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Members of the Ralstonia solanacearum species complex (RSSC) cause wilt in a wide range of hosts and are among the most destructive pathogens in the world. Despite the importance of bacterial wilt in Musaceae and Solanaceae for the state of Alagoas in Brazil, there are still no studies that determined the distribution of species and variability of RSSC in this region. To fill this gap in knowledge, the distribution, abundance, sequence variant (sequevar) identity, and genetic diversity of R. solanacearum (phylotype II) and R. pseudosolanacearum (phylotype I) were evaluated using 102 bacterial isolates. These isolates were recovered from symptomatic tissues of banana, eggplant, pepper and tomato in the agreste and eastern mesoregions of the state of Alagoas, Brazil. Repetitive sequence-based PCR (rep-PCR) genotyping revealed 11 clonal lineages among the 102 isolates tested, of which 22 isolates were selected for partial sequencing of the endoglucanase (egl) gene. In the agreste mesoregion, only R. solanacearum sequevar IIA-6/35 was detected. The expected 220-bp amplification product that is specific for sequevar IIA-6 via Moko multiplex PCR (Mmx-PCR) was absent, despite the banana host was expressing vascular discoloration that is characteristic of banana Moko disease. R. pseudosolanacearum (sequevars I-17 and I-18) was more abundant than R. solanacearum (sequevars IIA-36, IIA-41 and IIA-53) in the eastern mesoregion, with percentages of 55.7% and 44.3%, respectively. These results provide new insights into RSSC sequevar and genetic diversity, species abundance and distribution in one of the main Musaceae and Solanaceae producing regions in northeast Brazil.
Vol.:(0123456789) Keywords Bacterial wilt · Moko · Ralstonia solanacearum species complex · Diversity · Egl · mutS Bacterial wilts caused by members of the Ralstonia solanacearum species complex (RSSC) have a worldwide distribution, being often considered one of the most destructive diseases in Solanaceae and Musaceae. These pathogens have a wide host range and can infect approximately 450 plant species of 54 botanical families, including monocots and dicots (Wicker et al. 2007). Bacterial wilts were described for the first time in the USA by Erwin F. Smith in 1896, in potato (Solanum tuberosum), tomato (Solanum lycopersicum) and eggplant (Solanum melongena) (Hayward 1994). Unofficial reports of bacterial wilts in Brazil date back to 1922 infecting tobacco (Nicotiana tabacum) and potato in Rio Grande do Sul state; however, the first official report was only in 1976 in Para state (Takatsu and Lopes 1997; Tokeshi and Duarte 1976). In Alagoas state, northeastern Brazil, R. solanacearum was reported for the first time in 1987, causing Moko on banana (Musa spp.) in the municipality of Igreja Nova (Andrade et al. 2009). In 2000 a new outbreak was reported at the Yolanda de Melo de Oliveira and Lilia C. Carvalhais contributed equally. * Lilia C. Carvalhais l.carvalhais@uq.edu.au * Adriano Márcio Freire Silva adrianomfsilva@yahoo.com.br 1 Campus de Engenharia e Ciências Agrárias (CECA), Universidade Federal de Alagoas (UFAL), Maceió, Alagoas, Brazil 2 Centre for Horticultural Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia 3 Biotechnology Study Program, Faculty of Science and Mathematics, Diponegoro University, Central Java 50275, Indonesia 4 Departamento de Biologia e Pós-Graduação em Fitopatologia, Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil same locality and other outbreaks have been confirmed since then, although an eradication program has been implemented (Andrade et al. 2009). Traditionally, R. solanacearum has been divided into five races and six biovars according to the host range and ability to utilise different carbon substrates, respectively (Buddenhagen et al. 1962; Hayward 1964). Nevertheless, these terms have become obsolete as they do not reflect the genetic diversity of the isolates within this species complex (Pinheiro et al. 2011). In 2005 Fegan and Prior proposed a new classification system based on the phylogenetic analysis of the intergenic spacer region of the rDNA (ITS), the gene encoding the regulatory protein HrpB (hrpB), an endoglucanase encoding gene (egl), and the gene encoding the methyl-directed DNA mismatch repair protein (mutS). These analyses revealed clustering of isolates into four major groups, known as phylotypes. Isolates from Asia clustered within phylotype I, isolates from the Americas clustered within phylotype II, isolates from Africa and from islands of the Indian Ocean clustered within phylotype III, and isolates from Indonesia clustered within phylotype IV (Fegan and Prior 2005). Within each phylotype, sequence variants or sequevars were proposed based on partial sequences of