CAO Peng, CHEN Jia-lan, Ll Ning-ning, ZHANG Shuang-xi, WANG Rong-bo, Ll Ben-jin, LlU Pei-qing,AN Yu-yan#, ZHANG Mei-xiang#

1 National Engineering Laboratory for Endangered Medicinal Resource Development in Northwest China/Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry of Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710119, P.R.China

2 Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, P.R.China

3 Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Institute of Plant Protection, Fujian Academy of Agricultural Sciences, Fuzhou 350013, P.R.China

Abstract Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops, leading to huge losses in crop production every year.Understanding of plant-R.solanacearum interactions will aid to develop efficient strategies to control the disease.As a soilborne pathogen, R.solanacearum naturally infects plants via roots.A huge limitation in studying plant-R.solanacearum interactions is the large variation of R.solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.Here, we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.This method is easy to use, takes about only 10 days from seed germination to the completion of inoculation assay, and requires less inoculum of bacteria as well as growth chamber space.We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns, resistance of defense-related plant mutants, and virulence of R.solanacearum mutants.Furthermore, we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

Keywords: bacterial wilt, Ralstonia solanacearum, Petri-dish inoculation method, virulence, resistance, defense priming

1.lntroduction

Ralstoniasolanacearum, a soilborne phytopathogen with wide geographical distribution, causes a lethal bacterial wilt disease in more than 200 plant species including many important economic crops, leading to huge losses in crop production worldwide every year (Genin and Denny 2012; Qiet al.2022).It has been ranked the second most devastating bacterial phytopathogen in the world due to its extensive genetic diversity, broad host range, long persistence in soil and, most importantly,the lethality it causes to its numerous hosts (Mansfieldet al.2012).Although great progress has been made on plant-R.solanacearuminteractions, much still remains to be uncovered to formulate efficient strategies for plant resistance to bacterial wilt disease.

Ralstoniasolanacearumrelies on a type III secretion system (T3SS) to secrete lots of type III effectors (T3Es)into host cells, facilitating its infection (Genin and Denny 2012; Mansfieldet al.2012).Increasing numbers ofR.solanacearumstrains have been genome sequenced,including the model strain GMI1000 (Salanoubatet al.2002) and CQPS-1 isolated from wilted tobacco (Liuet al.2017).These resources contribute a lot to the identification of pathogenicity and host-range specificity determinants.Many T3Es inR.solanacearumhave been identified and characterized.For example, RipAC,a core T3E inR.solanacearumstrains, was previously suggested to play an important role in the establishment of the interaction at early stages of infection (Jacobset al.2012).Recently, its specific virulence activity and plant target were further demonstrated (Yuet al.2020).

To fight against various pathogens, plant has evolved a sophisticated immune system (Jones and Dangl 2006).The first layer of plant immunity is mediated by the pattern recognition receptors that perceive the molecular patterns such as the bacterial flagellin 22 peptide (flg22)and the plant elicitor peptide 1 (Pep1) (Gonget al.2019).The second layer of plant immunity is mediated by the intracellular receptors that recognize pathogen effectors(Jones and Dangl 2006).These two layers of immunity potentiate with each other, and they both trigger similar downstream defense responses, including production of reactive oxygen species, calcium influx, and activation of defense hormone signaling pathways (Tsuda and Katagiri 2010; Ngouet al.2021; Yuanet al.2021).Among the hormone signaling pathways, salicylic acid (SA) defense signaling pathway plays a critical role in both two layers of plant immunity.Nonexpressor of pathogenesisrelated gene 1 (NPR1), which is a SA receptor, positively modulates plant defense against biotrophic and hemibiotrophic pathogens (Fuet al.2012; Dinget al.2018).

