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Bacterial microorganisms which are latent in in vitro cultures can limit the efficiency of in vitro methods for the conservation of genetic resources. In this study we screened 2,373 accessions from the in vitro sweetpotato germplasm collection of the International Potato Center in Lima, Peru for bacteria associated with plantlets in tissue culture through a combination of morphological methods and partial 16S rDNA sequencing. Bacteria were detected in 240 accessions (10% of the accessions screened) and we were able to isolate 184 different bacterial isolates from 177 different accessions. These corresponded to at least nineteen Operational Taxonomic Units (OTUs) of bacteria, belonging to the genera Sphingomonas, Bacillus, Paenibacillus, Methylobacterium, Brevibacterium, Acinetobacter, Microbacterium, Streptomyces, Staphylococcus, and Janibacter. Specific primers were developed for PCR based diagnostic tests that were able to rapidly detect these bacteria directly from tissue culture plants, without the need of microbial sub-culturing. Based on PCR screening the largest bacterial OTUs corresponded to a Paenibacillus sp. closely related to Paenibacillus taichungensis (41.67%), and Bacillus sp. closely related to Bacillus cereus (22.22%), and Bacillus pumilus (16.67%). Since in vitro plant genetic resources must be microbe-free for international distribution and use, any microbial presence is considered a contamination and therefore it is critical to clean all cultures of these latent-appearing bacteria. To accomplish this, plantlets from in vitro were transferred to soil, watered with Dimanin® (2 ml/l) weekly and then reintroduced into in vitro. Of the 191 accessions processed for bacterial elimination, 100% tested bacteria-free after treatment. It is suspected that these bacteria may be endosymbionts and some may be beneficial for the plants.

La marchitez bacteriana causada por Ralstonia solanacearum E.F. Smith es una de las enfermedades bacterianas más importantes que ataca a cultivos agrícolas como papa, tomate, banana, entre otros, causando grandes pérdidas en la producción. Desafortunadamente, su control ha sido difícil por su amplio rango de hospederos alternativos, su supervivencia en el suelo y su variación biológica y genética; así como porque no hay variedades con altos niveles de resistencia y porque no existe un control químico efectivo. Quorum sensing (percepción de quorum) es el fenómeno mediante el cual la acumulación de unas moléculas permite a una bacteria saber el número de bacterias que se encuentran en el medio es decir la densidad poblacional. La bacteria R. solanacearum posee un sistema quorum sensing para la regulación de la expresión de genes de virulencia, y en la cual la molécula 3-OH-PAME es el autoregulador de esta señal. Se conoce que la molécula ΒHPMEH hidroliza a 3-OH-PAME, anulando así la señal de autorregulación y por tanto la comunicación quorum sensing en R. solanacearum. Con el objetivo de evaluar el gen βhpmeh, se diseñaron dos vectores que expresen este gen bajo el control de dos diferentes promotores, los cuales fueron verificados por análisis de restricción, secuenciamiento y posteriormente mediante técnicas de agroinfiltración, se observó su expresión y su efecto frente a R. solanacearum en hojas de papa de la variedad Desiree. Los resultados de la expresión transitoria, muestran que el gen βhpmeh retrasó la aparición de síntomas de la marchitez bacteriana y sería un candidato potencial para transformación genética de la planta entera., Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease that attacks important agricultural crops such as potato, tomato, banana, among others, causing serious yield losses. Control of R. solanacearum is difficult because of its wide range of alternate hosts, its long survival in soil, its biological and genetic variation, the lack of natural resistance sources and the insufficiency of the appropriate chemical control measures. Quorum sensing is the term that describes the phenomenon whereby the accumulation of molecules allows bacteria to know the number of bacteria found in the environment (population density). R. solanacearum has a quorum sensing system for the regulation of the expression of virulence genes; the molecule 3-OH-PAME is the self-regulatory signal. The molecule ΒHPMEH hydrolyzes 3-OH-PAME nullifying the signal of virulence, and thus, the quorum sensing communication in R. solanacearum. In order to evaluate the βhpmeh gene we designed two vectors that express this gene under the control of two different promoters. Both vectors were verified by restriction analysis and sequencing. Agroinfiltration assays were used to analyze gene expression and the effect against R. solanacearum in potato (Solanum tuberosum) leaves. The results of the transient expression experiments showed that the expression of gene βhpmeh caused a delay in the appearance of symptoms of bacterial wilt and thus is a good candidate for whole genetic plant transformation.

Ten potato genotypes with field resistance to bacterial wilt were evaluated for resistance to five strains of Ralstonia solanacearum belonging to biovars 1, 2A, 2T and 3 (phylotypes I and II) under greenhouse conditions. The plants were inoculated by pouring bacterial suspension into the soil and wilt incidence was recorded weekly. Stems and tubers of symptomless plants were tested for latent infection using post-enrichment NCM-ELISA. The analysis of variance of the percentage of infected plants indicated significant differences among potato genotypes and strains of R.solanacearum. Complete resistance to all tested strains was not observed in any of the potato genotypes. However, CIP 394895.7 was the most resistant genotype, showing hardly any wilting and low level of latent infections. The use of potato varieties with partial resistance like this genotype in combination with crop rotation, healthy seed and sanitation practices could significantly reduce losses due to bacterial wilt.

