Publications

4866

Abstract Antimicrobial peptides (AMPs) are widely used in disease-resistant breeding, and their application in citrus has recently been reported. However, the molecular mechanism by which AMPs inhibit the Huanglongbing (HLB) remains unclear. This study aimed to clarify the inhibitory mechanism of Hinnavin I (an AMP) against HLB-associated pathogens. We first demonstrated that Hinnavin I exhibits antibacterial activity against several tested Gram-negative bacterial strains. Using a transgenic citrus hairy root system, the heterologous expression of Hinnavin I was found to significantly reduce Candidatus Liberibacter asiaticus (CLas) colonization. Transcriptomic and phytohormone analyses further showed that constitutive Hinnavin I expression was associated with altered defense-related gene expression and enhanced SA-related responses in citrus hairy roots. Multiple interaction assays identified the CLas outer membrane lipoprotein Omlp as a direct interaction partner of Hinnavin I both in vitro and in vivo.

Abstract Purpose The objective of this study was to characterize Bradyrhizobium strains currently recommended as inoculants for Arachis pintoi in Brazil using comparative genomic and symbiotic analyses. In addition, this study aimed to expand current knowledge on the diversity, evolution, and bacterial symbiotic traits of Arachis -associated Bradyrhizobium while providing robust taxonomic evidence to support the description of a novel Bradyrhizobium species isolated from root nodules of Arachis . Methods Phylogenetic reconstruction was performed based on the 16S rRNA gene and by phylogenomics using 116 single- copy core gene sequences. Average Nucleotide Identity (ANI) analyses were conducted to evaluate the genomic relatedness of strains BR 1432ᵀ, BR 1433 and BR 14999 to 1,301 reference Bradyrhizobium genomes, including all available type strain strains. The results were validated using digital DNA–DNA hybridization (dDDH). Additional analyses included phylogenetic reconstruction of symbiotic genes ( nodC and nifH ) and, assessment of the denitrification pathway, and phenotypic characterization. Results Phylogenetic analyses based on 16S rRNA gene sequences and whole-genome data indicated that strains BR 1432ᵀ and BR 14999 form a distinct lineage within the genus Bradyrhizobium , clearly separated from BR 1433, which clustered with Bradyrhizobium brasilense . ANI and dDDH values between BR 1432ᵀ and BR 14999 and their closest related type strains, including B. zhanjiangense LMG 29279ᵀ, were below the species delineation thresholds of 96% and 70%, respectively, supporting the proposal of a novel species. In addition, the publicly available RefSeq genomes Bradyrhizobium sp. DN5 and Rc2d were found to be closely related to BR 1432ᵀ. Phylogenetic analyses of the nodC and nifH genes revealed distinct evolutionary patterns, while genomic analyses showed that the proposed novel species harbors a complete denitrification pathway, unlike strain BR 1433. Furthermore, phenotypic characterization revealed differences in carbon source utilization and enzymatic activities. Conclusion Based on genomic, phylogenetic, and phenotypic evidence, strain BR 1433 was identified as a member of B. brasilense , whereas strains BR 1432ᵀ (= UCCC 277ᵀ = LMG 34120ᵀ = CCP577ᵀ), BR 14999, DN5 and Rc2d represent a novel species within the genus Bradyrhizobium , for which the name Bradyrhizobium boddeyi sp. nov. is proposed.

Huanglongbing (aka: citrus greening) associated with ‘Candidatus Liberibacter asiaticus’ (CLas) is one of the most impactful citrus diseases throughout the world. The aim of this project was to support the sensitive and rapid detection of CLas to prevent the continued spread and destruction of US citrus crops by this psyllid vector-borne disease. Two multicopy targets specific to CLas were selected for assay development: the five-copy nrdB (ribonucleotide reductase-RNR) gene and a four-copy locus 4cp (4CP). Published RNR RPA primers were used to develop a one-pot RPA-CRISPR assay, and RPA primers for 4CP were designed. crRNAs for both RNR- and 4CP-RPA-CRISPR/Cas assays were designed, evaluated through preliminary testing, and the best candidate primers and crRNA combinations were selected. The 4CP assay was selected and subjected to a battery of validation tests and determined fit-for-purpose. A field extraction protocol was also developed using commercial reagents, syringes and filters to bring kit sensitivity to the field. When combined with the assay, the complete workflow was able to detect CLas in infected citrus, down to a single infected leaf per subsample; the field workflow showed similar sensitivity to the laboratory-based workflow. While the extraction and assay proved useful, particularly for symptomatic leaves, its performance with asymptomatic leaves for early detection was not evaluated. The complexity of the assay in its current form hinders widespread practical applicability. In the future, revalidation of lyophilized reagents in a convenient kit can be performed for practical deployment; preliminary testing has shown success.

Huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (CLas), poses a major threat to citrus production worldwide and has recently emerged as a serious constraint to sweet orange cultivation in Bangladesh. Despite widespread disease occurrence, information on the genetic diversity of CLas populations in the country is lacking. This study investigated the genetic structure of CLas across major sweet orange–growing regions of Bangladesh using tandem repeat number (TRN) variation at the CLIBASIA_01645 genomic locus. Field surveys were conducted in 17 districts between 2022 and 2023, and 89 HLB-symptomatic leaf samples were collected from commercial orchards. PCR and qPCR detection confirmed CLas infection in 67 (75%) and 74 (83%) samples, respectively. Thirty-seven representative isolates were sequenced and analyzed for TRN polymorphism. Ten TRN genotypes (TRN2–TRN12, excluding TRN3) were identified, with TRN6 being the most prevalent. Bangladeshi CLas isolates were further grouped into three classes based on TRN: Class I (TRN ≤ 5), Class II (5 < TRN ≤ 10), and Class III (10 < TRN ≤ 15), of which Class II was the predominant. All Bangladeshi isolates harbored the CTTGTGT repeat motif and clustered closely with Indian isolates in haplotype network and phylogenetic analyses, while isolates carrying the AGACACA motif from other countries formed a distinct clade. These results reveal intraspecific diversity of CLas in Bangladesh and suggest regional structuring and shared ancestry with neighboring Indian populations. This study provides the first population-level genetic insight into CLas in Bangladesh and establishes a foundation for improved epidemiological understanding and disease management.

The phloem-colonizing bacterial pathogen Candidatus Liberibacter asiaticus (CLas) causes the devastating citrus Huanglongbing (HLB). Despite the important roles of roots, how CLas affects roots remains largely unknown. Here we dissected host responses in leaf and root at different time points after CLas inoculation. Our data showed that CLas triggers less reactive oxygen species (ROS) accumulation and phloem callose deposition, which are known to contribute to HLB symptom development in leaves, in roots than leaves. Increased chloroplastic ROS accumulation was detected in CLas-infected leaf compared to uninfected controls, which may explain the increased ROS levels in leaves than roots infected by CLas. qRT-PCR and RNA-seq analyses showed global induction of immune related pathways in both roots and leaves, whereas photosynthesis related pathways were only downregulated in leaves, but not in roots, by CLas infection. The differential expression pattern related to immunity and photosynthesis in the leaf and root tissues suggests that chloroplasts might be involved in the differences in ROS accumulation and callose deposition. Root cell death was observed in HLB symptomatic trees regardless of CLas infection status of roots. Significantly more starch accumulation was first observed in CLas positive leaves than healthy controls followed by starch depletion in CLas positive roots later, suggesting a causative relationship between them. Taken together, root cell death caused by CLas infection maybe caused by carbon starvation rather than via a similar mechanism as in leaves. Our data suggests chloroplasts play a critical role in the different pathogenicity mechanisms of CLas in leaf and root.