Publications

4364

‘ Candidatus Phytoplasma pyri’, the pathogen associated with pear decline, affects pear trees across both the old and new worlds. However, research on this phytoplasma has been limited by the lack of genomic data. This study presents the first draft genome of ‘ Ca. P. pyri’ using a strain from Chile, with its genomic features analyzed in comparison with the closely related ‘ Ca. Phytoplasma’ species ‘ Ca. P. mali’ and ‘ Ca. P. prunorum’. The draft genome spans 456,478 bp with a GC content of 20.4%. Key genes possibly associated with pathogenicity and potential pathogenic effectors were identified, and they are notably lacking orthologs of known effectors. A single potential mobile unit similar to that of ‘ Ca. P. mali’ was identified. It is characterized by the absence of the transposase tra5 and the presence of the IS3 family transposase iSErh1. Multilocus sequence analysis of six genetic markers (16S rRNA gene, LSU36p, tuf, aceF, secA, and secY) from 10 Chilean and 10 Italian samples revealed high genetic uniformity among the Chilean strains, collected from five geographically distant orchards over a span of 13 months; by contrast, strains with greater diversity were detected among those from Italy, collected from a few localities over approximately 30 years. These findings suggest limited evolutionary divergence of this phytoplasma in Chile. This study provides a foundational framework for investigations into the pathogenic mechanisms and evolutionary dynamics of ‘ Ca. P. pyri’.

‘ Candidatus Phytoplasma prunorum’ is associated with European stone fruit yellows disease, affecting wild and cultivated species of Prunus at different degrees of susceptibility, and so far is being mainly restricted to Europe. Here, we report draft genome sequences for ‘ Ca. Phytoplasma prunorum’ strains ESFY1 and LNS1, which represent the first available for this species. Strain ESFY1 is the causal agent of the European stone fruit yellows disease of P. persica in Germany, and LNS1 is the causal agent of leptonecrosis disease of P. salicina in Italy. Their draft genomes consist of 480,123 and 490,930 bases assembled into 28 and 16 contigs, with an average GC content of 21.1 and 20.7%, respectively. Analysis of average nucleotide identity (ANI) values between genomes further supported the delineation of ‘ Ca. P. prunorum’ as a species distinct from ‘ Ca. P. mali’, in agreement with previous phylogenetic data. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Methanotrophic bacteria in peatlands mitigate emissions of methane (CH4), a potent greenhouse gas. Yet, the identity, physiology, and adaptive traits of methanotrophs inhabiting acidic peatlands are still not fully characterised. Using classical enrichment methods and single-cell sorting, we isolated a novel bacterial methanotroph species from Czech peatland soil: 'Candidatus Methylocystis sumavensis'. 'Ca. M. sumavensis' is moderately acidotolerant, growing optimally at pH 6.8 and 24 – 37°C, with a CH4 oxidation rate of 14.2 ± 0.51 nmol CH4 µg protein-1 · hr-1. The complete genome encodes two isozymes of particulate methane monooxygenase and genes providing the capacity for nitrous oxide reduction to di-nitrogen (N2O). This suggests a potential role as a N2O sink, possibly enabling the new species to oxidise CH4 under low-oxygen or anoxic conditions. The presence of four terminal oxidases, two of which are of a high-affinity type, and two different [NiFe]-hydrogenases (3b and a putative 4f group) suggests a capacity for diverse respiratory processes, likely including anaerobic metabolism. Several acid stress response systems, most strikingly a H+/Na+-translocating F-type ATP synthase in addition to a classical H+-translocating F-type ATP synthase, likely support survival in the isolates’ oligotrophic, acidic habitat. Our results reveal that the new methanotroph species combines unique metabolic traits, reflecting its adaptation to peat ecosystems and indicating a possible broader ecological role for peat-inhabiting methanotrophs beyond aerobic CH4 oxidation.

