Impaginato 413 Adv. Hort. Sci., 2020 34(4): 413­418 DOI: 10.13128/ahsc­8982 Identification and impact of phytoplas­ mas associated with greenhouse cucumber phyllody in Iran S.A. Esmaeilzadeh­Hosseini 1 (*), G. Babaei 2, S. Davoodi 2, A. Bertaccini 3 1 Plant Protection Research Department, Yazd Agricultural and Natural Resources Research and Education Center, AREEO, Yazd, Iran. 2 Plant Protection Research Department, Chaharmahal and Bakhtiari Agricultural and Natural Resources Research and Education Center, AREEO, Shahrekord, Iran. 3 Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy. Key words: Cucumis sativus, PCR, RFLP, 16SrVI­A, 16SrXII­A. Abstract: Cucumber phyllody symptoms were observed in greenhouse cucum­ ber plants during 2014­2018 in all surveyed areas of central and west of Iran where the highest disease incidence was up to 82% in Taft (Yazd province). Symptoms exhibited by diseased plants were virescence, phyllody and sterility of the flowers. For verification of phytoplasma presence and identity, total DNAs were extracted from 44 symptomatic and six asymptomatic plants that were subjected to PCR amplifying 16S rRNA genes of phytoplasmas. PCR ampli­ cons of the expected size were obtained only from the symptomatic plants. RFLP analysis of R16F2n/R2 amplicons showed patterns identical to those of the clover proliferation (16SrVI) and “stolbur” (16SrXII) phytoplasma groups. Consensus sequences corresponding to phytoplasma strains from the two local­ ities Taft and Shahrekord showed 99% identity with phytoplasmas enclosed in groups 16SrVI and 16SrXII, respectively. Phylogenetic analysis confirmed that these phytoplasmas cluster with ‘Candidatus Phytoplasma trifolii’ and ‘Ca. P. solani’, respectively. Virtual RFLP provided profiles identical to the patterns of 16SrXII­A and 16SrVI­A phytoplasma subgroups. These phytoplasma subgroups were previously reported in different plant species growing near to the green­ house cucumber areas in Iran, and play a possible role in the epidemiology of disease for its dissemination. 1. Introduction Among the cucurbitaceous plants grown in greenhouses, Cucumis sativus with 7,427 ha is considered the most economical important crop in Iran where about the 77% of the area under greenhouse cultivation is greenhouse cucumber (Iranian Ministry of Agriculture, 2019). These plants need less water than the species cultivated in the fields, and due to the water constraint, this production is expanding. The presence of phyllody disease was reported in cucumber up to 80% in Jiroft and Kahnooj (*) Corresponding author: phytoplasma.iran@gmail.com Citation: ESMAEILZADEH­HOSSEINI S.A., BABAEI G., DAVOODI S., BERTACCINI A., 2020 ­ Identification and impact of phytoplasmas associated with greenhouse cucumber phyllody in Iran. ­ Adv. Hort. Sci., 34(4): 413­418 Copyright: © 2020 Esmaeilzadeh­Hosseini S.A., Babaei G., Davoodi S., Bertaccini A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 3 June 2020 Accepted for publication 26 October 2020 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(4): 413­418 414 (Kerman province, Iran) (Azadvar et al., 2004). In some areas, due to high disease incidence and severi­ ty, infected plants did not bear fruits and farmers remove cultivated cucumbers and re­sown them. In 2004­2006 surveys in greenhouses the presence of cucumber phyllody was observed in Yazd, Varamin, and Larestan with about 35%, 80% and 3% of disease incidence respectively, and the phytoplasma pres­ ence was confirmed (Esmaeilzadeh­Hosseini et al., 2006). Phytoplasmas are destructive bacteria infect­ ing more than one thousand plant species world­ wide. They are transmitted mainly by leafhoppers and symptoms include yellowing, discoloration, dwarfing, witches’ broom, virescence and phyllody (Bertaccini et al., 2014). Their identification relies on molecular classification based on the amplification and/or RFLP analyses of their 16S rRNA gene (Lee et al., 1998; IRPCM, 2004). The aim of the present work was to identify the phytoplasmas associated with greenhouse cucumber phyllody in central and west parts of Iran in order to devise the appropriate dis­ ease management to reduce its economic impact. 