Areas were re-hydratedand antigen retrieval was performed by heating up slides in sodium citrate in a steamer

While phylotype II is classified as a separate genomic species, its name remains R. solanacearum. The Rssc comprises strains that are capable of causing wilt in Musaceae plants and that cluster into two distant phylogenetic groups: Moko disease-causing strains reported from Latin America, Asia, and the Philippines and the BDB originating in Indonesia and Malaysia. Systemic vascular infection by Rs induces symptoms that begin with the yellowing of leaves and tissue necrosis and that lead to a general collapse of the plant. The fruits are inedible and exhibit internal vascular discoloration. Specific symptoms can be observed, particularly with BDB, which produces a reddish coloration of the vascular ring in the fruit. Bugtok disease only affects the floral bud, leading to hardening of the fruit. Phylotype II harbors the largest number of epidemiologically active ecotypes, such as Brown rot, Moko, NPB, and Granville wilt. As a working definition, the phylotypes are further subdivided into sequevars. The Moko disease-causing strains are paraphyletic and have historically clustered into four sequevars: IIA-6, IIA-24, IIB-3, and IIB-4. The pathological variant IIB-4NPB was first reported in diseased anthurium in Martinique and was phylogenetically assigned to the Moko lineage IIB-4. These strains are variants that are not pathogenic to bananas but that demonstrate a host range that expands to Cucurbitaceae. The Moko-associated strains, in addition to being soil-borne and transmitted through wounds and cuttings, can also be actively transmitted by insects through the bud ; the pathogen then migrates down into the plant, leading to symptoms that start with fruit decay and end in plant collapse. In addition to these two groups, the epidemiological 4NPB lineage variant, which is grouped into the Moko sequevar IIB-4 lineage, does not cause wilt on Cavendish or plantain bananas; instead, this variant establishes itself and moves within the vascular tissues of plantains, even via soil-borne contamination, as it establishes a latent infection through the root system. Recently, unanticipated Moko disease-related strains from Brazil were reported that clustered into the previously described Solanaceae-related sequevars IIA-41 and IIB-25, which are not related to historic Moko lineages, and into a newly proposed sequevar, IIA- 53. This finding highlights the fact that the Moko ecotype benefits from a broad genetic basis and harbors far more genetic diversity than anticipated. Based on the whole-genome sequences of Moko strains, we developed a robust, simple, and affordable duplex PCR assay that is specific for phylotype II Rssc strains that can be retrieved from banana and plantain tissues. Here, we present an extensive characterization of the performance of the duplex PCR assay. The diagnostic method presented in this study for detecting Moko disease-causing strains and variant IIB-4NPB strains was developed to fit with the requirements of officially accredited diagnostic laboratories. The proposed protocol was fully evaluated, as required by the ISO 17025 standard, by following EPPO protocols, thus ensuring the highest confidence in the method development and validation. The focus of this study was on the development of a method for the detection of the Moko lineage, which is capable of causing wilt in Musaceae plants. This duplex PCR assay was able to detect the historical diversity of Moko strains and also the newly discovered Moko-related Brazilian strains that cluster into sequevars IIB-25, IIA-41, and IIA-53. The epidemiologically variant IIB-4NPB strains that cause latent infection within the vascular system of plantains were also detected in this duplex PCR assay framework. The protocol developed in this work appears suitable for research and diagnostic laboratories and showed reliable accuracy, detectability, and repeatability. Currently, no specific diagnostic protocol related to Rssc strains that are pathogenic to Musaceae plants has been defined by the European Commission. In addition, it is known that some strains within phylotype II that cause banana and Musaceae wilt are also able to wilt INCB28060 Solanaceae and may represent a threat to both plant families; phylotype II Musaceae-adapted strains might be carried by Solanaceae plants. Therefore, a general assay to detect Rssc strains and a specific Brown rot detection protocol for Solanaceae should be used along with a specific Rs banana wilt detection protocol to detect both types of strains. The performance assessment of the duplex PCR assay was fully compliant with its use as a reference method for diagnostic laboratories. Moko and the epidemiologically variant IIB- 4NPB strains were both specifically detected with confidence. Amplification of the 477-bp Moko band could rarely fail for the IIB-4NPB strains, positive detection status should be considered if only the specific 661-bp 4NPB band is amplified; additional confirmation might also be necessary.

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