Mechanisms Underlying the Overlooked Chiral Fungicide-Driven Enantioselective Proliferation of Antibiotic Resistance in Earthworm Intestinal Microbiome.

From a "One Health" perspective, the global threat of antibiotic resistance genes (ARGs) is associated with modern agriculture practices including agrochemicals application. Chiral fungicides account for a considerable proportion of wildly used agrochemicals; however, whether and how their enantiomers lead to differential proliferation of antibiotic resistance in agricultural environments remain overlooked. Focused on the soil-earthworm ecosystem, we for the first time deciphered the mechanisms underlying the enantioselective proliferation of antibiotic resistance driven by the enantiomers of a typical chiral fungicide mandipropamid (i.e., R-MDP and S-MDP) utilizing a multiomic approach. Time-series metagenomic analysis revealed that R-MDP led to a significant enhancement of ARGs with potential mobility (particularly the plasmid-borne ARGs) in the earthworm intestinal microbiome. We further demonstrated that R-MDP induced a concentration-dependent facilitation of plasmid-mediated ARG transfer among microbes. In addition, transcriptomic analysis with verification identified the key aspects involved, where R-MDP enhanced cell membrane permeability, transfer ability, biofilm formation and quorum sensing, rebalanced energy production, and decreased cell mobility versus S-MDP. Overall, the findings provide novel insights into the enantioselective disruption of microbiome and resistome in earthworm gut by chiral fungicides and offer significant contributions to the comprehensive risk assessment of chiral agrochemicals in agroecosystems.

Fungal Communities of Spring Barley from Seedling Emergence to Harvest During a Severe Puccinia hordei Epidemic.

All plant tissues from leaves, stems, and roots are hosting a wide diversity of fungal species. Our understanding of the assembly of this diversity of fungi during the plant growth cycle is limited. Here, we characterized the mycobiome of three spring barley cultivars grown in Zealand, Denmark, at weekly intervals during a growth season from seedling emergence to senescence and seed maturity. A notable proportion of members of the fungal communities were shared among different plant organs, but community dynamics were tissue-specific. A severe attack of Puccinia hordei occurring during the vegetative stage had profound effects on the mycobiome, and P. hordei biomass displaced that of other taxa. Plant tissue type was the most important factor determining the mycobiome, but also plant age was contributing significantly. Using a random forest model, we found that specific members of the mycobiome were responding differently to plant age, for instance, Olpidium and Articulospora in roots, Dioszegia and Sporobolomyces in leaves, Pyrenophora in stems, and Epicoccum in heads. A co-occurrence network analysis revealed complex interactions among fungal OTUs, and network connectivity was changing as per plant growth stage and plant tissue type. This study contributes to the understanding of assembly of fungal communities in cereals by providing a detailed description of fungal communities associated with barley. This knowledge will be vital for microbiome assisted plant health management and our study will serve as an important baseline for future efforts to harness microbiota in cereal health.

Rice diterpenoid phytoalexins are involved in defence against parasitic nematodes and shape rhizosphere nematode communities.

Rice diterpenoid phytoalexins (DPs) are secondary metabolites with a well known role in resistance to foliar pathogens. As DPs are also known to be produced and exuded by rice roots, we hypothesised that they might play an important role in plant-nematode interactions, and particularly in defence against phytoparasitic nematodes. We used transcriptome analysis on rice roots to analyse the effect of infection by the root-knot nematode Meloidogyne graminicola or treatment with resistance-inducing chemical stimuli on DP biosynthesis genes, and assessed the susceptibility of mutant rice lines impaired in DP biosynthesis to M. graminicola. Moreover, we grew these mutants and their wild-type in field soil and used metabarcoding to assess the effect of impairment in DP biosynthesis on rhizosphere and root nematode communities. We show that M. graminicola suppresses DP biosynthesis genes early in its invasion process and, conversely, that resistance-inducing stimuli transiently induce the biosynthesis of DPs. Moreover, we show that loss of DPs increases susceptibility to M. graminicola. Metabarcoding on wild-type and DP-deficient plants grown in field soil reveals that DPs significantly alter the composition of rhizosphere and root nematode communities. Diterpenoid phytoalexins are important players in basal and inducible defence against nematode pathogens of rice and help shape rice-associated nematode communities.

