Distinct <Emphasis Type="Italic">rpsC</Emphasis> single nucleotide polymorphism lineages of Flavescence Dorée subgroup 16SrV-D phytoplasma co-infect <Emphasis Type="Italic">Vitis vinifera</Emphasis> L.

During a survey on grapevine yellows disease complex in vineyards of Lombardy region (northern Italy), phytoplasmas associated with Flavescence dorée disease were identified in symptomatic grapevines. Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analyses of 16S rDNA revealed the prevalence of phytoplasmal subgroup 16SrV-D. Bioinformatic analyses of nucleotide sequences of rplV and rpsC genes, amplified from 16SrV-D phytoplasma infected grapevines and cloned, underscored the presence of five confirmed rpsC single nucleotide polymorphism (SNP) lineages, determined by different combination of SNPs at nucleotide positions 29, 365, 680, and 720 of rpsC gene. Virtual and actual RFLP analyses with the enzyme TaqI validated the presence of these SNPs. Co-infections by up to four distinct rpsC SNP lineages of 16SrV-D phytoplasma were found in grapevines. These results could open new perspectives for the study of the ecology and the epidemiology of Flavescence dorée.     

Occurrence of Phytoplasmas Related to Stolbur and to ‘Candidatus Phytoplasma japonicum’ in Woody Host Plants in China

During a survey in a limited area of the Shanxi province in China, phytoplasma symptoms were observed on woody plants such as Chinese scholar tree, apple, grapevine and apricot. The polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analyses on the phytoplasma 16S ribosomal gene confirmed that symptomatic samples from all these species were infected by phytoplasmas. The molecular characterization of the pathogen, performed also with sequencing of polymerase chain reaction amplified 16S rDNA, showed that the phytoplasmas detected in all plant species tested are closely related with stolbur, but two samples from a Chinese scholar tree were infected with phytoplasmas related to ‘Candidatus Phytoplasma japonicum’. The presence of RFLP polymorphism was found in the 16S rDNA amplicons with three of the six enzymes employed in the majority of phytoplasma strains studied.     

Identification of Phytoplasmas Associated with Grapevine Yellows in Serbia

The molecular identification and characterization of phytoplasmas from infected grapevines in four locations in Serbia are reported. Phytoplasmas were detected and identified by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rDNA. Grapevine yellows were associated with three molecularly distinguishable phytoplasmas: Flavescence dorée phytoplasmas (elm yellows group: 16SrV‐C subgroup) were present only in the Župa Aleksandrovac region; Bois noir phytoplasmas (stolbur group: 16SrXII‐A subgroup) were detected in the other surveyed regions; a mixed infection of European stone fruit yellows (apple proliferation group: 16SrX‐B subgroup) and Bois noir phytoplasmas was identified in one sample. A finer molecular characterization by RFLP analysis of rpS3 and SecY genes of Flavescence dorée phytoplasmas from Župa Aleksandrovac confirmed that the Serbian genotype is indistinguishable from a strain from the Veneto region, Italy. Characterization of the tuf gene of Bois noir phytoplasmas showed lack of amplification of samples from Erdevik. HpaII profiles of tuf gene PCR products of samples from Pali and Radmilovac were identical, and were indistinguishable from one of the two profiles produced by samples from Italian grapevines used as reference strains.     

Geographical Distribution of Bois Noir Phytoplasmas Infecting Grapevines in Croatia

Visual symptom assessment, polymerase chain reaction amplification and restriction fragment length polymorphism analyses were used to detect and identify phytoplasmas infecting grapevines in Croatia. Samples were collected from different viticultural areas in order to examine geographic distribution of phytoplasmas throughout the country. Only phytoplasmas belonging to Bois Noir (subgroup 16SrXII-A or stolbur) were found in vineyards of continental Croatia. The fact that no phytoplasmas were detected in Dalmatia and Istria was in accordance with the absence of grapevine yellows symptoms in these regions.     

Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines,bindweeds and vegetables

Aim: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Materials and Results: Phytoplasma isolates belonging to stolbur subgroup 16SrXII‐A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf‐a type and 68% by tuf‐b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf‐b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions. Conclusions: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Significance and Impact of the Study: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.     

