The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously ...reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of ‘Candidatus Phytoplasma’ species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by “flavescence dorée”, “bois noir” and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours.
•Phytoplasmas were isolated from infected field-collected grapevine samples.•Complex media for phytoplasma isolation and growth were tested.•Unreported microaerophilic conditions are settled for phytoplasma plate colony growth.•“Flavescence dorée”, “bois noir” and aster yellows phytoplasma colonies are obtained from field infected materials.•Phytoplasma presence in colonies was detected by nested-PCR and sequencing on two genes.
Symptoms of bud proliferation and aborted seed pods were observed in Iranian soybean fields in Golestan and Mazandaran provinces. Symptomatic soybean plants, possible insect vectors, and alternative ...plant host species were collected to verify the presence of possible pathogens. Symptomatic and asymptomatic soybean and herbaceous plant samples were subjected to ELISA for virus detection and all samples were subjected to nested PCR amplification of phytoplasma 16 S ribosomal RNA gene followed by sequence and phylogenetic analysis. A ‘
Candidatus
Phytoplasma trifolii’ strain was detected in the symptomatic soybean samples and in samples of willow and pampas grass. Some viruses were also detected in some the tested soybean samples. Biological assays such as insect and graft transmission were performed. Experimental infection of healthy soybean plants with symptomatic soybean shoots resulted in bud proliferation and seed pods abortion symptoms 8 weeks after grafting. The Hemiptera
Creontiades pallidus
known also as cotton shredder bug, transmitted ‘
Ca.
P. trifolii’ to healthy soybean plants under insect proof conditions. The reported identification of the phytoplasmas associated with this soybean disease, of their reservoir or alternative host plants and of an insect vector will help in the development of more effective strategies to manage this disease under the Iranian field conditions.
1 Dipartimento di Biologia Applicata alla Difesa delle Piante, University of Udine, 33100 Udine, Italy
2 Molecular Plant Pathology Laboratory, USDA, ARS, Beltsville, MD 20705, USA
3 Dipartimento di ...Scienze e Tecnologie Agroalimentari, University of Bologna, 40127 Bologna, Italy
4 FLREC, University of Florida, Fort Lauderdale, FL, USA
5 Dipartimento di Scienze Farmaceutiche, University of Salerno, I-84084 Fisciano, Italy
6 Department of Crop Science, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod 123, Oman
Correspondence I.-M. Lee leeim{at}ba.ars.usda.gov
Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV ( rpl22 ) and rpsC ( rps3 ), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.
Abbreviations: NJ, neighbour-joining; rp, ribosomal protein
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are given in Fig. 1.
Figures presenting the position of the degenerate primers and the analysis of putative restriction sites in ribosomal protein operon sequences are available as supplementary material with the online version of this paper.
The pigeon pea witches’-broom phytoplasma group (16SrIX) comprises diverse strains that cause numerous diseases in leguminous trees and herbaceous crops, vegetables, a fruit, a nut tree and a forest ...tree. At least 14 strains have been reported worldwide. Comparative phylogenetic analyses of the highly conserved 16S rRNA gene and the moderately conserved rplV (rpl22)–rpsC (rps3) and secY genes indicated that the 16SrIX group consists of at least six distinct genetic lineages. Some of these lineages cannot be readily differentiated based on analysis of 16S rRNA gene sequences alone. The relative genetic distances among these closely related lineages were better assessed by including more variable genes e.g. ribosomal protein (rp) and secY genes. The present study demonstrated that virtual RFLP analyses using rp and secY gene sequences allowed unambiguous identification of such lineages. A coding system is proposed to designate each distinct rp and secY subgroup in the 16SrIX group.
Between 2016 and 2017, symptoms of witches’ broom, proliferation, drying of twigs and ball-like structures formation were observed in
Pinus brutia
in urban green areas and parks in Isfahan (Iran). ...All samples from affected
P. brutia
trees resulted positive in nested-PCR using primer pairs targeting the phytoplasma 16S rRNA gene. Amplicons were obtained only from all the symptomatic pine tree samples and RFLP analyses of R16F2n/R16R2 amplicons with
Kpn
I,
Alu
I,
Hae
III,
Hha
I,
Hpa
II,
Mse
I,
Rsa
I,
Tha
I,
Bfa
I and
Taq
I restriction enzymes showed the presence of phytoplasmas enclosed in the 16SrVI group. Four amplicons were sequenced and showed 100% identity to each other. One of these sequences was deposited in the GenBank database (accession number MK158098) and the virtual RFLP pattern was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI, subgroup A confirming that the phytoplasma detected in pine is a member of 16SrVI-A subgroup. This is the first report of a 16SrVI-A phytoplasma strain associated with pine tree witches’ broom (
P. brutia
) disease.
