First Report of Beet necrotic yellow vein virus on Red Table Beet in Brazil.

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet (Beta vulgaris var. saccharifera) in several European, and Asian countries and in the United States (3). The virus is transmitted by the soil-inhabiting plasmodiophorid Polymyxa betae and causes the rhizomania disease of sugar beet. In November 2012, plants of B. vulgaris subsp. vulgaris cv. Boro (red table beet) exhibiting mainly severe characteristic root symptom of rhizomania were found in a commercial field located in the municipality of São José do Rio Pardo, State of São Paulo, Brazil. No characteristic virus-inducing foliar symptom was observed on diseased plants. The incidence of diseased plants was around 70% in the two visited crops. As the hairy root symptom is indicative of infection by BNYVV, the present study aimed to detect and identify this virus associated with the diseased plants. Preliminary leaf dip analysis by transmission electron microscopy revealed the presence of very few benyvirus-like particles. Total RNA was extracted from roots of three symptomatic plants and one asymptomatic plant according to Toth et al. (3). One-step reverse-transcription-polymerase chain reaction (RT-PCR) was performed as described by Morris et al. (2) with primers that amplify part of the coat protein gene at RNA2. The initial assumption that the hairy root symptom was associated with BNYVV infection was confirmed by the amplification of a fragment of ~500 bp from all three symptomatic samples. No amplicon was obtained from the asymptomatic control plant. Amplicons were directly sequenced, and the consensus nucleotide and deduced amino acid sequences showed 100% identity. The nucleotide sequence for one amplicon (Accession No. KM433683) was compared with other sequences deposited in GenBank. The nucleotide (468 nt) and deduced amino acid (156 aa) sequences shared 93 to 100 and 97 to 99% identity, respectively with the corresponding nucleotide and amino acid sequences for other isolates of type A of BNYVV. The virus was transmitted to three of 10 red table beet plants inoculated with contaminated soil, and infection was confirmed by nested RT-PCR, as described by Morris et al. (1), and nucleotide sequencing. This is the first report on the occurrence of BNYVV in Brazil, which certainly will affect the yield of red table beet in the producing region. Therefore, mapping of the occurrence of BNYVV in red table beet-producing areas in Brazil for containment of the spread of the virus is urgent. In the meantime, precautions should be taken to control the movement of contaminated soil and beet roots, carrots, or any vegetable grown on infested land that might introduce the virus to still virus-free regions. References: (1) J. Morris et al. J. Virol. Methods 95:163, 2001. (2) D. D. Sutic et al. Handbook of Plant Virus Diseases. CRC Press, Boca Raton, Florida, 1999. (3) I. K. Toth et al. Methods for the Detection and Quantification of Erwinia carotovora subsp. atroseptica (Pectobacterium carotovorum subsb. atrosepticum) on Potatoes: A Laboratory Manual. Scottish Crop Research Institute, Dundee, Scotland, 2002.

'Candidatus Phytoplasma brasiliense' (16SrXV-A Subgroup) Associated with Cauliflower Displaying Stunt Symptoms in Brazil.

Cauliflower stunt, caused by a phytoplasma of the group 16SrIII-J, was reported in the beginning of 2012 and has occurred with high incidences of infected plants (up to 90%) in crops located in the state of São Paulo in the southeast region of Brazil (3). Diseased plants exhibit general stunting, malformation of inflorescence, reddening leaves, and vessel necrosis (3). Further investigations with plants displaying identical symptoms collected in Nova Bassano, state of Rio Grande do Sul, Brazilian south region, have revealed the presence of a phytoplasma distinct from 16SrIII-J subgroup. Four symptomatic plus four asymptomatic samples were assayed from a field, and the presence of phytoplasma was evidenced by nested PCR assays performed with primers P1/Tint followed by R16F2n/16R2 in three affected plants, which amplified genomic fragments of 1.2 kb from the 16S rRNA gene. No amplification occurred in non-affected samples. Nested PCR products analyzed by conventional RFLP (2) using the enzymes AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI pointed to the presence of a phytoplasma belonging to group 16SrXV-A in all three phytoplasma-positive samples. Virtual RFLP analysis based on restriction patterns, derived from in silico digestion with 17 endonucleases (4), confirmed the previous results obtained from those samples by conventional RFLP. The 16S rDNA sequences of this phytoplasma identified in cauliflower (GenBank Accession No. JN818845) shared 99% sequence similarity with the reference phytoplasma for subgroup 16SrXV-A (Hibiscus witches'-broom phytoplasma, AF147708), designated 'Candidatus Phytoplasma brasiliense.' Analysis of putative restriction sites showed excellent identity between the phytoplasma studied here and the reference phytoplasma. In addition, the arrangement of branches of a phylogenetic tree constructed with phytoplasmas representing diverse 16Sr groups and subgroups supported that the phytoplasma found in cauliflower is closed related to the representative of the subgroup 16SrXV-A. Association of distinct phytoplasmas with the same kind of disease is not rare and the present pathosystem constitutes a new example. Members of this subgroup have been described almost exclusively in Brazil and previously reported in Sida sp., periwinkle, and hibiscus (1). In some European countries, as well as in the United States and Canada, phytoplasmas belonging to group 16SrI has been associated with this type of disease, which has been reported for various species of the genus Brassica, as published in previous works (3). However, a representative of the group 16SrVI was described in infected plants in Iran (3). Although the 16SrIII-J phytoplasma is currently the most important agent of cauliflower stunt in Brazil, and members of 16SrI are prevalent in other countries, this study revealed that a 16Sr XV-A phytoplasma may be also associated with this important disease of brassicas. Besides, the findings here reported expand the natural host range, including cauliflower as new host for phytoplasmas affiliated with 16SrXV-A. References: (1) B. Eckstein et al. Plant Dis. 95:363, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) M. C. C. Rappussi et al. Eur. J. Plant. Pathol. 133:829, 2012. (4) Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

