Publications

4693

Abstract Background Spirochetes are the most abundant bacterial group in the hindgut of termites. The largest species, with cell lengths of up to 100 µm, have been provisionally classified in the family “ Pillotinaceae ” based exclusively on morphological traits. However, in the absence of cultured representatives, their phylogenetic position and metabolism remain entirely unknown. Results We investigated phylogeny and metabolic capacities of “pillotinaceous” spirochetes using single-cell techniques, electron microscopy, and fluorescence in situ hybridization. All sequences of large spirochetes obtained from various termites fell into four distinct, well-supported clusters within the family Breznakiellaceae . Based on ultrastructural features, three of the clusters were assigned to the genera Pillotina , Hollandina , and the newly established genus Hollandinoides; a fourth cluster was tentatively assigned to the genus Clevelandina . Functional analysis of the single-cell genomes of Pillotina corrugata sp. nov., Hollandina grandis sp. nov., and Hollandinoides gharagozlouae gen. nov. sp. nov., combined with comparative genomics of other uncultured relatives, demonstrated differences in the capacity to degrade cellulose, hemicelluloses, and dextrins. While members of the genus Pillotina have a fermentative metabolism, members of the other genera encode a Wood–Ljungdahl pathway and, in the case of Hollandina , a group-III nitrogenase, suggesting roles in reductive acetogenesis and nitrogen fixation. Conclusions Our results provide the first molecular data on pillotinaceous spirochetes. We show that the three genera covered in our study belong to the family Breznakiellaceae , which harbors the majority of termite-gut spirochetes. Comparative genome analysis indicated that the large spirochetes in termite guts have distinct roles in symbiotic digestion.

Abstract ‘Candidatus Mycoplasma haemolamae’ (CMhl) is a hemotropic bacterium infecting South American Camelids (SAC), whose epidemiology and clinical significance are still not fully elucidated. This study investigated CMhl by qPCR in blood samples of alpacas ( Vicugna pacos ) and llamas ( Lama glama ) in Italy in 2021–2024, characterizing CMhl haplotypes based on partial 16 S rRNA sequences and defining haematological findings in a subset of CMhl-infected animals. Statistical analysis was performed to detect potential risk factors associated with CMhl positivity. Out of 206 animals (200 alpacas and 6 llamas), CMhl was detected by qPCR in 88 (42.7%) animals. CMhl haplotype #1 and #2 were detected in 42.2% and 0.5% of the animals, respectively. Binary logistic regression analysis showed that higher CMhl positivity was significantly associated with larger herd size ( P  = 0.011), Northern Italy area of origin ( P  = 0.031) and warmer seasons ( P  = 0.003). The detection of two CMhl haplotypes confirms the high diffusion of CMhl in SAC farmed in Italy. Results confirm that CMhl usually causes subclinical infections, suggesting that antimicrobial treatment is likely not necessary based on qPCR positivity alone. Results focusing on the association of CMhl positivity with warm temperatures and large herd size suggest the need of further studies on the genetic features, epidemiology and immune response in CMhl-positive SAC from Italy, with emphasis on understanding potential vector transmission.

Abstract Candidatus Liberibacter asiaticus ( C Las), the causal agent of Huanglongbing, is transmitted by the Asian citrus psyllid ( Diaphorina citri ) in a persistent, propagative manner. Reproducible transmission studies require not only controlled inoculation assays but also standardized propagation of citrus host plants, maintenance of D. citri colonies, and reliable pathogen detection. This study presents a comprehensive set of coordinated protocols that collectively support controlled C Las transmission experimentation. The workflow includes propagation of Citrus macrophylla for colony and assay use; generation and maintenance of C Las‐infected citrus and D. citri colonies; synchronization of D. citri populations to enable C Las acquisition; and a controlled single‐ D. citri transmission assay with defined inoculation access periods, and a Dellaporta‐based nucleic acid extraction method compatible with citrus and psyllid tissues. Quantitative PCR detection is integrated throughout to verify infection status, determine C Las titer, and monitor C Las movement and systemic colonization in citrus over time. By combining standardized plant production, vector infection systems, transmission assays, and molecular diagnostics, these coordinated methods provide a unified framework for testing hypotheses about vector competence, pathogen–vector interactions, microbial co‐infections, host susceptibility, and environmental effects on transmission. Together, the procedures enable consistent and comparable assessment of C Las acquisition, dissemination, and transmission outcomes under controlled laboratory conditions. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Controlled laboratory method for assessing Candidatus Liberibacter asiaticus transmission by Diaphorina citri Support Protocol 1 : Generation of Candidatus Liberibacter asiaticus‐infected Citrus macrophylla and Diaphorina citri Basic Protocol 2 : Dellaporta total nucleic acid extraction Basic Protocol 3 : Citrus macrophylla propagation

Areca palm yellow leaf phytoplasma (AYLP) and Areca palm velarivirus 1 (APV1) are two critical pathogens associated with yellow leaf in areca palm, resulting in devastating damage to areca production. However, evidence of their coinfection remains unclear. Areca palms showing yellow leaf symptoms (suspected to be caused by phytoplasma and velarivirus 1) were surveyed and sampled around Hainan Island of China, and the pathogens were identified and analyzed in the study. The results showed that infectious leaf yellowing symptoms of areca palms primarily occurred in eastern and southern cities and counties of Hainan Island. Molecular identification revealed coinfection of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup) and APV1 in individual areca palm samples. Among 520 samples tested by qPCR, AYLP was detected in 23.46% (n = 122), APV1 in 53.46% (n = 278), and a coinfection with both AYLP and APV1 in 10.96% (n = 57). A statistically significant difference in APV1 detection rate was observed between asymptomatic and symptomatic plantations (P < 0.0001), suggesting that APV1 infection likely represents an important risk factor for yellowing development in cultivated areca palm populations. These results confirm the coinfection of AYLP and APV1 on Hainan Island, offering epidemiological evidence for targeted disease management.