the egl gene (Fegan and Prior 2005). Sixty-three sequevars within the RSSC have been described so far (Albuquerque et al. 2020). Multilocus sequence analysis (MLSA) data have further supported the clustering of the RSSC in eight clades, being phylotypes I and III composed by one clade each, phytotype II comprising four clades, and phylotype IV two clades (Wicker et al. 2012). Safni et al. (2014) proposed the reclassification of this complex in three different species, being Ralstonia pseudosolanacearum composed by isolates belonging to phylotypes I and III, Ralstonia solanacearum by phylotype II and Ralstonia syzigii by phylotype IV. R. syzigii was further subdivided in three subspecies, namely R. syzigii subsp. indonesiensis, R. syzigii subsp. syzigii and R. syzigii subsp. celebesensis. The taxonomic revision of RSSC with three independent species was supported by Prior et al. (2016) through genomic and proteomic analyses of 29 and 73 isolates, respectively. Brazil is within the centre of diversity of R. solanacearum, where the genetic variability of these bacteria is high (Santiago et al. 2017). Nevertheless, knowledge on the diversity of this pathogen especially within Brazil is still in its infancy and is often limited to some regions and specific hosts, especially banana and potato. The high genetic and phenotypic variability within the RSSC is a limiting factor for the screening of plant varieties resistant to bacterial wilts in Brazil and worldwide. A better understanding of the population diversity of RSSC is of paramount importance for the development of strategies based on germplasm resistance and application of integrated control measures (Santiago et al. 2017). This study sought to characterise the diversity of the RSSC in the mesoregions of agreste and eastern of Alagoas state, Brazil, which are important areas for Solanaceae and Musaceae production. It was hypothesised that Ralstonia species occur at different relative abundances in distinct regions within the same state. The R. pseudosolanacearum species denomination was used to refer to phylotype I isolates, and R. solanacearum for phylotype II isolates, following the current taxonomy for the RSSC proposed by Safni et al. (2014). Field survey, sample collection, bacterial isolations, and DNA extraction: a survey was conducted from January 2018 to September 2019 to evaluate the distribution of bacterial wilts in tomato, eggplant, pepper (Capsicum frutescens) and banana plants in the mesoregions of agreste (municipalities of Arapiraca, Taquarana and Limoeiro de Anadia) and eastern Alagoas state (municipalities of Capela, Igreja Nova, Maceió, Murici e Teotonio Vilela), Brazil (Fig. 1). Bacterial isolations were carried out from symptomatic plant material on Kelman’s TZC medium (Kelman 1954). Pure bacterial cultures were obtained from single colonies and preserved in sterile distilled water (DW) at room temperature. Genomic DNA was extracted from pure bacterial cultures grown on TZC at 30 °C for 48 h as described in Ausubel et al. (2003). DNA integrity was verified by gel electrophoresis on a 1% agarose gel and DNA quantification was conducted via spectrometry at 260 nm wavelength (Biowave). DNA samples were stored at −20 °C. Identification of RSSC phylotypes and species: phylotypes and species were identified via phylotype-multiplex PCR (Pmx-PCR). This assay includes the primer set 759/760 that amplifies a 280-bp fragment specifically in isolates of the RSSC (Opina et al. 1997). This set is added in combination with Nmult primers that target the intergenic spacer region of the ribosomal DNA and amplify 144-pb, 372-pb, 91-pb and 213-pb products of phylotypes I, II, III and IV, respectively (Fegan and Prior 2005). Reactions had a total volume of 25 µL and were composed of a mixture of all primers (0.2 µM each), buffer (1X) MgCl2 (2 mM), DMSO (5%), Taq DNA polymerase (1U, Invitrogen®) and DNA (100 ng). Amplification cycles included the following steps: 96 °C for 10 min, 30 cycles of 94 °C for 30 s, 59 °C for 90 s and 72 °C for 90 s, and a final extension at 72 °C for 20 min. Amplification products were stained with SYBR Gold (Invitrogen®) and run on a 1.5% agarose gel (Tris–acetate-EDTA, TAE 0.5 X). Gel visualizations were carried out using the El Logic 200 Imaging documentation system. Genotyping of RSSC isolates: repetitive sequencebased PCR (rep-PCR) was used to conduct genetic profiling of isolates with REP primers (REPIR-I 5’IIICGICGICATCIGGC 3’ and REP2I 5’ ICGICTT ATG IGGC CTA C 3’). This allowed the determination of clonal lines and the selection of 22 isolates for egl sequencing. The reactions had a final volume of 25 µL and was composed of 2 μM of each primer, 0.1 mM dNTP, reaction buffer 1X, 1.5 mM MgCl2, 1U of Platinum Taq DNA polymerase (Invitrogen®), and 50 ng