A quick and efficient hydroponic potato infection method for evaluating potato resistance and Ralstonia solanacearum virulence
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.
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 optimum 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 screening 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.
We have established a quick and efficient in vitro potato infection system, which may facilitate breeding for new potato cultivars with high resistance to R. solanacearum.
KeywordsRalstonia solanacearum Potato In vitro infection Brown rot Bacterial wilt
Ralstonia solanacearum is the causal agent of bacterial wilt (also known as brown rot on potato), one of the most destructive plant diseases on many crops in tropical and subtropical areas, which leads to huge losses in food production . This soil-borne bacterium enters root through wounds or natural openings and multiplies in the vascular tissues, which results in xylem dysfunction and ultimately kills the plant host [2, 3]. The bacterium is also able to survive for years in soil and water retaining the ability to invade host plants .
R. solanacearum is a heterogeneous species composed of many genetic groups referred to as the R. solanacearum species complex (RSSC) [2, 3]. Phylogenetic analyses of several conserved genes revealed that the RSSC is divided into four phylotypes: phylotype I isolates are mainly from Asia, phylotypes II and III are, respectively, composed of American and African strains and phylotype IV strains originate from Indonesia, Japan, Australia and the Philippines [5, 6].
Besides its worldwide geographic distribution, R. solanacearum possesses an extraordinarily broad host range, causing disease on more than 200 plant species from 50 different botanical families . The pathogen not only infects solanaceous crops such as tomato, eggplant, peanut, pepper and potato, but also other plants from both the dicot and monocot families, and new hosts are being discovered continuously . Due to its wide geographical distribution, broad host range, long persistence in soil and highly aggressivity on plants, the bacterium was ranked by scholars as the second most important bacterial plant pathogen .
Potato is currently the third most important staple food crop for human direct consumption just after rice and wheat and it ranks first in energy and protein production per unit of water [8, 9]. In protein produced per acre land, potato ranks second to soybean. Importantly, potato is rich in microelements and vitamins essential for the human diet, such as vitamin C, potassium and in fiber . Brown rot caused by R. solanacearum is one of the most notorious potato diseases, estimated to result each year in 1 billion US$ economic losses worldwide . Breeding new potato cultivars with resistance to brown rot is essential for integrated management of this disease. To this end, the development of procedures to facilitate the screening for resistance in germplasm from wild potato collections, progenies from crosses between potato species and potato transgenic lines will help potato breeding programs.
Soil-drench and stem-puncture inoculation are the two methods most widely used to study plant resistance to R. solanacearum [10, 11, 12, 13]. However, adult full-size plants are needed in these procedures with the ensuing use of space, energy and time. Furthermore, since R. solanacearum is a soil-borne root pathogen, stem penetration bypasses potential root resistance mechanisms. The disadvantage of soil-drench inoculation is that the opacity of soil hinders the direct investigation of root responses to the pathogen. Vass et al. inoculated tomato plants grown in hydroponic conditions to study R. solanacearum root colonization . More recently, in vitro pathogenicity assays have been successfully established on tomato , Arabidopsis [16, 17], petunia  and M. truncatula . However, miniaturized in vitro infection assays have not been set up to screen for resistance in potato to the pathogen. We previously generated constitutively luminescent R. solanacearum reporter strains, a tool that we have used to characterize the colonization and the defense responses of potato breeding lines [20, 21]. Here we have established a faster, highly efficient, low-cost potato hydroponic infection method to study R. solanacearum-potato interactions. Using this new method, we successfully characterized the virulence of several R. solanacearum strains on potato and screened for potato varieties showing resistance to R. solanacearum. We also simplified the screening process using a luminescent pathogen that can be tracked in vivo in infected plants. This new method will promote the study of potato resistance to R. solanacearum and provide insights for investigating other root pathogens of potato under gnotobiotic conditions.
Development of an in vitro potato infection system for R. solanacearum
Hydroponic potato infection can be used to study R. solanacearum virulence in vitro
Evaluation of the resistance of potato varieties to R. solanacearum
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 investigate bacterial wilt disease progress on tomato, eggplant, potato, the model plants Medicago truncatula and Arabidopsis [10-13, 20, 30]. Using any of these two infection 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 . Chen and colleagues successfully identified three SSR alleles related to bacterial wilt resistance from Solanum tuberosum + S. chacoense somatic hybrids through in vitro inoculation of potato plants grown in solid medium . However, their infection protocol was not described explicitly. Here, we thoroughly described a quick, accurate and space-saving potato infection system to monitor R. solanacearum using plants grown under hydroponic in vitro conditions. In our system, four potato plants were directly propagated into a container and infected two weeks later with a R. solanacearum solution. Compared to soil drench inoculation  our method saves two-weeks. In our assay, 75% of plants were completely wilted at 2–3 days after the first wilting symptoms were recorded (Figs. 2a, 4a, 5a and 8a), showing that this assay is very stable and repetitive. Our recently established in vitro infection system for Arabidopsis grown on agar plates has shown that R. solanacearum infection changes the root architecture [16, 17, 33]. This phenomenon could not be observed and investigated by means of a traditional soil-drench or stem penetration inoculations. Thus, our assay provides the possibility to investigate early potato responses to R. solanacearum.