First Report of a Group 16SrVII-C Phytoplasma Associated with Shoot Proliferation of Sunn Hemp (Crotalaria juncea) in Brazil.

Sunn hemp (Crotalaria juncea) is widely grown in tropical and subtropical regions. In Brazil, this species is commonly used for green manure, since this legume is an efficient nitrogen fixer that produces organic residues for soil improvement. In July of 2012, C. juncea exhibiting intense shoot proliferation, leaf malformation, shortened internodes, and generalized yellowing were found in an experimental field located in Piracicaba, State of São Paulo, Brazil. The incidence was about 1 to 2% and the diseased plants were distributed at random. Since these symptoms are indicative of infection by phytoplasmas, the present study aimed to detect and identify the phytoplasma. Four symptomatic and two asymptomatic plants were sampled. Small segments of leaf veins were prepared for microscopy, as previously reported (1), and observations were made using a Jeol (Akishima/Japan) model Jem-1011 transmission electron microscope. Total DNA was extracted from leaves using a commercial kit (DNeasy Plant Mini, Qiagen Inc.), and nested PCR assays were performed with primers, P1/Tint followed by R16F2n/R16R2 (2). The initial assumption that disease symptoms were associated with phytoplasma was confirmed by PCR amplification of 1.2 kb DNA fragments from the 16S rDNA gene. In contrast, no amplicon was generated with PCR using template DNA from asymptomatic plants. The phytoplasma detected from each symptomatic sample was considered to be an isolate. PCR products were purified and cloned in Escherichia coli DH5α, using the pGEM-T Easy Vector System I (Promega). Three isolates were selected and the cloned 16S rDNA sequences from three colonies of each isolate were sequenced. Since no sequence polymorphisms were found, a majority consensus sequence was selected for each isolate. These sequences were identical and one of them, designated CrSP-Br01 (crotalaria shoot proliferation) with 1,249 bp (GenBank Accession KC756947), was used as representative of the sunn hemp phytoplasma. The 16S rDNA nucleotide sequence of this phytoplasma shared 100% sequence identity with the reference phytoplasma for subgroup VII-C (Argentinian Alfalfa witches'-broom phytoplasma, AY147038). According to the in silico RFLP analysis for delineation of subgroups (3), which is based on virtual RFLP patterns and similarity coefficient calculation, the C. juncea phytoplasma was classified as a member of group 16SrVII, subgroup C. Phylogenetic analysis supported that this phytoplasma is closely related to the representative of subgroup 16SrVII-C, since both phytoplasmas emerged from the same branch. Transmission electron microscopic examination revealed the presence of phytoplasmas by visualization of pleomorphic and round bodies 100 to 400 nm in diameter, in the phloem vessels of symptomatic plants. The present study reports the first occurrence of a 16SrVII-C phytoplasma in Brazil. In addition, C. juncea was identified as a new host for phytoplasmas belonging to this subgroup. References: (1) A. B. Maunsbach and B. A. Afzelius. Biomedical Electron Microscopy. Illustrated Methods and Interpretations. Page 381-426, San Diego, Academic Press, 1999. (2) M. C. C. Rappussi et al. Eur. J. Plant Pathol. 133:829, 2012. (3) W. Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

A Sida sp. Is a New Host for "Candidatus Phytoplasma brasiliense" in Brazil.

