roots of 2-week-old potato plants were cut and inoculated with 1 × ­108 cfu/ml GMI1000. a Workflow of the in vitro potato infection method. b Representative digital images of bacterial wilt development taken at the indicated days post-infection (dpi). The experiment has been repeated more than three times with similar results The amount of the pathogen in both conditions reached contained fivefold less bacteria (Fig. 4b). This suggested ­108 cfu/g at 9 dpi (Fig. 3b), which is 10-fold lower than that 1 × 107 cfu/ml is the optimum R. solanacearum the ­109 cfu/g attained in the root-cut plants at 5 dpi concentration to inoculate potato plants in hydroponic (Fig. 2b). Therefore, plant inoculation without root injury conditions. resulted in symptomless infections due to lower bacterial In the previous experiments, liquid MS medium con- numbers. Thus, the root-cutting treatment was selected taining many nutrients and vitamins (MS−) was used to for the following experiments based on its faster, repro- re-suspend R. solanacearum for infection. To rule out a ducible infection, which correlated with the development potential effect of the MS medium on R. solanacearum of wilting symptoms. growth or virulence, we substituted the MS− medium The bacterial concentration (1 × 108 cfu/ml) used in with tap water. Wilting symptoms developed faster and laboratory inoculations is usually far higher than that stronger on potato plants in tap water containing R. sola- occurring in nature [13, 20, 22]. To check whether lower nacearum than in MS− medium with the same bacterial inocula could be used for in vitro potato infection, dis- concentrations (Fig. 5a). In line with this observation, ease development in plants was investigated using a the amount of bacteria in potato plants treated with the series of bacterial suspensions. Eighty percent of plants water-resuspended pathogen was fivefold higher than in infected with the higher bacterial solution concentra- the potato plants treated with MS− resuspended bacteria tion (1 × 108 cfu/ml and 1 × 107 cfu/ml) showed wilting (Fig. 5b). symptoms at 8 dpi. At this time those plants infected with the lowest bacterial concentration (1 × 106 cfu/ Hydroponic potato infection can be used to study R. ml) just started wilting (Fig. 4a). Moreover, potato solanacearum virulence in vitro plants inoculated with 1 × 108 or 1 × 107 cfu/ml showed HrpG and HrpB, two key regulators of the bacterial similar amounts of bacteria in the aerial part of plant, type three secretion system, play critical regulatory while those inoculated with the 1 × 106 cfu/ml solution roles in R. solanacearum virulence. The hrpG and hrpB Wang et al. Plant Methods (2019) 15:145 Page 4 of 11 Fig. 2 Increased evapotranspiration promoted leaf wilting development on potato plants. a Representative plant pictures Fig. 3 Higher evapotranspiration does not accelerate wilting were taken at 4 dpi. “W/T” represents the number of wilted plants symptom appearance on plants with intact roots. a Representative with respect to the total number of infected plants. “Open” indicates pictures taken at 9 dpi. “W/T” represents the number of wilting air exchange conditions while “close” indicates no air exchange plants with respect to the total of infected plants. “Open” indicates conditions. b Bacterial proliferation in potato stems was counted air exchange conditions while “close” indicates no air exchange at 4 dpi. Roots of two-week-old potato plants were cut and then conditions. b Bacterial colonization in potato stems detected at 9 dpi. inoculated with 1 × ­108 cfu/ml of R. solanacearum strain GMI1000. Two-week-old potato plants were inoculated with 1 × ­108 cfu/ml The experiment was repeated twice using 12 plants in each of R. solanacearum strain GMI1000 without wounding the roots. The experiment experiment was repeated twice using 12 plants for each mutant strains lose the ability to invade tomato [14, 23, solanacearum strains different from GMI1000 to test 24]. To determine whether our in vitro infection system their aggressivity in our hydroponic potato infection was suitable to evaluate R. solanacearum pathogenesis, system. Brown rot of potato is most commonly caused we first infected potato plants with R. solanacearum in the field by a subgroup of R. solanacearum strains