Publications

4458

Abstract Background ‘ Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited bacterial pathogen causing significant diseases in solanaceous crops. In the United States, haplotypes A and B are transmitted by the potato psyllid Bactericera cockerelli . We previously identified differences in their acquisition and transmission between adults and nymphs. The present study characterized the dynamics of LsoA and LsoB acquisition and transmission by nymphs and examined the transcriptional responses of the nymphal gut upon their acquisition. Methods and results Nymphs were exposed to LsoA- or LsoB-infected plants for 1, 3, 5, or 7 days to measure the bacterial accumulation and for 8 days to assess the transmission efficiency following sequential inoculation of tomato plants. Quantitative PCR showed that LsoB accumulated to higher levels than LsoA after 3 days of acquisition. Following the sequential inoculation, LsoB was transmitted earlier than LsoA indicating a shorter latency period. RNA-seq analysis of the guts following a 1- and 5-day acquisition access periods revealed a greater transcriptional regulation at 5 days than at 1 day. Furthermore, the responses were haplotype-specific: LsoA primarily affected genes involved in protein translation, ER stress, and cell cycle regulation, whereas LsoB regulated genes involved in autophagy, apoptosis, and immune pathways. Conclusions This study revealed haplotype-specific gene regulation potentially leading to LsoB being transmitted more efficiently by psyllid nymphs.

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 Citrus huanglongbing (HLB) associated with Candidatus Liberibacter spp. is the most destructive bacterial disease of citrus crops worldwide. In this study, the response of three-year-old seedlings of 14 commercial citrus cultivars and rootstocks to the Asian form of HLB disease ( Ca . L. asiaticus) was investigated through graft-inoculation. The seedlings' reactions to the HLB disease were evaluated based on the PCR detection of CLas after grafting, the disease incubation period (time between graft-inoculation and appearance of the first disease symptoms), and disease severity. Depending on the time of the positive PCR detection of CLas and appearance of disease symptoms, Mexican lime, Persian lime and Volkamer lemon were ranked at the highest level of tolerance (lowest susceptibility), and Rough lemon and Local orange were at the next level, i.e., semi-tolerance (intermediate susceptibility). Alemow, Bakraei, Rangpur lime, Cleopatra, Clementine mandarin, Local mandarin, Blood orange, and Washington navel orange were classified as susceptible to the disease, followed by Valencia orange, which was the most susceptible. In conclusion, all citrus genotypes studied, were affected by HLB. However, symptoms varied by cultivar, with the most severe observed on Valencia sweet orange and Local mandarin, while less severe symptoms were observed on Mexican lime, Persian lime and Volkamer lemon.

Abstract Dermatophilaceae polyphosphate-accumulating organisms (PAOs), formerly classified as Tetrasphaera PAOs, play pivotal roles in enhanced biological phosphorus removal (EBPR). However, their phylogenetic diversity, ecological preferences, and metabolic traits remain poorly characterized, and a robust marker gene for their classification is lacking. Here, we performed an extensive phylogenomic and metabolic analysis of Dermatophilaceae PAOs utilizing 46 newly recovered metagenome-assembled genomes (MAGs) from a laboratory-scale EBPR reactor treating high-strength wastewater and full-scale wastewater treatment plants. These analyses revealed a previously uncharacterized PAO genus, named Candidatus Dermatophostum, which shows specific preference for high-phosphorus environments. Its representative species, Ca. Dermatophostum ammonifactor, was enriched in the EBPR reactor and its PAO phenotype was confirmed by polyphosphate staining and fluorescence in situ hybridization. Genomic, transcriptomic, and protein structure analyses revealed its specialized metabolic capabilities for phosphate metabolism, glycogen synthesis and dissimilatory nitrate reduction to ammonium. Moreover, Ca. Dermatophostum was found to be widely distributed across WWTPs worldwide, underscoring both its diverse metabolic capabilities and potential engineering implications for mitigating nitrous oxide (N2O) emissions for EBPR system. Finally, we propose a ppk1-based classification framework that resolves Dermatophilaceae PAOs into six distinct clades, consistent with whole-genome phylogeny, and demonstrates that ppk1 can serve as a reliable marker gene for tracking these populations. Together, these findings expand the ecological and functional understanding of Dermatophilaceae PAOs and highlight their promise for advancing sustainable wastewater treatment and resource recovery.