A novel phytoplasma species, ′Candidatus Phytoplasma zeae′, is proposed based on ecological distinctiveness, vector specificity, whole-genome comparisons, and community consensus. This phytoplasma is associated with maize bushy stunt (MBS) disease in corn (Zea mays) and is transmitted exclusively by Dalbulus maidis and D. elimatus, two leafhopper species endemic to the Americas. , and has been reported in Brazil, Colombia, Mexico, Peru, and several U.S. states. Here we sequenced and assembled the genome of MBS phytoplasma strains from Brazil, and U.S. to describe and propose this new species. Although the 16S rRNA gene sequence of the proposed reference strain, MBSP–BRRS, shares >99% identity with that of ′Ca. Phytoplasma asteris′, key nucleotide polymorphisms distinguish ′Ca. P. zeae′ from other 16SrI-related phytoplasma species. Average nucleotide identity (ANI) and average amino acid identity (AAI) values between ′Ca. P. zeae′ and ′Ca. P. asteris′ are 97.70–98.00% and 96.65–96.88%, respectively, both near the established species delineation thresholds. Comparative genomic analyses revealed unique gene clusters in ′Ca. P. zeae′ associated with amino acid transport, defence mechanisms, and protein turnover, which may contribute to its specialization in corn. The ecological profile of ′Ca. P. zeae′, including its narrow host range and restricted geographic distribution, supports its recognition as a novel species under Rule c of the IRPCM guidelines. The designation ′Candidatus Phytoplasma zeae′ is therefore proposed by members of the research community who have studied this pathogen for over a decade, with the MBSP-BrazilRS strain serving as the reference.

Abstract Axenic cultures are essential for studying microbial ecology, evolution, and genomics. Despite the importance of pure cultures, public culture collections are biased towards fast-growing copiotrophs, while many abundant aquatic prokaryotes remain uncultured due to uncharacterized growth requirements and oligotrophic lifestyles. Here, we applied high-throughput dilution-to-extinction cultivation using defined media that mimic natural conditions to samples from 14 Central European lakes, yielding 627 axenic strains. These cultures include 15 genera among the 30 most abundant freshwater bacteria identified via metagenomics, collectively representing up to 72% of genera detected in the original samples (average 40%) and are widespread in freshwater systems globally. Genome-sequenced strains are closely related to metagenome-assembled genomes (MAGs) from the same samples, many of which remain undescribed. We propose a classification of several novel families, genera, and species, including many slowly growing, genome-streamlined oligotrophs that are notoriously underrepresented in public repositories. Our large-scale initiative to cultivate the “uncultivated microbial majority” has yielded a valuable collection of abundant freshwater microbes, characterized by diverse metabolic pathways and lifestyles. This culture collection includes promising candidates for oligotrophic model organisms, suitable for a wide array of ecological studies aimed at advancing our ecological and functional understanding of dominant, yet previously uncultured, taxa.

