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Leaf symptoms of chlorosis and blotchy mottling, typically associated with agents of huanglongbing, were found in commercial citrus orchard located in the state of Sao Paulo, the main citrus producer region in Brazil. Affected trees tested negative for bacteria ‘Ca. Liberibacter americanus’ and ‘Ca. Liberibacter asiaticus’. So, the total DNA was submitted to nested PCR employing the primer pairs P1/Tint followed by R16F2n/R16R2. Genomic fragments of 1.2 kb were amplified, sequenced and analyzed by computer-simulated RFLP, which confirmed the presence of a 16SrVII phytoplasma in symptomatic plants. Collective restriction patterns allowed distinguish the detected phytoplasma from all other members of the 16SrVII group using enzymes BfaI and MseI. In addition, the citrus phytoplasma showed a similarity coefficient lower than 0.97, when compared with all representative strains of subgroups within of the 16SrVII group, characterizing, therefore, a new subgroup. In agreement, the phylogenetic tree displayed that this strain emerges from a distinct branch in relation to those 16SrVII phytoplasmas currently described. Based on these features, the strain identified in the present study may be delineated as a new phytoplasma representative of the novel 16SrVII-G subgroup. Moreover, the results implicate citrus as a new host for a phytoplasma belonging to the group 16SrVII.

Maize bushy stunt and corn stunt have emerged among the most important diseases of maize in Brazil. To evaluate the single or dual presence of the phytoplasma and spiroplasma associated with corn stunting diseases, maize samples were collected across several locations in four Brazilian states. Multiplex PCR was performed for simultaneous detection of the bacteria. Eighty-nine out of 100 samples were positive with percentage values of 40%, 35%, and 25% for phytoplasma, spiroplasma, and mixed infections, respectively. Temperature may be an important driver of the prevalence of these mollicutes as phytoplasma prevailed in areas with mild temperatures and spiroplasma prevailed in warmer areas. These results extend knowledge of factors associated with corn stunting diseases, such as the potential role of temperature shaping the composition of the regional plant pathogenic populations.

Acerola bushes were observed showing symptoms of shoot proliferation, generalized stunting, yellowing and decline. Since these symptoms are typically induced by phytoplasmas, this survey was carried out with the aim of detecting, identifying and classifying the supposed phytoplasma present in symptomatic bushes. Total DNA was extracted from symptomatic and asymptomatic samples and used in nested PCR conducted by the primer pairs R16mF2/mR1 followed by R16F2n/R2. Amplifications of expected genomic fragments of 1.2 kb revealed the presence of phytoplasma in 73 % of the symptomatic samples. Molecular analyses, using computer-simulated RFLP patterns, similarity coefficient calculation and phylogenetic analysis allowed for classifying the bacterium as a ‘Candidatus Phytoplasma pruni’ – related strain (subgroup 16SrIII-F). The phytoplasma induced the same symptoms in healthy acerola plants inoculated by grafting and showed molecular identity with the strain identified in naturally infected bushes. Although various strains belonging to distinct subgroups within the 16SrIII group have been previously identified in Brazil, this is the first report of the presence of a representative of the 16SrIII-F subgroup in the Brazilian agroecosystem. Considering that phytoplasmas can be systemically distributed throughout the plant and acerola plants are vegetatively propagated, it is recommended that propagation material be obtained from mother plants free of the pathogen.

Physalis is an herbaceous species native to the Andes region. Currently, it is cultivated in various Brazilian states due to the economic interest of growers for this new fruit. Physalis plants grown in the field showed symptoms of shoot proliferation, leaf malformation, and chlorosis. Since these symptoms are commonly induced by phytoplasmas, this study investigated to confirm the presence of these prokaryotes in symptomatic plants. After DNA extraction from symptomatic and asymptomatic plants, phytoplasmas were found in all affected plants through the nested PCR. Examination by transmission electron microscopy (TEM) using appropriately prepared segments of leaf veins allowed the visualization of typical pleomorphic cells of phytoplasmas in the phloem of symptomatic plants. The computer-simulated RFLP patterns and the phylogenetic analysis allowed identifying the detected phytoplasmas as a ‘Candidatus Phytoplasma fraxini’-related strain belonging to the 16SrVII-B subgroup. Moreover, physalis was identified as an additional host species for phytoplasmas in the 16SrVII group, expanding the current knowledge on the host range of phytoplasmas in this group.

