Molecular identification of ‘Candidatus Phytoplasma palmicola’ associated with coconut lethal yellowing in Equatorial Guinea

During the past two decades, a high mortality of coconut palms was observed in the coastal areas of Equatorial Guinea. Reportedly, the palm population has been reduced by 60%–70%, and coconut production has decreased accordingly. To identify the cause of the mortality, a survey was carried out in April 2021 in various localities of the coconut belt. Molecular analyses carried out on 16S rRNA and secA genes detected phytoplasma presence in the majority of the samples. Sequencing and BLAST search of the 16S rRNA gene sequences showed >99% identity of the detected phytoplasmas to ‘Candidatus Phytoplasma palmicola’. The RFLP analyses of 16S ribosomal gene using Tru1I and TaqI enzymes led to assign these phytoplasmas to subgroup 16SrXXII-A. In all samples that tested positive, including one from a hybrid coconut palm and two from oil palm the same phytoplasma was identified. The phylogenetic analyses of 16S rRNA and secA genes confirmed respectively 99.98%–100% and 97.94%–100% identity to ‘Ca. P. palmicola’. RFLP analyses using MboII enzyme on the secA gene amplicon differentiated the phytoplasma found in Equatorial Guinea from those present in Ghana and Ivory Coast. The Equatorial Guinean phytoplasma strain resulted to be identical to the strains from Mozambique, confirming the presence of a geographic differentiation among phytoplasma strains in the coastal areas of Western and Central Africa. The identified phytoplasma is different from the ‘Ca. P. palmicola’ strains found in Ghana and Ivory Coast and represents the first identification a 16SrXXII-A strain in Equatorial Guinea and in Central Africa. Strict monitoring and surveillance procedures for early detection of the pathogen are strongly recommended to reduce its impact and further spread in the country and permit the recovery of coconut plantations.     

The genome of ‘Candidatus Phytoplasma solani’ strain SA-1 is highly dynamic and prone to adopting foreign sequences

Bacteria of the genus ‘Candidatus Phytoplasma’ are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as ‘Ca. P. solani’, have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of ‘Ca. P. solani’ strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322 bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The ‘Ca. P. solani’ SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.     

Convenient Detection of the Citrus Greening (Huanglongbing) Bacterium ‘Candidatus Liberibacter asiaticus’ by Direct PCR from the Midrib Extract

A phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing.     

Validation of ‘Variable Number of Tandem Repeat’-Based Approach for Examination of ‘Candidatus Liberibacter asiaticus’ Diversity and Its Applications for the Analysis of the Pathogen Populations in the Areas of Recent Introduction

Citrus greening (Huanglongbing, HLB) is one of the most destructive diseases of citrus worldwide. In South Asia HLB has been known for more than a century, while in Americas the disease was found relatively recently. HLB is associated with three species of ‘Candidatus Liberibacter’ among which ‘Ca. Liberibacter asiaticus’ (CLas) has most wide distribution. Recently, a number of studies identified different regions in the CLas genome with variable number of tandem repeats (VNTRs) that could be used for examination of CLas diversity. One of the objectives of the work presented here was to further validate the VNTR analysis-based approach by assessing the stability of these repeats upon multiplication of the pathogen in a host over an extended period of time and upon its passaging from a host to a host using CLas populations from Florida. Our results showed that the numbers of tandem repeats in the four loci tested display very distinguishable “signature profiles” for the two Florida-type CLas haplotype groups. Remarkably, the profiles do not change upon passage of the pathogen in citrus and psyllid hosts as well as after its presence within a host over a period of five years, suggesting that VNTR analysis-based approach represents a valid methodology for examination of the pathogen populations in various geographical regions. Interestingly, an extended analysis of CLas populations in different locations throughout Florida and in several countries in the Caribbean and Central America regions and in Mexico where the pathogen has been introduced recently demonstrated the dispersion of the same haplotypes of CLas. On the other hand, these CLas populations appeared to differ significantly from those obtained from locations where the disease has been present for a much longer time.     

McrA primers for the detection and quantification of the anaerobic archaeal methanotroph ‘Candidatus Methanoperedens nitroreducens’

Applied Microbiology and Biotechnology - The nitrogen and methane cycles are important biogeochemical processes. Recently, ‘Candidatus Methanoperedens nitroreducens,’ archaea that...     

Unique Features of a Japanese ‘Candidatus Liberibacter asiaticus’ Strain Revealed by Whole Genome Sequencing

Citrus greening (huanglongbing) is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with phloem-limited bacteria of three species of α-Proteobacteria, namely, ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’. Recent findings suggested that some Japanese strains lack the bacteriophage-type DNA polymerase region (DNA pol), in contrast to the Floridian psy62 strain. The whole genome sequence of the pol-negative ‘Ca. L. asiaticus’ Japanese isolate Ishi-1 was determined by metagenomic analysis of DNA extracted from ‘Ca. L. asiaticus’-infected psyllids and leaf midribs. The 1.19-Mb genome has an average 36.32% GC content. Annotation revealed 13 operons encoding rRNA and 44 tRNA genes, but no typical bacterial pathogenesis-related genes were located within the genome, similar to the Floridian psy62 and Chinese gxpsy. In contrast to other ‘Ca. L. asiaticus’ strains, the genome of the Japanese Ishi-1 strain lacks a prophage-related region.     

