Publications

4467

Abstract Background Phytoplasma research encounters limitations due to the lack of availability of pure cultures of these microorganisms. In this study a culture-independent approach was employed to investigate the genome and pathogenic mechanisms of phytoplasma responsible for witches’ broom disease in Trema levigata (Yunnan province, China). The phytoplasma genome was assembled using Illumina sequencing data and the Phytoassembly pipeline based on mixed samples. Results Nested PCR analysis identified a 16Sr group I, ‘ Candidatus Phytoplasma asteris’ strain in Trema levigate showing witches’ broom disease. Comparative study between infected and healthy plants resulted in an 849.7 kb draft genome with 94.1% coverage, 27.6% GC content, encoding 1356 predicted genes, of which 587 were functionally annotated. Multilocus phylogenetic analysis showed that this phytoplasma is closely related to ‘ Ca. P. asteris’. The genome possesses complete pathways for glycolysis and pyruvate metabolism pathways and harbors specific transporters (spermidine/putrescine, lysine), indicating dependency on host metabolites. Thirty-one putative secreted proteins, including TENGU and SAP05/54-like effectors were identified along with three potential mobile units carrying replication/recombination genes. Conclusions This approach demonstrates the effectiveness of pathogen purification-free analysis for phytoplasma studies in naturally infected host species, thereby enhancing the understanding of phytoplasma-host interactions.

Huanglongbing (HLB), or citrus greening, remains one of the most destructive diseases affecting citrus production globally. Associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus ( C Las) and vectored by Diaphorina citri , HLB leads to canopy decline, fibrous root loss, and reductions in fruit yield and quality. Recently, the systemic delivery of oxytetracycline (OTC) via trunk injection was approved in Florida as a targeted therapy to reduce C Las titers and improve tree health. In parallel, Individual Protective Covers (IPCs) have been adopted to delay C Las infection in newly planted citrus trees by vector exclusion. This study investigates the combined use of IPCs and trunk injection of OTC for post-IPC therapy. ‘Valencia’ sweet orange trees grafted on US-812 and US-942 rootstocks were planted in December 2020 under HLB-endemic conditions in southwest Florida. IPCs were installed at planting and removed after 18 months. The first OTC injection was performed in May 2023, 10 months after IPC removal. A second injection was performed in May 2024. A 2 × 2 × 2 factorial experimental design evaluated the effects of infection history (early-infected and late-infected), rootstock cultivar (US-812 and US-942), and injection treatment (OTC-injected and non-injected) on tree responses over two consecutive production seasons. In year 1, infection history significantly influenced tree size, fruit yield, total soluble solids (TSS), TSS/titratable acidity ratio, and peel color. Late-infected trees outperformed early-infected trees, regardless of injection treatment and rootstock cultivar. In year 2, OTC-injected trees exhibited significantly higher yields, improved juice quality, and enhanced canopy health regardless of infection history and rootstock cultivar. Fibrous root microbiome analyses based on 16S rRNA sequencing revealed no significant effects of OTC injection on bacterial alpha or beta diversity, with stable community structure observed across treatments and time points. This suggests that targeted vascular delivery of OTC may not cause any major disruption to the root endorhizosphere microbiome. Together, the results from this study demonstrate the efficacy of integrating preventative (use of IPCs) and therapeutic (OTC vascular delivery) strategies for sustainable HLB management while preserving microbial integrity and offering a model for citrus production in parts of the world where HLB is prevalent.

ABSTRACT Methanol, the simplest alcohol, has long been known to be a key energy and carbon source for soil and plant-associated bacteria and fungi and is increasingly recognized as an important substrate for marine bacteria. Lanthanide-dependent methanol dehydrogenases (encoded by the gene xoxF ) have been shown to be key catalysts for methylotrophy in many environments, yet the identity of the most transcriptionally active methylotrophs in open ocean waters (“Clade X”) has remained elusive. Here, we show that “Clade X” methylotrophs belong to the deep-branching alphaproteobacterial order TMED127, which we propose be renamed “Methylaequorales”: “methyl” for “methylotrophic metabolism” and “aequor” for “ocean surface,” as these bacteria are most transcriptionally active near the sea surface. TMED127/Methylaequorales are present in surface waters of tropical and subtropical oceans throughout the global ocean. They have small, streamlined genomes (~1.5 Mb) and appear to be obligate methylotrophs that use the serine cycle for carbon assimilation. They display a diel pattern of xoxF5 and glucose dehydrogenase ( gdh ) transcription, peaking in the late afternoon, in oligotrophic surface water of the Sargasso Sea. Several other highly transcribed genes of unknown function had no homologs outside of TMED127/Methylaequorales genomes. Our findings illuminate an overlooked marine methylotrophic bacterium and predict a diel cycle of methanol production in surface seawater by an unknown pathway. IMPORTANCE Methanol metabolism is increasingly recognized as an important process in the marine carbon cycle, yet the identity and metabolism of the microorganisms mediating methylotrophy in the open ocean have remained unknown. This study reveals that bacteria in the TMED127 order of Alphaproteobacteria, renamed here as “Methylaequorales,” abundantly transcribe the key gene for lanthanide-dependent methylotrophy in oligotrophic surface waters of the world’s oceans. TMED127/Methylaequorales likely require methanol as a carbon and energy source and display a diel pattern of transcription of key genes for methylotrophy that peaks in the late afternoon. These findings motivate future studies on the mechanisms of methanol production in surface seawater.

Studies of the genetic diversity of ‘Candidatus Liberibacter asiaticus’ strains based on housekeeping genes have been unsuccessful. The increasing availability of complete genome sequences of several strains from different countries has allowed the identification of regions having greater variability, which have been successfully implemented for the bacterium characterization, including microsatellites, genes of prophage origin, and miniature transposable elements with inverted-repeats (MITEs). In the present work, the genetic structure of 147 ‘Ca. L. asiaticus’ strains from nine provinces of Cuba were investigated using two polymorphic regions, consisting of typing for prophages and MITEs. The results showed an important level of coexistence of type 1 and 2 prophages in the Cuban strains, while the type 3 prophage was not detected. Likewise, a high rate of co-occurrence of both types of MITEs (MCLas-A and -B) was also observed. However, the MITE MCLas-A was detected only in its empty form. The double-locus analysis allowed the identification of eight genotypes. Out of these, seven genotypes were present in the Western region, which constitutes the region with the highest genetic variability. This is the first report of a genetic characterization of Cuban strains of ‘Ca. L. asiaticus’ with polymorphic markers in orchards growing in commercial citrus regions.