Publications

4493

The endoplasmic reticulum (ER) stress response, or unfolded protein response (UPR), is crucial for cellular homeostasis and host defense. Its role in insect vectors of plant pathogens remains poorly understood. This study conducted a comprehensive molecular characterization of three core UPR genes—BiP, IRE1, and XBP1—in Diaphorina citri, the insect vector of the citrus huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas). Expression profiling showed distinct developmental and tissue-specific patterns for these genes. The IRE1-mediated unconventional splicing of XBP1 mRNA was identified in D. citri and predicted across diverse insect pests. A semi-quantitative RT-PCR assay was established to detect this splicing event for monitoring ER stress. Time-course analysis of CLas-infected D. citri revealed an early activation (upregulation of BiP, IRE1, XBP1 transcripts, and increased XBP1 splicing) followed by later suppression of the UPR. Functional studies demonstrated that induction of ER stress with thapsigargin increased CLas titer. RNAi-mediated silencing showed that IRE1 silencing increased CLas proliferation, whereas XBP1 silencing reduced it. These findings provide foundational insights into the ER stress pathway in D. citri and demonstrate that the IRE1-XBP1 branch of the UPR plays a critical role in modulating CLas infection dynamics within its insect vector.

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.

“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.