The current bacterial wilt infestation level in the potato fields in the Peruvian Andes was investigated by collecting stem samples from wilted plants and detecting Ralstonia solanacearum. In total 39 farmers’ fields located in the central and northern Peru between the altitudes 2111 and 3742 meters above sea level were sampled. R. solanacearum was detected in 19 fields, and in 153 out of the 358 samples analysed. Phylogenetic analysis using the partial sequence of the endoglucanase gene on strains collected in Peru between 1966 and 2016 from potato, pepper, tomato, plantain or soil, divided the strains in phylotypes I, IIA and IIB. The Phylotype IIB isolates formed seven sequevar groups including the previously identified sequevars 1, 2, 3, 4 and 25. In addition to this, three new sequevars of phylotype IIB were identified. Phylotype IIA isolates from Peru clustered together with reference strains previously assigned to sequevars 5, 39, 41 and 50, and additionally one new sequevar was identified. The Phylotype I strain was similar to the sequevar 18. Most of the Peruvian R. solanacearum isolates were IIB-1 strains. In the old collection sampled between 1966 and 2013, 72% were IIB-1 and in the new collection at 2016 no other strains were found. The pathogenicity of 25 isolates representing the IIA and IIB sequevar groups was tested on potato, tomato, eggplant and tobacco. All were highly aggressive on potato, but differed in pathogenicity on the other hosts, especially on tobacco. All IIA strains caused latent infection on tobacco and some strains also caused wilting, while IIB strains caused only few latent infections on this species. In conclusion, high molecular diversity was found among the R. solanacearum strains in Peru. Most of the variability was found in areas that are no longer used for potato cultivation and thus these strains do not pose a real threat for potato production in the country. Compared to the previous data from the 1990s, the incidence of bacterial wilt has decreased in Peru. The epidemics are likely caused by infected seed tubers carrying the clonal brown rot strain IIB-1.

This protocol is an updated version of ?Assessing potato clone field resistance to bacterial wilt? issued in The International Cooperators? Guide (CIP 2007). The first edition of the protocol presented a standard procedure for field assessment of resistance to bacterial wilt for documenting levels of resistance of advanced potato germplasm. This second edition has included a standardized procedure for greenhouse screening of potato seedlings for bacterial wilt resistance useful for perform genetic studies, parental selection or identification of new sources of resistance in accessions of wild species propagated or maintained as true seed.

The detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop, instead of tubers, would not only save highly valued planting material but would be less time-consuming and would also enhance farmers’ market decisions. Although pathogen detection in stems has been proven efficient for ring rot, this has never been investigated for bacterial wilt (BW). Therefore the possibility of detecting BW latent infection in stem pieces about three weeks before harvest was assessed in 57 fields of the Andean highlands of Peru. Two sensitive, specific and user-friendly serological methods were used to detect the pathogen in latently infected tubers and stems: double-antibody sandwich (DAS)-ELISA and indirect ELISA on nitrocellulose membrane (NCM) after enrichment of the plant extracts in a semi-specific broth. Optimum sample sizes of stems and tubers were evaluated for 37 potato crops showing between 0 and 0·1% BW incidence using a binomial distribution model to calculate the detection probabilities. Although results of detection using the two serological techniques had 100% concordance, detection probabilities were higher using DAS-ELISA, whatever the plant part tested. BW detection probabilities were higher for tubers than for stems; a 99% detection probability was obtained by analysing 400 stems sections or 250 tubers using DAS-ELISA. Detection of BW infection in symptomless plants 20 days before harvest using post-enrichment DAS-ELISA is a reliable and user-friendly technique that can easily be used by national plant protection services and seed programmes in developing countries.

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g−1 inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g−1 were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.

This paper reports results of a 3-year evaluation of CIP advanced potato clones in a bacterial wilt-infested field (race 3) in Peru. Clones resistant or moderately resistant to wilt were selected and all tubers harvested from each clone were tested for latent infection byRalstonia solanacearum using a sensitive serological technique developed at CIP. A sampling strategy to estimate accurately the frequency of infected tubers in the clones has been evaluated. This method will allow consideration of tuber latent infection as a new selection criterion in breeding for resistance to bacterial wilt. Thirteen clones were found resistant to wilt in all three evaluations (i.e.≤6% wilt), from which five had no wilt in all trials. However, all clones harboured latent infection in tubers averaging 30%. Analysing 30 tubers/clone provides an accurate estimation of the proportion of infected tubers with a high precision level.

A sampling strategy was evaluated in the Andean highlands of Peru to optimise the detection ofRalstonia solanacearum in seed tubers harvested from symptomless crops. A sensitive and specific serological method developed at CIP was used to detect the pathogen in latently infected tubers. Optimum sample size was evaluated for symptomless crops after analysing various numbers of composite samples and using a binomial distribution model to calculate the detection probabilities.R. solanacearum was detected in all lots from fields with visible symptoms, so validating the detection technique. About half of the seed lots from apparently healthy fields at altitudes of up to 3,100 m were found positive for the pathogen.R. solanacearum was detected with 99% probability in samples of 350 tubers from seed lots from symptomless crops. This number of seed tubers could feasibly be processed in a seed-health test without incurring too high a cost for labour and materials.

A post-enrichment enzyme-linked immunosorbent assay (ELISA) on nitrocellulose membrane (NCM-ELISA) is described for the detection of Ralstonia solanacearum in latently infected potato tubers. The polyclonal antiserum specificity was significantly improved by adsorption with cross-reacting bacteria. The detection efficiency after enrichment was compared with those of nucleic acid spot hybridization (NASH), double-antibody-sandwich immunoassay (DAS-ELISA) and plating on modified Kelman's medium. After 48 h of incubation of the tuber extracts with modified SMSA broth at 30°C, sensitivities of post-enrichment NCM-ELISA, DAS-ELISA and NASH were similar. As few as 10cells mL−1 were detected in either inoculated or naturally infected tuber extracts. Of 255 field samples, no cross-reactivity of NCM-ELISA was observed. Post-enrichment NCM-ELISA thus provides a reliable and sensitive low-cost method that is rapid and easy to use, making it suitable for assessing susceptibility of breeding lines to bacterial wilt, ecological studies and seed quality control in developing countries.