The HrpG and HrpB transcriptional regulators control the virulence of R. solanacearum through modulating the expression of the genes encoding the type three secretion system and its related effectors [25, 34]. The deletion of hrpG and hrpB abolished wilt symptom occurrence and restrained the pathogen proliferation in potato plants (Fig. 6), which is consistent with the mutant strains loss ability to infect on the tomato and Arabidopsis [14, 17]. However, while the △hrpG deletion mutant grew more than △hrpB in tomato stems , these two strains grew to similar levels in potato. This could be a host species-dependent phenomenon. In line with this hypothesis, the capacity of the △hrpB strain to colonize Arabidopsis seems to be stronger than that of the △hrpG strain .
R. solanacearum strains UW551 and IPO1609 belong to race 3 biovar 2, which causes potato brown rot at cool temperatures . In our potato infection assay UW551 was much more aggressive than IPO1609, causing stronger wilting symptoms and increased bacterial growth. In accordance with this, it has been reported that the pathogenicity of IPO1609 was strongly attenuated on tomato and potato relative to UW551 when using a soil-drench inoculation method, due to a major deletion present in its genome . We also found that strain CIP301, isolated from potato, did not display strong virulence on potato. Therefore we speculate it may be a hypoaggressive strain similar to IPO1609. CFBP2957 from tomato exhibited hypervirulence on potato in our assay. This is not surprising, as it has been known for long that host range in nature does not always correlate with aggressivity on different hosts under laboratory conditions. For instance, UW551, a potato strain, has been reported to cause strong bacterial wilt on tomato . All these data indicate that this in vitro infection assay is suitable for evaluating the pathogenicity of R. solanacearum strains on potato as accurately as when soil drench inoculation is used. In addition, our hydroponic infection also provides 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 established here can be effectively applied to high-throughput screening for bacterial wilt resistance in potato germplasm. Wilting symptoms are the simplest way to evaluate plant resistance to bacterial wilt. However, symptom recording is time consuming and latent symptomless infections that can cause havoc when environmental conditions change [20, 35, 36] escape detection. Thus, latent infection limits the application of leaf wilting to evaluate 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 bacteria colonization in the infected plant stem (Fig. 9). However, unlike our previous studies , bacterial colonization in the infected plant could not be visualized in this work using a light imaging system (ChemiDoc ™ XRST). One reason for this could that the luminescent 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 possible that the bacterial concentrations carried by the younger plants used here are below the detection limits of the light imaging system. In any case, we could effectively quantify the luminescent bacteria with a luminometer. Compared with colony counting after dilution plating, detection of bacterial luminescence from crushed stems using a 96-well plate luminometer is a faster, more reliable procedure.
In this study, a hydroponic potato infection assay in vitro has successfully been established for R. solanacearum. This assay is less time-consuming, low-cost, accurate and easier to handle comparing with the previously described and widely used infection assays. We demonstrated that it can also be applicable for large-scale screening of potato germplasm for resistance to brown rot disease, which will speed up and increase the efficiency of breeding resistance into potato cultivars.
Materials and methods
Plants and strains
Two centimeter shoot explants from Solanum tuberosum L. Désirée; B and N from S. tuberosum subsp. Andigenum; M, O, P from S. Raphanifolium; L from S. Pinnatisectum) were cut and inserted into paper holders which were immersed in 35 ml MS liquid medium (4.405 g/l MS salt including vitamins, 20 g/l sucrose, pH 5.8). Four plants were grown in each glass jar (diameter = 8 cm), containing 35 ml of MS- solution (or tap water after infection when indicated). Plants were grown in a chamber under long day conditions (16 h light, 8 h dark), 23 °C, 75% humidity and 10,000 lx light intensity conditions.
To prepare bacterial inocula, 2–3 single R. solanacearum colonies (strains GMI1000, UW551, IPO1609, CFBP2957 or CIP301) were transferred into 10 ml liquid B medium (10 g/l peptone, 1 g/l yeast extract and 1 g/l casamino acid) and incubated overnight at 28 °C in a shaker.
In vitro potato infection assay
Overnight R. solanacearum cultures were collected by centrifugation (4000 rpm, 5 min), washed once with MS-/tap water, diluted with MS−/tap water and adjusted to OD600 = 0.01. Then the bacterial suspensions were distributed into jars, using 35 ml per jar for infection.
Roots of 2-week-old potato plants were cut with scissors 2 cm below the stem and put into the bacterial suspension for inoculation. Inoculated potato plants were kept in the growth chamber under long day conditions (16 h light, 8 h dark), 25 °C and 10,000 lx light and 70% humidity. At 2 dpi, the lid of the jar containing the infected plants was loosened to allow air exchange. Wilting symptoms on the infected plants were recorded by taking digital images at the indicated times.
DAB staining assay
Plant leaves were directly infiltrated with R. solanacearum 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 distilled 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 homogenates from the aerial tissues of infected plants were transferred to a 96-well plate (Nunclone) and the luminescence 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.
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).
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.
This study was supported by the National Natural Science Foundation of China (No. 31601703), the Start-up Funds of Northwest A&F University (Z111021601), the Fundamental Research Fund for the Central Universities of China (Z109021706) and External Science and Technology Cooperation Program of Ningxia Academy of Agriculture and Forestry Sciences (DW-X-2018012). N.S.C. and M.V. work was funded by projects AGL2016-78002-R. (Spanish Ministry of Economy and Competitiveness) and financial support from the “Severo Ochoa Programme for Centres of Excellence in R&D” (SEV‐2015‐0533) and the CERCA Programme from the Catalan Government (Generalitat de Catalunya).
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The authors declare that they have no competing interests.
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