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 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.     

Identification of four distinct ‘Candidatus Phytoplasma’ species in pomegranate trees showing witches' broom,little leaf and yellowing in Jordan,and preliminary insights on their putative insect vectors and reservoir plants

During field surveys conducted in northern Jordan from June to November 2020, phytoplasma-like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non-crop plants (Plantago major and Capsicum annuum) resulted hosting ‘Ca. P. asteris’ strains, and one cicadellid (Balclutha incisa) was carrying a ‘Ca. P. solani’ strain. A new pomegranate disease complex associated with multiple phytoplasmas, including ‘Ca. P. aurantifolia’ and ‘Ca. P. ulmi’, never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country.     

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’.     

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.     

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.     

Association of CXCL10 and CXCL13 levels with disease activity and cutaneous manifestation in active adult-onset Still’s disease

IntroductionC-X-C motif chemokine 10 (CXCL10) is produced in response to interferon-γ, and tumor necrosis factor-α (TNF-α) triggers the accumulation of activated lymphocytes. CXCL13 is constitutively expressed in secondary lymphoid tissues, and the expression is upregulated by TNF-α, via T cell stimulation. It appears that CXCL10 and CXCL13 could play a potential role in the pathogenesis of adult-onset Still’s disease (AOSD), therefore, we investigated the associations between CXCL10 and CXCL13 levels and clinical manifestations in patients with active AOSD.MethodsBlood samples were collected from 39 active AOSD patients, 32 rheumatoid arthritis (RA) patients and 40 healthy controls (HC). Of the AOSD patients, follow-up samples were collected from 15 9.6 ± 9.2 months later. Serum levels of CXCL10 and CXCL13 were determined using enzyme-linked immunosorbent assay. CXCL10, CXCL13, and C-X-C chemokine receptor type 3 (CXCR3) expression levels in biopsy specimens obtained from 26 AOSD patients with skin rashes were investigated via immunohistochemistry.ResultsThe CXCL10 levels in AOSD patients (1,031.3 ± 2,019.6 pg/mL) were higher than in RA (146.3 ± 91.4 pg/mL, p = 0.008) and HC (104.4 ± 47.9 pg/mL, p = 0.006). Also, the CXCL13 levels of AOSD patients (158.8 ± 151.2 pg/mL) were higher than those of RA (54.4 ± 61.1 pg/mL, p < 0.001) and HC (23.5 ± 18.1 pg/mL, p < 0.001). Serum CXCL10 levels correlated with ferritin and systemic scores. Serum CXCL13 levels correlated with those of hemoglobin, C-reactive protein, ferritin, and albumin, and systemic scores. In follow-up AOSD patients, the levels of CXCL10 and CXCL13 fell significantly (153.7 ± 130.1 pg/mL, p = 0.002, and 89.1 ± 117.4 pg/mL, p = 0.001, respectively). On immunohistochemistry, the percentages of inflammatory cells expressing CXCL10 ranged from 1 to 85 %, CXCL13 from 1 to 72 %, and CXCR3 from 2 to 65 %. The percentage of CXCL10-positive inflammatory cells was higher in skin biopsy samples exhibiting mucin deposition than in those that did not (p = 0.01). CXCL13 levels were correlated with those of CD4 and CD68.ConclusionsSerum CXCL10 and CXCL13 levels may serve as clinical markers for assessment of disease activity in AOSD. CXCL10/CXCR3 and CXCL13 may contribute to the inflammatory response, especially skin manifestations thereof, in AOSD.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0773-4) contains supplementary material, which is available to authorized users.     

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.     

Incidence of and Risk Factors Associated with Pulmonary and Extra-Pulmonary Tuberculosis in Saudi Arabia (2010–2011)

Setting

National Tuberculosis Program, Department of Public Health, Ministry of Health, Kingdom of Saudi Arabia (KSA).

Objective

To summarize data on the incidence of tuberculosis and associated risk factors for cases reported during 2010–2011.

Design

Retrospective analysis of routinely collected data through an established national disease notification system of the Ministry of Health in KSA.

