During field surveys carried out from June to October 2020 and in January 2021 in orchards of northern Jordan, phytoplasma-like symptoms (early flowering along with evergreen pattern; witches’-broom, ...yellowing, and dieback; slim leaf and leaf rolling; stem fasciation) were observed in almond trees. In 23 investigated orchards, symptomatic almond trees ranged from 20 to 85%. PCR-based amplification of 16S rRNA gene detected phytoplasmas in 21% of 140 collected symptomatic almond trees. Sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma asteris’ (taxonomic subgroups 16SrI-B and -R), ‘Ca. P. aurantifolia’ (16SrII-B and -C), ‘Ca. P. omanense’ (16SrXXIX-A and -B), ‘Ca. P. phoenicium’ (16SrIX-B), ‘Ca. P. pyri’ (16SrX-C), ‘Ca. P. solani’ (16SrXII-A), and ‘Ca. P. ulmi’ (16SrV-A). Such phytoplasmas were found associated with specific symptoms and differentially distributed in the considered locations. Moreover, further investigation identified ‘Ca. P. asteris’ (subgroup 16SrI-R) in putative insect vectors such as Empoasca sp., Reptalus sp., and Hyalesthes obsoletus, ‘Ca. P. pyri’ in Cacopsylla bidens, and ‘Ca. P. omanense’ (subgroup 16SrXXIX-B) in the non-crop plant Amaranthus sp. In conclusion, this study described an almond disease complex associated with multiple phytoplasmas, including ‘Ca. P. pyri’, ‘Ca. P. omanense’, and ‘Ca. P. ulmi’ that were never reported before in this crop. Further studies are needed to survey the diffusion of this almond disease complex in the region, demonstrate the transmission capability of the identified putative vectors, and in-depth investigate the ecologies of all phytoplasmas associated with the disease.
•Seven ʻCandidatus Phytoplasmaʼ species were reported in Jordan in association with an almond disease complex.•ʻCa. P. omanenseʼ, ʻCa. P. ulmi’, and ʻCa. P. pyriʼ were firstly reported in almond around the world.•New subgroup XXIX-B was described.•Eight insect taxa were found positive to ʻCa. Phytoplasma pyriʼ and ʻCa. P. asterisʼ.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Phytoplasmas are insect-transmitted, uncultivable bacterial plant pathogens that cause diseases in hundreds of economically important plants. They represent a monophyletic group within the class ...Mollicutes (trivial name mycoplasmas) and are characterized by a small genome with a low GC content, and the lack of a firm cell wall. All mycoplasmas, including strains of 'Candidatus (Ca.) Phytoplasma asteris' and 'Ca. P. australiense', examined so far have circular chromosomes, as is the case for almost all walled bacteria.
Our work has shown that 'Ca. Phytoplasma mali', the causative agent of apple proliferation disease, has a linear chromosome. Linear chromosomes were also identified in the closely related provisional species 'Ca. P. pyri' and 'Ca. P. prunorum'. The chromosome of 'Ca. P. mali' strain AT is 601,943 bp in size and has a GC content of 21.4%. The chromosome is further characterized by large terminal inverted repeats and covalently closed hairpin ends. Analysis of the protein-coding genes revealed that glycolysis, the major energy-yielding pathway supposed for 'Ca. P. asteris', is incomplete in 'Ca. P. mali'. Due to the apparent lack of other metabolic pathways present in mycoplasmas, it is proposed that maltose and malate are utilized as carbon and energy sources. However, complete ATP-yielding pathways were not identified. 'Ca. P. mali' also differs from 'Ca. P. asteris' by a smaller genome, a lower GC content, a lower number of paralogous genes, fewer insertions of potential mobile DNA elements, and a strongly reduced number of ABC transporters for amino acids. In contrast, 'Ca. P. mali' has an extended set of genes for homologous recombination, excision repair and SOS response than 'Ca. P. asteris'.
The small linear chromosome with large terminal inverted repeats and covalently closed hairpin ends, the extremely low GC content and the limited metabolic capabilities reflect unique features of 'Ca. P. mali', not only within phytoplasmas, but all mycoplasmas. It is expected that the genome information obtained here will contribute to a better understanding of the reduced metabolism of phytoplasmas, their fastidious nutrition requirements that prevented axenic cultivation, and the mechanisms involved in pathogenicity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological ...properties, group 16SrXII encompasses several species, including ‘Candidatus Phytoplasma australiense’, ‘Candidatus Phytoplasma japonicum’ and ‘Candidatus Phytoplasma fragariae’. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, ‘Candidatus Phytoplasma solani’, but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the ‘Candidatus Phytoplasma’ genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated (‘Ca. Phytoplasma solani’) strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. ‘Ca. Phytoplasma solani’ is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse ‘Ca. Phytoplasma solani’ strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV–rpsC gene sequences supported this view and yielded congruent trees in which ‘Ca. Phytoplasma solani’ strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of ‘Ca. Phytoplasma australiense’-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, ‘Ca. Phytoplasma solani’; STOL11 is designated the reference strain.
JWB phytoplasma is a kind of insect-transmitted and uncultivable bacterial plant pathogen causeing a destructive Jujube disease. To date, no genome information about JWB phytoplasma has been ...published, which hindered its characterization at genomic level. To understand its pathogenicity and ecology, the genome of a JWB phytoplasma isolate jwb-nky was sequenced and compared with other phytoplasmas enabled us to explore the mechanisms of genomic rearrangement.