Under natural condition,R.solanacearumenters the host plants through root, grows, and colonizes in the xylem vessels, causing wilting and eventually the killing of the hosts (Genin 2010).Therefore, the well-known natural soil drenching inoculation method was developed and widely used to study plant-R.solanacearuminteractions (Vailleauet al.2007; Yuet al.2020; Anet al.2022; Qiet al.2022).However, adult plants are needed in this method and the inoculated plants should be observed for more than 20 days till completion of the pathogenicity assay.More importantly, variations are always unavoidable due to the uncontrolled influence of soil microbiota and the infection stochasticity (Machoet al.2010).Besides, limited by the need of sufficient space and labor, this method is not very convenient for large-scale evaluation of plant resistance or analysis ofR.solanacearumvirulence.Therefore, a robust, easy-touse and stable method saving labor and space is urgently needed to facilitate studies of plant-R.solanacearuminteractions.Several research groups have tried to develop new inoculation methods to overcome the limitations of the soil drenching inoculation method.Luet al.(2018) established a simple inoculation assay in which 6-day-oldArabidopsisseedlings were individually inoculated 1 cm above the root tip with 5 μL bacterial suspension and root phenotype was observed for another 9 days.Singhet al.(2018) reported another root inoculation method in which 6- to 7-day-old tomato seedlings were picked and inoculated one by one by dipping in the bacterial inoculum and disease progression was observed for 7 days.These two studies demonstrate that seedlings inoculated properly are equally efficient as adult plants in pathogenicity assay.Besides, Wanget al.(2019) developed a potato infection method in which 2-week-old plants were hydroponically inoculated in bacterial suspension after roots being cut at 2 cm below the stem.All these developed methods can be completed within 20 days, paving a way for developing a more rapid,easy-to-use and stableR.solanacearuminoculation system.

Here, a faster (about 10 days for the whole process),simple and highly efficient inoculation method was developed for studying plant-R.solanacearuminteractions.Since the whole process including seedling culture and inoculation was carried out in Petri dishes, we named this easy-to-operate method as the Petri-dish inoculation method.Using the previously well-characterized plant elicitor Pep1 (Boller and Felix 2009), susceptiblenpr1mutant (Xuet al.2022), and two virulence-reduced GMI1000 mutants, we validated this Petri-dish inoculation method and demonstrated that this method is suitable to study the defense priming by molecular patterns,evaluate plant resistance toR.solanacearum, and analyzeR.solanacearumvirulence.Finally, the developed inoculation method was used to evaluate tobacco resistance toR.solanacearum, and the results obtained by our method were consistent with previous reports,indicating that thisR.solanacearuminoculation method can be broadly applied for other host plants.

2.Materials and methods

2.1.Ralstonia solanacearum Petri-dish inoculation in Arabidopsis seedlings

Plant materials and growth conditionsArabidopsisseeds in a 1.5 mL microcentrifuge tube were surfacesterilized by 70% ethanol for 1 min and 30% sodium hypochlorite (containing 0.05% Silwet-L-77) for another 10 min with gentle shaking, washed with sterile distilled water at least five times, then maintained in a small amount of sterile distilled water, and stored at 4°C to improve the rate and synchrony of germination.Two days later, seeds were planted individually in low density (about 70 seeds per dish) using a 1 mL pipette on 1/2 Murashige and Skoog (MS) medium in 10 cm× 10 cm square Petri dishes.Seedlings were cultured at 23°C with a light intensity of 150 μmol m-2s-1and a 16 h light/8 h dark photoperiod for 6 days.

Bacterial growth and inoculum preparationA -80°C stock of bacterial culture was streaked on BG (Bacto agar-Glucose) medium and the plate was incubated inversely at 28°C for 2 days.A single colony of the culturedR.solanacearumstrain was transferred to BG liquid medium and incubated at 28°C, 200 r min-1in a shaker for 24 h.The bacterial colonies were collected by centrifugation at 5 000 r min-1for 5 min and then resuspended in an appropriate volume of sterile distilled water.The OD600of the bacterial suspension was measured using a spectrophotometer and diluted to appropriate concentration.Bacterial suspension was then immediately evenly dispensed onto 1/2 MS dishes, 3 mL per dish, and incubated for 1-2 min at room temperature.The plates were naturally dried for 15-20 min in the clean bench after removing the excess bacterial suspension by a pipette.Six-day-old healthy and uniform seedlings were transferred to the 1/2 MS dishes containingR.solanacearum, about 20 vertically grown seedlings in two rows per Petri dish.These Petri-dish inoculated seedlings were incubated under the same growth condition for 3 days and then sampled to evaluate bacterial growth.The whole process was displayed in Fig.1-A.