Infectious hematopoietic necrosis virus (IHNV) is the causative agent of IHN triggering a systemic syndrome in salmonid fish. Although IHNV has always been associated with low levels of mortality in Italian trout farming industries, in the last years trout farmers have experienced severe disease outbreaks. However, the observed increasing virulence of IHNV is still based on empirical evidence due to the poor and often confounding information from the field. Virulence characterization of a selection of sixteen Italian isolates was performed through in vivo challenge of juvenile rainbow trout to confirm field evidence. The virulence of each strain was firstly described in terms of cumulative mortality and survival probability estimated by Kaplan–Meier curves. Furthermore, parametric survival models were applied to analyze the mortality rate profiles. Hence, it was possible to characterize the strain-specific mortality peaks and to relate their topology to virulence and mortality. Indeed, a positive correlation between maximum mortality probability and virulence was observed for all the strains. Results also indicate that more virulent is the strain, the earliest and narrowest is the mortality peak. Additionally, intra-host viral quantification determined in dead animals showed a significant correlation between viral replication and virulence. Whole-genome phylogeny conducted to determine whether there was a relation between virulence phenotype and IHNV genetics evidenced no clear clustering according to phenotype. Moreover, a root-to-tip analysis based on genetic distances and sampling date of Italian IHNV isolates highlighted a relevant temporal signal indicating an evolving nature of the virus, over time, with the more virulent strains being the more recent ones. This study provides the first systematic characterization of Italian IHNV’s virulence. Overall results confirm field data and point out an abrupt increase in IHNV virulence, with strains from 2015–2019 showing moderate to high virulence in rainbow trout. Further investigations are needed in order to extensively clarify the relation between evolution and virulence of IHNV and investigate the genetic determinants of virulence of this viral species in rainbow trout.

The aim of this study is the molecular characterization of a phytoplasma associated with cabbage stunt and spatial analyses of symptomatic plants in the field. Detection by nested PCR, sequencing of 16S rRNA gene and conventional and virtual RFLP, as well as phylogenetic analysis revealed that a 16SrIII-J phytoplasma was associated with cabbage plants that exhibited stunting, reduced size, malformation or failure to form head, reddening leaves, sprout proliferation and vessel necrosis. The spatial analyses demonstrated that the dispersion pattern of symptomatic plants was aggregated and the pathogen introduced from adjacent areas. Phytoplasmas of 16SrIII group were also detected in leafhoppers of the species Atanus nitidus collected from cabbage fields and adjacent areas, evidencing that these insects are possibly potential vectors of this fastidious bacteria found in cabbage plants. These findings confirmed that a subgroup 16SrIII-J phytoplasma is associated with cabbage stunt. In addition, the spatial analyses indicated that the disease emerges from primary foci located in external areas and progress toward the center of the fields. The discovery of the etiological agent of the cabbage stunt together with the type of dispersion of the disease represent a relevant contribution to improve the management strategies aiming harm reduction.