Distinct chemical resistance-inducing stimuli result in common transcriptional, metabolic, and nematode community signatures in rice root and rhizosphere.

Induced resistance (IR), a phenotypic state induced by an exogenous stimulus and characterized by enhanced resistance to future (a)biotic challenge, is an important component of plant immunity. Numerous IR-inducing stimuli have been described in various plant species, but relatively little is known about 'core' systemic responses shared by these distinct IR stimuli and the effects of IR on plant-associated microbiota. In this study, rice (Oryza sativa) leaves were treated with four distinct IR stimuli (ß-aminobutyric acid, acibenzolar-S-methyl, dehydroascorbic acid, and piperonylic acid) capable of inducing systemic IR against the root-knot nematode Meloidogyne graminicola and evaluated their effect on the root transcriptome and exudome, and root-associated nematode communities. Our results reveal shared transcriptional responses-notably induction of jasmonic acid and phenylpropanoid metabolism-and shared alterations to the exudome that include increased amino acid, benzoate, and fatty acid exudation. In rice plants grown in soil from a rice field, IR stimuli significantly affected the composition of rhizosphere nematode communities 3 d after treatment, but by 14 d after treatment these changes had largely reverted. Notably, IR stimuli did not reduce nematode diversity, which suggests that IR might offer a sustainable option for managing plant-parasitic nematodes.

Phytohormones selectively affect plant parasitic nematodes associated with Arabidopsis roots.

Phytohormones may affect plant-nematode interactions directly as chemo-attractants or -repellents, or indirectly through the root-associated microbiome or through host defense mechanisms. However, the exact roles of phytohormones in these complex plant-soil-nematode interactions are not well understood. We used Arabidopsis thaliana mutants impaired in phytohormone synthesis or sensitivity to elucidate their role in root-nematode interactions. As root-associated microorganisms may modulate these interactions, we explored correlations between the relative abundances of root-associated nematodes, and bacteria and fungi using amplicon sequencing. We found distinct shifts in relative abundances of a range of nematode taxa in the A. thaliana phytohormone mutants. The root knot nematode Meloidogyne hapla, a sedentary endoparasitic species that is in intimate contact with the host, was highly enriched in JA-, SA- and SL-impaired lines, and in an ET-insensitive line. Positive or negative correlations between specific microbial and nematode taxa were observed, but, as the inference of causal relationships between microbiome responses and effects on nematode communities is premature, this should be studied in detail in future studies. In conclusion, genetic derailment of hormonal balances generally rendered plants vulnerable to endoparasitic nematode attack. Furthermore, preliminary data suggest that this effect may be partially modulated by the associated microbiome.

Benzoxazinoids selectively affect maize root-associated nematode taxa.

Although the effects of plant secondary metabolites on plant defence have been studied for decades, the exact roles of secondary metabolites in shaping plant-associated microbial and nematode communities remain elusive. We evaluated the effects of benzoxazinoids, a group of secondary metabolites present in several cereals, on root-associated nematodes. We employed 18S rRNA metabarcoding to compare maize root-associated nematode communities in a bx1 knockout maize line impaired in benzoxazinoid synthesis and in its parental wild type. Both genotype and plant age affected the composition of the nematode community in the roots, and the effects of benzoxazinoids on nematode communities were stronger in the roots than in the rhizosphere. Differential abundance analysis and quantitative PCR showed that the root lesion nematode Pratylenchus neglectus was enriched in the bx1 mutant line, while another root lesion nematode, Pratylenchus crenatus, was reduced. Correlation analysis showed that benzoxazinoid concentrations in maize roots mostly correlated negatively with the relative abundance of nematode sequence reads. However, positive correlations between benzoxazinoids and nematode taxa, including several plant-parasitic nematodes, were also identified. Our detailed nematode community analysis suggests differential and selective effects of benzoxazinoids on soil nematodes depending on both the nematode species and the benzoxazinoid compound.

Comparative Genomics, Pangenome, and Phylogenomic Analyses of Brenneria spp., and Delineation of Brenneria izadpanahii sp. nov.