Vitex agnus‐castus cannot be used as trap plant for the vector Hyalesthes obsoletus to prevent infections by ‘Candidatus Phytoplasma solani’ in northern Italian vineyards: Experimental evidence

Bois noir (BN), the most prevalent disease of the grapevine yellows complex, causes considerable yield loss in vineyards. BN is associated with phytoplasma strains of the species ‘Candidatus Phytoplasma solani’ (taxonomic subgroup 16SrXII‐A). In Europe, the BN phytoplasma is transmitted to grapevine mainly by Hyalesthes obsoletus, a polyphagous cixiid completing its life cycle on stinging nettle and field bindweed. As a result of the complexity of BN epidemiology, no effective control strategies have been developed. In previous studies conducted in the eastern Mediterranean coast of Israel, chaste tree (Vitex agnus‐castus) was found to be the preferred host plant of H. obsoletus but did not harbour BN phytoplasma. Thus, a ‘push and pull’ strategy was suggested based on the fact that chaste tree plants located at vineyard borders was an effective trap plant for H. obsoletus adults. However, in other studies carried out in the eastern Adriatic coast of Montenegro, chaste tree was found to be a key source plant for BN phytoplasma transmission to grapevine. This study aimed to investigate (i) the interaction between chaste tree and H. obsoletus through survival, attractiveness and oviposition experiments conducted comparing the behaviour of H. obsoletus in chaste tree versus stinging nettle and grapevine and (ii) the capability of chaste tree to harbor ‘Ca. P. solani’ in northern Italy through transmission trials. H. obsoletus adults were found to survive on chaste tree and grapevine over a 1 week period and prefer chaste tree to grapevine. Moreover, H. obsoletus produced eggs and overwintered as nymphs on chaste tree, even if at a lesser extent than on stinging nettle. H. obsoletus originating from nettle was found able to transmit ‘Ca. P. solani’ to chaste tree (2 plants of 16 were found infected by the BN phytoplasma strain St5 identified in H. obsoletus specimens). These results increased our knowledge about the role of Vitex agnus‐castus as host plant of H. obsoletus and BN phytoplasma in northern Italy and do not recommend considering chaste tree as trap plant at vineyard borders.     

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

A search for phytoplasma-associated diseases was conducted for the first time in the main grapevine-growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf-type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'-related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.     

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.     

Detection and Identification of Two Phytoplasmas (16SrIII‐B and 16SrXII‐A) From Alfalfa (Medicago sativa) in Serbia

Plants of alfalfa (Medicago sativa) exhibiting general stunting, proliferation and phyllody associated with leaf yellowing and reddening were observed in three localities of Central Serbia. Phytoplasma strains belonging to 16SrIII‐B and 16SrXII‐A groups were detected and identified by RFLP and sequence analysis of 16S rDNA. Stolbur phytoplasma tuf gene RFLP analysis showed the presence of the TufAY‐b‐type phytoplasma subgroup in 80% of symptomatic samples. This is the first report of 16SrIII‐B and 16SrXII‐A phytoplasma groups affecting alfalfa in Serbia.     

The Distribution of Grapevine Yellows Disease Associated with the Buckland Valley Grapevine Yellows Phytoplasma

Three blocks of Chardonnay in one vineyard in the Buckland Valley of Victoria, Australia, were surveyed over 4 years for grapevine yellows disease (GYd). Buckland Valley grapevine yellows phytoplasma (BVGYp) was the only phytoplasma detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in GYd affected grapevines. GYd affected many grapevines and was characterized by remission of disease, some recurrence and occurrences in previously unaffected grapevines. A regional survey of the Buckland Valley indicated that both GYd and BVGYp occurred in the same restricted grape growing area. Within this area, BVGYp was detected in two vineyards that had been established using planting material from different sources. One could therefore speculate that BVGYp was present in these grapevines as a result of aerial transmission and was not present in the original planting material.     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).     

Detection of a 16SrXII phytoplasma associated with sugarcane yellow leaf syndrome in India

Phytoplasma strain was detected in leaves of sugarcane in India exhibiting symptoms of yellowing of midribs. A phytoplasma characteristic 1.2 kb rDNA PCR product was amplified from DNAs of all diseased samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs P1/P7 and f5U/r3U. Restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA indicated that diseased sugarcane was infected by phytoplasma. The 16S rDNA sequence of the Indian sugarcane yellow leaf phytoplasma (SCYLP) showed the closest identity (99%) to that of SCYLP in Cuba identified as Macroptilium lathyroides (AY725233), which belongs to 16SrXII (Stolbur group). This is the first record of the detection of SCYLP, and identification of the 16SrXII group of phytoplasma associated with yellow leaf syndrome (YLS) in India.     