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated "Candidatus Phytoplasma brasiliense" (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). "Ca. Phytoplasma brasiliense" has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 85:1209, 2001. (4) E. G. Silva et al. Summa Phytopathol. 35:234, 2009.

First Report of a Group 16SrIII-B Phytoplasma Associated with Decline of China Tree in Brazil.

China tree (Melia azedarach L.), originally from Asia, is an exotic deciduous species in Brazil and is used as an ornamental shade tree in the southern region of the country. Since 2005, plants displaying yellowing, little leaves, witches' broom, and decline have been observed in the State of Rio Grande do Sul. In the streets and avenues of the capital city of Porto Alegre, there are approximately 173 tree species and China tree (6.57% of all trees) is among the top 10 (80,000 China trees and most are symptomatic). Plants with those symptoms are very distinctive and have been found also in the cities of Livramento, Rio Grande, Santa Maria, and Vacaria, places located in seashore areas, and along highways everywhere in the state. The high incidence seems to be related to drought during the last few years. These symptoms are typical of a disease identified by yellowing or decline of China tree associated with phytoplasma and previously reported in the neighboring countries of Argentina, Paraguay, and Bolivia (2). To demonstrate the presence of phytoplasma in diseased trees and to confirm its identity, total DNA was extracted from China tree leaf midribs collected from 10 symptomatic and three asymptomatic plants. Nested PCR was performed with the P1/P7 primer pair in the primary PCR to amplify a 1.8-kb fragment encompassing the 16S rRNA gene, the 16S-23S spacer region, and the 5' end of the 23S rRNA gene, while the secondary PCR was primed by the R16F2n/R16R2 primer pair to amplify a 1.2-kb fragment of the 16S rRNA gene from the 1.8-kb fragment (3,4). DNA fragments of 1.2 kb amplified from nested PCR were analyzed by restriction fragment length polymorphism with restriction enzymes AluI, HhaI, HpaII, KpnI, MboI, MseI, and RsaI, revealing identical profiles for each amplicon and demonstrating that a phytoplasma belonging to group 16SrIII, subgroup B (16SrIII-B) (1) was associated consistently with all symptomatic plants. BLAST analysis revealed 99% identity among these cloned 1.2-kb sequences and representative sequences of phytoplasmas affiliated with group 16SrIII (GenBank Accession Nos. AY081817 and AF147706). A majority consensus sequence representing the phytoplasma found in China trees was selected and deposited in GenBank (Accession No. FJ404775). These results were confirmed by observation with transmission electron microscopy of pleomorphic bodies 400 to 2,000 nm in diameter in the phloem sieve tubes of all symptomatic trees. No phytoplasma was detected or visualized in asymptomatic samples. These results corroborate those from studies conducted in neighboring countries that demonstrated the association between phytoplasmas of group 16SrIII and decline of China trees (1). In conclusion, the current study revealed that a phytoplasma affiliated with group 16SrIII-B is associated with the decline of China tree in Brazil, a disease previously described based solely on symptoms (2). The incidence and severity of the disease are enough to prevent further use of these trees as landscape plants in southern Brazil. References: (1) J. D. Arneodo et al. J. Phytopathol. 155:70, 2007. (2) M. Dalbosco et al. Fitopatol. Bras. 30(Suppl.):177, 2005. (3) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Role of Maize rayado fino virus in the Etiology of "Red Stunt" Disease in Brazil.