Buckwheat (Fagopyrum esculentum Moench) is a fast-growing, low-input crop in the Polygonaceae family, cultivated on about 2 million hectares worldwide, mainly in Russia, China, and Ukraine by FAO (2023). In Taiwan, it is grown as a winter cover crop during fallow periods and is valued for improving soil health, suppressing weeds, and supporting sustainable farming systems. It is also utilized in various food products due to its rich fiber content and gluten-free properties. In May 2024, 147 out of 3000 plants (Taichung No. 5) corresponding to an incidence rate of approximately 5% exhibiting floral abnormalities, including phyllody, virescence, and flower sterility, were observed in Xinshe District in Taichung, central Taiwan (Fig. 1). These symptoms were indicative of possible phytoplasma infection. For further investigation, floral and leaf tissues were randomly collected from 10 symptomatic and 10 asymptomatic buckwheat plants. Plant total DNA was extracted using the DNeasy Plant Mini Kit, and PCR was performed targeting the 16S rRNA gene using universal phytoplasma primers P1A/P7A, followed by a semi-nested reaction with P1A/16S-SR (Lee et al. 2004). All PCR amplicons of approximately 1.5 kb were obtained only from symptomatic samples. PCR amplicons were sequenced by Sanger sequencing, and sequences were assembled by BioEdit. All symptomatic samples yielded identical sequences and a representative sequence (1,543 bp, nearly full-length 16S rRNA gene) was deposited in GenBank (Accession no. PV487329). Based on virtual RFLP analysis using iPhyClassifier (Zhao et al. 2009), the phytoplasma strain detected in diseased buckwheat plants was identified as a ‘Candidatus Phytoplasma aurantifolia/citri’-related strain, sharing 98.27% sequence identity with the reference strain (witches broom disease of lime, WBDL, Accession no. U15442). The strain, designated as Buckwheat Phyllody (BWP) TSIPS 3-1, was classified within the 16SrII-A subgroup, with a similarity coefficient of 1.00 when compared to the reference strain of subgroup 16SrII-A (Accession no. L33765). A BLASTn search against the NCBI database revealed that BWP-TSIPS 3-1 shared 99.94% identity with the 16S rRNA gene of potato purple top phytoplasma (Accession no. KM212951, a ‘Ca. P. aurantifolia/citri’-related strain), identified in Guangdong, China (Cheng et al. 2019). To further confirm phytoplasma identity, an additional gene marker, secY gene was amplified by semi-nested PCR using group-specific primers SecYF1(II)/SecYR1(II) and SecYF2(II) /SecYR1(II) (Lee et al. 2010). The 1,263-bp amplicons were obtained from all symptomatic samples. Sequencing results revealed identical secY sequences across samples, and a representative sequence was deposited in GenBank (Accession no. PV567544). BLASTn analysis indicated 100% (1263/1263) identity with the secY gene of the phytoplasmas, including black gram witches’-broom phytoplasma in Myanmar (Accession no. AB703249), which is classified within the 16SrII-A subgroup (Win and Jung, 2012). Phytoplasma infections associated with buckwheat have been rarely reported, with only a single case documented in India, though the short 16S rRNA gene sequence limited subgroup classification (Bandakkanavar et al. 2023). Similar symptoms, such as phyllody, have also been observed in Ukraine, but the causal agent remains unconfirmed due to the absence of molecular diagnostics (Demchenko et al. 2016). Our study confirmed the first identification of a ‘Ca. P. aurantifolia/citri’-related strain (16SrII-A subgroup) in buckwheat in Taiwan. Phytoplasmas of the 16SrII-A subgroup have been widely documented across Taiwan, including in Taichung (Tseng et al. 2012). Given the rising popularity of buckwheat for its dietary benefits and its increasing incorporation into crop rotation systems, especially as a winter cover crop, the presence of phytoplasma presents potential concerns. Infected buckwheat plants may inadvertently serve as hosts or reservoirs for phytoplasmas and their insect vectors, indicating a need for ongoing monitoring, especially in areas where buckwheat is part of crop rotations.

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.

Infection of cranberry by the 16SrIII-Y group phytoplasma responsible for cranberry false blossom disease results in floral disfigurement, fruit abortion, and phyllody, ultimately sterilizing infected plants. The pathogen almost devastated the cranberry industry in the 1930s, until control measures involving resistant cultivars and insect vector management were implemented. Recent discoveries of the pathogen and its vector in Wisconsin have renewed interest in exploring the pathosystem to gain deeper insights into host-pathogen interactions. A qPCR assay was used to detect phytoplasma infection, both spatially and temporally, in both symptomatic and asymptomatic cranberry plants. Dissection of infected vines by tissue type allowed detection of the phytoplasma throughout the entirety of the plant, revealing the systemic nature of the infection in cranberry vines. Furthermore, sampling throughout the growing season demonstrated that phytoplasma detection is possible at multiple time points, suggesting that diagnosis may be feasible even in the absence of clear symptoms like flower abnormalities, thereby enhancing diagnostic capabilities. Additionally, the qPCR method proved effective in detecting the causal agent within the insect vector, the blunt-nosed leafhopper. Phytoplasma acquisition occurs during feeding, colonizes various organs of the insect, and ultimately infect salivary glands making transmission possible. While the rate of colonization of both the plant and insect hosts is unknown, these findings provide valuable insights in industry decision-making for control and detection, as well as expanding understanding of a returning threat.