Fig.1 Procedure of seedling Petri-dish inoculation method.A, the timeline required for Petri-dish inoculation method.B, schematic representation of the main steps for Petri-dish inoculation assay.dpi, days post-inoculation.

Determination of the proper inoculum concentrationBacterial suspension with an OD600of 0.05, 0.1 or 0.15 was dispensed onto 1/2 MS dishes, respectively.After seedlings were Petri-dish inoculated with different concentrations of bacteria, the bacterial growth in seedlings was monitored to compare and select the best inoculum concentration and the appropriate time of bacterial titer measurement (Fig.2).

Fig.2 Determination of the main parameters in Petri-dish inoculation assay.Ralstonia solanacearum cells with OD600 of 0.05, 0.1 and 0.15 were selected to confirm the proper inoculum concentration.Bacterial populations in Arabidopsis seedlings at 2, 3, or 4 days post-inoculation (dpi) were determined.Data are mean±standard error (SE) from at least 6 biological replicates.The same letters indicate no significant difference at P≤0.05.

2.2.Ralstonia solanacearum soil drenching inoculation

GMI1000 was grown overnight at 28°C in BG liquid medium till OD600to 2.0, centrifuged and then resuspended in distilled water (OD600=0.1).Four-weekold wild typeArabidopsisandnpr1mutant grown in jiffy pots were soil drenching inoculated with the bacterial suspension.After inoculation, the plants were kept in a growth chamber according to Yuet al.(2020).Scoring of visual disease symptoms on the basis of a scale ranging from ‘0’ (no symptoms) to ‘4’ (complete wilting) and the survival analysis were performed as previously described(Yuet al.2020).

2.3.Measurement of bacterial titers

Six seedlings were sampled as 1 replicate and 6 replicates were carried out for each test (Fig.1-B).After measuring the weight, seedlings were surface-sterilized by 70% ethanol for 10 s and rinsed three times with sterile distilled water to determine the internal bacterial population.Then seedlings were homogenized in 500 μL sterile distilled water by bead beater for 1 min at 4°C.The homogenized samples (100) were serially diluted from 10-1to 10-5, plated on BG agar (10 μL plate-1), air-dried and incubated at 28°C (Fig.1-B).A total of 36 h later, the bacterial colony forming units (CFU) were counted and normalized as CFU g-1tissue using the total weight of one pooled sample.

2.4.Evaluation of bacterial growth by luminescence

Aiming to evaluate the bacterial growth in Petri-dish inoculated seedlings more quickly, we employed a constitutively luminescent GMI1000 reporter strain GMI1000 (Ppsba-lux) whose luminescence can be used to quantify bacterial content inside plant tissues (Monteiroet al.2012; Cruzet al.2014).To detect the luminescence intensity inArabidopsisseedlings Petri-dish inoculated with GMI1000 (Ppsba-lux), seedlings were put into 96-well plates after being gently washed three times with sterile distilled water.One plant each well contained 100 μL sterile distilled water.The luminescence emitted from bacteria inside the seedlings was measured and quantified using a Glomax-96 Microplate Luminometer (Promega,USA).Bacterial abundance is presented as relative luminescence units (RLU) per seedling.This method can be an alternative for evaluation of bacterial growth if the bioluminescentR.solanacearumstain is available.

2.5.Defense priming by elicitor Pep1

Six-day-oldArabidopsisseedlings were pre-treated with 100 nmol L-1Pep1 (plant elicitor peptide 1), a 23-amino acid long peptide derived from the carboxyl end of the propeptide.Ater 24 h, the pre-treated seedlings were Petri-dish inoculated with GMI1000 or GMI1000 (Ppsbalux) to determine whether the Petri-dish inoculation assay can be used to study defense priming.

