First Report of Ralstonia pseudosolanacearum (Phylotype I) Causing Bacterial Wilt in Zingiber officinale in Slovenia

Ginger (Zingiber officinale) is an herbaceous perennial in the Zingiberaceae family grown primarily in tropical to subtropical biomes as a culinary spice, a traditional medicine, and a landscaping plant. While ginger grows at soil temperatures above 20°C, several farmers in the upper Midwestern US farmers grows short-season ginger in high tunnels. In 2023 and 2024, growers in southeastern Minnesota reported a new disease of ginger. USDA traced the origin of the ginger rhizomes imported by these growers to Peru (Soto-Heredia et al., 2024). Plants exhibited wilting, chlorosis, pseudostem collapse, and plant death. Rhizomes were squishy with a fetid, rotting odor. Disease incidence ranged from scattered plants to 50% of the crop. Symptomatic samples tested positive for Ralstonia solanacearum species complex (RSSC) using AgDia ImmunoStrips. Rhizome tissue extracts plated onto CPG + 1% tetrazolium chloride agar and mSMSA agar produced irregular, round-to-fluidal white colonies with pink centers after 48 h of incubation at 28°C (Kelman 1954; Engelbrecht 1994). Diagnostic RSSC phylotype-specific multiplex PCR primers Nmult21:1F, Nmult21:2F, Nmult23:AF, Nmult22:InF, Nmult22:RR, and 759f/760r confirmed isolate identity; two isolates (UMN24-1, UMN24-2) yielded bands at 280 bp and 144 bp, identifying them as RSSC and phylotype I respectively (Fegan and Prior 2005; Opina et al. 1997). Additionally, 16S rRNA PCR of these isolates was sequenced using primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3')/1492R (5'-TACGGYTACCTTGTTACGACTT-3') and shared >99% identity (isolate UMN24-1: 99.00% 27F, 99.53% 1492R; UMN24-2: 99.32% 1492R) with R. pseudosolanacearum (Rps) strain Gj707 (GenBank CP104497.1). Eight ginger plants in individual pots were grown in a Conviron growth chamber (28°C, 250 μmol/m2/s, 16 h photoperiod) and inoculated at the six-leaf stage (6 weeks old) by pipetting 109 CFU of the isolate, UMN24-1, suspended in sterile water into a 30°angled 1 cm incision on the pseudostem. We scored disease incidence based on wilted leaves per total leaves on each plant for 14 days post inoculation (dpi). While control plants remained healthy, inoculated plants showed symptoms (wilting, vascular discoloration, chlorosis) at 4 dpi, were 100% wilted by 6 dpi, and all pseudostems completely collapsed by 11 dpi (Fig. S1). Bacteria recovered from the inoculated plants matched the original isolate in appearance and positively identified as RSSC phylotype I using the PCR analyses described above. To confirm taxonomic identification of UMN24-1, the complete genome of this isolate was sequenced using hybrid Illumina and Oxford Nanopore sequencing (NCBI BioProject ID PRJNA1178296, release date upon publication) (Fig. S2). Average nucleotide identity (ANI) analysis of UMN24-1 using KBase's FastANI app reported 99.8% identity shared with Japanese RSSC phylotype I strain MAFF311693 (GenBank GCF 015698385.1). We also sequenced UMN24-2, and its phylogenetic analysis revealed that the two isolates are clonal (Fig. S2). Hence, we did not submit its sequence to the NCBI. This marks the first documented case of Rps in ginger in the United States. Similar to bacterial wilt in ginger closely related crop like turmeric has been repeatedly reported in northern Europe in recent years (EPPO 2024). While RSSC phylotype I is geographically limited by its minimal cold tolerance, the emergence of a pathogen with a potentially broad host range has worrying implications in a changing climate.

