Publications

4719

Date palm is a staple food crop and crucial export agricultural commodity in Arab countries. Date palm diseases caused by phytoplasmas are devastating diseases that severely constrain the sustainable development of date palm cultivation and industry. The phytoplasmas associated with date palm diseases mainly consist of three taxa: ‘Candidatus Phytoplasma asteris’ of the 16SrI group, ‘Ca. Phytoplasma citri’ of the 16SrII group, and ‘Ca. Phytoplasma fraxini’ of the 16SrVII group. Based on the 16S rRNA gene sequences of the three groups of phytoplasmas, two sets of primer pairs (Da-1 and Da-5) enabling simultaneous visual detection of date palm phytoplasmas were designed and screened. Universal simultaneous visual detection methods were successfully established for different groups and candidate species of date palm pathogenic phytoplasmas. The whole detection procedure of this optimized method can be accomplished via isothermal amplification at 64 °C within 30–50 min, and the detection results can be directly and intuitively judged by the color variation of the reaction system. Sensitivity verification showed that for the three groups of phytoplasmas, the minimum detection limit of the Da-1 primer set for simultaneous visual detection ranged from 1 fg/μL to 10 fg/μL, while that of the Da-5 primer set ranged from 1 fg/μL to 100 pg/μL. The established LAMP assay demonstrated high sensitivity and specificity using synthetic DNA templates. This method provides a promising laboratory tool for the simultaneous visual detection of three major date palm phytoplasma groups. Further validation on naturally infected field samples is required before practical application in field diagnosis, epidemic monitoring, and port quarantine.

Abstract The demand for D-amino acids is increasing as their unique physiological role continues to be elucidated. meso-Diaminopimelate dehydrogenase catalyzes the reversible NADP+-dependent oxidative deamination of meso-diaminopimelate to produce L-2-amino-6-oxopimelate. Moreover, a few enzymes show a broad range of reductive amination activity toward 2-oxo acids, synthesizing D-amino acids. Here, we report the identification and characterization of a novel NADP+-dependent meso-diaminopimelate dehydrogenase from Candidatus Syntrophocurvum alkaliphilum (CSaDAPDH). The purified enzyme exhibited oxidative deamination activity toward meso-diaminopimelate, as well as reductive amination activity toward several 2-oxo acids to produce the corresponding D-amino acids. The optimum pH and temperature for oxidative deamination of meso-diaminopimelate were 9.0 and 45°C, respectively. The optimum pH for reductive amination of pyruvate was 6.5, which was the lowest optimum pH among known enzymes, and thus, may lead to the development of new enzymatic methods for D-amino acid production. CSaDAPDH retained more than 60% of its activity after incubation for 30 min at 50°C (pH 7.0) or at pHs ranging from 5.5 to 7.0 (50°C). Moreover, using known enzymes as comparisons, the coenzyme and substrate recognition mechanisms of CSaDAPDH were elucidated based on a multiple sequence alignment and the homology model.

Bambusa multiplex, one of the most acclimatized clumping-bamboo species, has been broadly cultivated in China and has significant economical, ecological and ornamental importance (Yuan et al. 2009). In November 2025, more than 30% of B. multiplex plants exhibiting typical witches’ broom, internode shortening, leaf proliferation and clustering of leaves were observed in a horticultural landscaping field in Zhangjiang city, Guangdong Province, China. To confirm the presence of a phytoplasma, total DNA was extracted from fresh leaves of ten symptomatic and four asymptomatic plants using the CTAB method. Conventional nested PCR was performed using primer pairs P1/P7 followed by R16F2n/R16R2 (Gundersen and Lee, 1996), and expected fragment amplified only from the symptomatic plants. The PCR products were sequenced, and deposited in GenBank under the accession number PX998769. BLAST analysis revealed that the sequence shared 99.60% identity with that of the SPLL subgroup reference strain ‘Candidatus Phytoplasma australasiaticum subsp. Ipomoeae’ o7C (GenBank accession no. GCA_024425275.1, 1,244/1,249 bp) (Rodrigues Jardim et al. 2023). Further virtual RFLP pattern analysis of the 16S rRNA sequence by iPhyClassifier (Zhao et al. 2009) verified that the phytoplasma strain, designated as BMW-ZJ, was a member of the 16SrII-A subgroup with a 1.00 similarity coefficient to the reference phytoplasma strain (GenBank accession no. L33765). Moreover, the secY gene was amplified using primer pairs secYwbF1/secYwbR1 followed by secYwbF2/secYwbR2 (Wang et al. 2025), and the sequence(GenBank accession no. PZ135411) shared 100% identity to several ‘Ca. P. australasiaticum’ strains (NTU2011, BAWM-TWN and BAWM-THA-CLP). Phylogenetic analysis based on the 16S rRNA and secY genes conducted by MEGA 12 (Kumar et al. 2024), confirmed that BMW-ZJ was related to ‘Ca. P. australasiaticum’(Bertaccini et al. 2022; Rodrigues Jardim et al. 2023). Earlier, 16SrII phytoplasma associated with bamboo species Dendrocalamus giganteus (Che et al. 2024), and 16SrI phytoplasma associated with Sasa fortunei (Zhang et al. 2009) were reported in Hainan and Shaanxi Province, China, respectively. To our knowledge, this is the first report a ‘Ca. P. australasiaticum’ -related strain associated with Bamboo Witches’ Broom Disease (BWB) in Guangdong, China. The BWB caused by Ca. Phytoplasma (16SrI, 16SrII, 16SrVIII and 16SrXIV group), has been confirmed in several bamboo species in Southeast Asian countries such as India (Yadav et al. 2016; Ravi et al. 2022), Philippines (Dolores et al. 2023), Indonesia (Prasetya et al. 2020) and China (Zhang et al. 2009; Che et al. 2024). This study highlight the need to identify insect vectors and alternative hosts, which will provide theoretical guide to the control of Bamboo Witches’ Broom Disease.