2.6.Plant materials and R.solanacearum stains

Wild-type Col-0Arabidopsis,npr1mutants (Caoet al.1997),wild type GMI1000 and luminescent GMI1000 (Ppsbalux) were used to test whether resistance of plant materials toR.solanacearumcan be evaluated using the Petridish inoculation method.Wild-type Col-0Arabidopsisandnpr1mutants were grown half and half in each dish when inoculated.Four tobacco cultivars with different resistance to bacterial wilt, DB101, Honghuadajinyuan, Changbohuang and Yanyan 97 (Qianet al.2016; Zhanget al.2019; Shiet al.2022), and anotherR.solanacearumstain CQPS-1 (Liuet al.2017) were used to test whether the Petri-dish inoculation method is efficient in evaluating the tobacco resistance to bacterial wilt.Wild-type GMI1000 and its virulence-reduced mutants ΔhrcV(Sunet al.2017) and ΔRipAC(Yuet al.2020)were used to test whether the Petri-dish method can be used to analyzeR.solanacearumvirulence.

2.7.qRT-PCR analysis

After being Petri-dish inoculated with wild-type GMI1000,its virulence mutants ΔhrcVor ΔRipAC, the whole seedlings of wild-typeArabidopsisornpr1mutants were sampled and total RNA was extracted using the RNAsimple Total RNA Extraction Kit (TIANGEN,Beijing).The RNA sample was then reverse transcribed using the HiScript II Q Select RT SuperMix for qPCR Kit (Vazyme, Nanjing).The relative expression levels of a PTI-specific target geneFlg22-InducedReceptor Kinase1(FRK1) and SA-induciblepathogenesisrelatedprotein1acidic(PR1a) gene were measured by SYBR Green quantitative PCR.The gene expression level was normalized usingActin1as a control.The following primers were used for amplification: forFRK1,5′-CAAGCTTTGCTCTTCTGTTCTT-3′ and 5′-AACCACT TGATTGAAACTGAGC-3′; forPR1a, 5′-AATGGTGTGGA ACGAAACTCTT-3′ and 5′-TCGTAATTTTCCTTCTCCGT CA-3′; forActin1, 5′-TCTTGATCTTGCTGGTCGTG-3′and 5′-GAGCTGGTTTTGGCTGTCTC-3′.

2.8.Statistical analysis

All experiments were performed with at least three biological replicates.Student’st-test was performed along with analysis of variance to determine statistical difference using SPSS 20.0.All graphs were generated using GraphPad Prism 8.

3.Results

3.1.Development of a seedling Petri-dish inoculation assay to study plant-R.solanacearum interactions

Aiming to develop a robust and easy-to-use inoculation method, we established a Petri-dish inoculation method for studying plant-R.solanacearumsystem (Fig.1).In this method, theR.solanacearumstrain with appropriate concentration was evenly dispensed onto 1/2 MS dishes to establish an even inoculum exposure environment.Six-day-old seedlings were placed vertically in the above dishes so that plant roots developed at the surface of the medium and plants could be easily sampled after 2-4 days post-inoculation (dpi).Bacterial population in the seedlings were measured to evaluate the infection ofR.solanacearumin plants.The whole process of this Petri-dish inoculation can be finished within two weeks.Therefore, this method is pretty quick, space, labor and cost-saving, and consequently very easy-to-use.