The Ralstonia solanacearum Species Complex (RSSC) is a significant plant pathogen affecting various agricultural commodities, including ginger. Understanding the distribution and characteristics of RSSC in ginger plants is crucial for effective disease management. This study aims to determine the distribution of RSSC in ginger plants and identify the pathogen through morphological and biochemical characterization. The research was conducted in Banyubiru and Getasan districts, Semarang Regency, Indonesia. The study involved purposive sampling, isolation, and purification of isolates, followed by morphological characterization through observation and biochemical characterization using Gram staining with KOH, oxidase test, arginine activity test, hypersensitivity test, bacterial density calculation, Koch's postulates, and biovar characterization. Three isolates from Banyubiru displayed typical morphological characteristics of RSSC, including round, mucoid colonies with red centers and white edges on TZC medium, and rod-shaped bacterial cells. Biochemical characterization identified these isolates as RSSC strains biovar 3 and 4, capable of causing bacterial wilt in ginger plants. The study confirmed the presence of bacterial wilt in ginger in Banyubiru district. The findings reveal the spread of bacterial wilt caused by RSSC in ginger plants in Banyubiru, Semarang Regency. There is a need for measures to control the potential spread of RSSC in the surrounding host plant areas in Tlumpak Village, Banyubiru district.

The Ralstonia solanacearum species complex is a highly destructive plant pathogen with a remarkably broad range of hosts, and ongoing discoveries continue to expand its host list. In May 2023, a new type of bacterial wilt affecting ginger (Z. officinale) crops in Semarang Regency, Central Java was reported. Early symptoms included sudden withering of leaves in adult plants followed by complete wilting and darkening of the vasculature, ultimately leading to plant death. This research specifically investigates the spread of Ralstonia solanacearum Species Complex within ginger cultivation in Semarang Regency. Twenty bacterial isolates were collected from soil and diseased Z. officinale plants at twenty different locations. Physiological and biochemical analyses confirmed that the causative agent for Z. officinale bacterial wilt was Ralstonia solanacearum belonging to biovar 3 and 4. The study also revealed that the distribution of this pathogen remains focused in the Banyubiru and Sumowono sub-districts. These findings will enhance our understanding of how Ralstonia solanacearum Species Complex spreads among ginger crops and its impact on them.

Ralstonia solanacearum species complex (RSSC) consists of three species, including R. solanacearum, R. pseudosolanacearum, and R. syzygii. The K60-type strain of R. solanacearum was isolated from a wilted 'Marglobe' tomato in Raleigh, North Carolina (NC) in 1953 (Kelman 1954). It is classified as phylotype IIA, sequevar 7 (Prior and Fegan 2005). In July 2023, during a field visit in Eastern NC, patches of >50 eggplant (Solanum melongena cv. Pingtung Oriental) and tomato (Solanum lycopersicum cv. Saybrook) plants showing wilt symptoms were observed in a 1 ha field. Two plants from each host were collected from this site and tested positive for bacterial streaming in sterile deionized water for 2 min. One plant from each host was used for bacterial isolation by plating a 10 μl aliquot of the resulting bacterial streaming suspension on triphenyl tetrazolium chloride (TZC) medium (Kelman 1954) and incubated at 28°C for 48 hr. Multiple fluidal white colonies with a pink center and irregularly round morphology reminiscent of strains in the RSSC were observed on all plates. Only one colony from each plant host, NG-RL and EP-RL from tomato and eggplant, respectively, was selected for molecular characterization. Neither strain amplified the 357 bp band and was not R. solanacearum Select Agent (Opina et al. 1997). Genomic DNA from both NG-RL and EP-RL generated the 280 bp and 144 bp bands and confirmed as R. pseudosolanacearum phylotype I using the RSSC multiplex PCR (Fegan and Prior 2005). To determine sequevar, the primers Endo-F/Endo-R (Poussier et al. 2000; Fegan and Prior 2005) were used to sequence the partial endoglucanase (egl) gene from EP-RL and NG-RL (GenBank accessions: PQ554799 and PQ554800). These sequences were compared to publicly available egl sequences from GenBank and Cellier et al. (2023). A maximum likelihood phylogenetic tree showed that both NG-RL and EP-RL clustered with reference strains PSS81, MLI71-15, and Zo4 with 100% identity, confirming NG-RL and EP-RL are R. pseudosolanacearum phylotype I sequevar 14. To fulfill Koch's postulates, NG-RL and EP-RL inoculant was prepared from 48 h cultures grown on TZC plates at 28°C. Plates were flooded with sterile deionized water and then transferred to a falcon tube, adjusting O.D. 600 to 0.2 (~1×108 CFU/ml). The roots of six-week-old eggplants (cv. Black Beauty) and tomatoes (cv. Bonny Best) were wounded by running a scalpel through the soil 2 cm from the stem. This was repeated on six plants for both NG-RL and EP-RL. Mock-inoculated plants treated with sterile deionized water (SDW) served as controls. Plants incubated at 28°C in the greenhouse showed bacterial wilt symptoms nine days post-inoculation. R. pseudosolanacearum phylotype I sequevar 14 was confirmed from all bacteria-inoculated plants using the multiplex PCR and egl sequencing methods described above. No symptoms or bacteria were isolated from SDW mock-inoculated plants. There have been no prior reports of R. pseudosolanacearum phylotype I in North Carolina. To our knowledge, this is the first report on R. pseudosolanacearum phylotype I and the sequevar 14 in both tomato and eggplant in NC. This phylotype I is endemic to Asia and was first reported in the US in 2003 on pepper in Florida (Ji et al. 2006) and in 2015 on tomatoes in Louisiana (Jimenez Madrid et al. 2019). This finding highlights the need for a nationwide RSSC survey program, which can undoubtedly inform pathogen spread and management for crops in NC and beyond.