Olive trees exhibiting slow development, yellowing, and high intensity of shoot proliferation with small leaves were observed in commercial plantings, in the municipality of Extrema, Minas Gerais (MG) state in 2015. The incidence of symptomatic plants was about 70% and diseased trees presented yield reduction. Here we report the association of symptomatic olive trees with a phytoplasma and describe its molecular identification. Symptomatic plants (38 trees) were sampled in three growing areas located in the same municipality. The samples consisted of bunch of leaves and young shoots. The total DNA was extracted using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). Phytoplasma detection was conducted by nested PCR with primers P1/16S-SR (Lee et al. 2004) followed by R16F2n/R16R2 (Gundersen and Lee 1996). PCR assays generated amplicons (~1.2 kb) from 28 trees out of 38 symptomatic plants, confirming the association of phytoplasma with diseased plants. The disease was named olive witches' broom. The genomic fragments amplified by nested PCR were cloned into Escherichia coli DH5α and sequenced. The sequence representative of the olive phytoplasma was designated OWB-Br01 (Olive Wiches' Broom-Brazil 01) and deposited in GenBank under accession number MH141985. This sequence shared 99% sequence identity with phytoplasmas affiliated with 16SrVII group. According to the iPhyClassifier online tool (Zhao et al. 2009) the olive witches'-broom phytoplasma was classified as a variant of subgroup 16SrVII-B with a pattern similarity coefficient of 0.99. The phylogenetic tree showed that OWB-Br01 phytoplasma emerges from the same branch of the reference phytoplasma of the 16SrVII-B subgroup (Erigeron witches᾽-broom phytoplasma - GenBank AY034608), indicating that the olive tree phytoplasma is a member of the 16SrVII-B subgroup. The pathogenicity test was performed with 28 healthy plants (cultivar Arbequina) grown in pots, which were grafted by simple english forklift with scions obtained from olive plants (Arbequina) six years old, naturally infected by the phytoplasma. The initial symptoms were observed four months after grafting and at eight months 22 grafted plants exhibited slow growth, yellowing, and small leaves as those naturally observed in the fields. Molecular characterization allowed identify the phytoplasma as a member of the 16SrVII-B subgroup. In Brazil, representatives of the 16SrVII group were previously reported in association with diverse botanical species. Thus, a strain of 16SrVII-C subgroup was identified in sunn hemp (Flores et al. 2013); the reference phytoplasma of 16SrVII-D subgroup was found in erigeron plants (Flores et al. 2015); and the representative of 16SrVII-F was detected in the wild species Vernonia brasiliana. (Fugita et al. 2017). Specifically regarding subgroup 16SrVII-B, the reference phytoplasma of this subgroup was described from erigeron and periwinkle (Barros et al. 2002), while other members of this subgroup were reported in cauliflower (Pereira et al. 2016a) and ming aralia (Pereira et al. 2016b). The disease here studied is a threat since olive planting is in large expansion in Brazil. A potential control option could be use of propagative material from sources free of the pathogen. Based on our findings, olive tree represents a new host for subgroup 16SrVII-B phytoplasma, which is different from 16Sr groups previously reported as associated with olive witches' broom in other countries.

Made available in DSpace on 2021-06-25T12:23:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-27 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Plants of beggarticks (Bidens pilosa L.), honeyweed (Leonurus sibiricus L.), and citrus (Citrus ssp.) trees exhibiting phyllody and virescence, leaf distortion, and yellow shoots symptoms, respectively, were found in orchards at the state of Sao Paulo. Based on PCR assays, computer-simulated RFLP, and phylogenetic analyses, it was confirmed that these plants were infected with a phytoplasma of the subgroup 16SrIII-X, belonging to the 'Candidatus Phytoplasma pruni' species. This finding suggested that beggartick and honeyweed plants may be possible reservoirs of 16SrIII-X phytoplasma to citrus tree. The phytoplasma identified in this study presented perfect identity with the phytoplasma previously reported in association with citrus plants harboring HLB-like symptoms, in Brazil. Univ Sao Paulo, Dept Fitopatol, Escola Super Agr Luiz de Queiroz, Piracicaba, SP, Brazil Univ Estadual Paulista, Campus Expt Registro, Registro, SP, Brazil Embrapa Recursos Genet & Biotecnol, Brasilia, DF, Brazil Univ Estadual Paulista, Campus Expt Registro, Registro, SP, Brazil FAPESP: 2010/52045-0

Chrysanthemum plants showing symptoms typically induced by phytoplasmas, characterized by colour-breaking and virescence, were found with high incidences in commercial fields. Based on these symptoms, the present investigation aimed to confirm the association of phytoplasmas with affected plants. Expected genomic fragments of 1.2 Kb were generated by nested PCR revealing the occurrence of phytoplasma in all diseased plants. The presence of phytoplasma was confirmed by transmission electron microscopy that detected pleomorphic cells in the inner of phloem vessels. Nucleotide sequences from Chrysanthemum colour-breaking phytoplasma shared 99% similarity with the reference phytoplasma of the 16SrIII-X subgroup. The perfect identity was confirmed by computer-simulated RFLP analyses and similarity coefficient calculations (F equal to 1.0), as well as by the phylogenetic tree, which showed that both phytoplasmas emerge from the same branch. Our findings revealed for the first time the occurrence of a representative of the 16SrIII-X subgroup in Brazil. In addition, it also revealed that Chrysanthemum represents a novel host of a phytoplasma of the 16SrIII group in the world, since, to our knowledge, representatives of this group have not been reported inducing diseases in plants from this genus.