To determine the optimal inoculum concentration and the best time for bacterial population measurement using the model strain GMI1000, we compared the effects of three inoculum concentrations on the bacterial colonization inArabidopsisseedings at 2, 3 and 4 dpi.The result showed thatArabidopsisseedlings exposed to GMI1000 with an OD600of 0.15 showed significantly higher bacterial population at 2 dpi than that of OD600=0.05 or 0.1 at 3 dpi (Fig.2).More importantly, no significant difference was found between bacterial populations at 3 and 4 dpi with OD600of 0.15.This result indicates that inoculation with OD600of 0.15 is not appropriate to distinguish the differences in bacterial virulence or plant resistance due to the too fast colonization.For the concentration of OD600=0.05 and 0.1, seedlings showed increasing bacterial populations as the time progressed,among which concentration of 0.1 showed more striking results, facilitating better observation of bacterial colonization.Therefore, consistent with the traditional soil-drenching inoculation method (Anet al.2022; Qiet al.2022), OD600of 0.1 was chosen as the optimal inoculum concentration for the Petri-dish inoculation assay.At 2 and 3 dpi, bacterial populations in seedlings increased significantly with the inoculum concentrations, whereas no significant difference was found between OD600=0.1 and 0.15 at 4 dpi.This result indicates that 2 and 3 dpi are better than 4 dpi to compare the bacterial population in Petri-dish-inoculated seedlings.We chose 3 dpi for the Petri-dish inoculation assay, since the bacterial titers at 2 dpi was a bit low for quantification.

The bacterial titers of inoculated seedlings at 3 dpi from biological replicates were highly consistent (Fig.3),indicating our Petri-dish inoculation method is stable and uniform.

Fig.3 Bacterial titers of Ralstonia solanacearum in 6 biological replicates in Petri-dish inoculation assay.A, R.solanacearum colonies on BG plates.Samples were collected at 3 days post-inoculation.B, bacterial populations calculated according to A.

3.2.The Petri-dish inoculation method is suitable to evaluate defense priming by molecular pattern

Defense priming by Pep1, one of the best-characterized damage-associated molecular patterns, has been welldocumented (Boller and Felix 2009; Gonget al.2019).We investigated the priming effect of Pep1 using the Petridish inoculation assay to validate our new inoculation method.As expected, seedlings pretreated with Pep1 showed significantly higher transcripts ofFRK1(Fig.4-A),an early defense gene after pattern treatment (Majhiet al.2021), and exhibited more resistant to GMI1000 than the H2O pretreated control (Fig.4-B).These results indicate that the Petri-dish inoculation method is efficient for the study of plant defense priming by molecular patterns.

Fig.4 Analysis of plant defense priming by Petri-dish inoculation method.Six-day-old Arabidopsis seedlings pre-treated by 100 nmol L-1 Pep1 were Petri-dish inoculated with wild-type GMI1000 or luminescent GMI1000 (Ppsba-lux) with an OD600 of 0.1.A,determination of FRK1 expression by qRT-PCR analysis in Arabidopsis seedlings at 3 h post-inoculation (hpi).The bacterial titers(B) and the luminescence intensity (C) in seedlings were measured at 3 dpi.Data are mean±SE.＊＊＊ indicates significant difference at P≤0.001.

GMI1000 (Ppsba-lux) is a luminescent GMI1000 reporter strain (Monteiroet al.2012; Cruzet al.2014).We inoculatedArabidopsisseedlings with GMI1000(Ppsba-lux)viathe Petri-dish inoculation method and detected the luminescence intensity of seedlings using a luminometer immediately after sampling at 3 dpi.Result of the luminescence intensity was consistent with that of bacterial population (Fig.4-C), indicating that luminescence detection can be an alternative to evaluate bacterial growth when using the bioluminescentR.solanacearumstrain.Since determination of the bacterial titers needs to homogenize the seedlings,serially dilute the suspension and wait for 1-2 days to count the CFU, the luminescence detection method is more quickly and labor saving.