Physiological and genomic characterization of a new ‘Candidatus Nitrotoga’ isolate

Oxidation of nitrite to nitrate is an important process in the global nitrogen cycle. Recent molecular biology-based studies have revealed that the widespread nitrite-oxidizing bacteria (NOB) belonging to the genus ‘Candidatus Nitrotoga’ may be highly important for the environment. However, the insufficient availability of pure Nitrotoga cultures has limited our understanding of their physiological and genomic characteristics. Here, we isolated the ‘Ca. Nitrotoga’ sp. strain AM1P, from a previously enriched Nitrotoga culture, using an improved isolation strategy. Although ‘Ca. Nitrotoga’ have been recognized as cold-adapted NOB, the strain AM1P had a slightly higher optimum growth temperature at 23°C. Strain AM1P showed a pH optimum of 8.3 and was not inhibited even at high nitrite concentrations (20 mM). We obtained the complete genome of the strain and compared the genome profile to five previously sequenced ‘Ca. Nitrotoga’ strains. Comparative genomics suggested that lactate dehydrogenase may be only encoded in the strain AM1P and closely related genomes. While the growth yield of AM1P did not change, we observed faster growth in the presence of lactate in comparison to purely chemolithoautotrophic growth. The characterization of the new strain AM1P sheds light on the physiological adaptation of this environmentally important, but understudied genus ‘Ca. Nitrotoga’.     

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F₉ MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.     

Genomics and phylogeny of the proposed phylum ‘Candidatus Poribacteria’ associated with the excavating sponge Thoosa mismalolli

Members of the proposed phylum ‘Candidatus Poribacteria’ are among the most abundant microorganisms in the highly diverse microbiome of the sponge mesohyl. Genomic and phylogenetic characteristics of this proposed phylum are barely known. In this study, we analyzed metagenome-assembled genomes (MAGs) obtained from the coral reef excavating sponge Thoosa mismalolli from the Mexican Pacific Ocean. Two MAGs were extracted and analyzed together with 32 MAGs and single-amplified genomes (SAGs) obtained from NCBI. The phylogenetic tree based on the sequences of 139 single-copy genes (SCG) showed two clades. Clade A (23 genomes) represented 67.7% of the total of the genomes, while clade B (11 genomes) comprised 32.3% of the genomes. The Average Nucleotide Identity (ANI) showed values between 66 and 99% for the genomes of the proposed phylum, and the pangenome of genomes revealed a total of 37,234 genes that included 1722 core gene. The number of genes used in the phylogenetic analysis increased from 28 (previous studies) to 139 (this study), which allowed a better resolution of the phylogeny of the proposed phylum. The results supported the two previously described classes, ‘Candidatus Entoporibacteria’ and ‘Candidatus Pelagiporibacteria’, and the genomes SB0101 and SB0202 obtained in this study belong to two new species of the class ‘Candidatus Entoporibacteria’. This is the first comparative study that includes MAGs from a non-sponge host (Porites lutea) to elucidate the taxonomy of the poorly known Candidatus phylum in a polyphasic approach. Finally, our study also contributes to the sponge microbiome project by reporting the first MAGs of the proposed phylum ‘Candidatus Poribacteria’ isolated from the excavating sponge T. mismalolli.

     

Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province,Saudi Arabia

Typical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400?bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.     

Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees

‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘Ca. Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘Ca. Phytoplasma mali’ in order to develop sustainable approaches for managing AP.     

The flagella of ‘Candidatus Liberibacter asiaticus’ and its movement in planta

Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJ_Atu but not flgJ_Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJ_Las interacts with FlgB but not with FliE. Ectopic expression of flaA_Las in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.     

Effect of light wavelength on motility and magnetic sensibility of the magnetotactic multicellular prokaryote ‘Candidatus Magnetoglobus multicellularis’

‘Candidatus Magnetoglobus multicellularis’ is a magnetotactic microorganism composed of several bacterial cells. Presently, it is the best known multicellular magnetotactic prokaryote (MMP). Recently, it has been observed that MMPs present a negative photoresponse to high intensity ultraviolet and violet-blue light. In this work, we studied the movement of ‘Candidatus Magnetoglobus multicellularis’ under low intensity light of different wavelengths, measuring the average velocity and the time to reorient its trajectory when the external magnetic field changes its direction (U-turn time). Our results show that the mean average velocity is higher for red light (628 nm) and lower for green light (517 nm) as compared to yellow (596 nm) and blue (469 nm) light, and the U-turn time decreased for green light illumination. The light wavelength velocity dependence can be understood as variation in flagella rotation speed, being increased by the red light and decreased by the green light relative to yellow and blue light. It is suggested that the dependence of the U-turn time on light wavelength can be considered a form of light-dependent magnetotaxis, because this time represents the magnetic sensibility of the magnetotactic microorganisms. The cellular and molecular mechanisms for this light-dependent velocity and magnetotaxis are unknown and deserve further studies to understand the biochemical interactions and the ecological roles of the different mechanisms of taxis in MMPs.     