DNA. Amplification conditions included an initial denaturation of 95 °C for 7 min, followed by 30 cycles of 94 °C for 1 min, 44 °C for 1 min and 65 °C for 8 min, and final extension at 65 °C for 15 min. Amplification products were stained with SYBR Gold (Invitrogen®), run on a 1.5% agarose gel (Tris–acetate-EDTA, TAE, 0.5X) and visualised using the El Logic 200 Imaging documentation system. Amplification and partial sequencing of the genes egl and mutS: amplification products of the egl gene were generated with primers Endo-F (5′-ATG CAT GCC GCT GGT CGC CGC-3′) and Endo-R (5′-GCG TTG CCC GGC ACG AAC ACC-3′) (Poussier et al. 2000) and of those of the mutS gene with primers mutSRsF1570 (5′-ACA GCG CCT TGA GCC GTA CA-3′) and mutSRsR1926 (5′GCT GAT CAC CGG CCC GAA CAT-3′) (Toukam et al. 2009). For both genes, 25 μL reactions were prepared by adding Amplitaq Gold Master Mix (1X) (Thermo Scientific®), 0.4 μM of each primer, 100 ng of DNA (Albuquerque et al. 2014). Cycling conditions for the partial egl amplification included an initial denaturation step at 96 °C for 9 min, followed by 30 cycles of 95 °C for 1 min, 70 °C for 1 min and 72 °C for 2 min and a final extension step at 72 °C for 10 min (Fegan and Prior 2006). Cycling conditions for the mutS amplification included an initial denaturation step at 96 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 66 °C for 1 min and 72 °C for 90 s, with a final extension step of 72 °C for 5 min (Toukam et al. 2009). Amplification products were stained with SYBR Gold (Invitrogen®) and run on a 1.5% agarose gel (TAE 0.5 X). PCR products were purified with a PCR purification kit (Norgen Biotek®) and Sanger sequenced by Macrogen (Seoul, South Korea). Phylogenetic analysis: analysis of chromatograms generated for egl and mutS genes by bidirectional Sanger sequencing of the amplification products and contig assembly of forward and reverse reads were carried out with Staden Package software (version 2.0; Medical Research Council, Cambridge, England) (Staden et al. 1998). Sequences were analysed using the algorithm ClustalW in MEGA software (version 5.0; MEGA, Tempe, Arizona). Phylogenetic trees were inferred using Maximum Likelihood (ML) using the software RaxML (https:// www. phylo. org/ porta l2/), with the model GTR + G including an estimate of invariable sites for all analyses. Phylogeny was inferred using the GAMMA AUTO model, which allows RaxML to automatically determine the substitution model of nucleotides with the best score (Stamatakis 2014). Phylogenetic trees were generated with the portal Cipres visualised using Figtree software (http:// tree. bio. ed. ac. uk/ softw are/ figtr ee/). From the egl sequences, a haplotype network was built to select the isolates whose mutS amplicons were sequenced. Nucleotide divergence was calculated for egl sequences using P-DISTANCE in MEGA. Ralstonia isolates were considered as belonging to different sequevars when nucleotide divergence values of partial egl sequeces were higher than 1%. Reference sequences for egl and mutS genes that were representative of sequevars were included in the phylogenetic analyses. Moko-multiplex PCR (Mmx-PCR) and pathogenicity in banana: based on the results of the phylogenetic tree generated from the egl gene sequences and the calculation of nucleotide divergence, it was considered necessary to determine whether isolates of sequevar IIA-6/35 were potentially pathogenic to Musa spp. To this end, the Musa multiplex PCR assay using only the primer sets 759/760 and SI28F/ SI28R was performed. Reactions had a total volume of 25 µL and were composed of a mixture of all primers (0,2 µM each), buffer (1X), MgCl2 (2 mM), DMSO (5%), Taq DNA polymerase (1U, Invitrogen®) and DNA (100 ng). Amplification cycles included the following steps: 96 °C for 10 min, 30 cycles of 92 °C for 15 s, 59 °C for 15 s and 72 °C for 30 s, and a final extension at 72 °C for 10 min. Isolate BD1109, belonging to sequevar IIA-6, a Moko ecotype strain, was used as a positive control. Amplification products were stained with SYBR Gold (Invitrogen®), run on a 1.5% agarose gel (TAE 0.5 X) and visualized using the El Logic 200 Imaging documentation system. To verify the pathogenicity of tomato isolates of sequevar IIA-6/35 in banana, seedlings of the ‘Prata ana’ (AAB) variety were propagated in vitro, acclimated and were transplanted to 400 mL polyethylene pots with Basaplant® potting mix in a glasshouse (temperatureMin/Max: 23/46.4 °C; relative humidityMin/Max: 23.5/82.8%). Five weeks after transplanting, four plants were inoculated with a bacterial suspension of R. solanacearum sequevar IIA-6/35 diluted in water to the concentration of 5 × 109 CFU.mL−1 using the pseudostem injection method (Albuquerque et al. 2014). Control treatments composed of four plants were