Brenneria species are bacterial plant pathogens mainly affecting woody plants. Association of all members with devastating disorders (e.g., acute oak decline in Iran and United Kingdom) are due to adaptation and pathogenic behavior in response to host and environmental factors. Some species, including B. goodwinii, B. salicis, and B. nigrifluens, also show endophytic residence. Here we show that all species including novel Brenneria sp. are closely related. Gene-based and genome/pangenome-based phylogeny divide the genus into two distinct lineages, Brenneria clades A and B. The two clades were functionally distinct and were consistent with their common and special potential activities as determined via annotation of functional domains. Pangenome analysis demonstrated that the core pathogenicity factors were highly conserved, an hrp gene cluster encoding a type III secretion system was found in all species except B. corticis. An extensive repertoire of candidate virulence factors was identified. Comparative genomics indicated a repertoire of plant cell wall degrading enzymes, metabolites/antibiotics, and numerous prophages providing new insights into Brenneria-host interactions and appropriate targets for further characterization. This work not only documented the genetic differentiation of Brenneria species but also delineates a more functionally driven understanding of Brenneria by comparison with relevant Pectobacteriaceae thereby substantially enriching the extent of information available for functional genomic investigations.

Fusarium Head Blight Modifies Fungal Endophytic Communities During Infection of Wheat Spikes.

Fusarium head blight (FHB) is a devastating disease of wheat heads. It is caused by several species from the genus Fusarium. Several endophytic fungi also colonize wheat spikes asymptomatically. Pathogenic and commensal fungi share and compete for the same niche and thereby influence plant performance. Understanding the natural dynamics of the fungal community and how the pre-established species react to pathogen attack can provide useful information on the disease biology and the potential use of some of these endophytic organisms in disease control strategies. Fungal community composition was assessed during anthesis as well as during FHB attack in wheat spikes during 2016 and 2017 in two locations. Community metabarcoding revealed that endophyte communities are dominated by basidiomycete yeasts before anthesis and shift towards a more opportunistic ascomycete-rich community during kernel development. These dynamics are interrupted when Fusarium spp. colonize wheat spikes. The Fusarium pathogens appear to exclude other fungi from floral tissues as they are associated with a reduction in community diversity, especially in the kernel which they colonize rapidly. Similarly, the presence of several endophytes was negatively correlated with Fusarium spp. and linked with spikes that stayed healthy despite exposure to the pathogen. These endophytes belonged to the genera Cladosporium, Itersonillia and Holtermanniella. These findings support the hypothesis that some naturally occurring endophytes could outcompete or prevent FHB and represent a source of potential biological control agents in wheat.

Molecular and biological characterisation of two novel pomo-like viruses associated with potato (Solanum tuberosum) fields in Colombia.

Potato is the fourth most important crop worldwide that is used as a staple food, after rice, wheat and maize. The crop can be affected by a large number of pathogens, including fungi, oomycetes, bacteria and viruses. Diseases caused by viruses are among the most important factors contributing to reduced quality and yield of the crop. Potato mop-top virus (genus Pomovirus) induces necrotic flecks in the tuber flesh and skin of potato in temperate countries. Spongospora subterranea is the vector of PMTV. Both the virus and its vector cause disease in potato. In Colombia, PMTV has been detected throughout the country together with a novel pomo-like virus in the centre (Cundinamarca and Boyacá) and south west (Nariño) of the country. We studied the molecular and biological characteristics of this novel virus. Its genome resembles those of members of the genus Pomovirus, and it is closely related to PMTV. It induces mild systemic symptoms in Nicotiana benthamiana (mosaic, branch curling), but no symptoms in N. tabacum, N. debneyi and Chenopodium amaranticolor. The proposed name for the virus is "Colombian potato soil-borne virus" (CPSbV). Additionally, another pomo-like virus was identified in Nariño. This virus induces severe systemic stem declining and mild mosaic in N. benthamiana. The tentative name "soil-borne virus 2" (SbV2) is proposed for this virus. No vectors have been identified for these viruses despite several attempts. This work focused on the characterisation of CPSbV. The risk posed by these viruses if they are introduced into new territories is discussed.

Effect of Verticillium dahliae Soil Inoculum Levels on Spinach Seed Infection.