GMI1000 wild type (wt), and the same strain carry- belonging to phylotype IIB [26]. UW551 and IPO1609 ing a precise deletion of the hrpG (ΔhrpG) or the hrpB from this group and the related CFBP2957 and CIP301 coding sequence (ΔhrpB). The hrpB and hrpG mutants strains from phylotype IIA, were selected to investi- did not cause any bacterial wilt disease when com- gate their virulence [6]. UW551 and CFBP2957 infec- pared with the strong wilting symptoms caused on tion resulted in a strong leaf wilt symptom while potato plants by the wild type strain (Fig. 6a). Bacterial IPO1609 and CIP301 did not cause any visible symp- growth analysis showed that the potato colonization tom (Fig. 7a). In addition, the multiplication of UW551 was similar in hrpG and hrpB mutants, but this bacte- and CFBP2957 in potato was 100 fold higher than that rial content in stems was 100 fold lower than that of the of IPO1609 and CIP301 (Fig. 7b). Our results indicate wild type GMI1000 (Fig. 6b). These data indicate that that UW551 and CFBP2957 are much more aggressive mutations on HrpB and HrpG abolish R. solanacearum on potato than IPO1609 and CIP301. Hence the in vitro virulence on potato, which is consistent with the fact infection system we have established here can be used that both mutants are non-pathogenic on tomato and to measure differences in the aggressivity of R. solan- Arabidopsis [23, 25]. Next, we selected wild type R. acearum stains on potato. Wang et al. Plant Methods (2019) 15:145 Page 5 of 11 Fig. 5 Tap water-resuspended bacterial solutions triggered stronger Fig. 4 Potato plants infected with different R. solanacearum bacterial wilt disease symptoms than MS-resuspended bacterial concentrations. a Representative pictures taken at 8 dpi. “W/T” solutions. a Wilting symptoms in representative photographs at 4 dpi. represents the number of wilting plants with respect to the total of “W/T” represents the number of wilting plants with respect to the infected plants. b Bacterial colonization in potato stems counted total of infected plants. b Bacterial content in potato stem recorded at at 8 dpi. The root-cut potato plants were infected with different 4 dpi. This experiment was repeated three times with similar results. concentrations of bacterial solutions as indicated. Each experiment Student’s t test was performed with respect to MS- solution (P < 0.05) was repeated three times using 12 plants each with similar results. Asterisk indicates P < 0.01 (Student’s t test) with respect to the 1 × ­108 cfu/ml bacteria innocula pathogen population in M was twofold lower than that in Désirée (Fig. 8b), but significantly delayed wilting symp- Evaluation of the resistance of potato varieties to R. tom development, indicating that M is a tolerant potato solanacearum variety towards strain GMI1000. Reactive oxygen species Easy identification of potato varieties with high resist- (ROS) play a key role in plant defense [27]. To ascertain ance to R. solanacearum is a prerequisite for potato whether ROS production was triggered in the resist- breeding programs. Hence, we performed a small scale ance plants O and P in response to R. solanacearum, we experiment to test if our pathosystem could be used measured it in leaves of O, P and Desirée infiltrated with to efficiently screen for potato resistance to R. solan- the pathogen. Comparing with Desirée and O, higher acearum. Thirty-two varieties of Solanum tuberosum L. ROS level was detected in P plants at 3 dpi (Fig. 8c). This S. tuberosum subsp. andigenum, S. raphanifolium and suggests that ROS signaling may be part of the defense S. pinnatisectum were grown and inoculated using our responses of P leading to resistance to R. solanacearum. hydroponic conditions. The results obtained from six of Moreover, while the leaf-infiltrated Desirée plants exhib- these varieties and the control susceptible variety Desirée ited wilting symptoms, O and P plants did not (Fig. 8c). are shown in Fig. 8. Wilting symptoms were clearly abol- This indicates that the resistance of O against R. solan- ished on varieties O and P, and delayed on B, M and N acearum seems to be controlled by alternative, ROS- species, compared to variety L and the control suscep- independent mechanisms. tible variety Désirée (Fig. 8a). Consistent with this, the The observation