2. Materials and Methods During 2014 to 2018, greenhouse cucumber growing areas of central and west of Iran were sur­ veyed for evaluation of phytoplasma disease pres­ ence. Symptomatic and symptomless cucumber plants were collected in greenhouses located in Akramia, Chah Shahrdar and Taft (Yazd province) and Shahrekord region (Chaharmahal and Bakhtiari province) and subjected to molecular studies for phytoplasma detection and identification. Sampling was carried out randomly in five 1,000 m2 green­ houses and the disease incidence was calculated by counting the number of symptomatic plants exhibit­ ing phyllody out of the total number of greenhouse cucumber plants in each greenhouse multiplied by 100. Nucleic acid extraction was carried out as described by Zhang et al. (1998) using 0.2 g of fresh midrib tissue from 44 symptomatic greenhouse cucumber plants and from 6 asymptomatic seed grown greenhouse cucumber plants. The DNA from pot marigold phyllody phytoplasma (16SrII­D sub­ group) (Esmaeilzadeh­Hosseini et al., 2018) was used as positive control. A total of 100 ng of nucleic acid was used for the PCR to amplify the 16S rRNA gene of the phytoplasmas with primers P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16mF2/R16mR2 and R16F2n/R16R2 (Gundersen and Lee, 1996) primers in a total volume of 50 µl. One µL of the products from direct amplification was diluted in 29 µL of ste­ rile deionized water for the nested amplifications. The PCR reaction was performed in 50 µL mixtures containing 0.4 µM of each primer, 0.2 mM of each dNTP, 1.25 U Taq DNA polymerase and 1X Taq poly­ merase buffer (CinnaGen, Iran). PCR protocols were done as reported by Salehi et al. (2015). Following PCR, 2 µL of each reaction mixture was electropho­ resed in a 1% (w/v) agarose gel containing 0.3 µg/mL ethidium bromide in 0.5 X TBE buffer (22.5 mM Tris­ borate, 1 mM EDTA, pH 8.0). The amplicons obtai­ ned with R16F2n/R16R2 primers were analyzed by single restriction endonuclease digestion with AluI, HaeIII, TaqI, HpaI, HpaII, MseI, RsaI, KpnI and HhaI (Thermo Scientific). The digestion products were analyzed through an 8% polyacrylamide gel elec­ trophoresis and the visualization of DNA bands was carried out with a UV transilluminator after staining by ethidium bromide. Direct sequencing in both directions of twelve samples (three per each greenhouse area) was car­ ried out using R16mF2/R16mR2 amplicons and the same primers. The resulting sequences were trimmed to the R16F2n/R2 fragment (about 1,240 bp) and submitted to GenBank. A database search of homologous sequences was performed by BLAST analyses at the NCBI (www.ncbi.nlm.nih.gov). The R16F2n/R2 sequence of the 16S rRNA gene of green­ house cucumber phyllody phytoplasma strains SGCP (Shahrekord greenhouse cucumber phyllody), TGCP (Taft greenhouse cucumber phyllody), Fars (GenBank accession number JN574839) and Tehran (GenBank accession number MH004460) and of 16S rRNA gene of selected ‘Candidatus Phytoplasma’ species or phy­ toplasma strains enclosed in the subgroups of groups 16SrVI and 16SrXII were aligned (Table 1). A phyloge­ netic tree was constructed with the phytoplasma sequences obtained and others retrieved from the GenBank using the neighbor­joining method with MEGA software