injected with sterile distilled water. A total of 102 bacteria were isolated, 27 from symptomatic banana, three from eggplant, 31 from pepper and 41 from tomato plants. Approximately 76.7% of isolates affiliated to the RSSC came from the mesoregion of the eastern Alagoas state, while 23.3% came from the Agreste mesoregion (Supplementary Table 1). In the municipalities Arapicara and Limoeiro de Anadia, no bacterial wilt was observed. The 280 bp-product that is indicative of RSSC was amplified for all isolates. Approximately 44.7% of the isolates were identified as phylotype I, which showed an amplification product of 144-bp with Nmult primers, while 57.3% of isolates were identified as phylotype II by the presence of a 372-bp amplicon, which is indicative of R. solanacearum identity (Supplementary Fig. S1). R. pseudosolanacearum was present at higher abundance (55.7%) than R. solanacearum (44.3%) in the mesoregion of the eastern Alagoas state, while R. solanacearum was the only species detected in the Agreste region (Fig. 3A). This is the first record of R. pseudosolanacearum phylotype I in Alagoas state, Brazil. This bacterial species probably spread from Pernambuco, a bordering state located to the north of Alagoas (Albuquerque et al. 2020). The introduction pathway of R. pseudosolanacearum phylotype I to Brazil is unclear (Garcia et al. 2013). Asia is the centre of origin this species and a significant immigration of Japanese growers who settled in several states occurred throughout Brazil, including in Pernambuco state (Lu 2022). Nonetheless, true seed transmission is not considered important in the dispersion of species within the RSSC, despite its reports in tomato, eggplant, and peanut (Kelman et al. 1994; Momol et al. 2008). As expected, populations within the RSSC were diverse, including sequevars of R. solanacearum and R. pseudosolanacearum, with the former being slightly more abundant than the latter when considering both east and agreste mesoregions. This prevalence probably derives from the origin of R. solanacearum, which is native to Brazil (Wicker et al. 2012). Furthermore, the introduction of R. pseudosolanacearum in northern Brazil is quite recent, consequently this species may not have been dispersed and properly established in all producing regions in northeastern Brazil (Garcia et al. 2013). These findings are in line with previous reports that showed prevalence of R. solanacearum in northern Brazil, which corresponded to 67.8% of all studied isolates (Coelho Netto et al. 2003). Although R. pseudosolanacearum has been detected in several states in Brazil, most isolates sampled in surveys were identified as R. solanacearum, and these abundances are similar to those reported for other countries in Latin America (Santiago et al. 2017). The presence of different Ralstonia species in distinct countries are associated with various transmission modes, which includes infected plant material dispersed through human activities, contaminated soil and water, and insect dissemination (Sequeira 1958; Kelman 1965; Wenneker et al. 1999; Coelho Netto et al. 2003; Sanchez Perez et al. 2008; Ramsubhag et al. 2012). Within RSSC, phylotype II is composed of two groups, IIA and IIB. In the present study only R. solanacearum isolates of phylotype IIA were detected, and none of phylotype IIB (Fig 2). Phylotype IIA is highly diverse, recombinogenic and vast, while IIB is mostly clonal. Both subgroups originated in South America but have spread to other regions of the world, probably through infected planting material of banana, ornamental plants, and potato tubers (Buddenhagen and Kelman 1964; Milling et al. 2009). The construction of a haplotype network based on egl sequences led to the definition of seven haplotypes, three of which were classified within R. pseudosolanacearum (haplotypes 3, 4 and 5) and four within R. solanacearum (haplotypes 1, 2, 6 and 7). To better support the sequevar identification, two isolates of each haplotype were chosen for the mutS gene sequencing. The phylogenetic reconstruction of RSSC isolates and sequevar identification was based on partial sequences of the egl gene, using the reference sequences deposited on Genbank (https:// www. ncbi. nlm. nih. gov/ nucco re/). The egl and mutS trees were built based on partial sequences of 22 and 14 sequenced isolates, respectively, and compared with 71 egl reference sequences and 42 mutS sequences (Supplementary Table 2). The egl and mutS-based trees presented genealogical concordance and phylogenetic relationships that were sufficiently similar to allow sequevar identification, although some differences were observed (Fig 2 and Supplementary Fig. S2). The egl phylogenetic analysis revealed that in the eastern mesoregion of Alagoas state, a higher sequevar diversity