Correlation Analysis of Differences of Photoinhibitory Sensitivity of D1 Proteins in Oryza sativa ssp. japonica and indica and Structural Features of the Sequences of the Coding Genes

Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psbA , the gene encoding D1 protein of O. sativa ssp. japonica cv. 9516, and that of O. sativa ssp. indica cv. Shanyou 63 was cloned. As revealed by homology comparison of their sequences, the sequences are identical in the regions of promoter and 5′-UTR; differences are found in individual bases in the coding region all of which, being in the third position of respective codons, however, do not affect the amino acids coded finally; a difference is noted in the length of the oligo-U sequence in the region of 3′-UTR. It is thus apparent that, rather than a result of any difference in the amino acid sequences, the differences in the sensitivity to photoinhibition of D1 proteins between japonica and indica rice may be related to the upstream factors that regulate expression of psbA or to differences of photoprotective mechanisms.     

Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators?

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO₂ as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.     

Genome-Wide Identification and Analysis of MicroRNAs Involved in Witches’-Broom Phytoplasma Response in Ziziphus jujuba

MicroRNAs (miRNAs) play an important role in responding to biotic and abiotic stresses in plants. Jujube witches’-broom a phytoplasma disease of Ziziphus jujuba is prevalent in China and is a serious problem to the industry. However, the molecular mechanism of the disease is poorly understood. In this study, genome-wide identification and analysis of microRNAs in response to witches’-broom was performed. A total of 85 conserved miRNA unique sequences belonging to 32 miRNA families and 24 novel miRNA unique sequences, including their complementary miRNA* strands were identified from small RNA libraries derived from a uninfected and witches’-broom infected Z. jujuba plant. Differentially expressed miRNAs associated with Jujube witches’-broom disease were investigated between the two libraries, and 12 up-regulated miRNAs and 10 down- regulated miRNAs identified with more than 2 fold changes. Additionally, 40 target genes of 85 conserved miRNAs and 49 target genes of 24 novel miRNAs were predicted and their putative functions assigned. Using the modified 5’-RACE method, we confirmed that SPL and MYB were cleaved by miR156 and miR159, respectively. Our results provide insight into the molecular mechanisms of witches’-broom disease in Z. jujuba.     

The effect of methane and methanol on the terrestrial ammonia-oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’

The ammonia monooxygenase (AMO) is a key enzyme in ammonia-oxidizing archaea, which are abundant and ubiquitous in soil environments. The AMO belongs to the copper-containing membrane monooxygenase (CuMMO) enzyme superfamily, which also contains particulate methane monooxygenase (pMMO). Enzymes in the CuMMO superfamily are promiscuous, which results in co-oxidation of alternative substrates. The phylogenetic and structural similarity between the pMMO and the archaeal AMO is well-established, but there is surprisingly little information on the influence of methane and methanol on the archaeal AMO and terrestrial nitrification. The aim of this study was to examine the effects of methane and methanol on the soil ammonia-oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus C13’. We demonstrate that both methane and methanol are competitive inhibitors of the archaeal AMO. The inhibition constants (K_i) for methane and methanol were 2.2 and 20 μM, respectively, concentrations which are environmentally relevant and orders of magnitude lower than those previously reported for ammonia-oxidizing bacteria. Furthermore, we demonstrate that a specific suite of proteins is upregulated and downregulated in ‘Ca. Nitrosocosmicus franklandus C13’ in the presence of methane or methanol, which provides a foundation for future studies into metabolism of one-carbon (C1) compounds in ammonia-oxidizing archaea.     

Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sapporoensis’

We successfully enriched a novel anaerobic ammonium-oxidizing (anammox) bacterium affiliated with the genus ‘Candidatus Brocadia’ with high purity (>90%) in a membrane bioreactor (MBR). The enriched bacterium was distantly related to the hitherto characterized ‘Ca. Brocadia fulgida’ and ‘Ca. Brocadia sinica’ with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The bacterium exhibited the common structural features of anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of anammox activity was 20–45 °C with a maximum activity at 37 °C. The maximum specific growth rate (μ_max) was 0.0082 h^?1 at 37 °C, corresponding to a doubling time of 3.5 days. The half-saturation constant (K_S) for nitrite was 5 ± 2.5 μM. The anammox activity was inhibited by nitrite (IC₅₀ = 11.6 mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched anammox bacterium shared nearly half of genes with ‘Ca. Brocadia sinica’ and ‘Ca. Brocadia fulgida’. The enriched bacterium showed all known physiological characteristics of anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name ‘Ca. Brocadia sapporoensis’ sp. nov.