Results

The estimated incidence of all forms of tuberculosis fell from 15.8/100000 (95% CI: 15.3/100,000–16.3/100,000) in 2010 to 13.8/100,000 (95% CI: 13.4/100,000–14.2/100,000) in 2011. Saudis experienced a decrease from 11.8/100,000 (95% CI: 11.3/100,000 to 12.3/100,000) in 2010 to 9.9/100,000 (95% CI: 9.5/100,000–10.4/100,000) in 2011 while the incidence in non-Saudis declined from 24.7/100,000 (95% CI: 23.6/100,000 to 25.7/100,000) in 2010 to 22.5/100,000 (95% CI: 21.5/100,000 to 23.4/100,000) in 2011. The proportion of Extra Pulmonary TB (EPTB) which increased minimally from 30% in 2010 to 32% in 2011 was higher than global figures and strongly associated with age, sex, nationality and occupation.

Conclusion

The current estimated incidence of about 14/100,000 in 2011 is less than half its estimated value of 44/100000 in 1990. Without prejudice to any under-reporting, the KSA appeared to be on the course for TB elimination by 2050 having reached the first milestone set by WHO. The proportion of EPTB remains higher than global figure and age, sex, nationality and occupation were significant independent predictors of EPTB.     

Breeding biology of Saunders’s tern (Sterna saundersi) in the Farasan Islands,Kingdom of Saudi Arabia

Saunders’s terns (Sternula saundersi) are a small, ground-nesting marine bird species that have a massive rearing range, including the shores and islands of Asia and Africa adjacent to the north Indian Ocean. Despite occupying a large breeding range, little is known about the breeding ecology of this species. This research explored aspects of Saunders’s terns’ breeding ecology and predation rate in 2013 on the Farasan Islands of Saudi Arabia. The outcomes confirm that the mean clutch size of a Saunders’s tern was 1.77 ± 0.08 (n = 31) eggs per clutch and the mean egg size was 31.05 × 23.15 mm. The results of this study show a remarkable relationship between clutch size and egg volume and length (p = .002, p = .004, respectively). Predation was the major reason for nest damage (62.5%). Evidence from cameras at nests showed that the predators of Saunders’s tern nests on the Farasan Islands were white-tailed mongoose (Ichneumia albicauda) and Egyptian vultures (Neophron percnopterus). This is the first study on the breeding ecology of Saunders’s terns, and it shows that predator control is essential to the existence of the species. The results of this study suggest that fencing some breeding sites may help to minimize human disturbance and decrease the risk of nest predation from mammalian predators. Further research is needed to compare the predation rates on the mainland and islands and to develop efficient strategies to conserve this ground-nesting species.     

First record of the ant subfamily Aenictinae (Hymenoptera,Formicidae) from Saudi Arabia,with?the description of a new species

The ant subfamily Aenictinae is recorded for the first time from the Kingdom of Saudi Arabia and for the second time from the Arabian Peninsula. A new species Aenictus arabicus sp. n., is described from the worker caste. Aenictus arabicus belongs to the Aenictus wroughtonii-group and appears to be most closely related to Aenictus rhodiensis Menozzi, but can be easily distinguished from the latter by the following characters: overall smaller size; cephalic index (head width/head length) small; occipital corners in lateral view rounded; antennal scape when laid back surpassing approximately two-thirds of head length; funicular segments 2–8 each at least 2× as long as broad; subpetiolar process well developed; petiole and postpetiole distinctly imbricate; gaster and clypeus entirely yellow, teeth of mandibles reddish- brown. Aenictus arabicus was collected from leaf litter, next to a tree of Psidium guajava L. The new species also is similar to Aenictus sagei and Aenictus wroughtonii. Affinities and a key to related species of the species group are given.     

A preliminary study on the insect fauna of Al-Baha Province,Saudi Arabia,with descriptions?of?two?new?species

