Ecology

Huanglongbing (HLB, or citrus greening disease) affects all citrus varieties worldwide. In the United States, Asia, and South America, the causal agent is ‘ Candidatus Liberibacter asiaticus’ ( CLas), a phloem-limited, uncultured alphaproteobacterium. The hemipteran insect vector Diaphorina citri (Asian citrus psyllid) acquires and transmits CLas in a circulative, propagative manner. In addition to CLas, D. citri hosts multiple symbiotic bacterial species including Wolbachia (wDi). In D. citri, wDi has been sequenced and studied, but specific roles in D. citri biology are unknown. Using well-established quantitative PCR methods, we measured CLas titer in D. citri collected from four groves in central Florida with distinct HLB management strategies and tested whether CLas and wDi titer were correlated in a subset of these insects. Grove site had the largest effect on CLas titer. Sex had no effect on CLas titer, whereas a higher wDi titer was correlated with noninfected insects. Our results suggest that more directed follow-up research is necessary and important to clarify whether field management tactics influence CLas titer in D. citri and to better understand gene-by-environment interactions among D. citri, wDi, and CLas. Now that millions of trees in Florida have been treated with injectable formulations of oxytetracycline, which is likely to decrease bacterial populations in D. citri, this study may represent the last biologically meaningful snapshot of grove-level vector-pathogen ecology in the state during the HLB epidemic.

ABSTRACT “ Candidatus Liberibacter asiaticus” (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars (“Shatangju” mandarin, “Guanxi” pomelo, and “Shatian” pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/ cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth. IMPORTANCE Citrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, “ Candidatus Liberibacter asiaticus” (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

ABSTRACT Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense ( Ca . M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca . M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca . M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca . M. massiliense and possible transfer between hosts. IMPORTANCE Methanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca . M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

<p><strong>Background.</strong> Climate change models have projected an increase in the distribution of certain crop pests of economic importance by forecasting more favorable future conditions for these organisms. In citrus farming, Huanglongbing is one of the most devastating diseases worldwide, since it has caused the death of millions of trees. <strong>Objetive.</strong> The objective of this study was to estimate the current and future distribution of <em>Candidatus </em>Liberibacter asiaticus in Mexico, under the climate change scenarios SSP2 4.5 and SSP5 8.5, for the years 2050 and 2070. <strong>Methodology.</strong> Distribution models were generated with MaxEnt and R, using uncorrelated bioclimatic variables from eight General Circulation Models (GCM) derived from CMIP6 and disease presence data. <strong>Results.</strong> The results indicate that the current suitability is 44.6 %. The future distribution depended on how model predictions were pooled. An optimistic approach that considered the intersection of all models showed a small reduction of 4.1% while, considering the union of all the GCM models, the increase will vary from 12.3 to 20.1 % of the Mexican territory depending on the particular scenario and time projection. <strong>Implications.</strong> The zones of potential occurrence of <em>Candidatus </em>Liberibacter asiaticus include most of the citrus-growing areas in Mexico. <strong>Conclusion. </strong>In some regions, future scenarios show a reduction in the potential occurrence of the species in citrus plantations. However, the risk remains because its surroundings include suitable areas that can be sources of dissemination of the disease. </p>

‘Candidatus Phytoplasma ulmi’ (Ca. P. ulmi) belongs to the ribosomal subgroup 16SrV-A and is associated with dieback, shoot proliferation and yellows disease on various Ulmus spp. Other plant species, such as Carpinus betulus and Prunus spp. have also been reported infected by the same pathogen. In 2021, in the frame of research activities focused on grapevine’s Flavescence dorée (FD), one specimen of Orientus ishidae - an East Palearctic leafhopper that was identified as an alternative vector of FD phytoplasmas - was found harboring Ca. P. ulmi in southern Switzerland. No phytoplasmas were detected in plant samples taken in the same location. Orientus ishidae has already been reported to be able to acquire diverse phytoplasmas associated with other plant diseases, such as Peach X-disease. This is the first report of Ca. P. ulmi in Switzerland, as well as in O. ishidae. Ca. P. ulmi may potentially be present in the wild compartment of the Swiss Pre-alpine and Alpine range, but no dedicated survey has so far been conducted. In the case of O. ishidae, this finding highlights the broad affinity of such a species for the acquisition of several phytoplasmas. This calls for a further investigation regarding its potential role as a vector on various pathosystems of agronomic importance.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.

This survey presents the first precise records of Leucomigus candidatus candidatus (Pallas, 1781) in Romania, confirming thus the presence of this species almost a century after its first and single record. Furthermore, ecology, habitat preference and distribution are discussed. We illustrate this taxon with high quality macro photographs, including the male genitalia with the everted and inflated endophallus. Comparative plates with similar-looking species are provided. New localities from Romania and Republic of Moldova are recorded for Cleonis pigra (Scopoli, 1763) and Cyphocleonus dealbatus (Gmelin, 1790).

Huanglongbing (HLB) is a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas). Since its initial outbreak in Guangdong Province, China, it has spread to 10 provinces and caused significant economic losses. Hence, assessing CLas genetic diversity and demographic history is crucial for HLB epidemic prevention and control. In this study, we collected 500 leaf samples of CLas-infected plants from 10 provinces. We performed multi-loci sequence analysis on four gene fragments (omp, DnaA, GroEL, and SDE1) to explore the genetic differentiation and diversity of CLas in China. Our results indicated low nucleotide diversity (0.00005 ± 0.00001) in CLas, with the absence of significant systematic geographic structure in its distribution. Molecular variance analysis revealed predominant (81.7%) genetic variations within the population, with a minor variation (18.3%) occurring between populations as well as Yunnan provinces. In the Fujian population, significant gene exchange occurred with the other nine populations. Significant negative values in Tajima’s D and Fu’s FS neutrality tests indicated historical population expansions. The nucleotide mismatch distribution curve exhibits a single peak pattern, further supporting the expansion events. Our findings hold potential for advancing epidemiological research and providing suggestions for effective strategies to mitigate the spread of CLas and control HLB.