of symptomless plants does not always population of the pathogen in O and P was 1000 fold correlate with potato resistance to R. solanacearum lower than that in Désirée, suggesting O and P are highly as symptomless latent infections often occur [20]. resistant to R. solanacearum (Fig. 8). Interestingly, the Thus, bacterial counts must be also assayed. However, Wang et al. Plant Methods (2019) 15:145 Page 6 of 11 hydroponic potato infection system for evaluation of potato germplasm resistance to bacterial wilt. Discussion The interaction between R. solanacearum and its plant hosts has been established as a model system to study plant resistance to soil-borne bacterial phytopathogens for more than two decades [10, 24]. Soil-drench and/or stem penetration inoculations are mostly used to inves- tigate bacterial wilt disease progress on tomato, egg- plant, potato, the model plants Medicago truncatula and Arabidopsis [10-13, 20, 30]. Using any of these two infec- tion methods requires a large amount of time and space, with the ensuing high costs. Moreover, the growth in soil prevents the investigation of early root responses to the pathogen [17, 20]. To overcome these problems, we set up here an in vitro inoculation assay on potato. The in vitro potato inoculations have previously been used to quantify blackleg disease on shoots, showing results comparable to greenhouse assays [31]. Chen and col- leagues successfully identified three SSR alleles related Fig. 6 GMI1000 exhibited stronger virulence on potato than to bacterial wilt resistance from Solanum tuberosum + S. GMI1000(ΔhrpB) and GMI1000(ΔhrpG) mutants in our infection chacoense somatic hybrids through in vitro inoculation of system. a Wilting symptoms observed in representative photos taken potato plants grown in solid medium [32]. However, their at 4 dpi. “W/T” represents the number of wilting plants with respect infection protocol was not described explicitly. Here, we to the total of infected plants. b The bacterial content in potato stems recorded at 4 dpi. This experiment was repeated twice with similar thoroughly described a quick, accurate and space-saving results. **P < 0.001 (Student’s t test) with respect to GMI1000 potato infection system to monitor R. solanacearum using plants grown under hydroponic in vitro conditions. In our system, four potato plants were directly propa- measuring bacteria in plants is a laborious task that can- gated into a container and infected two weeks later with not be applied at high scale to screen germplasm for a R. solanacearum solution. Compared to soil drench resistance to R. solanacearum. Stable insertion of a lux- inoculation [20] our method saves two-weeks. In our CDABE luminescence reporter operon in the R. solan- assay, 75% of plants were completely wilted at 2–3 days acearum genome has facilitated real-time monitoring after the first wilting symptoms were recorded (Figs. 2a, of bacterial growth in plant hosts and has been used to 4a, 5a and 8a), showing that this assay is very stable and evaluate potato resistance in plants grown in pots [20, repetitive. Our recently established in vitro infection sys- 28]. To improve this screening method, we applied the tem for Arabidopsis grown on agar plates has shown that luxCDABE luminescence reporter in our in vitro infec- R. solanacearum infection changes the root architecture tion system. To this end, we used the strain GMI1000 [16, 17, 33]. This phenomenon could not be observed (Pps-Lux) carrying the entire Lux operon under the con- and investigated by means of a traditional soil-drench or trol of the PpsbA chloroplast promoter, which exhibits stem penetration inoculations. Thus, our assay provides strong, constitutive expression when introduced into the possibility to investigate early potato responses to R. R. solanacearum [20, 29]. As expected, potato plants solanacearum. infected with either GMI1000 or GMI1000 (Ppsba-lux) The HrpG and HrpB transcriptional regulators control showed comparable wilting symptoms (Fig. 9a) and bac- the virulence of R. solanacearum through modulating the terial counts (Fig. 9b) in potato at 5 dpi. In addition, light expression of the genes encoding the type three secretion detection with a luminometer showed a strong lumi- system and its related effectors [25, 