First Report of Dickeya dadantii Causing Soft Rot in Cariru in Brazil.

Cariru (Talinum fruticosum) is a non-conventional food plant and a source of vitamins A, B2, B5, and C, calcium, phosphorus, and iron. It is an important crop for producers of the Amazon region and it is used as a leafy green vegetable, similar to spinach, as well as for medicinal purposes. It is cultivated by producers in the states of Pará and Amazonas (Amorim et al. 2014). In February 2020, in gardens of approximately 1.0 and 0.18 ha in the municipality of Ananindeua (01° 34' 12" S - 48° 38' 11" W), Pará, Brazil, we observed approximately 10% of the plants exhibited soft rot symptoms, bacterial oozing, and stem pith disintegration. From cariru infected leaves, the bacteria were selectively isolated using healthy pepper fruit, as described by Moraes et al. (2017). Two strain (UFRADD17 and UFRADD18) were obtained and both showed white, small, "broken glass" colonies on CPG (peptone-casamino acid-glucose) medium when observed in a stereoscope under oblique lighting. Pathogenicity tests were performed on 20-day-old cariru seedlings by depositing 10 ul of the bacterial suspension (106 CFU/ml) onto a wound made on the leaf blade with an entomological pin. After inoculation, seedlings were incubated in a greenhouse for 48 h at 30 ± 2°C and 90 ± 2% RH. Plants treated similarly with sterile distilled water were used as a negative control. Symptoms of soft rot were observed between 12 and 24 h after inoculation. Seedlings used as negative control remained symptomless. Forty-eight hours after inoculation, we reisolated the pathogen and performed rep-PCR (REP, ERIC, and BOX-PCR) analyses (Gama et al. 2018) with the strains used for inoculation and those reisolated from the pathogenicity test to confirm the identity of the strains and to fulfill Koch's postulates. The two re-isolated strains showed the same REP, ERIC, and BOX-PCR profiles as the strains used for inoculation. In addition, there were no differences between the rep-PCR profiles of the isolates UFRADD17 and UFRADD18. These isolates were Gram-negative, grew at 37°C, and were positive for maceration in pepper fruit and potato tubers. Molecular identification of the isolate UFRADD18 was performed from sequencing fragments from the 16S rDNA region and dnaX, fusA, gyrA, and mdH housekeeping genes as previously described (Van der Wolf et al. 2014; Ma et al. 2007). Sequences were deposited in Genbank under accessions OP142347, OP191704, OP191705, OP191706, and OP191707. Blastn analysis showed 96.15% identity with the 16S rDNA region of accession KY231142.1 (400/416 bp), 99.5% identity with dnaX of accession KC844490.1 (396/398 bp), 99.86% identity with fusA of accession CP023467.1 (714/715 bp), 99.74% identity with gyrA of accession KC844598 (387/388 bp), and 99.10% identity with mdH of accession GQ891979.1 (563/560 bp) from the type strain of Dickeya dadantii. A phylogenetic analysis performed by Bayesian inference with the dnaX, fusA, gyrA, and mdH genes grouped the isolate UFRADD18 along with NCPPB898T, with a 1.00 posterior probability. To our knowledge, this is the first report of D. dadantii causing soft rot in cariru in the Brazilian territory. In addition, this report increases understanding of the host range of this bacterium, which is important for adopting management strategies based on the control of alternative hosts.

Characterization and Association of Rips Repertoire to Host Range of Novel Ralstonia solanacearum Strains by In Silico Approaches.

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.

Comparative genomics and phylogenomics of the Ralstonia solanacearum Moko ecotype and its symptomatological variants.

Banana tree bacterial wilt is caused by the Ralstonia solanacearum Moko ecotype. These strains vary in their symptom progression in banana, and are classified as typical Moko variants (phylotype IIA and IIB strains from across Central and South America), Bugtok variant (Philippines), and Sergipe facies (the states of Sergipe and Alagoas, Brazil). This study used comparative genomic and phylogenomic approaches to identify a correlation between the symptom progression of the Moko ecotypes based on the analysis of 23 available genomes. Average nucleotide identity and in silico DNA-DNA hybridization revealed a high correlation (>96% and >78%, respectively) between the genomes of Moko variants. Pan-genome analysis identified 21.3% of inheritable regions between representatives of the typical Moko and Sergipe facies variants, which could be traced to an abundance of exclusive homolog clusters. Moko ecotype genomes shared 1,951 orthologous genes, but representatives with typical symptoms did not display unique orthologues. Moreover, Bugtok disease and Sergipe facies genomes did not share any unique genes, suggesting convergent evolution to a shared symptom progression. Overall, genomic and phylogenomic analyses were insufficient to differentiate the Moko variants based on symptom progression.