Brachiaria spp. are the most important and widely used warm season grasses for pasture, but are also important weeds for most economic crops in the tropics. During phytoplasma surveys in pasture fields, asymptomatic plants of Brachiaria decumbens were collected for inspection. Nested PCR detected phytoplasmas in more than 90% of sampled plants. Based on computer-simulated RFLP and a similarity coefficient calculation, a subgroup 16SrIII-J phytoplasma was molecularly characterized. Phylogenetic analysis was consistent with virtual RFLP identification. Representatives of this subgroup have been identified in diverse Brazilian regions encompassing various commercial crops. Our study revealed B. decumbens as a new host and reservoir of 16SrIII-J phytoplasma.

Momordica charantia (bitter melon) presents two distinct types or varieties, known as wild type and commercial type. Plants of the wild type are hosts of a phytoplasma of the group 16SrIII-J, which is associated with a disease known as witches’ broom. However, this disease has not yet been reported in commercial bitter melon. Thus, symptomatic plants of the commercial type were analyzed in order to demonstrate the association between phytoplasmas and disease. In further assays, strains found in symptomatic plants of the commercial type were subjected to analysis of sequences of the secY gene to determine the extent of genetic diversity. Amplification of DNA fragments from genes 16Sr rRNA (1.2Kb) and secY (1.6Kb) revealed association of phytoplasma with symptomatic plants of the commercial type. Virtual Restriction Fragment Length Polymorphism (RFLP) analysis identified this phytoplasma as a member of the subgroup 16SrIII-J. Phylogenetic analysis showed that the phytoplasma was closely related to the representative of the 16SrIII-J subgroup. Molecular analysis indicated that the secY gene, in spite of the greater genetic variation compared with 16S rRNA gene, did not separate strains of the phytoplasma of the subgroup 16SrIII-J among those strains present in M. charantia.

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.

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.

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.

In Brazil, phytoplasmas belonging to seven distinct groups and occurring in over 50 hosts belonging to commercial crops, weeds and wild plant species. The present chapter focuses on three economically relevant diseases occurring in three crops extensively grown in Brazilian fields: corn, brassicas and cassava. Brazil is one of the largest world producers of corn. The first epidemics of corn stunt diseases occurred in the beginning of 1990s and since this decade, the diseases has been reported as a major limiting factor of corn production, due its high level of incidence. General recommendations for control include the use of genetically resistant varieties and appropriate planting periods, eradication of volunteer plants and insecticides treatments. Brassicas, mainly cabbage, cauliflower and broccoli, are extensively cultivated and consumed by Brazilian population. Diseases associated with phytoplasmas are responsible for significant damage that reduce yield and quality of production and cause serious financial losses for growers. Prevention and control measures include the use of heathy seedlings, elimination of weeds from adjacent areas of crops, chemical applications in the first stages of crop development and maintenance of fields free of weeds after harvest. Cassava is an important source of carbohydrate for inhabitants of tropical areas, including Brazil. A disease known as witches’-broom is occurring with high incidence in the main producer regions, causing underdevelopment of roots and, consequently, low starch production per area. The most usual and efficient control is the use of healthy propagative materials for planting or resistant varieties.

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.