version 7 (Kumar et al., 2016). Acholeplasma laidlawii was used as an out­group to root the tree and bootstrapping was performed 1,000 times to estimate the stability and support for the branches. The ribosomal subgroup affiliation of the detected phytoplasmas was confirmed by virtual RFLP analysis with the iPhyclassifier (Zhao et al., 2009). http://www.ncbi.nlm.nih.gov Esmailzadeh‐Hosseini et al. ‐ Phytoplasmas infecting greenhouse cucumber 415 3. Results and Discussion Greenhouse cucumber diseased plants showed flower virescence, phyllody and sterility (Fig. 1), the disease was named greenhouse cucumber phyllody (GCP). The symptoms were observed in all the green­ houses of the four surveyed areas. The disease rate was up to 11.2%, 33.5%, 82% and 8.7% in Akramia, Chah Shahrdar, Taft (Yazd province) and Shahrekord region (Chaharmahal and Bakhtiari province), respec­ tively. PCR amplicons of about 1.8, 1.4 and 1.25 kb were obtained from all the symptomatic greenhouse cucumber samples but not from the symptomless ones. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using AluI, HaeIII, TaqI, HpaI, HpaII, MseI, RsaI, KpnI and HhaI restriction enzymes showed two RFLP pattern identical to those reported for the 16SrVI and 16SrXII phytoplasma groups, respectively (Fig. 2). The DNA fragments obtained from twelve sam­ ples after direct sequencing were aligned and the consensus sequences corresponding to a representa­ Table 1 ­ Phytoplasma sequences used for comparison with the reported greenhouse cucumber strains from Iran Disease or phytoplasma GenBank accession number Country 16S ribosomal subgroup ‘Ca. P. trifolii' AY390261 Canada 16SrVI­A Strawberry multiplier disease AF190224 Canada 16SrVI­B Illinois elm yellows AF409069 USA 16SrVI­C Periwinkle little leaf AF228053 Bangladesh 16SrVI­D Centarurea solstitialis virescence AY270156 Italy 16SrVI­E Catharanthus phyllody EF186819 Sudan 16SrVI­F Portulaca little leaf EF651786 India 16SrVI­H ‘Ca. P. sudamericanum’ GU292081 Brazil 16SrVI­I 'Ca. P. solani’ AF248959 Serbia 16SrXII­A ‘Ca. P. australiense’ L76865 Australia 16SrXII­B Strawberry lethal yellows AJ243045 Australia 16SrXII­C ‘Ca. P. japonicum’ AB010425 Japan 16SrXII­D ‘Ca. P. fragariae’ DQ086423 Lithuania 16SrXII­E “Bois noir” strain BN­Op30 EU836652 Italy 16SrXII­F “Bois noir” strain BN­Fc3 EU836647 Italy 16SrXII­G ‘Ca. P. convolvuli’ JN833705 Italy 16SrXII­H Fig. 1 ­ Flower virescence, phyllody and sterility in a greenhouse cucumber plant from Yazd (A) and Chaharmahal and Bakhtiari (B, C, D) provinces. Fig. 2 ­ Real and virtual RFLP patterns (Zhao et al., 2009) respec­ tively of 1.2 kb amplicons from TGCP (A and B) and SGCP (C and D) phytoplasma strains 16S ribosomal gene sequence. Lane M, 100 bp DNA ladder (Biobasic, Canada). DNA products were digested with the enzymes listed at the top of the figures. Adv. Hort. Sci., 2020 34(4): 413­418 416 using online iPhyClassifier program exhibited virtual RFLP profiles identical to the reference pattern of 16SrVI­A and 16SrXII­A, respectively (Fig. 2). Phytoplasmas belonging to diverse ribosomal groups have been detected in Cucurbitaceae species worldwide showing different arrays of symptoms. In particular, 16SrI in Cucurbita pepo L. in Italy (Minucci et al., 1995) and in Sechium edule (Jacq.) Sw. in Costa Rica (Villalobos et al., 2002), 16SrII in C. sativus and C. pepo in Australia, Egypt and Iran (Davis et al., 1997; Omar and Foissac, 2012; Salehi et al., 2015), 16SrIII in Luffa cylindrica L. (Rox.) and Sicana odorifera (Vellozo) Naud in Brazil (Montano et al., 2000, 2007a, 2007b) and 16SrVIII in L. cylindrica in Taiwan (Davis et al., 2017). Phyllody is an important phytoplasma disease of cucurbitaceous plants in Iran (Salehi et