3.3.The Petri-dish inoculation assay confirmed the contribution of NPR1 to plant resistance against R.solanacearum

It has been showed thatnpr1, an NPR1 loss-of-function mutant, is susceptible toR.solanacearum(Xuet al.2022).Here, we soil drenching inoculated 4-week-old wild-typeArabidopsis(Col-0) andnpr1mutant with GMI1000, and found that thenpr1mutant showed significantly higher disease index (Fig.5-A) and lower survival percent(Fig.5-B) than wild-type Col-0.This result confirmed the positive role of NPR1 in regulating plant resistance toR.solanacearum.We employednpr1mutant to examine whether the Petri-dish inoculation method can be used to quantify plant resistance toR.solanacearum.After being inoculated with wild-type GMI1000,npr1showed extremely lower expression ofPR1a, a marker gene in SA defense signaling pathway (Delaneyet al.1994), and significantly higher bacterial population than wild-typeArabidopsisseedlings (Fig.5-C and D).Consistently,the luminescence intensity innpr1mutants was strikingly higher than that in wild-type seedlings when seedlings were inoculated with GMI1000 (Ppsba-lux) (Fig.5-E).These results demonstrated the suitability of Petri-dish inoculation method for evaluating plant resistance.

Fig.5 Evaluation of npr1 resistance to Ralstonia solanacearum by Petri-dish inoculation method.A, disease index of 4-week-old wild-type Arabidopsis (Col-0) and npr1 mutant after soil drenching inoculation of GMI1000 (OD600=0.1).Fifteen plants per genotype were used for one biological replicate.B, survival analysis of the data in A.Statistical analysis was performed using a Log-rank(Mantel-Cox) test (n=45), and the corresponding P-value is shown.C, expression of salicylic acid (SA)-inducible pathogenesisrelated protein 1 acidic (PR1a) in Arabidopsis seedlings at 12 hpi by qRT-PCR analysis.D, the bacterial titers of R.solanacearum after Petri-dish inoculation.E, the luminescence intensity in seedlings after Petri-dish inoculation at 3 dpi.Six-day-old wild-type Arabidopsis (Col-0) and npr1 mutant were Petri-dish inoculated with wild-type GMI1000 (D) or luminescent GMI1000 (Ppsba-lux)(E) with an OD600 of 0.1.Data are mean±SE.＊＊ and ＊＊＊ indicate significant differences at P≤0.01 and P≤0.001, respectively.

3.4.The Petri-dish inoculation assay is efficient to study R.solanacearum virulence

HrcV is a component of type III secretion system(Galanet al.2014).RipAC, a core type III effector inR.solanacearum, contributes toR.solanacearumvirulence (Yuet al.2020).To determine whether our Petri-dish inoculation system is suitable to evaluateR.solanacearumvirulence, we inoculatedArabidopsisseedlings with the wild-type GMI1000, and ΔhrcV(Sunet al.2017) or ΔRipACmutant (Yuet al.2020),respectively, using this system.As anticipated, the bacterial populations of ΔhrcVmutant were significantly lower than that of wild-type GMI1000 (Fig.6-A).Consistently, expression of PTI marker geneFRK1was significantly higher in seedlings inoculated with ΔhrcVthan those inoculated with wild-type GMI1000 (Fig.6-B).Similarly, deletion ofRipACsignificantly reducedR.salanacearumvirulence as indicated in our Petridish inoculation assay (Fig.6-C), and the expression of defense-related genePR1awas upregulated in ΔRipACinoculated plants (Fig.6-D).These results confirmed that the Petri-dish inoculation assay can be used for investigatingR.solanacearumvirulence.

Fig.6 Determination of Ralstonia solanacearum virulence by Petri-dish inoculation method.Six-day-old Arabidopsis seedlings were inoculated with wild-type GMI1000 and its virulence mutants ΔhrcV and ΔRipAC, and samples were collected at 3 days post-inoculation (dpi).A, bacterial titers of ΔhrcV mutant in Arabidopsis seedlings.B, relative expression of FRK1 in inoculated Arabidopsis seedlings.C, bacterial titers of ΔRipAC mutant in Arabidopsis seedlings.D, relative expression of PR1a in inoculated Arabidopsis seedlings.The expression levels of defense-related genes FRK1 and PR1a were measured at 3 dpi.Data are mean±SE.＊, ＊＊ and ＊＊＊ indicate significant differences at P≤0.05, P≤0.01 and P≤0.001, respectively.

