Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

A new DNA extraction method and a new multiplex real‐time TaqMan PCR test for detection of Ralstonia solanacearum, Ralstonia pseudosolanacearum and Clavibacter michiganensis subsp. sepedonicus in asymptomatic potato tubers are presented. This new multiplex PCR and three published TaqMan PCRs for detection of R. solanacearum and/or R. pseudosolanacearum and/or R. syzygii spp. and/or C. michiganensis subsp. sepedonicus were validated using linear regression analysis for estimating the Ct values and its variation at 5 × 10³ bacteria mL^?1. The three published PCRs that have been validated are Massart et al. (2014, detecting R. solanacearum and C. michiganensis subsp. sepedonicus), Weller et al. (1999, detecting R. solanacearum, R. pseudosolanacearum and R. syzygii spp.) and Gudmestad et al. (2009, detecting C. michiganensis subsp. sepedonicus). All tested PCRs were fit for purpose for their target organisms. The PCR tests have different target genes, allowing one of the sets to be used as first screening test and another as second screening test for the detection of R. solanacearum and/or R. pseudosolanacearum and/or C. michiganensis subsp. sepedonicus in asymptomatic potato tubers.     

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

This paper describes a comparison study of test methods and supports the use of real‐time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real‐time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real‐time PCR tests was compared with that of immunofluorescence (IF). Real‐time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real‐time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).     

Euphresco inter‐laboratory comparison (2009–2012) on detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers: proposal to include TaqMan® real‐time PCR as a primary (core) screening test in EU/EPPO standard methods

In the European Union (EU) potato production is surveyed for Clavibacter michiganensis subsp. sepedonicus (potato ring rot) and Ralstonia solanacearum (potato brown rot) under Commission Directives 93/85/EEC with its amendment 2006/56/EC and 98/57/EEC with its amendment 2006/63/EC. A regular update of the Directives is required in view of developments in understanding of the biology of these organisms and the diagnostics recommended for their detection and identification. Three inter‐laboratory tests (ILT1, ILT2 and ILT3) were performed from 2009 to 2012 as part of a Euphresco Phytosanitary ERA‐NET project to assess performance of current official methods for C. michiganensis subsp. sepedonicus and R. solanacearum. A major aim of the ILTs was to generate data on the performance of real‐time PCR protocols to support their introduction as primary (core) screening tests for both pathogens. In ILT1, 29 laboratories from 23 countries participated, in ILT2, 23 laboratories from 18 countries and in ILT3 42 laboratories from 24 countries. Relative accuracies for real‐time PCR tests averaged 92% for R. solanacearum and 96% for C. michiganensis subsp. sepedonicus) and compared with existing primary (core) screening tests (immunofluorescence, conventional PCR, semi‐selective plating and bioassay) in terms of analytical sensitivity, analytical specificity and robustness. It was concluded that all methods tested, including real‐time PCR, can be considered as equivalent. Therefore TaqMan ^® real‐time PCR is recommended for inclusion in EU Directives and EPPO Standards as a reliable primary (core) screening method.     

Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by Multiplex PCR with Coamplification of Host DNA

A new multiplex PCR assay was developed for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers. The assay combines two different tests in one reaction mixture. First, a highly specific and sensitive detection of the pathogen and second, an indicator test for successful amplification (internal PCR control), which monitors potentially false-negative PCR results, caused by inhibition of the PCR. For the simultaneous amplification of two different targets in one reaction mixture, a mix of two different primer sets was used. For the detection of C. michiganensis subsp. sepedonicus, a pathogen-specific primer set PSA-1/PSA-R was used, based on the intergenic spacer region of the 16S–23S rRNA genes of C. michiganensis subsp. sepedonicus. For the simultaneous amplification of the internal PCR control, the plant-specific primer set NS-7-F/NS-8-R was employed, permitting amplification of target sequence from plant DNA present in DNA extractions from potato core fluid. The applicability of the multiplex PCR was verified in 3500 composite samples of 200 seed potato tubers from 143 different cultivars in a survey for C. michiganensis subsp. sepedonicus by parallel testing using immunofluorescence, a bioassay in eggplant seedlings and multiplex PCR.     

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.     