“Candidatus Sarcina troglodytae” (“Ca. S. troglodytae”), a proposed bacterial species in the family Clostridiaceae, has been epidemiologically associated with a lethal disease in sanctuary chimpanzees in Sierra Leone, epizootic neurologic and gastroenteric syndrome (ENGS). As sarcinae are known to be difficult to culture, a lack of viable in vitro growth conditions has limited further characterization of this bacterium. Such studies are critical to elucidating the relationship between “Ca. S. troglodytae” and ENGS. Here, we isolate a gram-positive, endospore-forming, coccus and anaerobic bacterial strain, designated JB3T, from brain tissue of a western chimpanzee (Pan troglodytes verus) using a reinforced clostridial medium. We describe the growth of strain JB3T at a pH range of 6 to 9 (optimum 6) at 37°C in an atmosphere composed of 5% H2, 5% CO2, and 90% N2 and demonstrate endospore production under alkaline conditions, suggesting the possibility of an environmental reservoir. We identify ethanol as the primary non-gaseous fermentation byproduct which we hypothesize to have relevance for ENGS. Antibiotic susceptibility testing shows susceptibility to common antimicrobial agents with notable resistance to ceftriaxone. By establishing in vitro conditions for the viable culture, propagation, and storage of “Ca. S. troglodytae”, this study lays the groundwork for future infection studies to test hypotheses about pathogenic mechanisms and prevention/treatment measures for ENGS.

Coconut lethal yellowing disease (CLYD) caused by phytoplasma is a destructive disease affecting coconut and other palm species. The disease has been reported in Mozambique, Tanzania, and some West African countries, where millions of coconut trees have been affected. Although the disease was first reported in 1999 in Northern Coast of Kenya, no further studies have been conducted, leaving the giant coconut industry at risk of destruction. It is for this reason that a surveillance was undertaken to study the extent of the disease spread and the specific Candidatus phytoplasma strain causing the disease. A total of 125 coconut tree samples were collected in Kwale, Kilifi, Lamu, and Tana River counties by drilling the tree trunks aseptically. DNA extractions were done, followed by PCR process done using P1/P7 universal primers, gel electrophoresis and amplicon sequencing. For the coconut tree drillings results, the BLAST comparison with existing data in the NCBI database revealed it to be Candidatus phytoplasma palmae strain isolate Tanz08, under gene accession No. GU952107.1. Further phylogenetic analysis using the maximum likelihood method indicated that the strain is closely related to those reported in Tanzania. The CLYD positive samples were identified from Lamu and Kwale counties, with 6 cases from each County. Although there were no positive cases reported in Kilifi and Tana River counties, the presence of the vectors suggests that more studies need to be done on the disease. Further studies should be done on the alternative hosts, varieties prone to the disease, characterization and diversity of the vectors, the incubation period and the host pathogen relationship.

The Clostridium estertheticum complex (CEC) consists of closely related bacterial species that are mostly isolated from meat processing environment. Genome mining studies have recently established CEC as a source of class I bacteriocins. However, up until now, class II bacteriocins have not been reported from CEC. In the present study, we determined the presence of class II bacteriocin biosynthetic clusters in 33 CEC genomes through genome mining followed by bacteriocin production through heterologous expression in Escherichia coli . Six biosynthetic gene clusters belonging to class IIa ( n  = 1), IIb ( n  = 2), IId ( n  = 2) and an undefined class containing three precursor peptides were identified in six different CEC strains. Using molecular biology, we developed dedicated expression vectors for the class IIa bacteriocin, clesteriocin A. Its precursor peptide, CleA, and protease, CleB150, were initially expressed in insoluble form in E. coli . Through a systematic analysis of suitable solubility enhancing protein tags, both CleA and CleB150 were expressed in significant amounts of soluble fractions using tandem tags derived from Small Ubiquitin-like Modifier, superfolder Green fluorescent protein, Maltose binding protein and N-utilization substance. Mature clesteriocin A was obtained following in vitro maturation reactions between the tagged CleA and CleB150. The novel bacteriocin displayed antimicrobial activity against Listeria monocytogenes , which was mediated by the mptC gene. By combining genome mining and molecular biology, we have shown CEC is a source of novel class II bacteriocins that have potential for application as food biopreservatives.