34]. The deletion of nescence signal only in plants infected with GMI1000 hrpG and hrpB abolished wilt symptom occurrence and (Ppsba-Lux) (Fig. 9c). These data corroborate that inser- restrained the pathogen proliferation in potato plants tion of the PpsbA::luxCDABE in the R. solanacearum (Fig. 6), which is consistent with the mutant strains loss genome affect neither its colonization ability nor its ability to infect on the tomato and Arabidopsis [14, 17]. capacity to cause disease symptoms. The application of However, while the △hrpG deletion mutant grew more a luminescence reporter increases the efficiency of our than △hrpB in tomato stems [14], these two strains grew Wang et al. Plant Methods (2019) 15:145 Page 7 of 11 the possibility to directly investigate the interaction between potato root and other soil-borne pathogens. Three wild type potato lines were identified with higher bacterial wilt resistance among 32 tested candidate lines. This indicates that the in vitro infection system estab- lished here can be effectively applied to high-throughput screening for bacterial wilt resistance in potato germ- plasm. Wilting symptoms are the simplest way to evalu- ate plant resistance to bacterial wilt. However, symptom recording is time consuming and latent symptomless infections that can cause havoc when environmental con- ditions change [20, 35, 36] escape detection. Thus, latent infection limits the application of leaf wilting to evalu- ate potato resistance to the pathogen. To overcome this problem, we employed a luminescent reporter strain [20, 29] in our infection system to be able to quantify bacteria inside the plant, which may not have caused symptoms. Luminescence intensity was positively correlated with Fig. 7 R. solanacearum strains showed differential virulence in in vitro bacteria colonization in the infected plant stem (Fig. 9). potato infection system. a Representative picture of inoculated plants However, unlike our previous studies [20], bacterial col- in this work using a light imaging system (ChemiDoc ™ at 4 dpi. “W/T” indicates the number of wilting plants with respect to onization in the infected plant could not be visualized the total of infected plants. b The bacterial content in potato stems measured at 4 dpi. This experiment was repeated three times with similar results. **P < 0.001 (Student’s t test) comparing with UW551 XRST). One reason for this could that the luminescent strain GMI1000 strain originally isolated from tomato is less aggressive than the luminescent UY031 strain on potato that we used in previous reports [37, 38]. In addition, it is to similar levels in potato. This could be a host species- possible that the bacterial concentrations carried by the dependent phenomenon. In line with this hypothesis, younger plants used here are below the detection limits of the capacity of the △hrpB strain to colonize Arabidopsis the light imaging system. In any case, we could effectively seems to be stronger than that of the △hrpG strain [17]. quantify the luminescent bacteria with a luminometer. R. solanacearum strains UW551 and IPO1609 belong Compared with colony counting after dilution plating, to race 3 biovar 2, which causes potato brown rot at detection of bacterial luminescence from crushed stems cool temperatures [26]. In our potato infection assay using a 96-well plate luminometer is a faster, more reli- UW551 was much more aggressive than IPO1609, caus- able procedure. ing stronger wilting symptoms and increased bacterial growth. In accordance with this, it has been reported that Conclusion the pathogenicity of IPO1609 was strongly attenuated on In this study, a hydroponic potato infection assay in vitro tomato and potato relative to UW551 when using a soil- has successfully been established for R. solanacearum. drench inoculation method, due to a major deletion pre- This assay is less time-consuming, low-cost, accurate and sent in its genome [26]. We also found that strain CIP301, easier to handle comparing with the previously described isolated from potato, did not display strong virulence on and widely used infection assays. We demonstrated potato. Therefore we speculate it may be a hypoaggres- that it can also be applicable for large-scale screening of sive strain similar to IPO1609. CFBP2957 from tomato