Tomato (Solanum lycopersicum), pepper (Capsicum annum), and gboma (Solanum macrocarpon) are major vegetables in Togo, with many people depending on these crops for their livelihood. In December 2018, during the dry season with temperatures between 21°C to 35°C, tomato ('Petomech'), pepper ('Gboyebesse') and gboma (local landrace) showing wilt symptoms without foliar yellowing were collected from two locations, Tchouloum and CECO-AGRO sites in the Sotouboua Prefecture of Togo, ~300 km from the capital city of Lome. Disease incidence ranged between 10% to 50% in multiple fields. Cut stems of most wilting tomato, pepper and gboma plants produced bacterial ooze in water and vascular discoloration was visible in longitudinal stem sections. Ground cut stem tissue tested positive with Rs ImmunoStrips specific to the Ralstonia solanacearum species complex (RSSC) (Agdia Inc., Elkhart, IN, USA). Collected samples were stored at ambient temperature and cultured within 36 hr. Culturing sap from cut stems plated on modified SMSA medium (Engelbrecht 1994) yielded colonies with typical RSSC morphology: slow-growing, irregular, mucoid, and white with red centers. Genomic DNA was extracted from thirteen isolates: two from gboma, five from tomato and six from pepper. The expected 280-bp band was amplified from all 13 genomic DNAs following polymerase chain reaction (PCR) using the 759/760 RSSC-specific primer pair (Opina et al. 1997). PCR with the 630/631 primers, which identify the Race 3 biovar 2 RSSC subgroup, did not yield a product from any Togo isolate (Opina et al. 1997). The phylotype multiplex PCR identified all Togo isolates as belonging to the phylotype I subgroup, also called R. pseudosolanacearum (Prior et al. 2016; Fegan and Prior 2005). Phylotype control DNAs were from strains GMI1000 (phylotype I, Asia), K60 (phylotype II, Americas), CMR15 (phylotype III, Africa), and PSI07 (phylotype IV, Indondesia). Comparative genomic analysis of the partial endoglucanase (egl) gene, amplified with the Endo primer pairs (Poussier et al. 2000), revealed all Togo strains belonged to sequevar 17, a group known to cause bacterial wilt of peanut in China. (Xu et al. 2009). The egl sequences are in NCBI GenBank accessions MT572393 to MT572405. Koch's postulates were completed by inoculating 28-day-old bacterial wilt-susceptible 'Bonny Best' tomato plants by soil soak (Khokhani et al. 2018). Briefly, soil around each unwounded plant was drenched with 50 ml of a 108 CFU/mL suspension of bacteria grown from a single colony. Five plants were inoculated with each of four randomly selected Togo strains. RSSC phylotype I strain GMI1000 served as a positive control and water treated plants as negative controls. Plants were kept in a 28°C growth chamber with a 12 hr photoperiod. All RSSC inoculated plants were fully wilted within a week; symptoms resembled to those observed in the field. Water treated control plants did not wilt. Culturing sap from all inoculated plants on SMSA medium yielded colonies with typical RSSC morphology that tested positive with the Rs ImmunoStrips. This is the first identification of RSSC in Togo. These results will guide development of disease management strategies and regionally appropriate breeding of vegetable lines with resistance to the phylotype I RSSC strains present in Togo.

