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Cassava frogskin disease (CFSD) is a particular threat in cassava because symptoms remain hidden until harvest and losses can be total. The information related to the etiological agent of this disease is contradictory, because some authors believe it is caused by phytoplasmas while others believe that it is caused by a virus. In order to refine detection protocols and to characterize organisms associated with CFSD in Brazil, 32 symptomatic and 20 asymptomatic cassava plants were collected in Minas Gerais state. Total DNA was extracted and used for nested polymerase chain reaction (PCR) to detect phytoplasmas. Because endophytic Bacillus spp. led to false positives, primers were designed to facilitate the detection of phytoplasma in the presence of bacteria. In addition, double-stranded (ds)RNA was extracted from tubers and used in reverse-transcription PCR for the detection of the RNA-dependent RNA polymerase gene from Cassava frogskin virus segment 4. The detected phytoplasma was identified as belonging to the group 16SrIII-A by restriction fragment length polymorphism (RFLP), sequencing, and RFLP in silico. This is the first report of a phytoplasma belonging to the 16SrIII-A group associated with cassava plants, the first molecular characterization of a phytoplasma associated with CFSD in Brazil, and a first report of phytoplasma and a dsRNA virus (possible reovirus) co-infecting cassava plants with CFSD symptoms.

At present, the principal bacterial disease of citrus in Brazil is Huanglongbing, caused by the alpha-proteobacterium ‘Candidatus Liberibacter spp.’ (although a phytoplasma of the 16SrIX group is also associated with this disease [4]). While there is a wide diversity of phytoplasmas in crop species in Brazil (3), there have been no reports of symptoms associated with phytoplasma in Brazilian citrus. Asymptomatic infections of citrus cannot be excluded as a possibility and such plants could serve as a reservoir of phytoplasma inoculum. The aim of this study was to assess the presence of phytoplasma in asymptomatic Citrus aurantifolia (acid lime) in Brazil. Thirty-three leaf samples (young leaves from the upper canopies) were randomly collected from different plants in the states of Minas Gerais (n = 23), Santa Catarina (n = 2), and São Paulo (n = 8). Two additional samples of C. limonia (‘Rangpur’ lime) and one of C. latifolia (‘Persian’ or ‘Tahiti’ lime) were collected in Minas Gerais. Total DNA extraction was performed using NucleoSpin Plant II Kit (Macherey-Nagel) according to the manufacturer's recommendations. PCR was carried out with a universal P1/P7 primer set followed by nested primers R16F2n/R16R2 (2). Additionally, direct PCR was performed using primers specific for phytoplasma immune-dominant membrane protein IMP3F/IMP3R (1). ‘Rangpur’ and ‘Tahiti’ lime were not infected with phytoplasma. Of the C. aurantifolia samples, 52% were positive for phytoplasma in the direct and nested PCR assays. The numbers of positive samples in Minas Gerais, Santa Catarina, and São Paulo states were 12, 1, and 4, respectively. Of these, five were selected for DNA purification and 1,246-bp fragments were ligated to the pGEM T-easy vector (Promega) and partial 16Sr DNA was sequenced. Nucleotide sequences of Brazilian phytoplasma strains BR:MG:FNS10:2011, BR:MG:FNS53:2011, BR:SP:FNS73:2011, BR:SC:FNS86:2011, and BR:MG:FNS126:2012 (GenBank Accession Nos. KJ158173, KJ158174, KJ158175, KJ158176, and KJ158177, respectively) were subjected to RFLP analyses. The 16S rDNA RFLP in silico patterns for the five strains were identical to each other and to Cactus witches'-broom phytoplasma (16SrII-C subgroup, AJ293216). In addition, the highest similarity coefficient (5) and nucleotide sequence identity of Brazilian phytoplasma strains were 0.99 and 99%, respectively, with Cactus witches'-broom phytoplasma. PCR-RFLP analyses using the enzymes Bstu I, EcoR I, and Hpa II were consistent with RFLP in silico results, showing the same pattern as the 16SrII-C subgroup. Phylogenetic analyses based on 16S rDNA sequences (1,246 bp) demonstrated that all the Brazilian strains grouped in the same clade with other representative sequences from the 16S rDNAII group. To confirm the absence of any macroscopic symptoms, morphological characteristics of 10 uninfected and 10 phytoplasma-infected plants randomly selected from a single field in Minas Gerais were analyzed. There were no significant differences in leaf area, stalk diameter, or numbers of leaves, flowers, or fruits per branch. To our knowledge, this is the first report of the 16SrII-C subgroup phytoplasma associated with C. aurantifolia in Brazil, and the first report of asymptomatic citrus plants infected with phytoplasma. References: (1) N. Askari et al. J. Microbiol. Biotechnol. 21:81, 2011. (2) I. M. Lee et al. Phytopathology 84:559, 1994. (3) H. G. Montano et al. Bull. Insectol. 60:129, 2007. (4) D. C. Teixeira et al. Phytopathology 98:977, 2008. (5) Y. Zhao et al. Meth. Mol. Biol. 938:329, 2013.

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