The complete genome sequence of JWB phytoplasma (jwb-nky) was determined, which consisting of one circular chromosome of 750,803 bp with a GC content of 23.3%. 694 protein-encoding genes, 2 operons for rRNA genes and 31 tRNA genes as well as 4 potential mobile units (PMUs) containing clusters of DNA repeats were identified. Based on PHIbaes analysis, a large number of genes were genome-specific and approximately 13% of JWB phytoplasma genes were predicted to be associated with virulence. Although transporters for maltose, dipeptides/oligopeptides, spermidine/putrescine, cobalt, Mn/Zn and methionine were identified, KEGG pathway analysis revealed the reduced metabolic capabilities of JWB phytoplasma. Comparative genome analyses between JWB phytoplasma and other phytoplasmas shows the occurrence of large-scale gene rearrangements. The low synteny with other phytoplasmas indicated that the expansion of multiple gene families/duplication probably occurred separately after differentiation.
In this study, the complete genome sequence of a JWB phytoplasma isolate jwb-nky that causing JWB disease was reported for the first time and a number of species-specific genes were identified in the genome. The study enhanced our understandings about genomic basis and the pathogenicity mechanism of this pathogen, which will aid in the development of improved strategies for efficient management of JWB diseases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Field surveys were conducted in wine and table grape vineyards from June to October 2020 in 13 locations belonging to five governorates in North and South Jordan. Typical grapevine yellows ...symptoms, including leaf reddening/yellowing and rolling were observed on 10% to 55% of vines. Nested PCR‐based amplification of the 16S rRNA gene detected phytoplasmas in 22% and 15.7% of the analysed symptomatic wine and table grape cultivar plants, respectively. Amplicon nucleotide sequence analyses identified the detected phytoplasmas as “
Candidatus
Phytoplasma solani” (taxonomic subgroup 16SrXII‐A), “
Ca
. P. omanense” (16SrXXIX‐A and ‐B), “
Ca
. P. aurantifolia” (16SrII‐C) and “
Ca
. P. asteris” (16SrI‐R) in 72.4%, 17.2%, 6.9% and 3.4% of infected plants, respectively. Such phytoplasmas were found differentially distributed in wine and table grape cultivar vineyards surveyed. Further investigation identified “
Ca
. P. solani” in the putative insect vectors
Orosius cellulosus
(first report in Jordan),
Euscelidius mundus
,
Laodelphax striatellus
, and
Circulifer
sp., and in bindweed; “
Ca
. P. aurantifolia” in the insect
O. cellulosus
and in bindweed; “
Ca
. P. omanense” in the insect
Psammotettix striatus
; and “
Ca
. P. asteris” in the insects
Arboridia adanae
,
Cicadulina bipunctata
,
Circulifer
sp.,
L. striatellus
,
Hyalesthes obsoletus
, and
P. striatus
. Based on this preliminary data, ecological cycles of such phytoplasmas are discussed. Results suggest that the diversity and ecology of grapevine yellows phytoplasmas in Jordan are more complex than previously known, leading to a potential risk of disease outbreaks.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Bois noir, a disease of the grapevine yellows complex, is associated with 'Candidatus Phytoplasma solani' and transmitted to grapevines in open fields by the cixiids Hyalesthes obsoletus and Reptalus ...panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing by stamp gene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized by stamp gene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi, Dicranotropis hamata, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Laodelphax striatella, Philaenus spumarius, and Psammotettix alienus/confinis) are alternative vectors of 'Candidatus Phytoplasma solani' to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Flower malformation represented by phyllody is a common symptom of phytoplasma infection induced by a novel family of phytoplasma effectors called phyllogens. Despite the accumulation of functional ...and structural phyllogen information, the molecular mechanisms of phyllody have not yet been integrated with their evolutionary aspects due to the limited data on their homologs across diverse phytoplasma lineages. Here, we developed a novel universal PCR‐based approach to identify 25 phytoplasma phyllogens related to nine “Candidatus Phytoplasma” species, including four species whose phyllogens have not yet been identified. Phylogenetic analyses showed that the phyllogen family consists of four groups (phyl‐A, ‐B, ‐C, and ‐D) and that the evolutionary relationships of phyllogens were significantly distinct from those of phytoplasmas, suggesting that phyllogens were transferred horizontally among phytoplasma strains and species. Although phyllogens belonging to the phyl‐A, ‐C, and ‐D groups induced phyllody, the phyl‐B group lacked the ability to induce phyllody. Comparative functional analyses of phyllogens revealed that a single amino acid polymorphism in phyl‐B group phyllogens prevented interactions between phyllogens and A‐ and E‐class MADS domain transcription factors (MTFs), resulting in the inability to degrade several MTFs and induce phyllody. Our finding of natural variation in the function of phytoplasma effectors provides new insights into molecular mechanisms underlying the aetiology of phytoplasma diseases.
Comparison of phyllogen, a phyllody‐inducing effector family, revealed its molecular evolution and functional variation attributed to a single amino acid polymorphism governing the phyllody symptoms of phytoplasma infection.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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.
PCR‐based amplification and sequence analyses of 16S rRNA gene detected ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’ in pomegranate trees showing phytoplasma‐like symptoms (leaf yellowing/reddening and rolling, little leaf and witches' broom) in Jordan. 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. Further studies are needed to verify the diffusion of pomegranate phytoplasma‐associated diseases in the region and demonstrate the transmission capability of the identified phytoplasma positive insects.
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BFBNIB, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
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