Around 90% of Peru's ginger (Zingiber officinale) production is concentrated in the Junín region, due to the optimal agroecological conditions for its cultivation. In March 2024, fields with ginger plants (cultivar Criollo) in Junín region, provinces Chanchamayo and Satipo, specifically in the cities of Pichanaqui and Satipo respectively, exhibited approximately 40% of plants with severe symptoms of a disease characterized initially by plant yellowing and rapid progressing to necrosis. Affected rhizomes showed dark vascular bundles with milky white exudates upon cutting, while stems displayed vascular necrosis hindering water and nutrient transport, often resulting in plant death. Fifteen plants were sampled and diseased vascular tissues from rhizomes and stems were cultured on nutrient agar (NA) and incubated at 28°C. After 72 h, all isolations resulted in colonies with typical characteristics of Ralstonia solanacearum species complex (RSSC) were produced, with appearing fluid, irregularly round, and creamy white. Three isolates were selected for the identification steps (UNALM-RP01 to 03) were identified by PCR using primers 759/760 (Opina et al. 1997) confirming as RSSC with a 282 bp amplification product. Additionally, isolates were assigned to biovar 3 based on their ability to metabolize three acid-producing disaccharides (maltose, lactose, cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) (Hayward, 1964). Phylotype I was identified by multiplex PCR (primers Nmult) with a 114 bp amplification product (Fegan and Prior 2005). For the identification of the sequevars of the three isolates, DNA was extracted and PCR with primers ENDO-F/R (Ji et al. 2007) were performed to amplify and sequence the partial gene sequence of egl gene with 681 bp in length. The phylogeny by Neighbor joining with 10,000 bootstraps clustered the UNALM isolates along other sequevar 30 of R. pseudosolanacearum. The sequences were deposited in Genbank under accessions PQ213016, PQ213017 and PQ213018. For pathogenicity tests, bacterial colonies of isolate UNALM-RP01 were scraped from the culture media with a sterile needle and introduced into the stems of three 2-month-old ginger plants (cultivar Gigante). The plants subsequently exhibited yellowing seven days post-inoculation. Additionally, the rhizomes showed internal discoloration and bacterial exudation. Three plants were used as a control, which were pierced with a sterilized needle and showed no symptoms. All tested plants were kept in a greenhouse with controlled temperature (20-40 °C) The pathogen was successfully re-isolated from infected plants on NA medium, presenting typical colonies of RSSC and identified via PCR with primers 759/760, fulfilling Koch's postulates. This represents the first case in Peru of ginger plants infected with a Ralstonia species, specifically R. solanacearum phylotype I, corresponding to R. pseudosolanacearum. This species of RSSC and sequevar is known for causing disease in ginger. Its presence in Peru, however, may be the result of the pathogen's introduction, as its geographical origin is associated with Asia (Fegan and Prior 2005). To our knowledge, this is the first report of R. pseudosolanacearum causing ginger wilt disease in Peru. In 2024, an estimated average yield loss of 30% has been attributed to wilt disease in the Junín region, posing a significant threat to cultivation. Urgent and effective disease management strategies are essential to control and mitigate further losses.