3.5.The Petri-dish inoculation assay can be applied to study tobacco-R.solanacearum interactions

Theoretically, our Petri-dish inoculation method could be used for studying interactions ofR.solanacearumwith other plants.To verify this, 10-day-old seedlings of four tobacco cultivars were Petri-dish inoculated with virulentR.solanacearumstrain CQPS-1.The hypersusceptible cultivar Honghuadajinyuan showed the highest bacterial population, the susceptible Changbohuang exhibited the second highest, and the two resistant cultivars showed significantly lower bacterial population (Fig.7).This result is consistent with previous findings that DB101 and Yanyan 97 are resistant toR.solanacearuminfection, and Honghuadajinyuan and Changbohuang are susceptible toR.solanacearum(Qianet al.2016; Zhanget al.2019; Shiet al.2022).Therefore, our Petri-dish inoculation method can be applied for other plant materials, and also may facilitate large-scale screening of resistance germplasms in the future.

Fig.7 Evaluation of tobacco resistance to bacterial wilt by seedling Petri-dish inoculation method.Ten-day-old seedlings of 4 tobacco cultivars were inoculated with Ralstonia solanacearum strain CQPS-1 (OD600=0.1).The bacterial titers were measured at 3 days post-inoculation(dpi).Data are mean±SE.Different letters indicate significant differences at P≤0.01.

4.Discussion

Ralstoniasolanacearumis one of the most destructive soilborne plant pathogens because of its worldwide geographical distribution, large host range and lethal pathogenicity (Mansfieldet al.2012).In order to develop efficient strategies to control bacterial wilt disease, lots of studies have been devoted to understand the underlying mechanisms of plant-R.solanacearuminteractions(Vallset al.2006; Genin and Denny 2012; Landryet al.2020).One of the limitations in the study of plant-R.solanacearuminteractions is the large variation ofR.solanacearuminfection assay due to the variable soil conditions and uneven inoculum exposure (Machoet al.2010; Luet al.2018).Here, we developed a robust and simple Petri-dish inoculation method with even inoculum exposure and uniform infection conditions.This method takes only about 10 days from seed germination to the completion of bacterial growth assessment, thus providing a more rapid system than traditional soil drenching method (Yuet al.2020) and other newly established root inoculation protocols (Luet al.2018; Singhet al.2018;Wanget al.2019).In addition, the Petri-dish inoculation method is very easy to use.Bacterial suspensions(OD600=0.1) were dispensed onto 1/2 MS Petri dishes to create a uniform inoculation environment.Six-day-old seedlings transferred to these dishes can be successfully and naturally inoculated through the inevitable wounds in the root (Figs.2 and 3).There is no need to specifically injury the seedlings or inoculate the seedlings one by one individually in this inoculation system, saving lots of time and labor.Therefore, although maybe bothering by the possible culture contamination problem, this system has potential to be utilized as a standard inoculation method for studying plant-R.solanacearuminteractions, and it is especially useful for labs with limited plant growth facilities.

Defense priming is an important process in systemic plant immunity.Primed plants often show enhanced defense responses when challenged by pathogens(Conrathet al.2015; Mauch-Maniet al.2017).Molecular patterns, such as the well-known plant-derived damageassociated molecular pattern Pep1, are well-documented to induce defense priming of plants (Boller and Felix 2009;Gonget al.2019).Here, we inoculated Pep1-pretreatedArabidopsisseedlings using the Petri-dish method and found that Pep1-pretreated seedlings showed higher expression of the early defense geneFRK1and were indeed more resistant to GMI1000 than H2O-pretreated control (Fig.4), confirming the effectiveness of the Petridish inoculation method.Since defense priming is an adaptive strategy to improve plant defensive capacity, the robust Petri-dish inoculation assay has the potential to be applied for screening natural or synthetic chemicals,beneficial microbes, as well as stimuli from pathogens that can induce plant defense priming.