Evaluation of a diagnostic microarray for the detection of major bacterial pathogens of potato from tuber samples

Potato can be infected with many bacterial pathogens, the detection of which is necessary in seed certification. In this study, a diagnostic microarray previously tested for specificity of probes for detecting the potato bacteria causing blackleg and soft rot (Pectobacterium atrosepticum, Pectobacterium carotovorum, and Dickeya spp.), ring rot (Clavibacter. michiganensis subsp. sepedonicus), scab (Streptomyces scabies and Streptomyces turgidiscabies) and brown rot (Ralstonia solanacearum) from pure culture was evaluated for analytical sensitivity when testing directly from tuber samples. The microarray readily detected all the bacterial species when 100 ng of the target bacterial DNA from pure culture was mixed with DNA from soil microbes and potato. However, detection was inconsistent when total DNA isolated directly from infected tubers or enriched bacterial culture was used. While the high specificity of the probes could be confirmed from the results of the DNA cocktail experiment used as a control, the study demonstrated that the level of analytical sensitivity of the microarray under the tested condition was not sufficient to detect bacteria directly from tubers. Therefore, in addition to the cost and organizational complexities, the low analytical sensitivity and limited reproducibility of the microarray are constraints for establishing the platform for routine detection of potato bacterial pathogens from tuber samples.     

The MeloTuber Test: a real‐time TaqMan® PCR‐based assay to detect the root‐knot nematodes Meloidogyne chitwoodi and M. fallax directly in potato tubers

Potato is one of the many important hosts for the root‐knot nematodes Meloidogyne chitwoodi and M. fallax that can infest roots as well as tubers. In the latter they may cause surface galls and necrotic spots below the skin. In the EU these pathogens are categorized as quarantine organisms and are therefore regulated. Phytosanitary measures (PMs) are implemented and one aspect involves diagnostic procedures to detect these pathogens. To date, visual screening of external and internal symptoms is combined with the specific identification of these pests, either through microscopy, biochemical or molecular tests. A disadvantage of all these tests is the requirement of the prior extraction of nematodes from the tubers, which is not suitable for high‐throughput screening. This paper describes the MeloTuber Test developed to simultaneously detect M. chitwoodi and M. fallax by triplex real‐time TaqMan^® PCR directly in secondary potato tuber peelings. DNA extraction is carried out in a 96‐well plate of pooled secondary peelings from one hundred tubers. The analytical sensitivity is such that a single female can be readily detected in such a sample size. The validation data, described here, prove the suitability of this molecular test for the detection of M. chitwoodi and M. fallax in large scale screening tests.     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

Accreditation process in the National Phytosanitary laboratory in Latvia1

The National Phytosanitary laboratory is the only laboratory in Latvia that provides testing of samples for regulated pests in the phytosanitary field. In 2001 a decision was made to implement Quality Assurance systems in the laboratory and obtain accreditation. From 2001 to 2003 preparation for accreditation took place in the laboratory. During this time a client of the laboratory, Sanitary Border Inspection of Latvia (SBI), arranged three audits to assess the activities of the laboratory in accordance with the requirements of ISO 17025. After a time and labour‐consuming preparation process on 2003‐12‐29 all the necessary documents including a Quality manual, two methods and procedures were submitted to the Latvian National Accreditation Bureau (LATAK). The first audit by LATAK was carried out in March 2005. After elimination of all non‐conformities, accreditation for two methods was received in 2005 (Method for detection and identification of Clavibacter michiganensis subsp. sepedonicus in potato tubers and Method for detection and identification of Ralstonia solanacearum in potato tubers). The Quality Assurance System was implemented and applies to the whole laboratory. Every year LATAK experts carry out monitoring visits and each time imperfections are found they must be eliminated or improved.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads

Using quantitative PCR, DNA of Plasmodiophora brassicae, the causal agent of clubroot, was detected and quantified on canola, pea and wheat seeds, as well as on potato tubers, all harvested from clubroot‐infested fields in Alberta, Canada. Quantifiable levels of infestation were found on seven of the 46 samples analysed, and ranged from <1·0 × 10³ to 3·4 × 10⁴ resting spores per 10 g seeds; the vast majority (80–100%) of resting spores on these samples were viable, as determined by Evan’s blue vital staining. However, the levels of infestation found were generally lower than that required to cause consistent clubroot symptoms in greenhouse plant bioassays. While the occurrence of P. brassicae resting spores on seeds and tubers harvested from clubroot‐infested fields suggests that seedborne dissemination of this pathogen is possible, practices such as commercial seed cleaning may be sufficient to effectively mitigate this risk.     