Verticillium dahliae is a soilborne pathogen and a threat to spinach seed production. The aim of this study was to understand the relation between V. dahliae soil inoculum and infection in harvested seed. Quantitative polymerase chain reaction was used for quantification of the pathogen. Semifield experiments in which spinach was grown in soils with different inoculum levels enabled us to determine a threshold level for V. dahliae DNA of 0.003 ng/g of soil for seed infection to occur. Soils from production fields were sampled in 2013 and 2014 during and before planting, as well as the harvested seed. Seed from plants grown in infested soils were infected with V. dahliae in samples from both the semifield and open-field experiments. Lower levels of pathogen were found in seed from spinach grown in soils with a scattered distribution of V. dahliae (one or two positive of three soil subsamples) than in soils with a uniform distribution of the pathogen (three of three positive soil subsamples). Our results showed that infection of V. dahliae in harvested seed strongly depended on the presence of pathogen inoculum in the soil.

Host genotype is an important determinant of the cereal phyllosphere mycobiome.

The phyllosphere mycobiome in cereals is an important determinant of crop health. However, an understanding of the factors shaping this community is lacking. Fungal diversity in leaves from a range of cultivars of winter wheat (Triticum aestivum), winter and spring barley (Hordeum vulgare) and a smaller number of samples from oat (Avena sativa), rye (Secale cereale) and triticale (Triticum × Secale) was studied using next-generation sequencing. The effects of host genotype, fungicide treatment and location on fungal communities were explored. In total, 635 251 fungal internal transcribed spacer (ITS) reads were obtained from 210 leaf samples. Visual disease assessments and relative read abundance of Zymoseptoria tritici and Ramularia collo-cygni were strongly positively related. Crop genotype at the species level explained 43% of the variance in the total dataset, followed by fungicide treatment (13%) and location (4%). Indicator species, including plant pathogens, responding to factors such as crop species, location and treatment were identified. Host genotype at both the species and cultivar level is important in shaping phyllosphere fungal communities, whereas fungicide treatment and location have minor effects. We found many host-specific fungal pathogens, but also a large diversity of fungi that were relatively insensitive to host genetic background, indicating that host-specific pathogens live in a 'sea' of nonspecific fungi.

Diversity and evolution of potato mop-top virus.

Nearly complete sequences of RNA-CP and 3'-proximal RNA-TGB were determined for 43 samples of potato mop-top virus (PMTV) originating from potato tubers and field soil from Sweden, Denmark and the USA. The results showed limited diversity and no strict geographical grouping, suggesting only a few original introductions of PMTV from the Andes. Two distinguishable types of RNA-CP and RNA-TGB were found in the samples, but no specific combination of them correlated with spraing symptoms in tubers. Lack of positive selection in the coding sequences indicates that there is no specific molecular adaptation of PMTV to new vectors or hosts.

High-throughput sequencing of nematode communities from total soil DNA extractions.

BACKGROUND: Nematodes are extremely diverse and numbers of species are predicted to be more than a million. Studies on nematode diversity are difficult and laborious using classical methods and therefore high-throughput sequencing is an attractive alternative. Primers that have been used in previous sequence-based studies are not nematode specific but also amplify other groups of organisms such as fungi and plantae, and thus require a nematode enrichment step that may introduce biases. RESULTS: In this study an amplification strategy which selectively amplifies a fragment of the SSU from nematodes without the need for enrichment was developed. Using this strategy on DNA templates from a set of 22 agricultural soils, we obtained 64.4% sequences of nematode origin in total, whereas the remaining sequences were almost entirely from other metazoans. The nematode sequences were derived from a broad taxonomic range and most sequences were from nematode taxa that have previously been found to be abundant in soil such as Tylenchida, Rhabditida, Dorylaimida, Triplonchida and Araeolaimida. CONCLUSIONS: Our amplification and sequencing strategy for assessing nematode diversity was able to collect a broad diversity without prior nematode enrichment and thus the method will be highly valuable in ecological studies of nematodes.

Harnessing root-soil-microbiota interactions for drought-resilient cereals.