A preliminary study was carried out on the insect fauna of Al-Baha Province, south-western part of Saudi Arabia. A total number of 582 species and subspecies (few identified only to the genus level) belonging to 129 families and representing 17 orders were recorded. Two of these species are described as new, namely: Monomorium sarawatensis Sharaf & Aldawood, sp. n. [Formicidae, Hymenoptera] and Anthrax alruqibi El-Hawagry sp. n. [Bombyliidae, Diptera]. Another eight species are recorded for the first time in Saudi Arabia, namely: Xiphoceriana arabica (Uvarov, 1922) [Pamphagidae, Orthoptera], Pyrgomorpha conica (Olivier, 1791) [Pyrgomorphidae, Orthoptera], Catopsilia florella (Fabricius, 1775) [Pieridae, Lepidoptera], Anthrax chionanthrax (Bezzi, 1926) [Bombyliidae, Diptera], Spogostylum near tripunctatum Pallas in Wiedemann, 1818 [Bombyliidae, Diptera], Cononedys dichromatopa (Bezzi, 1925) [Bombyliidae, Diptera], Mydas sp. [Mydidae, Diptera], and Hippobosca equina Linnaeus, 1758 [Hippoboscidae, Diptera]. Al-Baha Province is divided by huge and steep Rocky Mountains into two main sectors, a lowland coastal plain at the west, known as “Tihama”, and a mountainous area with an elevation of 1500 to 2450 m above sea level at the east, known as “Al-Sarat or Al-Sarah” which form a part of Al-Sarawat Mountains range. Insect species richness in the two sectors (Tihama and Al-Sarah) was compared, and the results showed that each of the two sectors of Al-Baha Province has a unique insect community. The study generally concluded that the insect faunal composition in Al-Baha Province has an Afrotropical flavor, with the Afrotropical elements predominant, and a closer affiliation to the Afrotropical region than to the Palearctic region or the Eremic zone. Consequently, we tend to agree with those biogeographers who consider that parts of the Arabian Peninsula, including Al-Baha Province, should be included in the Afrotropical region rather than in the Palaearctic region or the Eremic zone.     

Study of dynamic microcirculatory problems in ‘blackfoot disease’ -emphasizing its differences from arteriosclerosis

The cutaneous microcirculation can be divided into thermoregulatory shunt vessels and nutritive skin capillaries. Flux in nonnutritional shunt vessels dominates the signal recorded by the laser Doppler flowmeter. Computerized videophotometric capillaroscopy is a sensitive method for assessing cutaneous nutritive microcirculation. Using patients with blackfoot disease and arteriosclerosis as disease models, we evaluated the sensitivity and clinical usefulness of these relatively new techniques for peripheral vascular disorders. In blackfoot disease, blood flux measured by the laser Doppler flowmeter in the affected toe was lower than that in the nonaffected toe. In symptom-free fingers, blood flow was not significantly different between blackfoot disease and arteriosclerosis. However, blood flow in both diseases was lower than that of the control group. Patients who had the same status of thermoregulatory flow and eyeground arteriosclerotic classification underwent a 1-min arterial occlusion of the digits. The postocclusive reactive hyperemia response (PRH) of nailfold capillary loops was evaluated. All parameters for PRH for the cutaneous nutrient microcirculation including resting capillary blood cell velocity (rCBV), peak capillary blood cell velocity (pCBV) and time to pCBV were more significantly disturbed in the blackfoot disease group than in the arteriosclerotic group. On the basis of the results of this study, dynamic capillaroscopy provides a new approach for the early detection of circulatory disturbances resulting from different mechanisms.     

The ‘requirement of total evidence’ and its role in phylogenetic systematics

The question of whether or not to partition data for the purposes of inferring phylogenetic hypotheses remains controversial. Opinions have been especially divided since Kluge's (1989, Systematic Zoology 38, 7–25) claim that data partitioning violates the requirement of total evidence (RTE). Unfortunately, advocacy for or against the RTE has not been based on accurate portrayals of the requirement. The RTE is a basic maxim for non-deductive inference, stipulating that evidence must be considered if it has relevance to an inference. Evidence is relevant if it has a positive or negative effect on a given conclusion. In the case of ℈partitioned’ phylogenetic inferences, the RTE is violated, and the basis for rational belief in any conclusion is compromised, unless it is shown that the partitions are evidentially irrelevant to one another. The goal of phylogenetic systematics is to hypothesize past causal conditions to account for observed shared similarities among two or more species. Such inferences are non-deductive, necessitating consideration of the RTE. Some phylogeneticists claim the parsimony criterion as justification for the RTE. There is no relation between the two – parsimony is a relation between a hypothesis and causal question(s). Parsimony does not dictate the content of premises prior to an inference. ℈Taxonomic congruence,’ ℈supertrees,’ and ℈conditional combination’ methods violate the RTE. Taxonomic congruence and supertree methods also fail to achieve the intended goal of phylogenetic inference, such that ℈consensus trees’ and ℈supertrees’ lack an empirical basis. ℈Conditional combination’ is problematic because hypotheses derived from partitioned data cannot be compared – a causal hypothesis inferred to account for a set of effects only has relevance to those effects, not any comparative relevance to other causal hypotheses. A similar problem arises in the comparisons of hypotheses derived from different causal theories.