Publications

4329

ABSTRACT Huanglongbing (HLB) is a devastating citrus disease associated with the gram-negative, phloem-limited, and unculturable bacterium “ Candidatus Liberibacter asiaticus ( C Las),” which is transmitted by the Asian citrus psyllid Diaphorina citri . Despite extensive research, effective, long-term, and sustainable solutions for managing HLB remain elusive. Oxytetracycline (OTC) is currently used as an emergency measure, but there is an urgent need for alternative compounds to complement or replace OTC. In this study, we identified amicoumacins, a class of antimicrobial compounds produced by the bacterium Bacillus safensis CB729 isolated from the citrus microbiome, and demonstrated their ability to suppress C Las. Genome mining of B. safensis CB729, combined with metabolomic analysis and bioassay-guided fractionation, revealed the presence of amicoumacins and related derivatives in fractions inhibitory to Liberibacter crescens , a culturable surrogate for C Las. We tested commercially available synthetic amicoumacins A and B, along with a B. safensis -derived amicoumacin mixture, against L. crescens and C Las. We determined the MICs of amicoumacin A (1.25 µg/mL) and amicoumacin B (10 µg/mL) against L. crescens . Furthermore, amicoumacin B and the amicoumacin mixture significantly reduced C Las populations in ex vivo citrus hairy root assays. This study highlights the potential of amicoumacins as a promising group of natural products for the management of HLB, offering valuable insights for the development of novel and sustainable disease control strategies. IMPORTANCE For two decades, the citrus industry has been severely impacted by Huanglongbing (HLB), a devastating disease caused by “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by the Asian citrus psyllid ( Diaphorina citri ). Despite extensive research, effective, long-term, and sustainable solutions remain unavailable for growers. Currently, medically relevant antibiotics, such as oxytetracycline (OTC), are used as an emergency response to combat HLB in Florida, the most affected citrus-producing state in the U.S. This underscores the urgent need for alternative treatments that can be used in rotation or as replacements for OTC. Here, we present amicoumacins, a group of bioactive secondary metabolites with antibiotic properties. We identified amicoumacin B and its derivatives from the culture broth of a Bacillus safensis isolate, native to citrus, and demonstrated their ability to inhibit Liberibacter spp. and reduce C Las populations in citrus tissue. This study highlights how microbial discovery can lead to the identification of antimicrobial compounds with potential applications in plant disease management.

Phytoplasmas ('Candidatus Phytoplasma') are intracellular pleomorphic plant pathogens belonging to the class Mollicutes. They colonize both plant hosts and insect vectors in their life cycle. Apple proliferation (AP) is one of the most important phytoplasmoses present in Europe causing significant economic losses in apple production. The causal agent, ‘Ca. P. mali', was identified in apple and Cacopsylla picta samples using both real-time PCR and nested PCR based on the amplification of 16S rDNA. The objective of this study was to gain deeper insights into the epidemiology of apple proliferation in Croatia. Variability of genetic markers other than 16S rRNA was used for characterization of strains. Four molecular markers differing in level of conservation, aceF, pnp, imp and secY, were selected in line with previously typed fruit tree phytoplasmas. New genotypes were discerned for each genetic marker and 20 different sequence types were revealed in the Croatian isolates of ‘Ca. P. mali’. On the basis of this comprehensive analysis, the founder sequence type ST1 (A13-P10-S12-I21) can be proposed. This is the first extensive research and multigene typing performed on Croatian ‘Ca. P. mali’ isolates. Obtained results reveal considerable genetic diversity of epidemiological relevance limited to only two locations in north-western Croatia. Additionally, novel primers were constructed to amplify fragments larger than the entire coding region for all four genes in order to further expand phytoplasma multi locus sequence typing scheme.

A Gram-stain-negative, aerobic, non-motile and short rod-shaped bacterium, designated strain SCSIO 64248T, was isolated from the stony coral Fungia sp. collected from South China Sea. Phylogenetic analysis indicated that strain SCSIO 64248T belonged to the family Geminicoccaceae and showed the highest 16S rRNA gene similarities with the types of Marinimicrococcus flavescens (90.5%), Arboricoccus pini (90.2%) and Benzoalithermus flavus (89.8%). The whole genome of strain SCSIO 64248T was 4.9 Mbp with a 68.8mol% G+C content. It shared average amino acid identity (AAI) values of 52–56.2%, which is below the proposed AAI threshold at the genus level and when compared to the known species of Geminicoccaceae. Colonies of strain SCSIO 64248T are circular, pink and convex. It was able to grow in the range of 4–37 °C, at pH 6.0–10.0 and in the presence of up to 8.0% (w/v) NaCl concentration. The major cellular fatty acid (>9%) was summed feature 8, C19 : 0 cyclo ω8c and C16 : 0, and the sole respiratory quinone was Q-10. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified phospholipid. Data of polyphasic characteristics revealed that strain SCSIO 64248T represented a novel species of a new genus within the family Geminicoccaceae, for which the name Marinivivus gen. nov. is proposed with the type strain of the type species Marinivivus vitaminiproducens SCSIO 64248T (=MCCC 1K08013T=KCTC 92503T).