Cereal plants form complex networks with their associated microbiome in the soil environment. A complex system including variations of numerous parameters of soil properties and host traits shapes the dynamics of cereal microbiota under drought. These multifaceted interactions can greatly affect carbon and nutrient cycling in soil and offer the potential to increase plant growth and fitness under drought conditions. Despite growing recognition of the importance of plant microbiota to agroecosystem functioning, harnessing the cereal root microbiota remains a significant challenge due to interacting and synergistic effects between root traits, soil properties, agricultural practices, and drought-related features. A better mechanistic understanding of root-soil-microbiota associations could lead to the development of novel strategies to improve cereal production under drought. In this review, we discuss the root-soil-microbiota interactions for improving the soil environment and host fitness under drought and suggest a roadmap for harnessing the benefits of these interactions for drought-resilient cereals. These methods include conservative trait-based approaches for the selection and breeding of plant genetic resources and manipulation of the soil environments.

Early assessment of fungal and oomycete pathogens in greenhouse irrigation water using Oxford nanopore amplicon sequencing.

Water-borne plant pathogenic fungi and oomycetes are a major threat in greenhouse production systems. Early detection and quantification of these pathogens would enable us to ascertain both economic and biological thresholds required for a timely treatment, thus improving effective disease management. Here, we used Oxford nanopore MinION amplicon sequencing to analyze microbial communities in irrigation water collected from greenhouses used for growing tomato, cucumber and Aeschynanthus sp. Fungal and oomycete communities were characterized using primers that amplify the full internal transcribed spacer (ITS) region. To assess the sensitivity of the MinION sequencing, we spiked serially diluted mock DNA into the DNA isolated from greenhouse water samples prior to library preparation. Relative abundances of fungal and oomycete reads were distinct in the greenhouse irrigation water samples and in water samples from setups with tomato that was inoculated with Fusarium oxysporum. Sequence reads derived from fungal and oomycete mock communities were proportionate in the respective serial dilution samples, thus confirming the suitability of MinION amplicon sequencing for environmental monitoring. By using spike-ins as standards to test the reliability of quantification using the MinION, we found that the detection of spike-ins was highly affected by the background quantities of fungal or oomycete DNA in the sample. We observed that spike-ins having shorter length (538bp) produced reads across most of our dilutions compared to the longer spikes (>790bp). Moreover, the sequence reads were uneven with respect to dilution series and were least retrievable in the background samples having the highest DNA concentration, suggesting a narrow dynamic range of performance. We suggest continuous benchmarking of the MinION sequencing to improve quantitative metabarcoding efforts for rapid plant disease diagnostic and monitoring in the future.

Molecular identification of a new begomovirus associated with mosaic disease of Jatropha curcas L. in Nigeria.

We report the complete nucleotide sequence of DNA-A of a begomovirus naturally infecting Jatropha curcas L. in Nigeria. Symptoms observed on infected plants were severe mosaic, mottling and blistering of leaves. The virus, which we provisionally name "jatropha mosaic Nigeria virus" (JMNV), has a monopartite genome of 2,779 to 2,789 nucleotides. Pairwise comparisons of DNA-A sequences showed that JMNV had maximum nucleotide sequence identity (72%) with a strain of tomato yellow leaf curl virus. Since there are widespread infections of jatropha in Nigeria showing similar symptoms as those investigated in the present study, JMNV may represent a significant threat to a promising bioenergy crop.

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants.

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

Fusarium oxysporum Disrupts Microbiome-Metabolome Networks in Arabidopsis thaliana Roots.