Corn production in several areas of Brazil recently has been seriously afflicted by a disease commonly referred to as "red stunt," characterized by stunting and leaf reddening. Early observations that a phytoplasma was associated with the disease were confirmed through molecular analysis, which revealed the presence of maize bushy stunt phytoplasma (MBS) (1). Another disease of corn, corn stunt, is considered to be caused by one or more of a complex of MBS, corn stunt spiroplasma (CSS), and Maize rayado fino virus (MRFV), which can all be transmitted simultaneously by their leafhopper vector Dalbulus maidis (Delong & Wolcott). The contributions of CSS and MRFV to the recently described "red stunt" disease in Brazil are unknown. A virus serologically related to MRFV, Brazilian corn streak virus, was first identified in Sao Paulo State, Brazil, in the early 1970s; serological studies indicated that isolates of Brazilian corn streak virus were related to, but distinguishable from, MRFV isolates from other Latin American countries (4). Therefore, there was a high probability that MRFV would be found in maize tissues collected from plants exhibiting symptoms of "red stunt" disease. Maize leaf samples exhibiting symptoms of "red stunt" disease were collected and preserved by drying from four Brazilian States during 1995 and 1996 (1). Total nucleic acid extracts were prepared from dried leaf tissue and aliquots of the extracts were spotted onto a nylon membrane, which subsequently was hybridized to an MRFV-specific cRNA probe (3). Of the 37 samples tested for the presence of MRFV by nucleic acid hybridization, 16 were positive for MRFV. It was present in some, but not all, samples that were positive for MBS (1). MBS was detected in six, and CSS was detected by polymerase chain reaction (PCR) (2) in 12 samples. In 8 of the 37 samples, both CSS and MRFV were present, 4 of 37 were positive for MBS and MRFV, and in 3 of 37 samples, all three pathogens were detected. Therefore, there is not a clear correlation between the presence of MRFV and the symptoms of "red stunt." The coat protein gene and adjacent 3' nontranslated region of MRFV were amplified from infected tissues by reverse transcription-polymerase chain reaction (RT-PCR) using MRFV-specific primers (3). Three cloned PCR products were sequenced (deposited at GenBank under accession nos. AF186177 to AF186179), which revealed that the nucleotide sequences of the Brazilian isolates were 98% sequence identical and shared 90 to 97% identity with other MRFV isolates (3). Phylogenetic analysis of the sequences revealed close relationships to MRFV isolates from Peru and Bolivia, which neighbor Brazil (3). The contribution of MRFV to the stunting and leaf reddening symptoms exhibited by maize plants with "red stunt" disease is unknown. Of the 37 samples examined in this study, MRFV was detected in 16. A more complete epidemiological study of the association of MBS, CSS, MRFV, and their insect vector with "red stunt" disease will provide information on the significance of these pathogens in the current disease outbreak. References: (1) I. P. Bedendo et al. Plant Dis. 81:957, 1997. (2) R. E. Davis and E. L. Dally (Abstr.), Phytopathology 88:S20, 1998. (3) R. W. Hammond et al. J. Gen. Virol. 78:3153, 1997. (4) E. W. Kitajima et al. Proc. Am. Phytopathol. Soc. 2:76, 1975.

Molecular Evidence for the Presence of Maize Bushy Stunt Phytoplasma in Corn in Brazil.

Previously, electron microscopy revealed that the corn (Zea mays L.) disease characterized by stunting and leaf reddening and commonly known as "red stunt" in Brazil is associated with plant infection by an unidentified phytoplasma (formerly mycoplasmalike organism) (1). During recent years, corn production in Brazil has been seriously affected by the increasing prevalence of a disease exhibiting symptoms similar to those of "red stunt." The present investigation was initiated to determine the possible association of a phytoplasma with the current disease problem and to attain definitive molecular identification of any associated phytoplasma. To detect the possible presence of a phytoplasma in diseased corn in Brazil, plants exhibiting symptoms of stunting and leaf reddening in the field in 1995 and 1996 were assayed for the presence of phytoplasma DNA sequences by the use of polymerase chain reactions (PCR). We used primer pairs R16mF2/R16mR1 and R16F2n/R16R2 in nested PCRs (2) to prime phytoplasma-universal amplification of 16S ribosomal (r) DNA. Oligonucleotide pair rpF1/rpR1 (3) was used to prime phytoplasma-universal amplification of ribosomal protein (rp) gene operon sequences. Phytoplasma identification was accomplished by restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA and rp gene operon sequences. Primer pair MBS-F1/MBS-R1 (4) was used to prime amplification of a maize bushy stunt (MBS)-specific chromosomal DNA sequence. Preparation of template DNAs, PCR conditions, and RFLP analyses of PCR products were as previously described (2-4). DNA amplification was observed in all PCRs containing template DNAs derived from symptomatic plants, indicating phytoplasmal infection of corn in Brazil. No DNA amplification was observed in PCR containing template DNA from healthy control corn plants. Polymorphisms in amplified 16S rDNA were those characteristic of phytoplasmas classified in 16S rRNA gene group 16SrI, subgroup I-B, of which MBS phytoplasma is a member (3). Collective RFLP patterns of amplified rp gene operon sequences were similar or identical to those observed in parallel tests for a known reference strain of MBS phytoplasma, indicating that the Brazilian corn plants were infected by MBS phytoplasma. Amplification of MBS-characteristic DNA was observed in PCRs containing MBS-specific primer pair MBS-F1/MBS-R1 and DNA from diseased corn, confirming infection of the plants by MBS phytoplasma. This work provides the first firm evidence for association of maize bushy stunt phytoplasma with the current disease problem of corn in Brazil. References: (1) A. S. Costa et al. Rev. Soc. Bras. Fitopatol. Piracicaba 4:39, 1971. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35: 144,1996. (3) D. E. Gundersen et al. Int. J. Syst. Bacteriol. 46:64, 1996. (4) N. A. Harrison et al. Plant Dis. 80:263, 1996.