potato germplasm for resistance to brown rot disease, exhibited hypervirulence on potato in our assay. This is which will speed up and increase the efficiency of breed- not surprising, as it has been known for long that host ing resistance into potato cultivars. range in nature does not always correlate with aggressiv- ity on different hosts under laboratory conditions. For Materials and methods instance, UW551, a potato strain, has been reported to Plants and strains cause strong bacterial wilt on tomato [26]. All these data Two centimeter shoot explants from Solanum tuberosum indicate that this in vitro infection assay is suitable for L. Désirée; B and N from S. tuberosum subsp. Andige- evaluating the pathogenicity of R. solanacearum strains num; M, O, P from S. Raphanifolium; L from S. Pinnati- on potato as accurately as when soil drench inoculation is sectum) were cut and inserted into paper holders which used. In addition, our hydroponic infection also provides were immersed in 35 ml MS liquid medium (4.405 g/l Wang et al. Plant Methods (2019) 15:145 Page 8 of 11 Fig. 8 Potato varieties exhibited differential resistance to GMI1000 in the in vitro infection system. a Representative image of inoculated plants taken at 4 dpi. “W/T” represents the number of wilted plants over the total of infected plants. Tested varieties include: Solanum tuberosum L. Désirée; S. tuberosum subsp. andigenum (varieties B and N); S. raphanifolium (varieties M, O, P) and S. pinnatisectum (variety L). b Bacterial content in potato stems measured at 4 dpi. *P < 0.01 (Student’s t test) with respect to Désirée. c ROS production in the infiltrated leaves of potatos measured at 3 dpi. Left: DAB staining; Right: representative image of the plants for DAB staining assay taken at 6 dpi. These experiments were repeated at least twice with similar results MS salt including vitamins, 20 g/l sucrose, pH 5.8). Four In vitro potato infection assay plants were grown in each glass jar (diameter = 8 cm), Overnight R. solanacearum cultures were collected by containing 35 ml of MS- solution (or tap water after centrifugation (4000 rpm, 5 min), washed once with infection when indicated). Plants were grown in a cham- MS-/tap water, diluted with MS−/tap water and adjusted ber under long day conditions (16 h light, 8 h dark), 23 °C, to ­OD600 = 0.01. Then the bacterial suspensions were dis- 75% humidity and 10,000 lx light intensity conditions. tributed into jars, using 35 ml per jar for infection. To prepare bacterial inocula, 2–3 single R. solanacearum Roots of 2-week-old potato plants were cut with scis- colonies (strains GMI1000, UW551, IPO1609, CFBP2957 sors 2 cm below the stem and put into the bacterial or CIP301) were transferred into 10 ml liquid B medium suspension for inoculation. Inoculated potato plants (10 g/l peptone, 1 g/l yeast extract and 1 g/l casamino acid) were kept in the growth chamber under long day condi- and incubated overnight at 28 °C in a shaker. tions (16 h light, 8 h dark), 25 °C and 10,000 lx light and Wang et al. Plant Methods (2019) 15:145 Page 9 of 11 DAB staining assay Plant leaves were directly infiltrated with R. solan- acearum solution at OD = 0.001. Infiltrated leaves were detached at the indicated time and immediately immersed into 1 mg/ml DAB solution for overnight in the dark. Then the leaves were de-stained with absolute ethanol and boiled for 10 min and photographed. Bacteria counting and bacteria luminescence quantification The aerial part of the infected plants was harvested 1 cm above the level of the liquid in the jars and weighed, then homogenized with pestle and mortar. Two ml double dis- tilled water ­(ddH2O) was added and mixed with the plant material and the homogenates were serially diluted in water and plated on solid B medium. Plates were kept in the 28 °C incubator for 48 h and bacterial colonies were counted. The bacterial contents in the stem (cfu/fresh weight of aerial part of the infected plants) was used to evaluate bacterial virulence or plant resistance. For luminescence measurement assays, the homoge- nates from the aerial tissues of infected plants were transferred to a 96-well plate (Nunclone) and the lumi- nescence emitted from the pathogen was measured and quantified with a plate reader infinite 200 Pro (Tecan). Luminescence