Phytoplasma disease “Bois noir” in Crimea: diagnosis of the pathogen

In 2018-2019, grape leaf samples with symptoms of yellowing (reddening) and leaf rolling collected from grapevines of different regions of Crimea were tested for the presence of phytoplasma using the “nested”PCR with primer pairs P1/16S-Sr and R16F2n/R16R2. Phytoplasmas were detected in 91% grape samples of 2018 and 46% samples of 2019. Genotyping performed by restriction fragment length polymorphism analysis for three genes: ribosomal (16S rRNA), the tuf gene encoding the elongation factor EF-Tu, and the vmp1 gene encoding a membrane protein, showed that the phytoplasmas isolated from all samples were related to the species Candidatus Phytoplasma solani, type "b". Polymorphism was observed only for the vmp1 gene. The electrophoretic profiles of grape phytoplasma of 2018 (‘Chardonnay’ variety) differed from those of grape phytoplasma of 2019 (‘Pinot Noir’ and ‘Verdelho’ varieties), as well as from the profiles published in well-known works, which indicates the genetic diversity.


Introduction
Yellows caused by phytoplasmas are among the most common and harmful diseases of grapes. Phytoplasmas are bacteria without a cell wall. They parasitize in cells of plant phloem and are carried by insects of Hemiptera order, Auchenorrhyncha and Sternorrhyncha suborders. Some of species within the family are vectors, others are not. Not all of individuals and species with identified phytoplasma can be the competent vectors [1,2].
The most dangerous phytoplasma diseases in the main grape growing areas of Europe are the Grapevine Flavescence Dorée (FD) and the Bois Noir Phytoplasma (BN). Both diseases are common in the same European countries of viticulture, but FD is a quarantine object. This disease caused by phytoplasma of the group 16SrV has not been identified in Russia yet. BN is associated with phytoplasma of the stolbur group, subgroup 16SrXII-A -Candidatus Phytoplasma solani, the symptoms of which are yellowing or reddening of leaves and leaf blade rolling inwards are similar with FD. In nature the BN is transferred to grapes from field bindweed (Convolvulus arvensis L.), hedge bindweed (Calystegia sepium (L.) R.BR.), greater nettle (Urtica dioica L.) or planting material infected with planthoppers Hyalesthes obsoletus Sign., Reptalus panzeri Löw and others that are distrubuted in all viticultural areas around the world [2][3][4][5][6][7][8][9].
Stolbur is a dangerous disease of tomatoes and other crops of the Solanaceae family known since the 1930s in the South of Russia, but it has never been registered on grapevine cultivars before. However, since the second decade of the XXI century, grapevine Bois Noir Phytoplasma has become a real danger to the vineyards of Russia [3,4,10]. The reason of its distribution may be the introduced planting material, since the pathogen was first-ever discovered in such material in Crimea [3]. Therefore, it is so important to conduct a detailed study of phytoplasma genotypes peculiar for the regional flora and the corresponding local vectors.
The aim of the research was to determine the taxonomic inhering of the pathogen causing yellowing and rolling of grape leaves in Crimea.
Commonly used for detection of phytoplasma of a particular group highly conservative 16Sp rRNA gene, is not suitable for a detailed study of genetic diversity and phylogenetic relationships of isolates, as well as the relationships between isolates, host and geographical distribution, since the genetic variability within the stolbur subgroup, which is detected based on RFLP analysis of this gene, is very low. To obtain information on the molecular variability of stolbur phytoplasma isolates, causing diseases of grape, other crops and wildlife plant species, the researchers began to use nonribosomal genes, more suitable to differ and classify closely related phytoplasmas due to their greater variability, including the constitutive tuf gene -a gene that codes elongation factor EF-Tu (elongation factor Tu), which, like the 16S rRNA, is a highly conservative gene, often used to differ and classify phytoplasmas. In 1997 Schneider et al. designed two pairs of primers that could be used in the direct reaction of sequence amplification of the tuf gene of group majority of phytoplasmas fTufl / rTufl, and several internal primers for the "nested" PCR. With the aid of one of these pairs, namely: ftufAY / rtufAY, it was possible to distinguish three strains (a, b and c) inside the 16SrXII-A stolbur subgroup after RFLP analysis using HpaII for endonuclease restriction.
The parameter of stolbur genetic diversity significantly increases if the analysis of encoding a membrane protein gene vmp1 is carried out. It is considered that the variability of the vmp1 may be the result of protein location on the cell surface, therefore it can play a certain role in the interaction of phytoplasma with the host. Genes encoding the surface proteins show greater variability than the rest of the genome, and therefore they can present suitable markers for molecular epidemiology. Based on PCR RFLP analysis using RsaI endonuclease restriction in the area of the European Mediterranean, 23 vmp1 genotypes were identified on different cultures and wild-growing grass plants [11].