al., 2015) and it was associated with the presence of a peanut witches’ broom phytoplasma (16SrII) in greenhouse cucumber plants showing phyllody (Dehghan et al., 2014) and of a clover proliferation phytoplasma (16SrVI) in Tehran (Ghayeb Zamharir and Azimi, 2019). Molecular assays confirmed the phytoplasma presence in the symptomatic greenhouse cucumber analyzed in this work and allow their identification as ‘Ca. P. trifolii’ (16SrVI­A) and ‘Ca. P. solani’ (16SrXII­ A)­related strains (Esmaeilzadeh Hosseini et al., 2019). In the present work the identification of phyto­ plasmas in subgroups 16SrVI­A and 16SrXII­A allows epidemiological considerations. The 16SrVI­A­related phytoplasma strain was identified in the central areas of Iran, in the Yazd province where the most impor­ tant plant species harboring 16SrVI phytoplasmas are tomato and eggplant (Salehi et al., unpublished) and alfalfa (Esmaeilzadeh Hosseini et al., 2015a, 2015b; Purmohammadi et al., 2017). Greenhouse cucumber phyllody associated with the presence of 16SrXII­A (“stolbur”) phytoplasmas was present in Chaharmahal and Bakhtiari province where this phy­ toplasma was detected also in grapevine showing diverse symptoms (Mirchenari et al., 2015) and alfal­ fa showing witches’ broom (Esmaeilzadeh Hosseini et al., 2016a, 2016b). Phytoplasma diseases associated with “stolbur” were present in plant host species adjacent to greenhouses cucumber areas but their role in the epidemiology of the disease needs to be proved. Due to the problems of water constraint, the cucumber greenhouse cultivation in Iran has been widely increased. In the majority of cucumber pro­ duction greenhouses, aeration valves are usually not tive of GCP phytoplasmas in Taft (TGCP) and Shahrekord (SGCP) were deposited in GenBank under the accession numbers MF438041 and MK402983, respectively. The BLAST search of these sequences showed that TGCP (1,251 bp) and SGCP (1,243 bp) phytoplasmas had 99.60% and 99.28% identity with phytoplasmas enclosed in subgroups 16SrVI­A (‘Ca. P. trifolii’, GenBank accession number AY390261) and 16SrXII­A (‘Ca. P. solani’, GenBank accession number AF248959), respectively. The phylogenetic analysis confirmed that TGCP phytoplasmas cluster with phy­ toplasmas classified in the 16SrVI group and were therefore confirmed as closely related to ‘Ca. P. tri­ folii’, while the SGCP phytoplasmas cluster with those enclosed in the 16SrXII group and were therefore related to ‘Ca. P. solani’ (Fig. 3). The R16F2n/R2 amplified regions from TGCP and SGCP phytoplas­ mas digested in silico with 17 restriction enzymes Fig. 3 ­ Phylogenetic tree constructed by the Neighbor­Joining method of the R16F2n/R16R2 sequence of 16S rRNA gene of 38 phytoplasmas including the cucumber strains SGCP, TGCP, Tehran and Fars, and phytoplasmas enclo­ sed in subgroups of the 16SrVI, 16SrII and 16SXII groups. The greenhouse cucumber phyllody phytoplasmas are in color and bolded (in red those sequenced in this work). Numbers at the nodes are bootstrap values based on 1,000 repetitions. ‘Ca. P.’: ‘Candidatus Phytoplasma’. GenBank accession numbers for sequences are given in parentheses while the phytoplasma ribosomal grouping is before the strain name. Esmailzadeh‐Hosseini et al. ‐ Phytoplasmas infecting greenhouse cucumber 417 covered with netting by greenhouse owners (Fig. 4) which allows also the possible entry of insect vectors. Furthermore, the major cucumber greenhouses are located close to the agricultural fields and rangelands where during the recent droughts, the insect vectors are attracted and possibly transmitted the phytoplas­ mas from sources outside the greenhouse. It is therefore probable that infection in these plants play a role in the epidemiology