Publications

4364

Abstract Aims Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Methods and results Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. Conclusions In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Citrus greening disease was first reported in Saudi Arabia during the 1970s when characteristic foliar and fruit symptoms were observed in commercial citrus groves. However, ‘Candidatus Liberibacter asiaticus’ (CLas) was not detected in symptomatic trees until 1981 to 1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the 13 citrus-growing regions of the country. Citrus trees were surveyed for the presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults revealed that trees were positive for CLas detection in 10 of the 13 survey regions; however, CLas was undetectable in ACP adults. Phylogenetic and single nucleotide polymorphism (SNP) analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (∼1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, U.S.A., isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.

Ciliates are unicellular eukaryotes, regularly involved in symbiotic associations. Symbionts may colonize the inside of their cells as well as their surface as ectosymbionts. Here, we report on a new ciliate species, designated asZoothamnium mariellasp. nov. (Peritrichia, Sessilida), discovered in the northern Adriatic Sea (Mediterranean Sea) in 2021. We found this ciliate species to be monospecifically associated with a new genus of ectosymbiotic bacteria, here proposed asCandidatusFusimicrobium zoothamnicola gen. nov., sp. nov. To formally describe the new ciliate species, we investigated its morphology and sequenced its 18S rRNA gene. To demonstrate its association with a single species of bacterial ectosymbiont, we performed 16S rRNA gene sequencing, fluorescencein situhybridization, and scanning electron microscopy. Additionally, we explored the two partners’ cultivation requirements and ecology.Z. mariellasp. nov. was characterized by a colony length of up to 1 mm. A consistent number of either seven or eight long branches alternated on the stalk in close distance to each other. The colony developed three different types of zooids: microzooids (“trophic stage”), macrozooids (“telotroch stage”), and terminal zooids (“dividing stage”). Viewed from inside the cell, the microzooids’ oral ciliature ran in 1 ¼ turns in a clockwise direction around the peristomial disc before entering the infundibulum, where it performed another ¾ turn. Phylogenetic analyses assignedZ. mariellasp. nov. to clade II of the family Zoothamnidae. The ectosymbiont formed a monophyletic clade within theGammaproteobacteriaalong with two other ectosymbionts of peritrichous ciliates and a free-living vent bacterium. It colonized the entire surface of its ciliate host, except for the most basal stalk of large colonies, and exhibited a single, spindle-shaped morphotype. Furthermore, the two partners together appear to be generalists of temperate, oxic, marine shallow-water environments and were collectively cultivable in steady flow-through systems.

Abstract Background Hemotropic mycoplasmas or hemoplasmas are bacteria that attach to the erythrocyte surface and cause bovine hemoplasmosis. Two species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been identified and shown to be distributed worldwide. However, there is currently no information available on hemoplasmas in cattle in the Republic of Korea. The aim of this study was to investigate the presence of hemoplasmas in Korean native cattle and to evaluate the association between hemoplasma infection and anemia. Methods One farm was selected, at which blood samples were collected from 104 Korean native cattle [grazing cattle (n = 89) and housed cattle (n = 15)]. Hemoplasmas were detected via polymerase chain reaction analysis and complete blood counts were also performed. Results The overall prevalence of hemoplasmas was 34% (35/104); 20.2% (21/104) for M. wenyonii, 3.8% (4/104) for C. M. haemobos, and 9.6% (10/104) for co-infection. Candidatus Mycoplasma haemobos was detected only in grazing cattle. Of red blood cell (RBC) parameters, C. M. haemobos-infected cattle had lower RBC and hematocrit, and higher mean cell volume than hemoplasma-negative cattle, although none of these differences were statistically significant. This is the first study to report the occurrence of M. wenyonii and C. M. haemobos. Mycoplasma wenyonii is more prevalent than C. M. haemobos in Korean native cattle. The results did not show an association between hemoplasma infection and anemia. Conclusions Considering the infection rate of hemoplasmas shown in this study, further studies, such as on the pathogenicity and clinical significance of hemoplasmas are necessary.