The Ralstonia solanacearum Species Complex (RSSC) is the most significant plant pathogen group with a wide host range. It is genetically related but displays distinct biological features, such as restrictive geography occurrence. The RSSC comprises three species: Ralstonia pseudosolanacearum (phylotype I and III), Ralstonia solanacearum (phylotype IIA and IIB), and Ralstonia syzygii (phylotype IV) (Fegan and Prior 2005). In Brazil, eucalyptus production has been growing as one of the leading industries to produce cellulose, energy, and other products from renewable sources, covering approximately 7.8 million hectares, representing 76% of all planted forests in 2023 (IBÁ, 2024). The eucalyptus bacterial wilt (BW), caused by Ralstonia solanacearum and Ralstonia pseudosolanacearum, members of RSSC (Lopes and Rossato 2018), is one of the most important eucalyptus diseases, causing damage to nurseries and eucalyptus plantations (Alfenas et al., 2009). We collected Eucalyptus urophylla clone plants showing typical wilt symptoms in a commercial nursery with an average size of 400 m2 harboring 300k cuttings plant in rooting, showing 5% symptom incidence in February 2024 in the Bahia State, Brazil. Affected plants showed wilting of leaves and branches, reduced growth, and death at a higher level of disease severity. The vascular tissues showed darkening and intense bacterial exudation. Next, we aimed to determine the presence of RSSC bacteria in these plants by using the isolation method. For this, the stem macerate of symptomatic plants was plated on a semi-selective SMSA medium (ELPHINSTONE et al. 1996) and incubated for 72h at 28oC. It was possible to isolate bacteria with typical morphology of RSSC in all samples. Using multiplex PCR (Fegan & Prior, 2005), 96.77% of isolates were classified as phylotype II and 2.58% as phylotype I. Notably, we identified one isolate (0,65%), BR19, which exhibited an amplification pattern characteristic of phylotype IV. This isolate demonstrated typical RSSC growth, forming a slimy colony with a red internal region on CPG medium supplemented with tetrazolium. To fulfill Koch's postulates, eight plants of Eucalyptus urophylla were inoculated with bacterial suspension (20 µL adjusted to 108 CFU/mL of saline solution), and eight plants injected with distilled water were used as controls. The bacterial inoculum or the distilled water was injected at the base of the stem, and the plants were kept in a greenhouse (28 ± 2 °C), according to the methodology described by Fonseca et al. (2016). Then, after 30 days of post-inoculation with the bacterium, all plants showed wilt symptoms, and it was re-isolated from plating stem tissue macerates on SMSA medium. The identity of the bacteria was confirmed using RSSC species phylotype multiplex PCR (Fegan & Prior, 2005). Using Long-read Nanopore technology, we were able to sequence the draft complete genome composed of a chromosome and a megaplasmid, which were deposited at GenBank with assembly accession number GCF_042494895.1. The completeness of the BR19 genome was found to be 92.1%, according to the BUSCO program. Phylogenomic analysis was performed according to Subedi et al. (2024), revealing that BR19 is genetically closer to two other isolates of Ralstonia syzygy subsp. syzygii (GenBank assemblies: ASM1591059v1 and ASM2921996v1) isolated from clove (Syzygium aromaticum) in Indonesia. It is the first report of the detection of R. syzygii subsp. syzygii phylotype IV away from Southeast Asia and the first report of R. syzygii subsp. syzygii infecting eucalyptus. This result expands our knowledge about the geographic distribution and host range of RSSC.

Specific scopeThis Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Ralstonia solanacearum Species Complex (RSSC).It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentApproved in 2003‐09. First revised in 2018‐02.

Ralstonia solanacearum species complex (RSSC) cause several phytobacteriosis in many economically important crops around the globe, especially in the tropics. In Brazil, phylotypes I and II cause bacterial wilt (BW) and are indistinguishable by classical microbiological and phytopathological methods, while Moko disease is caused only by phylotype II strains. Type III effectors of RSSC (Rips) are key molecular actors regarding pathogenesis and are associated with specificity to some hosts. In this study, we sequenced and characterized 14 newly RSSC isolates from Brazil's Northern and Northeastern regions, including BW and Moko ecotypes. Virulence and resistance sequences were annotated, and the Rips repertoire was predicted. Confirming previous studies, RSSC pangenome is open as α≅0.77. Genomic information regarding these isolates matches those for R. solanacearum in NCBI. All of them fit in phylotype II with a similarity above 96%, with five isolates in phylotype IIB and nine in phylotype IIA. Almost all R. solanacearum genomes in NCBI are actually from other species in RSSC. Rips repertoire of Moko IIB was more homogeneous, except for isolate B4, which presented ten non-shared Rips. Rips repertoire of phylotype IIA was more diverse in both Moko and BW, with 43 common shared Rips among all 14 isolates. New BW isolates shared more Rips with Moko IIA and Moko IIB than with other public BW genome isolates from Brazil. Rips not shared with other isolates might contribute to individual virulence, but commonly shared Rips are good avirulence candidates. The high number of Rips shared by new Moko and BW isolates suggests they are actually Moko isolates infecting solanaceous hosts. Finally, infection assays and Rips expression on different hosts are needed to better elucidate the association between Rips repertoire and host specificities.