Biofilm formation by strains of Burkholderia cenocepacia lineages IIIA and IIIB and B. gladioli pv. alliicola associated with onion bacterial scale rot.

The Burkholderia genus has high ecological and nutritional versatility, having species capable of causing diseases in animals, humans, and plants. During chronic infections in humans, biofilm formation is a characteristic often associated with strains from different species of this genus. However, there is still no information on the formation of biofilms by plant pathogenic strains of B. cenocepacia (Bce) lineages IIIA and IIIB and B. gladioli pv. alliicola (Bga), which are associated with onion bacterial scale rot in the semi-arid region of northeast Brazil. In this study, we performed an in vitro characterization of biofilm formation ability in different culture media by the phytopathogenic strains of Bce and Bga and investigated its relationship with swarming motility. Our results indicated the existence of an intraspecific variation in biofilm formation capacity in vitro by these bacteria and the existence of a negative correlation between swarming motility and biofilm formation for strains of Bce lineage IIIB. In addition, histopathological analyses performed using optical microscopy and scanning electron microscopy revealed the formation of biofilm in vivo by Bce strains in onion tissues.

First Report of Xanthomonas citri subsp. citri causing Citrus Canker on lime in Rio Grande do Norte, Brazil.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important citrus diseases in the world (Gottwald et al. 2002), mainly for citrus-producing countries with humid sub-tropical regions such as United States, Argentina, and Brazil, where losses may be significant (Behlau et al. 2020). In the state of Rio Grande do Norte (RN), Brazil, citrus production is expanding and shows social and economic importance for small farmers, which produced approximately 297 tons of lime in this state in 2019 (IBGE 2021). In December 2019, we observed symptoms of erumpent lesions with margins surrounded by yellow haloes on leaves and fruit of the lime (Citrus aurantifolia cv. 'Galego') (about 5% incidence) in a plantation located in the municipality of Mossoró, RN (05°12'21.1"S, 37°19'16"W). Samples were collected from the lime orchard, and five bacterial strains (CCRMXC01 to CCRMXC05) showing yellow, convex, mucoid colonies were isolated in a nutrient-yeast-dextrose-agar medium (NYDA). Pathogenicity tests were performed on sweet orange (C. sinensis cv. 'Pêra') and lime (C. latifolia cv. 'Tahiti') seedlings. Four wounds per leaf (upper side) were carried out with an entomological pin and 10 µl of a bacterial suspension (108 CFU mL-1) were deposited on each wound. The negative control consisted of leaves treated with sterile distilled water (SDW). For each citrus species, we used four replicates per strain and one leaf with four wounds per replicate. Inoculated leaves developed erumpent lesions with margins surrounded by yellow haloes six days after inoculation (DAI) in both citrus species, while leaves treated with SDW remained symptomless. Nine DAI, we reisolated the pathogen and performed rep-PCR (REP, ERIC, and BOX-PCR) analyses (Gama et al. 2018) with the strains inoculated and reisolated to confirm the identity of the strains and to fulfill Koch's postulates. The strains were stored at the Culture Collection Rosa Mariano (CCRM) of the Phytobacteriology Laboratory at the Universidade Federal Rural de Pernambuco. The five strains reisolated showed the same REP, ERIC, and BOX-PCR profiles as the strains used for inoculations. The molecular identification was performed sequencing the dnaK, fyuA, gyrB, and rpoD genes (Young et al. 2008). Each fragment was sequenced in both the forward and reverse directions. Using the BLASTn tool, we observed that sequences of the dnaK (GenBank MW218913 to MW218917), fyuA (GenBank MW218918 to MW218922), and rpoD (GenBank MW218928 to MW218932) genes of the strains CCRMXC01 to CCRMXC05 showed 100% of identity with the sequences of these genes from the type strain (ICMP 24T) and of other strains of X. citri subsp. citri (ICMP 21 and ICMP 7493), while sequences of gryB (GenBank MW218923 to MW218927) of the former strains showed 100% identity with the gyrB sequence of the strains ICMP 24T and ICMP 7493 and 99,85% identity with strain ICMP 21. This short variation in the sequence of the gyrB gene also may be observed among strains of X. citri subsp. citri available in NCBI database (https://www.ncbi.nlm.nih.gov/). The phylogenetic analysis performed using Bayesian inference and the concatenated sequence of all the type or representative strains of species and pathovars of Xanthomonas available in GenBank showed that the strains CCRMXC01 to CCRMXC05 clustered together with strain ICMP 24T with 1.0 posterior probability. To our information, this is the first report of X. citri subsp. citri causing citrus canker on lime in RN state, Brazil.

Genomic sequencing of different sequevars of Ralstonia solanacearum belonging to the Moko ecotype.

