Publications

4495

‘ 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’.

ABSTRACT The phyllody severity in sesame crop ranged from 9% to 20% under field conditions, and the maximum was recorded in Thiruvannamalai district of Tamil Nadu, India. The observed morphological characters of the transmitting insect vector Orosius albicinctus were light ochraceous colour with irregular striations and dark brown with black mottling. The infected sesame samples were subjected to PCR amplification by using universal primer pair P1/P6, followed by nested PCR using R16F2n/R16R2 primers, which amplified ~1.25 kb, which was also occurred in the Parthenium weed plant. Further, BLAST analysis showed that the sequences were aligned with the 16SrII‐B group phytoplasma consisting of Candidatus Phytoplasma aurantifolia (94.87%), sesame phyllody (97.97%), Blainvillea acmella phytoplasma (97.70%) and Mollugo disticha phyllody phytoplasma (98.28%). Since there is no adorus impact despite the vast host range, polyphagous insect vectors, lack of environmentally acceptable effective insecticide, and unavailability of resistant sources, management of phyllody disease has been attempted with biotic inducers (salicylic acid [SA], methyl jasmonate [MeJA] and beta amino butyric acid [BABA]) individually (50, 100 and 150 ppm) and in combination. Among the inducers tested in vitro, SA 50 ppm showed a higher vigour index (1401) in the seed infiltration method of seed treatment, further confirmed in blotter paper technique. The same treatment showed a high germination percentage (86%) with more shoot length on the 60th day (67.0 cm) and five branches/plant under glasshouse experiment. In addition, SA 50/100 ppm increased the phenolic activity and plant defence enzymes; hence it has been further test verified at two different field locations, and the results revealed that seed infiltration treatment with SA 50/100 ppm, followed by foliar application of SA (50/100 ppm) at 30, 45 and 60 DAS, was shown to be effective by recording lesser disease severity compared to control (42.2%). Also, salicylic acid treatment showed higher germination (87.6%), more capsules/plant, thereby incorporation of inducers like salicylic acid in the integrated disease management will serve as a viable and sustainable management option.

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.

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.

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.

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.

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.