Characterisation and phylogeny of a phytoplasma inducing sandal spike disease in sandal (Santalum album)

Sandal (Santalum album) is an industrially important forest species in India, where it is devastated by sandal spike (SAS) disease. Diseased S. album trees show characteristic witches’ broom symptoms suspected to be caused by phytoplasma. Since the first report of occurrence of this disease at the end of 19th century, studies mainly have been carried out to detect SAS phytoplasma through various approaches. The causative agent, however, has remained poorly characterised at a molecular level. The present investigation was aimed to characterise the pathogen at this level. In nested PCR, a 1.4‐kb 16S rDNA fragment was amplified and analysed by restriction fragment length polymorphism using 17 restriction enzymes. The patterns were identical to those of strains AY1 and APh of the aster yellows subgroup 16SrI‐B, except for BfaI, which gave a different pattern. After cloning and sequencing, a phylogenetic analysis revealed the closest relationship to aster yellows subgroup 16SrI‐B members. Nucleotide sequence identity ranged from 99.2% to 99.5% with this subgroup. On the basis of these results, the SAS phytoplasma was classified as a member of subgroup 16SrI‐B.     

Sequence Heterogeneity in the 16S rDNA of Tomato Big Bud Phytoplasma Belonging to Group 16SrIII

Phytoplasmas are associated with several plant diseases occurring in Brazil. A phytoplasma of group 16SrIII found in tomato plants with symptoms of big bud was identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA. RFLP patterns using HhaI and RsaI endonucleases were distinct from those exhibited by phytoplasmas representatives of diverse subgroups of group 16SrIII. Nucleotide sequence analyses demonstrated sequence heterogeneity expressed through a few base positions and restriction site among cloned fragments, revealing lineages different from members of currently known subgroups. The detection of lineages within tomato big bud phytoplasma present in Brazil revealed the diversity of representatives of group 16SrIII in tropical ecosystem and confirmed the genetic diversity of phytoplasmas of that group around the world.     

The groEL gene as an additional marker for finer differentiation of ‘Candidatus Phytoplasma asteris'‐related strains

Phytoplasma classification established using 16S ribosomal groups and ‘Candidatus Phytoplasma’ taxon are mainly based on the 16S rDNA properties and do not always provide molecular distinction of the closely related strains such as those in the aster yellows group (16SrI or ‘Candidatus Phytoplasma asteris'‐related strains). Moreover, because of the highly conserved nature of the 16S rRNA gene, and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, more variable single copy genes, such as ribosomal protein (rp), secY and tuf, were shown to be suitable for differentiation of closely related phytoplasma strains. Specific amplification of fragments containing phytoplasma groEL allowed studying its variability in 27 ‘Candidatus Phytoplasma asteris'‐related strains belonging to different 16SrI subgroups, of which 11 strains were not studied before and 8 more were not studied on other genes than 16S rDNA. The restriction fragment length polymorphism (RFLP) analyses of the amplified fragments confirmed differentiation among 16SrI‐A, I‐B, I‐C, I‐F and I‐P subgroups, and showed further differentiation in strains assigned to 16SrI‐A, 16SrI‐B and 16SrI‐C subgroups. However, analyses of groEL gene failed to discriminate strains in subgroups 16SrI‐L and 16SrI‐M (described on the basis of 16S rDNA interoperon sequence heterogeneity) from strains in subgroup 16SrI‐B. On the contrary, the 16SrI unclassified strain ca2006/5 from carrot (showing interoperon sequence heterogeneity) was differentiable on both rp and groEL genes from the strains in subgroup 16SrI‐B. These results indicate that interoperon sequence heterogeneity of strains AY2192, PRIVA (16SrI‐L), AVUT (16SrI‐M) and ca2006/5 resulted in multigenic changes – one evolutionary step further – only in the latter case. Phylogenetic analyses carried out on groEL are in agreement with 16Sr, rp and secY based phylogenies, and confirmed the differentiation obtained by RFLP analyses on groEL amplicons.     

Grapevine yellows in Northern Italy: molecular identification of Flavescence dorée phytoplasma strains and of Bois Noir phytoplasmas

AIMS: Verify the presence and the molecular identity of phytoplasmas in Northern and Central Italy vineyards where yellows diseases are widespread. METHODS AND RESULTS: Phytoplasma presence and identity were determined by PCR/RFLP analyses on 16S ribosomal gene testing 1424 symptomatic samples. The 65% of samples resulted phytoplasma infected; in particular 256 samples were found positive to phytoplasmas belonging to group 16SrV (mainly Flavescence dorée associated), and the remaining 37% was infected by phytoplasmas belonging to ribosomal subgroup 16SrXII-A (Stolbur or Bois Noir associated). 16SrV ribosomal group representative strains were further typed for variability in SecY and rpS3 genes. The results showed the presence of phytoplasmas belonging to 16SrV-C, 16SrV-D and to a lesser extent, 16SrV-A subgroup. CONCLUSIONS: Possible relationships between genetic polymorphisms of phytoplasma strains belonging to subgroup 16SrV-C and their geographic distribution and/or epidemic situations were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: Bois Noir and Flavescence dorée phytoplasmas are present in significant percentages in the areas under investigation. Molecular tools allowed to identify phytoplasma-infected plants and the genes employed as polymorphism markers resulted useful in distinguishing and monitoring the spreading of the diseases associated with diverse phytoplasmas belonging to 16SrV subgroup in vineyards.     