Abstract The critically endangered Caribbean staghorn coral Acropora cervicornis hosts microbiomes frequently dominated by the putatively parasitic intracellular bacterium Candidatus Aquirickettsia rohweri, which is associated with reduced coral growth and heightened disease susceptibility. Whether this dominance can be disrupted through antibiotic treatment and a sequential disturbance of thermal stress, remains unknown. In this study, we exposed disease-susceptible A. cervicornis fragments to broad-spectrum antibiotics, sub-bleaching thermal stress, or the combination of an antibiotic pre-treatment followed by thermal stress, and tracked changes in microbiome composition and diversity across all experimental phases using 16S rRNA amplicon sequencing and quantitative PCR (qPCR). We find that while the minor microbial fraction exhibits sustained compositional shifts in response to treatment, Ca. Aquirickettsia rohweri is resilient to antibiotic and thermal perturbation and may in fact increase in abundance following antibiotic exposure, suggesting that its dominance is actively maintained and not readily displaced by current disease mitigation strategies.These results indicate that antibiotic intervention is unlikely to be a viable strategy for disrupting Ca. A. rohweri dominance in disease-susceptible A. cervicornis , underscoring the urgency of understanding its transmission routes to inform microbiome rescue efforts.

Rapid and accurate diagnosis of ‘Candidatus Phytoplasma’ is critical for disease management but remains challenging due to the pathogen's unculturable nature, low titers, uneven distribution and genetic complexity. While current official guidelines provide a highly adaptable taxonomic foundation for species assignment, translating these broad recommendations into standardized routine diagnostics remains difficult. This study implements the established species-assignment guidelines by developing a structured, proof-of-concept diagnostic framework utilizing a Multi-Locus Sequence Analysis (MLSA) workflow coupled with Oxford Nanopore Technologies (ONT) sequencing. The developed workflow targets four barcodes — 16S, secA, rplV and tufB — and replaces fixed demarcation criteria with a best-match principle for non-16S barcodes. Furthermore, the study offers guidance for barcode selection and challenges the use of 16S rRNA sequencing as a sole screening assay. Moreover, a read-based quantification system incorporating control thresholds was implemented to provide an objective and reliable assessment of the results. The workflow was validated using spiked dilution series and naturally infected samples, demonstrating that the ONT workflow achieves sensitivity and accuracy comparable to, or exceeding, that of traditional Sanger sequencing. Although PCR multiplexing was found to reduce sensitivity, limiting the immediate throughput advantages over Sanger sequencing, the proposed framework offers significant value through flexible species assignment and stringent contamination controls. Consequently, this method serves as a robust, high-resolution tool for resolving complex phytoplasma infections.

Abstract Anammox bacteria require large amounts of iron for hydrazine synthase (HZS) and hydrazine oxidoreductase (HZO). By analyzing 8 anammox genomes across four genera, we found that only Candidatus Brocadia sinica harbors a compact genomic island (<10 kb) where hzs co-localizes with iron uptake ( TonB , FeoAB ) and Fe-S cluster assembly ( NifU / NifS ) genes. All other species show dispersed architectures (>100 kb separation). In the dispersed species Ca. Kuenenia stuttgartiensis , transcriptomic data revealed a 300- to 1500-fold excess of hzs over iron genes, indicating severe expression uncoupling. Thus, physical co-localization of iron support genes with anammox core enzymes is rare but exists in one Brocadia lineage, potentially enabling better co-regulation. These findings provide a genomic basis for predicting iron responsiveness across anammox species in engineered systems.