Most micro-organisms remain unculturable under standard laboratory conditions, limiting our understanding of microbial diversity and ecological interactions. One major cause of this uncultivability is the loss of access to essential cross-fed metabolites when bacteria are removed from their natural communities. During a bioprospecting campaign targeting actinomycetes of an Apis mellifera beehive, we identified five isolates (DT32, DT45T, DT55, DT59 and DT194) that required co-cultivation for growth recovery, suggesting a dependence on microbial interactions in their native habitat. Whole-genome sequencing and phylogenetic analysis positioned these isolates within a distinct lineage of Micromonosporaceae, separate from the five officially recognized clades of the Micromonospora genus. A combination of microscopic, chemotaxonomic and physiological characterizations further supported their uniqueness. Notably, they exhibited high auxotrophy, being unable to use all carbon sources tested, likely due to genome reduction (4.6 Mbp) compared to other Micromonosporaceae. Pangenomic comparisons with their closest Micromonospora relatives revealed gene losses in key metabolic pathways, including the glyoxylate bypass and the Entner–Doudoroff pathway, which may explain their metabolic reliance. These findings reveal a highly specialized, ecologically adapted lineage with deep evolutionary divergence and further support microbial interdependence isolation strategies to explore the microbial dark matter. We propose Melissospora conviva as a novel genus and species within the Actinomycetota phylum, with isolate DT45T as the representative type species and type strain, which has been deposited in public collections under the accession numbers DSM 117791 and LMG 33580.

Abstract ‘Candidatus Liberibacter asiaticus’ (CLas), the prevalent agent associated with Huanglongbing (HLB), poses a severe threat to global citrus production. This study aimed to investigate the occurrence of psyllid species (other than Diaphorina citri) landing on citrus trees, evaluate their survival on citrus over optimal acquisition access periods (AAP), assess their ability to acquire CLas, and infer their potential role in pathogen transmission. Field surveys were conducted in Citrus sinensis plants located in backyards and poorly managed groves in two locations of São Paulo State, Brazil. Psyllids from twelve genera were identified, with Isogonoceraia divergipennis and Heteropsylla cubana being among the most constant species, showing greater abundance particularly in late winter and early spring. Controlled acquisition assays demonstrated that I. divergipennis, H. cubana, and Triozoida limbata could acquire CLas following a 72-h AAP, post-acquisition latency of 14 days and a subsequent 72-h post-latency period, with acquisition rates of 8.1%, 3.2%, and 2.4%, respectively, compared to 85.7% in D. citri. Notably, CLas was detected in the 'head-mesothorax' region only in T. limbata and D. citri, suggesting potential bacterial presence in the salivary glands and possible transmission competence. CLas titers were consistently higher in the 'metathorax-abdomen' than in the 'head-mesothorax'. While the data indicate occasional CLas acquisition by psyllids other than D. citri, there is currently no evidence supporting their role as natural vectors. Nonetheless, these species may serve as valuable models for elucidating transmission barriers in non-competent psyllids and highlight the need for further investigation into their epidemiological relevance.

A Gram-stain-negative, anaerobic and rod-shaped bacterium was isolated from gut of rainbow trout in Japan. The temperature range for the growth of 9N8T was 10–30 °C with an optimum at 20 °C. The organism produced acetate and butyrate when grown on AccuDia™ Gifu Anaerobic Medium broth, Modified. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain 9N8T was related to the genus Fusobacterium (<92.9% similarity) and formed a distinct phylogenetic lineage from existing genera within the family Fusobacteriaceae. Strain 9N8T has a genome size of 2.60 Mb. The genomic DNA G+C content was 29.4 mol%. Average amino acid identity values between strain 9N8T and strains in the family Fusobacteriaceae ranged from 39.2 to 57.5%. The major fatty acids of strain 9N8T were C14 : 0, C16 : 1 ω7c and C16 : 0. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Piscibacter tructae, is proposed, with 9N8T (=NBRC 116745T=DSM 118263T) as the type strain.

ABSTRACT Psyllids (Psylloidea) are host-specific, phloem-feeding insects that are associated with the transmission of destructive plant diseases caused by the bacterial genus “ Candidatus Liberibacter”. While psyllid-liberibacter interactions have been studied in great detail, most research mainly focused on species associated with important crop diseases. Therefore, a more general understanding of the associations between these two groups of organisms remains limited. The present study investigated the occurrence of infection by “ Ca . Liberibacter” across undercharacterized psyllid lineages in Taiwan. Samples of 46 psyllid species, representing six of the seven known Psylloidea families from host plants spanning 22 families, were obtained. The presence of “ Ca . Liberibacter” in adult psyllid DNA samples was determined by PCRs targeting 16S rDNA of “ Ca . Liberibacter”. PCR tests amplifying mitochondrial 16S DNA of the psyllids were also carried out for quality confirmation. The assays detected “ Ca . Liberibacter” in five of the psyllid species tested, namely Calophya nigridorsalis , Homotoma radiata , Cacopsylla tobirae , Epipsylla albolineata , and Trioza quadrimaculata . BLASTn searches and phylogenetic analysis on near-full-length 16S rDNA sequences identified the strain detected in C. tobirae as “ Ca . Liberibacter europaeus” and showed that E. albolineata carried a strain closely related to a “ Ca . Liberibacter” strain found in Bhutan, while the remaining detected strains could potentially be novel “ Ca . Liberibacter” species. The findings from this study showed that a large diversity of “ Ca . Liberibacter” could exist among underexplored psyllid and plant species, opening new avenues for investigating the ecology of “ Ca . Liberibacter”. IMPORTANCE Bacteria of the genus “ Candidatus Liberibacter” can cause some of the most devastating plant diseases. Gaining a broader perspective on the diversity of associations between these bacteria and their psyllid vectors is crucial for both fundamental and applicative purposes. By taking advantage of the biological diversity in Taiwan, the present study conducted one of the broadest surveys on the presence of “ Ca . Liberibacter” in psyllids, in terms of the diversities of the psyllids examined. The data from this work indicated that previously unknown “ Ca . Liberibacter” genotypes and perhaps even novel species may be more prevalent among psyllid species than previously known. These findings highlight the potential importance of exploring psyllid-liberibacter associations from a broader ecological perspective.