for the dissemi­ nation of this bacterium in the greenhouses also con­ sidering that the plants are not completely isolated from the environment. The presence of consistent populations of Orosius albicinctus and Circulifer haematoceps both recognized vectors of the cucum­ ber phyllody disease was detected in plants grown adjacent to these greenhouses (Salehi et al., 2015). Their feeding activity during the year, especially in the Yazd province, leads to the widespread dissemination of phytoplasmas; therefore, preventing the entry of insect vectors into the greenhouses is the most recom­ mended management to reduce the disease incidence. Acknowledgements This paper present part of the results of the pro­ ject no. 2­64­16­94189 approved and supported by Agricultural Research, Education and Extension Organization (AREEO), Ministry of Agriculture, Iran. References IRANIAN MINISTRY OF AGRICULTURE, 2019 ­ Agricultural Statistics. ­ Iranian Ministry of Agriculture, Vol. 2, pp. 425. AZADVAR M., SALEHI M., IZADPANAH K., HOSSEINI POUR A., 2004 ­ First report of greenhouse cucumber phyllody dis‐ ease in Iran. ­ Proc. 16th Iran. Plant Prot. Cong. Tabriz, Iran, pp. 252. BERTACCINI A., DUDUK B., PALTRINIERI S., CONTALDO N., 2014 ­ Phytoplasmas and phytoplasma diseases: a severe threat to agriculture. ­ Am. J. Pl. Sci., 5: 1763­ 1788. DAVIS R.E., ZHAO Y., WEI W., DALLY E.L., LEE I­M., 2017 ­ ‘Candidatus Phytoplasma luffae’, a novel taxon associ‐ ated with witches’ broom disease of loofah, Luffa aegyptica Mill. ­ Int. J. Syst. Evol. Microbiol., 67: 3127­ 3133. DAVIS R.I., SCHNEIDER B., GIBBS K.S., 1997 ­ Detection and differentiation of phytoplasmas in Australia. ­ Aus. J. Agric. Res., 48: 535­544. DEHGHAN H., SALEHI M., KHANCHEZAR A., AFSHAR H., 2014 ­ Biological and molecular characterization of a phytoplasma associated with greenhouse cucumber phyllody in Fars province, Iran. ­ J. Plant. Path., 50(4): 185­186. DENG S., HIRUKI C., 1991 ­ Amplification of 16S rRNA genes from culturable and non‐culturable mollicutes. ­ J. Microbiol. Meth., 14: 53­61. ESMAEILZADEH HOSSEINI S.A., BABAIE G., PURMOHAMADI S., BERTACCINI A., 2019 ­ Phytoplasmas infecting greenhouse cucumber in Iran. ­ Phytopath. Moll., 9(1): 31­32. ESMAEILZADEH HOSSEINI S.A., KHODAKARAMIAN G., SALEHI M., BERTACCINI A., 2016 a ­ First report of 16SrVI‐A and 16SrXII‐A phytoplasmas associated with alfalfa witches’ broom diseases in Iran. ­ J. Pl. Pathol., 98(2): 369. ESMAEILZADEH HOSSEINI S.A., KHODAKARAMIAN G., SALEHI M., BERTACCINI A., 2016 b ­ Molecular identifi‐ cation and phylogenetic analysis of phytoplasmas asso‐ ciated with alfalfa witches’ broom diseases in the west‐ ern areas of Iran. ­ Phytopath. Moll., 6(1): 16­22. ESMAEILZADEH HOSSEINI S.A., KHODAKARAMIAN G., SALEHI M., FANI S.R., BOLOK YAZDI H.R., RAOUFI D., JADIDI O., BERTACCINI A., 2015 a ­ Status of alfalfa witches’ broom phytoplasma disease in Iran . ­ Phytopath. Moll., 5(1­Suppl.): 65­66. ESMAEILZADEH HOSSEINI S.A., SALEHI M., BABAIE G., BERTACCINI A., 2018 ­ Characterization of a 16SrII sub‐ group D phytoplasma strain associated with Calendula officinalis phyllody in Iran. ­ 3 Biotech, 8(7): 295. ESMAEILZADEH HOSSEINI S.A., SALEHI M., KHODAKARAMI­ AN G., MIRCHENARI S.M., BERTACCINI A., 2015 b ­ An up to date status of alfalfa witches’ broom disease in Iran. ­ Phytopath. Moll., 5(1­Suppl.): 9­18. ESMAEILZADEH HOSSEINI S.A., SALEHI M., SHAHRIARI D., GHAUMI M., 2006 ­ Occurrence ofgreenhouse cucumber phyllody in Yazd, Tehran and Fars province. ­ Proc. 17th Iran. Pl. Prot. Cong. Karaj, Iran, pp. 228. GHAYEB ZAMHARIR M., AZIMI H., 2019 ­ Detection and char‐ Fig. 4 ­ Aeration valves are usually not covered with netting by greenhouse owners and this allows the entry of insect vectors. acterisation of a phytoplasma associated with cucumber (Cucumis sativus) regional yellows disease in Iran. ­ Arch. Phytopath. Pl. Protect., 51(15­16): 889­893. GUNDERSEN D.E., LEE I.