Materials and methods
Studies were carried out during 2018-2019. Grapevine leaf samples with Bois Noir disease symptoms were collected by staff scientists of the Laboratory of Plant Protection of FSBSI Institute Magarach of the RAS on 25 th and 30 th of July, 2018; 8 th of July, 2 nd and 9 th of August, 2019.
The leaves were flattened out and dried under a small press between layers of filter paper at a temperature of 27-30 °C. Central and lateral leaf ribs were deleted, dry material was finely chopped with scissors to the homogeneous condition (about 0.5 mm) and stored in paper bags at a room temperature.
The total DNA was isolated from 50-100 μg of dry material according to the method of Maixner et al., described in detail by Marcone [12]. Phytoplasma DNA was detected using «nested" PCR with a pair of primers P1/16S-SR in the first amplification and R16F2n/R16R2 -in the second. The resulting products were treated with restriction endonucleases, DNA fragments were separated using electrophoresis, and RFLP-profiles were used to identify a group (subgroup) of phytoplasmas [13,14]. Samples that gave a positive result for the presence of 16Sr gene, being a part of 16SrXII-A subgroup, were genotyped for nonribosomal tuf and vmp1 genes. To amplify the tuf gene, we completed a PCR with primers fTuf1 and rTuf1 in direct PCR and with primers fTufAY and rTufAYin the "nested" one. Restriction was performed with endonucleases AluI, HpaII, and Sau3AI. A fragment of vmp1 gene was amplified using PCR with primers Stol H10F1 and Stol H10R1 in direct PCR and TYPH10F and TYPH10R in the "nested" one [15]. The final PCR product (1.4 Kb) was subject to RsaI endonuclease restriction.