While the plant host metabolome drives distinct enrichment of detrimental and beneficial members of the microbiome, the mechanistic interomics relationships remain poorly understood. Here, we studied microbiome and metabolome profiles of two Arabidopsis thaliana accessions after Fusarium oxysporum f.sp. mathioli (FOM) inoculation, Landsberg erecta (Ler-0) being susceptible and Col-0 being resistant against FOM. By using bacterial and fungal amplicon sequencing and targeted metabolite analysis, we observed highly dynamic microbiome and metabolome profiles across FOM host progression, while being markedly different between FOM-inoculated and noninoculated Col-0 and Ler-0. Co-occurrence network analysis revealed more robust microbial networks in the resistant Col-0 compared to Ler-0 during FOM infection. Correlation analysis revealed distinct metabolite-OTU correlations in Ler-0 compared with Col-0 which could possibly be explained by missense variants of the Rfo3 and Rlp2 genes in Ler-0. Remarkably, we observed positive correlations in Ler-0 between most of the analyzed metabolites and the bacterial phyla Proteobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, and Verrucomicrobia, and negative correlations with Actinobacteria, Firmicutes, and Chloroflexi. The glucosinolates 4-methyoxyglucobrassicin, glucoerucin and indole-3 carbinol, but also phenolic compounds were strongly correlating with the relative abundances of indicator and hub OTUs and thus could be active in structuring the A. thaliana root-associated microbiome. Our results highlight interactive effects of host plant defense and root-associated microbiota on Fusarium infection and progression. Our findings provide significant insights into plant interomic dynamics during pathogen invasion and could possibly facilitate future exploitation of microbiomes for plant disease control. IMPORTANCE Plant health and fitness are determined by plant-microbe interactions which are guided by host-synthesized metabolites. To understand the orchestration of this interaction, we analyzed the distinct interomic dynamics in resistant and susceptible Arabidopsis ecotypes across different time points after infection with Fusarium oxysporum (FOM). Our results revealed distinct microbial profiles and network resilience during FOM infection in the resistant Col-0 compared with the susceptible Ler-0 and further pinpointed specific microbe-metabolite associations in the Arabidopsis microbiome. These findings provide significant insights into plant interomics dynamics that are likely affecting fungal pathogen invasion and could possibly facilitate future exploitation of microbiomes for plant disease control.

Genetic disruption of Arabidopsis secondary metabolite synthesis leads to microbiome-mediated modulation of nematode invasion.

In-depth understanding of metabolite-mediated plant-nematode interactions can guide us towards novel nematode management strategies. To improve our understanding of the effects of secondary metabolites on soil nematode communities, we grew Arabidopsis thaliana genetically altered in glucosinolate, camalexin, or flavonoid synthesis pathways, and analyzed their root-associated nematode communities using metabarcoding. To test for any modulating effects of the associated microbiota on the nematode responses, we characterized the bacterial and fungal communities. Finally, as a proxy of microbiome-modulating effects on nematode invasion, we isolated the root-associated microbiomes from the mutants and tested their effect on the ability of the plant parasitic nematode Meloidogyne incognita to penetrate tomato roots. Most mutants had altered relative abundances of several nematode taxa with stronger effects on the plant parasitic Meloidogyne hapla than on other root feeding taxa. This probably reflects that M. hapla invades and remains embedded within root tissues and is thus intimately associated with the host. When transferred to tomato, microbiomes from the flavonoid over-producing pap1-D enhanced M. incognita root-invasion, whereas microbiomes from flavonoid-deficient mutants reduced invasion. This suggests microbiome-mediated effect of flavonoids on Meloidogyne infectivity plausibly mediated by the alteration of the abundances of specific microbial taxa in the transferred microbiomes, although we could not conclusively pinpoint such causative microbial taxa.

Phytoplasma PMU1 exists as linear chromosomal and circular extrachromosomal elements and has enhanced expression in insect vectors compared with plant hosts.

Phytoplasmas replicate intracellularly in plants and insects and are dependent on both hosts for dissemination in nature. Phytoplasmas have small genomes lacking genes for major metabolic pathways. Nevertheless, their genomes harbour multicopy gene clusters that were named potential mobile units (PMUs). PMU1 is the largest most complete repeat among the PMUs in the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB). PMU1 is c. 20 kb in size and contains 21 genes encoding DNA replication and predicted membrane-targeted proteins. Here we show that AY-WB has a chromosomal linear PMU1 (L-PMU1) and an extrachromosomal circular PMU1 (C-PMU1). The C-PMU1 copy number was consistently higher by in average approximately fivefold in insects compared with plants and PMU1 gene expression levels were also considerably higher in insects indicating that C-PMU1 synthesis and expression are regulated. We found that the majority of AY-WB virulence genes lie on chromosomal PMU regions that have similar gene content and organization as PMU1 providing evidence that PMUs contribute to phytoplasma host adaptation and have integrated into the AY-WB chromosome.