“Flavescence dorée” (FD) is a grapevine quarantine disease associated with phytoplasmas and transmitted to healthy plants by insect vectors, mainly Scaphoideus titanus. Development of efficient ...methods for its control has been hampered by the lack of knowledge about phytoplasma biological properties, linked also to difficulties in its in vitro cultivation. Conventional management strategies rely mainly on the application of insecticide treatments, roguing of infected plants and production of phytoplasma‐free propagation material. However, these strategies are costly and could have undesirable environmental impacts. Novel approaches are being investigated using transcriptomic and proteomic tools that can assist in identifying key regulators expressed by diseased, recovered and healthy plants. These studies allowed the identification of molecular profiles linked to the grapevine cultivar‐diverse susceptibility that are of great interest for the development of FD less susceptible plants by breeding programmes. Other promising FD management strategies include the use of grapevine endophytic microorganisms with known biocontrol properties and endophytes living inside specialized insect cells, which can be potential candidates for FD vector control. Finally, the application of plant defence elicitors might be an interesting tool for FD containment, but more research is needed before it can be implemented. In this review, the methodologies used for detecting and confining FD diffusion are discussed, focusing mainly on conventional tools, current research perspectives and knowledge gaps.
Sophora alopecuroides
dwarfing and yellowing symptoms were observed at the edge of several Damaneh and Fereydun Shahr (Isfahan province, Iran) fields. Polymerase chain reaction using primers P1/P7 ...and nested PCR using the same primers followed by R16mF2/R16mR2 and R16F2n/R16R2 primer pairs amplified the 16S ribosomal RNA-encoding gene in all the symptomatic plants tested, but not in the asymptomatic ones. RFLP analysis of R16F2n/R16R2 amplicons with
Alu
I,
Hae
III,
Hha
I,
Hpa
I,
Hpa
II,
Rsa
I,
Mse
I and
Taq
I restriction enzymes showed profiles referable to 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX phytoplasma ribosomal groups indicating also the presence of mixed infection in several samples. The identity percentage and the phylogeny obtained after direct sequencing of selected 16S rRNA-encoding gene sequences (R16F2n/R2 amplicons) from symptomatic
S. alopecuroides
confirmed the presence of phytoplasma strains related to ‘
Candidatus
Phytoplasma omanense’ (group 16SrXXIX), ‘
Ca
. P. australasia’ (group 16SrII), and ‘
Ca.
P. trifolii’ (16SrVI). Moreover, in a few samples/amplicons, 16SrXII-A, 16SrIX-H, 16SrVI-F and 16SrXXIX-A phytoplasma subgroups were identified by real and virtual RFLP analyses. The other phytoplasmas detected were classified in subgroups 16SrII-D and 16SrVI-A. This is the first report of the presence of 16SrII and 16SrXXIX phytoplasma groups and of mixed infection of phytoplasma strains belonging to groups 16SrII, 16SrVI, 16SrIX, 16SrXII and 16SrXXIX in
S. alopecuroides
worldwide.
Aims
To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8‐year period and to assess the relative contribution of ...mutation and recombination in shaping their diversity.
Methods and Results
By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed.
Conclusions
The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains.
Significance and Impact of the Study
We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.
During 2010–14 surveys in the major sesame growing areas of Fars, Yazd and Isfahan provinces (Iran), genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody were ...studied. Virtual RFLP, phylogenetic, and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to three ribosomal subgroups, 16SrII-D, 16SrVI-A, and 16SrIX-C. The same analyses using 16S rDNA sequences from sesame phyllody-associated phytoplasmas retrieved from GenBank database showed the presence of phytoplasmas clustering with strains in the same subgroups in other Iranian provinces including Bushehr and Khorasan Razavi.
Circulifer haematoceps
and
Orosius albicinctus
, known vectors of the disease in Iran, were tested for transmission of the strains identified in this study.
C. haematoceps
transmitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas, while
O. albicinctus
only transmitted 16SrII-D strains. Based on the results of the present study and considering the reported presence of phytoplasmas belonging to the same ribosomal subgroups in other crops, sesame fields probably play an important role in the epidemiology of other diseases associated with these phytoplasmas in Iran.
The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible ...inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.
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