ABSTRACTPlant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30 s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field.IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Following a request from the European Commission, the EFSA Panel on Plant Health performed a pest categorisation of the Ralstonia solanacearum species complex (RSSC), a distinguishable cosmopolitan group of bacterial plant pathogens (including R. solanacearum, Ralstonia pseudosolanacearum and two subspecies of Ralstonia syzygii) of the family Burkholderiaceae. The RSSC causes bacterial wilt in solanaceous crops, such as potato, tomato and pepper, but can also cause wilts in other important food crops such as fruit banana, plantain banana and cassava. The pest survives in the soil, and a number of weed species can also be infected by the pest, often asymptomatically. The RSSC is regulated in Council Directive 2000/29/EC (Annex IAII) (indicated by its former name R. solanacearum, as delimited by Yabuuchi et al.) as a harmful organism whose introduction into the EU is banned. In addition, Council Directive 1998/57/EC (amended by Commission Directive 2006/63/CE) concerns the measures to be taken within EU Member States (MS) against the RSSC to (a) detect it and determine its distribution, (b) prevent its occurrence and spread, and (c) control it with the aim of eradication. The pest is present in several EU MS, but in all cases with a restricted distribution and under official control. New phylotypes of the RSSC could enter the EU primarily via host plants for planting (including seed tubers). The pest could establish in the EU, as climatic conditions are favourable, hosts are common and the pathogen has high adaptability. Spread is mainly via plants for planting. Substantial crop losses in the EU would occur in the presence of RSSC epidemics. The RSSC is regarded as one of the world's most important phytopathogenic bacteria due to its broad geographical distribution, large host range, aggressiveness, genetic diversity and long persistence in soil and water. The list of hosts and commodities for which the pest is regulated is incomplete due to the high diversity of hosts and the lack of knowledge of the complete host range. Moreover, the comparative epidemiology of the different pathogen species has not yet been studied. The criteria assessed by the Panel for consideration of the RSSC as potential quarantine pest are met, while, for regulated non‐quarantine pests, the criterion on the widespread presence in the EU is not met.

Bacterial wilt (BW), caused by Ralstonia solanacearum species complex (RSSC), leads to substantial potato yield losses in Rwanda. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with BW of potato, (ii) generate an RSSC distribution map for epidemiological inferences, and (iii) test the pathogenicity of predominant RSSC phylotypes on six commercial potato cultivars. In surveys conducted in 2018 and 2019, tubers from wilting potato plants were collected for pathogen isolation. DNA was extracted from 95 presumptive RSSC strain colonies. The pathogen was phylotyped by multiplex PCR and typed at sequevar level. Phylotype II sequevar 1 strains were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. Pathogenicity of one phylotype II strain and two phylotype III strains were tested on cultivars Kinigi, Kirundo, Victoria, Kazeneza, Twihaze, and Cruza. Two RSSC phylotypes were identified, phylotype II (95.79%, n = 91) and phylotype III (4.21%, n = 4). This is the first report of phylotype III strains from Rwanda. Phylotype II strains were identified as sequevar 1 and distributed across potato growing regions in the country. The TRST scheme identified 14 TRST haplotypes within the phylotype II sequevar 1 strains with moderate diversity index (HGDI = 0.55). Mapping of TRST haplotypes revealed that a single TRST '8-5-12-7-5' haplotype plays an important epidemiological role in BW of potato in Rwanda. None of the cultivars had complete resistance to the tested phylotypes; the level of susceptibility varied among cultivars. Cultivar Cruza, which is less susceptible to phylotype II and III strains, is recommended when planting potatoes in the fields with history of BW.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is the most destructive potato disease in Kenya. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with bacterial wilt of potato in Kenya, (ii) generate an RSSC distribution map for epidemiological inference, and (iii) determine whether phylotype II sequevar 1 strains exhibit epidemic clonality. Surveys were conducted in 2018 and 2019, in which tubers from wilting potato plants and stem samples of potential alternative hosts were collected for pathogen isolation. The pathogen was phylotyped by multiplex PCR and 536 RSSC strains typed at a sequevar level. Two RSSC phylotypes were identified, phylotype II (98.4%, n = 506 [sequevar 1 (n = 505) and sequevar 2 (n = 1)]) and phylotype I (1.6%, n = 30 [sequevar 13 (n = 9) and a new sequevar (n = 21)]). The phylotype II sequevar 1 strains were haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. The TRST scheme identified 51 TRST profiles within the phylotype II sequevar 1 strains with a modest diversity index (HGDI = 0.87), confirming the epidemic clonality of RSSC phylotype II sequevar 1 strains in Kenya. A minimum spanning tree and mapping of the TRST profiles revealed that TRST27 '8-5-12-7-5' is the primary founder of the clonal complex of RSSC phylotype II sequevar 1 and is widely distributed via latently infected seed tubers. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ralstonia pseudosolanacearum (Rps), which causes bacterial wilt disease of many crops, must integrate environmental signals to successfully transition from soil to its pathogenic niche in host plant xylem tissue. Mutating a gene encoding a putative sensing/signaling protein had little transcriptomic effect on Rps strain GMI1000 in culture. However, when the mutant grew in tomato, over 180 genes were differentially expressed relative to the wild type. The gene was therefore named rprR for Ralstonia plant-responsive regulator. In planta, the ∆rprR mutant dysregulated genes for diverse traits, including stress response, degradation of phenolic compounds, motility, attachment, and production of extracellular polysaccharide (EPS), which is a key bacterial wilt virulence factor. Quantifying Rps EPS by ELISA found increased levels in stems of plants infected with ∆rprR as compared to the wild type. Functional assays showed that ∆rprR is defective in attachment to tomato roots, colonization of tomato stems, and bacterial wilt virulence. In a rich medium, ∆rprR formed biofilm normally, but the mutant formed less biofilm in tomato stem homogenate and in tomato xylem sap under flow. This phenotype correlates with the mutant's altered expression of EPS biosynthetic genes and aberrant extracellular matrix. When grown in tomato stem homogenate, ∆rprR produced 57% more of the bacterial signal cyclic di-GMP (c-di-GMP) than the wild type. This is consistent with the presence, in RprR, of predicted c-di-GMP-modulating domains. Together, these findings reveal that RprR, which is highly conserved across plant pathogenic Ralstonia, modulates several bacterial wilt virulence traits in response to the plant host.IMPORTANCEMembers of the Ralstonia solanacearum species complex (RSSC) cause bacterial wilt, a globally destructive disease of market and subsistence crops. Like other plant-associated microbes, bacteria in the RSSC must integrate a complex array of biotic and abiotic signals to successfully infect plant hosts. All RSSC genomes encode an unusual protein, termed RprR, that contains multiple sensing and signaling domains, including two putative modulators of the secondary messenger c-di-GMP. Deleting RprR in Ralstonia pseudosolanacearum affected many virulence properties, including production of biofilm and exopolysaccharide, and increased intracellular c-di-GMP levels, all in a strictly plant-dependent fashion. While c-di-GMP has been investigated in other plant pathogenic bacteria, this is the first report of its role in the RSSC. Most importantly, rprR was required for Ralstonia to effectively colonize plants and cause wilt disease. Thus, RprR is a plant-responsive sensor-regulator that controls pathogen adaptation to the host environment and virulence.

Bacterial wilt (BW) is caused by Ralstonia solanacearum species complex (RSSC) and can lead to severe losses in a wide range of crops, including many traditional African vegetables (TAV). Given the critical role of TAV in African food security, investigations of BW incidence, distribution, and effective breeding strategies are needed to support public and private TAV breeding programs. In this review, we address key questions related to the diversity of BW pathogens, susceptible TAV hosts, distribution, incidence, breeding strategies, sources of resistance, and gaps in the development of resistant TAV varieties in Africa. We also discuss the potential of multiomics integration to enhance our understanding of the host plant defense system against BW in Solanaceae crops. We curated BW strain databases obtained from several online platforms, representing a total of 948 BW strains. Using a refined database, we highlighted the diversity of RSSC and TAV crops affected by RSSC in different regions of Africa. Out of 29 species documented to be affected by BW in Africa, ten are TAV, including widely consumed TAV such as Amaranths and nightshades. In addition, phylotypes I and III are reported to affect TAV, and the incidence can reach up to 72.4% in farmers’ fields. An overview of the first reports revealed that the disease has become a serious threat to TAV in the past decade. Finally, this review proposes a schematic map of possible avenues for successful breeding of BW-resistant TAV using Gboma eggplant as a case study.