and wider host range were observed, as sequevars I-17 and I-18 were detected in tomato and pepper, sequevar IIA36 in tomato, sequevar IIA-41 in tomato and eggplant, and sequevar IIA-53 in banana (Fig. 3B). In different states in the north and northeast of Brazil, sequevars I-17 e I-18 have been reported in a wide host range within Solanaceae (Santiago et al. 2017; Albuquerque et al. 2016). Sequevars IIA-41 and IIA-36 have been reported in the north, northeast, and central-west regions of Brazil, and have been associated with different hosts, mainly tomato and eucalypt (Eucalyptus spp.) (Fonseca et al. 2014; Santiago et al. 2017). The occurrence of these sequevars in Alagoas state represents a threat to the growing eucalypt industry. Losses caused by bacterial wilts in eucalypt nurseries can reach approx. US2.7 million per year (Alfenas et al. 2006). The only sequevar that was found in the agreste region was IIA-6/35 (Fig. 3B). The isolates of this sequevar presented a nucleotide divergence of 0.2% relative to the reference isolate CFBP 2972 of sequevar 35 (GenBank accession number AF295264), and a null nucleotide divergence when compared with the reference isolate IBSBF 2661of sequevar IIA-6 (Genbank accession number KF875432). This sequevar was solely isolated from tomato but caused vascular discoloration in the banana variety ‘Prata ana’. MmxPCR revealed that IIA-6/35 isolates were not classified into sequevar IIA-6 as they have not generated the 220-bp PCR product. These results concur with those reported in Florida where tomato isolates belonged to sequevar IIA-6/5 (Hong et al. 2012). Furthermore, these isolates showed a 167-bp amplification product using the Mmx-PCR and colonised banana plants systemically, having a deleterious effect on their development. Similarly, a previous study classified as sequevar IIA-35 isolates that have been previously classified as sequevar IIA-6 using the Mmx-PCR (Deberdt et al. 2014). The primer set SI28 targets a conserved protein in sequevar IIA-6 with unknown function that contain three phage derived domains and is localised in a region that has genomic plasticity. Although the phylogenetic placement of the previously mentioned sequevars within phylotype IIA is consistent, the ecotype classification differs: sequevar IIA-6 isolates cause Moko in banana plants, while sequevar IIA-35 is only pathogenic to Solanaceae. Incorrect diagnostics can lead to serious consequences and cause erroneous reports of Moko. For this reason, it is recommended that the diagnostics of sequevar IIA-6 isolates is confirmed with egl and mutS sequencing, followed by phylogenetic analyses (Deberdt et al. 2014). All Moko-associated isolates were identified as IIA-53. Based on these results and those of Albuquerque et al. (2014), it is possible to conclude that only isolates of R. solanacearum belonging to sequevar IIA-53 cause Sergipe facies on banana in a limited area in the northeast of Brazil. This syndrome is associated with atypical inflorescence symptoms with the infection occurring via male flowers, similarly to Bugtok in the Philippines. In this study, it was confirmed that sequevar IIA-53 isolates were only found in the low Sao Francisco River basin region of northeastern Brazil, hence being endemic to this area. The reported findings support the proposed hypothesis that Ralstonia species occur at different relative abundances in distinct regions within the Alagoas state, where the bacterial diversity within the RSSC was evaluated for the first time. These results have paramount importance to the management of bacterial wilts and to breeding programs aiming to select resistant plant varieties. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s40858- 025- 00749-6. Acknowledgements We thank the National Council for Scientific and Technological Development (CNPq) for awarding Yolanda de Melo de Oliveira master's scholarship. We also extend our thanks to the Ministerio de Agricultura e Pecuária (MAPA) and Agência de Defesa e Inspeção Agropecuária de Alagoas for their invaluable help in collecting samples in the field. Author contributions Conceptualization: YMO, IPA, AMFS; Performed research: YMO, SLS, WOV; Formal analysis: YMO, LCC, SLS; Writing, review, and editing: all authors; Funding acquisition: GSAL, AMFS, IPA, EBS, LCC; Supervision: AMFS, IPA, GSAL, EBS. Funding Open Access funding enabled and organized by CAUL and its Member Institutions Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, process number 88882452247/2019–01. Data availability The data supporting this study’s findings are available from the corresponding author upon reasonable request.
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Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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