Evidence for presence of the founder Ia mtDNA haplotype of Phytophthora infestans in 19th century potato tubers from the Rothamsted archives

Late blight remained a significant disease for potato growers in Europe long after the famine of the 1840s. Of the four mitochondrial haplotypes of Phytophthora infestans, only the Ia mitochondrial DNA (mtDNA) haplotype has been identified previously in infected potato leaves from famine‐era herbarium specimens collected in England, Ireland and Europe in the 19th century. Long‐term soil fertility experiments were conducted on potato between 1876 and 1901 in Rothamsted to investigate effects of combinations of organic manures and mineral fertilizers on disease and yield. This report identifies for the first time the same Ia mtDNA haplotype of P. infestans in three diseased tubers from 1877 from the long‐term Rothamsted trials, thus providing the earliest evidence of the presence of the founder Ia mtDNA haplotype of P. infestans in potato tubers in England. Soil amendments had a significant impact on disease and yield. A real‐time PCR assay was used to detect and quantify P. infestans in tubers. The level of pathogen DNA was greatest in tubers from highest yielding plots that received combinations of inorganic nitrogenous and mineral fertilizers and least in tubers from plots with organic farmyard manures or non‐nitrogenous mineral fertilizers. The Ia mtDNA haplotype was also confirmed from diseased potato leaves during the same time period. Thus, the founder Ia mtDNA haplotype survived in potato tubers after 1846 and was present over 30 years later in the UK.     

Conventional PCR and Real-time Quantitative PCR Detection of Helminthosporium Solani in Soil and on Potato Tubers

Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g^–1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.     

Evaluation of antagonistic bacteria for suppression of bacterial ring rot of potato

Bacterial strains with potential for biological control of bacterial ring rot of potato caused byClavibacter michiganensis subsp.sepedonicus were isolated from the surface of potato tubers. Eighty-eight potential biocontrol candidates, selected on the basis ofin vitro antibiosis toC. m. sepedonicus, produced inhibition zones with radii ranging from 0.5 to 16 mm on test plates. All antagonistic isolates were screened in the greenhouse for biocontrol activity on micropropagated potato plantlets root-inoculated withC. m. sepedonicus. Eight strains consistently prevented infection of plantlets but there was no significant correlation between the width of the inhibition zone in thein vitro assay and ring rot suppression in the plant bioassay. Three strains that showed a high level of biological control potential were identified as a saprophytic enteric bacterium (strain 7G), anArthrobacter sp. (strain 16C), and a soil coryneform bacterium (strain 18A). These were tested in a field plot by co-inoculating cut seed potato tubers withC. m. sepedonicus and antagonists. Strains 7G and 18A significantly increased plant stand whereas 16C decreased disease incidence. The relative number of ostensibly ring rot-free progeny tubers was generally greater when antagonists were present.     

Development of a quantitative real‐time PCR assay for Phytophthora infestans and its applicability to leaf,tuber and soil samples

A sensitive real‐time polymerase chain reaction (PCR) assay was developed for the quantification of Phytophthora infestans, the cause of foliar and tuber late blight in potato. A primer pair (PinfTQF/PinfTQR) and a fluorogenic probe (PinfTQPR) were designed to perform a quantitative assay for the detection of P. infestans in leaves, tubers and soils. The assay was shown to be specific to P. infestans and the very closely taxonomically related non‐potato pathogen species P. mirabilis, P. phaseoli and P. ipomoea, but did not detect the potato pathogens P. erythroseptica and P. nicotianae. The assay was able to reliably detect P. infestans DNA at 100 fg per reaction and was effective in quantifying P. infestans in infected leaf tissue from 24 h after inoculation and also in infected symptomless tubers and diseased tubers. Attempts to detect oospores of P. infestans in naturally and artificially infested soil samples are described and compared with baiting tests and previous literature. It was not possible to detect oospores in soil samples due to problems with DNA extraction from the oospores themselves. However, the assay was shown to detect even very low levels of asexual inoculum (sporangia and mycelium) in soil. This work assembles all the necessary features of a quantitative P. infestans assay, which have previously been somewhat disparate: the sensitivity, specificity and quantitation are fully validated, the assay is shown to work in common applications in leaf and tuber tissue and the problems with P. infestans oospore detection are explored and tested experimentally.     

Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low-temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real-time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.     

Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab

Infection by Spongospora subterranea of roots of two potato (Solanum tuberosum) cultivars, either very resistant or very susceptible to powdery scab on their tubers, was studied in a glasshouse experiment. Plants grown in sand/nutrient solution culture were inoculated with S. subterranea sporosori 2 weeks after planting. Plant parameters, the intensity of zoosporangium infection in roots, numbers of Spongospora root galls and amounts of Spongospora DNA in roots, measured using quantitative PCR (qPCR), were assessed at sequential harvests. Inoculation with S. subterranea reduced water use (56 days after planting) by 26% in the tuber resistant cultivar compared with uninoculated plants, and by 60% in the susceptible cultivar. Inoculation did not affect growth of the resistant cultivar, nor shoot mass of the susceptible cultivar, but caused a 38% reduction in root mass of the susceptible cultivar. The intensities of zoosporangium development in both cultivars were similar. The susceptible cultivar had approximately four times more Spongospora root galls g^?1 root mass than the resistant cultivar. Quantitative PCR detected S. subterranea DNA in roots 1 week after inoculation, and indicated a twofold greater amount of pathogen DNA in roots of the susceptible than the resistant cultivar. This study suggests that the S. subterranea zoosporangium stage in host roots is affected differently by host resistance factors than the sporosorus (root gall and tuber scab) stages. The study has also demonstrated the usefulness of qPCR for sensitive and consistent detection of S. subterranea across the duration of potato root infection.     