Banana vascular wilt or Moko is a disease caused by Ralstonia solanacearum. This study aimed to sequence, assemble, annotate, and compare the genomes of R. solanacearum Moko ecotypes of different sequevar strains from Brazil. Average nucleotide identity analyses demonstrated a high correlation (> 96%) between the genome sequences of strains CCRMRs277 (sequevar IIA-24), CCRMRs287 (IIB-4), CCRMRs304 (IIA-24), and CCRMRsB7 (IIB-25), which were grouped into phylotypes IIA and IIB. The number of coding sequences present in chromosomes and megaplasmids varied from 3,070 to 3,521 and 1,669 to 1,750, respectively. Pangenome analysis identified 3,378 clusters in the chromosomes, of which 2,604 were shared by all four analyzed genomes and 2,580 were single copies. In megaplasmids, 1,834 clusters were identified, of which 1,005 were shared by all four genomes and 992 were identified as single copies. Strains CCRMRsB7 and CCRMRs287 differed from the others by having unique clusters in both their chromosomes and megaplasmids, and CCRMRsB7 possessed the largest genome among all Moko ecotype strains sequenced to date. Therefore, the genomic information obtained in this study provides a theoretical basis for the identification, characterization, and phylogenetic analysis of R. solanacearum Moko ecotypes.

Genomic sequencing of two isolates of Ralstonia solanacearum causing Sergipe facies and comparative analysis with Bugtok disease isolates.

Ralstonia solanacearum is the causal agent of Moko disease in bananas, which in the state of Sergipe in northeastern Brazil causes "Sergipe facies". This disease induces atypical symptoms similar to those of Bugtok disease in the Philippines. This study was conducted to sequence, assemble, and annotate the genomes of the Sergipe facies-causing isolates SFC and IBSBF2570 (sequevar IIA-53) and compare their genomes with two representative isolates causing Bugtok disease. The genomes were sequenced and assembled, resulting in lengths of 5.58 Mb (SFC) and 5.46 Mb (IBSBF2570) in 185 and 174 contigs, respectively. The isolates of Sergipe facies and Bugtok disease showed similarities in their gene contents. We identified 5,668 information clusters, 3,752 of which were shared by all genomes (core genes). Moreover, 3,585 single-copy genes were identified. Isolates causing Bugtok disease exclusively shared 266 more information clusters than the isolates causing Sergipe facies. These results suggest that Sergipe facies and Bugtok disease isolates show high genomic similarity. However, the similarity is even greater between the Bugtok disease isolates. This may be because of their longer period of interaction compared to Sergipe facies isolates.

Taxonomic Repositioning of Xanthomonas campestris pv. viticola (Nayudu 1972) Dye 1978 as Xanthomonas citri pv. viticola (Nayudu 1972) Dye 1978 comb. nov. and Emendation of the Description of Xanthomonas citri pv. anacardii to Include Pigmented Isolates Pathogenic to Cashew Plant.

Grapevine bacterial canker, which is caused by Xanthomonas campestris pv. viticola, is one of the most important grapevine diseases in the northeastern region of Brazil. This disease causes severe damage and represents a high potential risk to the development of Brazilian viticulture. In turn, pigmented isolates pathogenic to cashew plant, making cashew fruit unfit for sale, also have been detected in Northeastern Brazil. Given that the taxonomic position of these bacteria is unclear, the multilocus sequence analysis (MLSA) technique, average nucleotide identity (ANI) values and tetranucleotide frequency correlation coefficients (TETRA) were used to analyze their phylogenetic relationship in relation to other Xanthomonas species. X. campestris pv. viticola was closely related to X. citri pv. mangiferaeindicae (repetitive-polymerase chain reaction [rep-PCR], MLSA, and ANI) and X. citri subsp. citri (MLSA and ANI). Pigmented isolates pathogenic to cashew plant were closely related to X. citri pv. anacardii (rep-PCR, MLSA, ANI, and TETRA). The results obtained in this study support the emendation of the description of X. citri pv. anacardii to include pigmented isolates of Xanthomonas pathogenic to cashew plant. In addition, the reclassification of X. campestris pv. viticola as X. citri pv. viticola comb. nov. is suggested.

Complete Genome Sequence of Xanthomonas citri pv. anacardii Strain IBSBF2579 from Brazil.

The bacterium Xanthomonas citri pv. anacardii is the agent of angular leaf spot of the cashew tree (Anacardium occidentale L.). The complete genome sequencing of the strain IBSBF2579 was done on an Illumina HiSeq 2500 platform. The de novo assembly of the X. citri pv. anacardii strain IBSBF2579 genome yielded 133 contigs, with a size of 5,329,247 bp and a G+C content of 64.03%. The prediction was performed by GeneMarkS and the automatic annotation by Rapid Annotations using Subsystems Technology (RAST), with 4,406 identified genes.