Selecting or breeding for resistant plants is the primary control measure for bacterial wilt disease (Huet 2014;Vincelli 2016; Thomaset al.2020).Effective and reliable assays could speed up the screening for new sources of resistance againstR.solanacearum.NPR1 is a SA receptor, playing an important role in plant defense signaling (Fuet al.2012; Dinget al.2018).npr1mutant has been indicated to be susceptible toR.solanacearum(Xuet al.2022).Here, after confirming the susceptibility ofnpr1to GMI1000 using the traditional soil-drenching inoculation method (Fig.5), we checked the suitability of the Petri-dish inoculation method to discriminate plant resistance againstR.solanacearumusing wild-type plants andnpr1.The higher bacterial population at 3 dpi in Petri-dish inoculatednpr1clearly demonstrated that the Petri-dish method is sensitive and efficient for evaluating plant resistance toR.solanacearum.In addition, our method successfully distinguished the relative resistance differences of four tobacco cultivars to CQPS-1 (Fig.7),demonstrating the potential of our method in identifying resistant plant germplasms and studying interactions ofR.solanacearumwith other host plants.

T3SS allowsR.solanacearumto inject a large set of T3Es to plant cells, and plays a crucial role in bacterial pathogenicity (Genin and Denny 2012), which has been well-demonstrated by characterization of T3SS deletion or T3E loss-of-function mutants (Landryet al.2020;Schreiberet al.2021).Here, pathogenicity of the T3SSdeficient strain ΔhrcVand a mutant strain of virulence T3E RipAC was found significantly lower than that of the wild type GMI1000 strain using Petri-dish inoculation method(Fig.6).This result is completely consistent with previous reports using other inoculation systems (Sunet al.2017;Yuet al.2020), indicating that our method can clearly detect differences in the pathogen’s virulence.Therefore,the Petri-dish inoculation method not only is useful in plant resistance evaluations but also facilitates screening ofR.solanacearumvirulence mutants at early stages.The Petri-dish inoculation system can be a robust way to detect contribution of individual T3E to bacterial fitness in a much more sensitive manner than the traditional method which monitors symptom development in pathogenicity assays of adult plants.

To visualize the pathogens inside infected plants,GMI1000 (Ppsba-lux), a luminescent GMI1000 reporter strain has been successfully generated with no influence on bacterial growth and pathogenicity (Monteiroet al.2012; Cruzet al.2014).The luminescence inside plant tissues was proved to be linearly related to the bacterial concentration (Monteiroet al.2012; Cruzet al.2014), indicating the luminescence is a good indicator of bacterial concentration.We inoculatedArabidopsisseedlings with GMI1000 (Ppsba-lux) using the Petridish inoculation system and found that the luminescence results were consistent and even more sensitive than results of bacterial titers (Figs.4 and 5).Quantification of luminescence is a faster assessment of bacterial colonization, while determination of bacterial titers does not need any special equipment.Therefore, researchers could choose either of them for their convenience.

5.Conclusion

In this study, a Petri-dish inoculation system has been successfully developed forR.solanacearum.We demonstrated that this system is stable and highly efficient in the study of plant-R.solanacearuminteractions including defense priming assay, plant resistance evaluation and virulence assessment.Due to its robustness, user-friendly nature, space and budget savings, as well as the requirement for less bacterial inoculum, this Petri-dish inoculation method offers an excellent system for high-throughput screening of virulence-related mutants ofR.solanacearumor for assessing the resistance of plant germplasm to bacterial wilt.This method also provides a reference for inoculation methods of other soil-borne pathogens.

Acknowledgements

We thank professor Ding Wei in Southwest University,China for providing tobacco seeds.This work was supported by the National Natural Science Foundation of China (32072399 and 32272641), the Fundamental Research Funds for the Central Universities(GK202201017 and GK202207024), and the Program of Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, China (MIMCP-202203).

Declaration of competing interest

The authors declare that they have no conflict interest.