Results and discussion
In 2018, 23 samples of the most sensitive to phytoplasma infection 'Chardonnay' grape variety were tested for the presence of phytoplasma. Grape leaves with symptoms of disease were collected from different parts of vineyards of the South Coast of Crimea (SCC). In DNA isolated from 19 samples, the 1.2 Kb amplicon was detected directly in the reaction mixture obtained after the "nested" PCR. Four samples gave negative result. Considering that the reason could be the inhibition of PCR with admixtures contained in DNA solution, these samples were re-amplified using ten-fold diluted total DNA as a templet for direct PCR. Two samples were positive, in other two the target product was not detected (Table 1). In 2019, 13 grape samples of five varieties were tested for the presence of phytoplasma DNA, collected from three regions of Crimea: 'Pinot Noir', 'Aligote' and 'Merlot' (Bakhchisaray district of Crimea), 'Bastardo' and 'Pinot Noir' (Sevastopol city) and 'Verdelho' (SCC, Yalta). Two samples of weed plants were also tested -field bindweed (Convolvulus arvensis L.) and clematis (Clematis vitalba L.), collected in a vineyard (SCC, Yalta). Only 6 out of 13 samples were positive (Table 1). Of the 4 tested samples of 'Pinot Noir' grape variety (Bakhchisaray district of Crimea), phytoplasma was found in 3 samples, as well as in both 2 tested samples of 'Merlot' variety. DNA of phytoplasma was not found in 2 samples of 'Aligote' grapes from the same region.
Phytoplasma was not detected in a sample of 'Bastardo' grapes, presence of pathogen was also seen in only one asymptomatic specimen out of 2 samples of 'Pinot Noir' grapes (Sevastopol city). Among 2 samples of 'Verdelho' grapes, one contained phytoplasma DNA, detected only after additional purification: dissolution in a citrate buffer containing 4M guanidine thiocyanate and 0.5% lauryl sarcosinate, followed by precipitation with isopropanol. Phytoplasma was not found in samples of weed plants from this vineyard (field bindweed and grape-leaved clematis).
An attempt to use ten-fold diluted DNA in direct PCR for samples in which it was not possible to detect the presence of phytoplasma DNA was unsuccessful.
As a rule, up to 17 endonucleases are used to identify the group pertain of phytoplasmas using RFLP analysis. Since it is known that the Bois Noir disease is caused by phytoplasma of a stolbur group, in order to identify phytoplasma found in samples, it was enough to analyze the electrophoregrams obtained after amplicon restriction of the 16Sr gene (1.2 kb) by three restriction endonucleases -AluI, MseI (Tru1I) and TaqI. Restriction of DNA by the endonuclease AluI allows the phytoplasma found in grapes to be attributed to the subgroup 16SrI-A or subgroup 16SrXII-A. The profile of fragments obtained with endonuclease restriction MseI (Tru1I) and TaqI excludes the presence of 16SrI-A subgroup and confirms the belonging of phytoplasma to the stolbur group, the 16SrXII-A subgroup. Figure 1 shows typical profiles of DNA fragments obtained as a result of restriction by three endonucleases, specific for all grape samples of 2018 (21 samples) and 2019 (6 samples). Restriction analysis of the tuf gene amplicons by endonucleases AluI, HpaII and Sau3AI in 5% PAGE of phytoplasma from grape varieties 'Chardonnay' of 2018, 'Pinot Noir' and 'Verdelho' of 2019 is presented in Figures 2 and 3 (in Fig. 3 the Sau3AI restricts are not shown, as similar to those in Fig. 2). It is known that electrophoregrams obtained with endonuclease restriction HpaII make it possible to distinguish three types of the tuf genea, b, and c. Comparison of the profiles shown in Figures 2 and 3 with the published electrophoregrams allows to include the tuf phytoplasma gene of Crimean grapes in the group of "b" type [15]. Thus, the phytoplasmas found in the Crimean vineyards in 2018 and 2019 belonged to the Ca. Phytoplasma solani species. Genotyping for the tuf gene, which included RFLP analysis using the restriction endonuclease HpaII, showed that they belong to the same type of this gene -type 5 "b", which is a characteristic of phytoplasma genotypes found in the countries of Eastern Europe. Analysis of the vmp1 gene revealed two different genotypes: the first is a characteristic of all samples collected in 2018, the second -for samples of 2019. The efficiency of amplifying the DNA sequence with primers specific for the tuf and vmp1 genes was generally lower than with primers for ribosomal 16Sr genes, as was also confirmed by other researchers [16]. In the case of the vmp1 gene, this difference was even more significant. We failed to find absolutely identical profiles of the RsaI gene endonuclease restriction identified by us in the works of other authors.

Conclusion
Samples of grape leaves, with symptoms of yellowing or reddening and leaf rolling, collected from grapevine cultivars in various regions of Crimea in 2018 and 2019, were analyzed for the presence of phytoplasma. Phytoplasmas were detected in 91% grape samples of 2018 and in 46% of 2019. Genotyping performed using polymorphism analysis of restriction fragment length for three genes: ribosomal (16S rRNA), the tuf gene encoding the elongation factor EF-Tu, and the vmp1 gene encoding a membrane protein, showed that phytoplasmas isolated from all samples belonged to the species Candidatus Phytoplasma solani, type "b". Polymorphism was observed only for the vmp1 gene. The electrophoretic phytoplasma profiles of 2018 ('Chardonnay' variety) differed from those of 2019 ('Pinot Noir' and 'Verdelho' varieties), and also differed from the profiles published in well-known works, indicating the genetic diversity. At the same time, only two types of nucleotide sequence of the vmp1 gene have been identified in Crimea so far based on the length polymorphism of restriction fragments during the RsaI endonuclease restriction.