Report of ‘Candidatus Phytoplasma hispanicum’ (16SrXIII-E) Associated with Cauliflower Stunt in São Paulo State, Brazil, and Balclutha hebe as Its Potential Vector

Abstract

A cauliflower stunt disease associated with phytoplasmas occurs in crops of cauliflower (Brassica oleracea var. botrytis) in Sao Paulo state (SP), southeastern region of Brazil. The symptoms are characterized by stunting, extensive malformations of floral parts, leaf reddening, and vessel necrosis. 16SrIII-J and 16SrXV-A phytoplasmas have already been reported in association with this disease in this region (Canale and Bedendo 2013; Rappussi et al. 2012). Herein we report the involvement of 16SrXIII-E phytoplasma with cauliflower stunt disease and the presence of this bacteria in Balclutha hebe leafhopper. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from leaf veins of seven symptomatic and seven asymptomatic cauliflower samples collected in May 2008 from a farm located in Morungaba (SP, –22.882603, –46.755783). In addition, in an attempt to identify a possible vector for the phytoplasma, insects were captured using a sweep net from a cauliflower farm in Sorocaba (SP, –23.391314, –47.507474), where the disease occurred in high incidence levels (Rappussi et al. 2012). The most abundant leafhopper species collected with the net was identified as B. hebe Kirkaldy, 1900 (Hemiptera: Cicadellidae). DNA was also obtained from whole insects using the same extraction kit as we used for plants. Phytoplasma detection was performed with a nested-PCR assay using P1/Tint and R16F2n/R16R2 universal primers specific to the phytoplasma 16S rRNA gene (Lee et al. 1998; Smart et al. 1996). PCR products of the expected size (∼1.2 kb) were obtained from all of the symptomatic cauliflower but not from the collected symptomless samples. We amplified phytoplasma DNA from four out of 15 B. hebe specimens. Two plant amplicons and two leafhopper amplicons were sequenced (Macrogen, Seoul, Korea). Nucleotide sequence alignment revealed that 16S rDNA of the phytoplasma from cauliflower and insect samples shared ˃99% sequence similarity with Papaya apical curl necrosis phytoplasma, a member of the 16SrXIII-E subgroup and previously described by our group (Melo et al. 2013). Restriction fragment length polymorphism analysis using AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI restriction enzymes and in silico analysis using the iPhyClassifier online tool (Zhao et al. 2009) also allowed confirmation of the identification of the phytoplasma affecting cauliflower plant and leafhopper samples as member of the 16SrXIII-E subgroup (F = 1.00). A phylogenetic tree was generated with sequences of 16S rDNA of distinct subgroups affiliated with the 16SrXIII group, using the neighbor-joining method and 1,000 bootstrap replicates (MEGA software, Tamura et al. 2011), using Acholeplasma laidlawii as an outgroup. The phylogenetic analysis supported the identification of cauliflower and B. hebe phytoplasmas reported in this study as members of 16SrXIII-E subgroup. Sequences of the 16SrXIII-E phytoplasmas were deposited in GenBank as the accession JN818844 (Cauliflower stunt phytoplasma) and JN818843 (B. hebe phytoplasma). The 16SrXIII phytoplasma group (‘Candidatus Phytoplasma hispanicum’) is diverse and apparently restricted to the American continent, affecting a wide range of plant hosts (Davis et al. 2016). B. hebe is a polyphagous generalist species omnipresent in southern and south Brazil. It is reported as a putative vector for Xylella fastidiosa in plum orchards in south Brazil (Hickel et al. 2001). Also, this species was found in grasses within citrus rows in orchards in Sao Paulo, and one specimen was infected with 16SrIX phytoplasma (Marques et al. 2012). B. hebe was abundant within the cauliflower crops in both locations of this study, and its abundance along with our research results indicate that this leafhopper may play a role as a vector for 16SrXIII-E to cauliflower. However, more refined transmission assays employing phytoplasma and insects maintained in a laboratory are needed to prove B. hebe capacity as a phytoplasma vector. Here, we report ‘Ca. P. hispanicum’ strain as one of the associated phytoplasmas with cauliflower stunt disease in Brazil and B. hebe leafhopper as a putative vector.