Artemisia vulgaris,a new host of 16SrV-C phytoplasma related strains infecting black alder in Poland

Yellowing of leaf tissue and strongly deformed shoots were observed in common mugwort (Artemisia vulgaris L.) growing in a nature reserve in Southern Poland. Similar foliage chlorosis together with abnormal shoot proliferation was noticed on alder tree (Alnus glutinosa Gaertn.) growing next to the common mugwort. DNA specific fragments coding 16S rRNA and ribosomal proteins (rp) were amplified from mugwort and alder samples using direct and nested PCR (Polymerase Chain Reaction) assays. Phylogenetic relationships inferred from 16S and rps3 genes indicated that strains infecting mugwort and alder were most closely related to phytoplasmas of subgroups 16SrV-C and 16SrV-D. Based on the restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA, the investigated phytoplasma strains were classified to subgroup 16SrV-C. Two sequence variants of the rps3 gene which differed by a single nucleotide were detected in all analysed samples by pairwise analysis of the aligned reads. Taking into account that this single-nucleotide polymorphism (SNP) occurs among 16SrV-C and 16SrV-D related phytoplasmas and that the phytoplasmas have a single copy of rp operon, we concluded that each plant species was infected by two distinct, closely related phytoplasma strains. To the best of our knowledge, this is the first report of group 16SrV-C related phytoplasmas infecting common mugwort worldwide, adding a new host species that is possibly linked to the spread of the alder pathogen in Eastern Europe. Although alder yellows phytoplasma has been frequently found in Europe, this is the first detection of phytoplasmas associated with alder in Poland.     

Molecular Identification of a Candidatus Phytoplasma asteris Associated with Cabbage Witches’‐Broom in China

In 2010, cabbages (Brassica oleracea L.) showing symptoms of proliferated axillary buds, crinkled leaves and plant stunting with shortened internodes typical to phytoplasma infection were found in a breeding facility in Beijing, China. Three symptomatic plants and one symptomless plant were collected, and total DNA was extracted from the midrib tissue and the flowers. With phytoplasma universal primers R16F2n/R16R2, a special fragment of 1247 bp (16S rDNA) was obtained from all three symptomatic cabbage plants, but not from the one symptomless cabbage plant. The 16S rDNA sequence showed 99% similarity with the homologous genes of the aster yellows group phytoplasma (16SrI group), and the phytoplasma was designed as CWBp‐BJ. Phylogenetic and computer‐simulated restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA gene revealed that CWBp‐BJ belongs to subgroup 16SrI‐B. This is the first report of a phytoplasma associated with cabbage witches’‐broom in China.     

Invasion biology and host specificity of the grapevine yellows disease vector Hyalesthes obsoletus in Europe

Within the past 10 years, the yellows disease ‘bois noir’ (BN) has become one of the commercially most important diseases of grapevine [Vitis vinifera L. (Vitaceae)] in Europe. Infection pressure is caused by phytoplasmas of the stolbur 16SrXII‐A group that are transmitted by a planthopper vector, Hyalesthes obsoletus Signoret (Homoptera: Auchenorrhyncha). Infestation happens as an accidental side‐effect of the feeding behaviour of the vector, as vector and pathogen proliferation is dependent on other plants. In Germany, the increase of BN is correlated with the use of a new host plant by the vector, increase in abundance of the vector on the new host plant, and dissemination of host plant‐specific pathogen strains. In this article, we investigate geographic and host‐associated range expansion of the vector. We test whether host‐plant utilization in Germany, hence the increase in BN, is related to genetic host races of the vector and, if so, whether these have evolved locally or have immigrated from southern populations that traditionally use the new host plant. The genetic population analysis demonstrates a recent expansion and circum‐alpine invasion of H. obsoletus into German and northern French wine‐growing regions, which coincides with the emergence of BN. No H. obsoletus mitochondrial DNA haplotype host‐plant affiliation was found, implying that the ability to use alternative host plants is genetically intrinsic to H. obsoletus. However, subtle yet significant random amplified polymorphic DNA (RAPD) genetic differentiation was found among host plant populations. When combined, these results suggest that a geographic range expansion of H. obsoletus only partly explains the increase of BN, and that interactions with host plants also occur. Further possible beneficial factors to H. obsoletus, such as temperature increase and phytoplasma interactions, are discussed.