­M., 1996 ­ Ultrasensitive detection of phytoplasmas by nested‐PCR assays using two uni‐ versal primer sets. ­ Phytopath. Medit., 35: 144­151. IRPCM, 2004 ­ ‘Candidatus Phytoplasma’, a taxon for the wall‐less, non‐helical prokaryotes that colonize plant phloem and insects. ­ Int. J. Syst. Evol. Microbiol., 54: 1243­1255. KUMAR S., STECHER G., TAMURA K., 2016 ­ MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. ­ Mol. Biol. Evol., 33(7): 1870­1874. LEE I.­M., GUNDERSEN­RINDAL D.E., DAVIS R.E., BAR­ TOSZYK I.M., 1998 ­ Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. ­ Int. J. Syst. Evol. Microbiol., 48: 1153­1169. MINUCCI C., RAMASSO E., DELLAVALLE G., LISA V., MASEN­ GA V., BOCCARDO G., 1995 ­ Caratterizzazione di un organismo simile a micoplasmi in zucchino in Liguria. ­ Inf.tore Fitopat., 45: 61­64. MIRCHENARI S.M., MASSAH A., ZIRAK L., 2015 ­ “Bois noir”: new phytoplasma disease of grapevine in Iran. ­ J. Pl. Prot. Res., 55: 88­93. MONTANO H.G., BRIOSO P.S.T., CUNHA JUNIOR J.O., FIGUEIREDO D.V.,PIMENTEL J.P., 2007 a ­ First report of group 16SrIII phytoplasma in loofah (Luffa cylindrica). ­ Bull. Insectol., 60(2): 277­278. MONTANO H.G., BRIOSO P.S.T., PEREIRA R.C., PIMENTEL J.P., 2007 b ­ Sicana odorifera (Cucurbitaceae) a new phytoplasma host. ­ Bull. Insectol., 60(2): 287­288. MONTANO H.G., DAVIS R.E., DALLY E.L., PIMENTEL J.P., BRIOSO P.S.T., 2000 ­ Identification and phylogenetic analysis of a new phytoplasma from diseased chayote in Brazil. ­ Pl. Dis., 84: 429­436. OMAR A.F., FOISSAC X., 2012 ­ Occurrence and incidence of phytoplasmas of the 16SrII‐D subgroup on solanaceous and cucurbit crops in Egypt. ­ Eur. J. Pl. Pathol., 133: 353­360. PURMOHAMMADI S., ESMAEILZADEH HOSSEINI S.A., GHO­ LAMPOOR H., MIRCHENARI S.M., 2017 ­ Occurrence of a 16SrVI phytoplasma strain associated with alfalfa witches’ broom disease in Yazd, Iran. ­ 2nd Int. and 10th Nation. Biotech. Congr. Islamic Republic of Iran, Karaj, Iran. SALEHI M., SIAMPOUR M., ESMAEILZADEH HOSSEINI S.A., BERTACCINI A., 2015 ­ Characterization and vector identification of phytoplasmas associated with cucum‐ ber and squash phyllody in Iran. ­ Bull. Insectol., 68(2): 311­319. SCHNEIDER B., SEEMÜLLER E., SMART C.D., KIRKPATRICK B.C., 1995 ­ Phylogenetic classification of plant patho‐ genic mycoplasma‐like organisms or phytoplasmas, pp 369­380. ­ In: RAZIN R., and TULLY G.J. (eds.) Molecular and diagnostic procedures in Mycoplasmology . Academic Press, San Diego, CA, USA, pp. 466. VILLALOBOS W., MOREIRA L., RIVERA C., BOTTNER K.D., LEE I­M., 2002 ­ First report of an aster yellows sub‐ group 16SrI‐B phytoplasma infecting chayote in Costa Rica. ­ Pl. Dis., 86: 330. ZHANG Y.P., UYEMOTO J.K., KIRKPATRICK B.C., 1998 ­ A small‐scale procedure for extracting nucleic acids from woody plants infected with various phytoplasmas for PCR assay. ­ J. Virol. Meth., 71: 45­50. ZHAO Y., WEI W., LEE I­M., SHAO J., SUO X., DAVIS R.E., 2009 ­ Construction of an interactive online phytoplas‐ ma classification tool, iPhyClassifier, and its application in analysis of the peach X‐disease phytoplasma group (16SrIII). ­ Int. J. Syst. Evol. Microbiol., 59: 2582­2593. 418 Adv. Hort. Sci., 2020 34(4): 413­418 https://www.ncbi.nlm.nih.gov/pubmed/?term=Kumar%20S%5BAuthor%5D&cauthor=true&cauthor_uid=27004904 https://www.ncbi.nlm.nih.gov/pubmed/?term=Stecher%20G%5BAuthor%5D&cauthor=true&cauthor_uid=27004904 https://www.ncbi.nlm.nih.gov/pubmed/?term=Tamura%20K%5BAuthor%5D&cauthor=true&cauthor_uid=27004904 https://www.ncbi.nlm.nih.gov/pubmed/27004904