Ralstonia solanacearum as a potato pathogen in Serbia: Characterization of strains and influence on peroxidase activity in tubers

Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During 6 years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from seven cultivars among 12 monitored, and in five localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLI-1/Y-2), as well as the following tests: restriction fragment length polymorphism analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdhA, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato were homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2–22 times in vascular necrotic tissues compared to non-necrotic ones, and depended on disease severity but not on cultivar. Native polyacrylamide gel electrophoresis analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.     

Virulence of Pectobacterium carotovorum subsp. brasiliense on potato compared with that of other Pectobacterium and Dickeya species under climatic conditions prevailing in the Netherlands

In western Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as a causal agent of blackleg disease. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium species. In 2013 and 2014, seed potato tubers were vacuum infiltrated with high densities of bacteria (10⁶ CFU mL^?1) and planted in clay soil. Inoculation with P. carotovorum subsp. brasiliense and P. atrosepticum resulted in high disease incidences (75–95%), inoculation with D. solani and P. wasabiae led to incidences between 5% and 25%, but no significant disease development was observed in treatments with P. carotovorum subsp. carotovorum, D. dianthicola or the water control. Co‐inoculations of seed potatoes with P. carotovorum subsp. brasiliense and D. solani gave a similar disease incidence to inoculation with only P. carotovorum subsp. brasiliense. However, co‐inoculation of P. carotovorum subsp. brasiliense with P. wasabiae resulted in a decrease in disease incidence compared to inoculation with only P. carotovorum subsp. brasiliense. In 2015, seed potatoes were inoculated with increasing densities of P. carotovorum subsp. brasiliense, D. solani or P. atrosepticum (10³–10⁶ CFU mL^?1). After vacuum infiltration, even a low inoculum density resulted in high disease incidence. However, immersion without vacuum caused disease only at high bacterial densities. Specific TaqMan assays were evaluated and developed for detection of P. carotovorum subsp. brasiliense, P. wasabiae and P. atrosepticum and confirmed the presence of these pathogens in progeny tubers of plants derived from vacuum‐infiltrated seed tubers.     

Detached leaf inoculation of germplasm for rapid screening of resistance to citrus canker and citrus bacterial spot

A detached leaf protocol for rapid screening of germplasm for resistance to citrus canker (Xanthomonas citri subsp. citri, Xcc) and citrus bacterial spot (Xanthomonas alfalfae subsp. citrumelonis, Xac) was developed to evaluate limited quantities of leaf material. Bacterial inocula of Xcc or Xac at 10⁴, 10⁵, or 10⁸ cfu ml⁻¹ were injection-infiltrated into the abaxial surface of disinfested, immature leaves of susceptible and resistant genotypes. Inoculated detached leaves were placed on the surface of 0.5% water agar plates and incubated at 28°C under a 12 h photoperiod. Likewise, inocula were infiltrated into attached leaves of greenhouse plants. At high inoculum concentrations of Xcc or Xac (10⁸ cfu ml⁻¹), resistant cultivars of kumquat developed a hypersensitive-like reaction within 3 days post inoculation (dpi). At 10⁵ cfu ml⁻¹, populations 14 dpi were <10⁴ per inoculation site. In canker-susceptible Citrus spp. (‘Duncan’ grapefruit and ‘Rough’ lemon), water-soaked areas occurred by 3 dpi and typical canker lesions developed by 7 to14 dpi. Concentration of Xcc recovered from inoculation sites was approximately 10⁵ cfu ml⁻¹ by 14 dpi. In citrus bacterial spot-susceptible citrus (‘Swingle’ citrumelo and grapefruit), symptoms developed within 7 dpi. Populations of Xac after inoculation at 10⁵ cfu ml⁻¹ were comparable to Xcc in susceptible hosts 14 dpi (>10⁵). The detached leaf assay is useful for the characterization and differentiation of lesion phenotype for each Xanthomonas pathogen permitting rapid screening of germplasm resistance based on the quantification of number of lesions and bacterial concentration.