readings were normalized to the fresh weight of each sample and presented as RLU (relative luminescence units) per gram of fresh tissue. Acknowledgements We are grateful for helps from Prof. Changgen Xie (Northwest A & F University) and the crop biology innovation center of college of agronomy (Northwest A & F University). Authors’ contributions CZ, QC and HL designed the experiments. HW, JH, YL, MZ, NQ and RZ carried out the experiments. YH and DW prepared materials for in vitro system. YC, CZ and HL analyzed the data. CZ, NSC, MV and HL wrote the paper. All authors read and approved the final manuscript. Funding This study was supported by the National Natural Science Foundation of China (No. 31601703), the Start-up Funds of Northwest A&F University Fig. 9 The growth of luminescent R. solanacearum was easily (Z111021601), the Fundamental Research Fund for the Central Universities detected in plants. a Representative picture of infected potato plants of China (Z109021706) and External Science and Technology Cooperation taken at 5 dpi. b Bacterial content in the stem counted at 5 dpi. c Program of Ningxia Academy of Agriculture and Forestry Sciences (DW-X- Luminescence of R. solanacearum (Ppsba-lux) detected with a 96-well 2018012). N.S.C. and M.V. work was funded by projects AGL2016-78002-R. (Spanish Ministry of Economy and Competitiveness) and financial sup- plate reader using the luminometer mode. This experiment was port from the “Severo Ochoa Programme for Centres of Excellence in R&D” performed at least twice with similar results. **P < 0.001 (Student’s t (SEV‐2015‐0533) and the CERCA Programme from the Catalan Government test) with respect to GMI1000 (Generalitat de Catalunya). Availability of data and material All data generated or analysed during the this study are included in this 70% humidity. At 2 dpi, the lid of the jar containing the published article. infected plants was loosened to allow air exchange. Wilt- ing symptoms on the infected plants were recorded by Ethics approval and consent to participate Not applicable. taking digital images at the indicated times. Wang et al. Plant Methods (2019) 15:145 Page 10 of 11 Consent for publication 14. Vasse J, Genin S, Frey P, Boucher C, Brito B. The hrpB and hrpG regulatory Not applicable. genes of Ralstonia solanacearum are required for different stages of the tomato root infection process. MPMI. 2000;13:259–67. Competing interests 15. Singh N, Phukan T, Sharma PL, Kabyashree K, Barman A, Kumar R, Sonti The authors declare that they have no competing interests. RV, Genin S, Ray SK. An innovative root inoculation method to study Ralstonia solanacearum pathogenicity in tomato seedlings. Phytopathol- Author details ogy. 2018;108:436–42. 1 College of Agronomy and State Key Laboratory of Crop Stress Biology 16. Digonnet C, Martinez Y, Denance N, Chasseray M, Dabos P, Ranocha P, for Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China. Marco Y, Jauneau A, Goffner D. Deciphering the route of Ralstonia solan- 2 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, acearum colonization in Arabidopsis thaliana roots during a compatible Campus UAB, Bellaterra, 08193 Barcelona, Catalonia, Spain. 3 Department interaction: focus at the plant cell wall. Planta. 2012;236:1419–31. of Genetics, University of Barcelona, 08028 Barcelona, Catalonia, Spain. 4 State 17. Lu H, Lema AS, Planas-Marques M, Alonso-Diaz A, Valls M, Coll NS. Type III Key Laboratory of Crop Stress Biology for Arid Areas, College of Food Science secretion-dependent and -independent phenotypes caused by Ralstonia and Engineering, Northwest A & F University, Yangling 712100, Shaanxi, China. solanacearum in Arabidopsis Roots. MPMI. 2018;31:175–84. 18. Zolobowska L, Van Gijsegem F. Induction of lateral root structure forma- Received: 1 August 2019 Accepted: 18 November 2019 tion on petunia roots: a novel effect of GMI1000 Ralstonia solanacearum infection impaired in Hrp mutants. MPMI. 2006;19:597–606. 19. Vailleau F, Sartorel E, Jardinaud MF, Chardon F, Genin S, Huguet T, Gen- tzbittel L, Petitprez M. Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume References plant Medicago truncatula. MPMI. 2007;20:159–67. 1. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow 20. 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