Symptoms of fruit phyllody and slow growth, which are suggestive of phytoplasma infection, were observed in strawberry plants cultivated in commercial fields. In order to provide evidence of association of phytoplasma with affected plants, assays for detecting and identifying were performed through computer-simulated restriction fragment length polymorphism (RFLP) and phylogenetic analysis. Total DNA was extracted from symptomatic and asymptomatic samples and used as template in nested PCR primed by the primers P1/Tint followed by R16F2n/16R2. Amplified DNA fragments of 1.2 kb from the 16S rRNA gene revealed the presence of phytoplasma in all symptomatic samples. Molecular detection was confirmed by electron transmission microscopy, which evidenced pleomorphic bodies in the phloem vessels. Nucleotide sequence representative of the strawberry phytoplasma shared 97.2 to 99 % similarity with phytoplasmas currently classified as members of the distinct subgroups within the 16SrXIII group. Similarity coefficient (F) values ranged from 0.70 to 0.92, indicating that strawberry phytoplasma delineates a new strain in addition to ‘Candidatus Phytoplasma hispanicum’-related strains. The evolutionary tree displayed that this strain emerges as a new branch in relation to those previously described. The novel strain, designated SFP (strawberry fruit phyllody) phytoplasma represents the new 16SrXIII-J subgroup and its sequence, denominated SFP-Br02, was deposited in the GenBank database (EU719108). These findings contribute for the knowledge of the genetic diversity existing among members of the group 16SrXIII and establishes strawberry as an additional host of representatives of this group in Brazil.

The symptoms of the main cereal diseases associated with phytoplasma presence such as barley deformation, maize bushy stunt, maize redness, green ear, oat proliferation, oat stunt, oat yellows, rice orange leaf, rice yellow dwarf, triticosecale stunt, wheat blue dwarf and wheat streak, yellowing, and stunting are described. Moreover the phytoplasmas associated with each disease are described together with their geographical distribution. In some cases, factors that affect the occurrence, development, and severity of these diseases are discussed. The insect vectors involved in transmission of some of these diseases are presented, along with the main morphological characteristics adopted for their identification.

Aegiphila verticillata (parrot fruit) is a wild plant that commonly grows in Brazilian savanna fields, which are also denominated Cerrado. Trees and shrubs of this species were observed exhibiting profuse shoot proliferation that produced small leaves with mild chlorosis and flowers and fruit of reduced size which fell off prematurely. In the present study, PCR assays demonstrated the association of a phytoplasma with naturally affected plants. Identification performed with group-specific primers indicated this phytoplasma as belonging to the 16SrIII group. Molecular analysis based on virtual RFLP patterns and similarity coefficient calculations allowed to classify the phytoplasma as a representative of the 16SrIII-J subgroup. Phylogenetic analysis confirmed this result and evidenced that this phytoplasma was closely related to the reference strain for the 16SrIII-J subgroup. The disease was denominated Aegiphila witches’ broom (AegWB). In South America, representatives of the 16SrIII-J subgroup are frequently found in association with diseases of phytoplasmal etiology that occur in several wild and cultivated species. This study reveals for the first time that A. verticillata, a typical plant from the Brazilian Cerrado region, is a host of phytoplasmas, specifically of a member of the 16SrIII-J subgroup.

This study fully describes a severe disease outbreak occurred in 2016 in black bullhead catfish farmed in Italy. Affected fish showed nervous clinical signs as well as emaciations and haemorrhagic petechiae on the skin at the fin bases, abdomen and gills. Viral isolation in cell culture allowed the subsequent identification of a rhabdovirus, tentatively named ictalurid rhabdovirus (IcRV), through electron microscopy, immunofluorescence and whole genome sequencing (WGS). The newly isolated virus, together with 14 additional viral strains stored in our repository and detected during similar mortality episodes in the period 1993-2016, was phylogenetically analysed on the basis of the nucleoprotein and the glycoprotein nucleotide and amino acid sequences. The genetic distances among Italian IcRV strains were also estimated. Our results show that all the IcRV strains belong to the genus Sprivivirus and are closely related to the tench rhabdovirus (TenRV). Italian catfish production is constantly decreasing, mainly due to viral infections, which include the newly characterized IcRV. Data presented in this work will assist to investigate the molecular epidemiology and the diffusive dynamics of this virus and to develop adequate surveillance activities.

Phytoplasmas were detected using nested PCR with extracts of soybean plants which exhibited delayed maturity. PCR assays, phylogenetic and virtual RFLP analysis identified the phytoplasma as a member of the l6Srl-B subgroup.

Vernonia brasiliana is a wild perennial shrub frequently found in pasture areas. Plants of this species have been observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, deformed leaves and leaf chlorosis. The present study confirmed the presence of phytoplasmas in association with affected plants. Sequencing of the 16S rRNA gene, computer-simulated RFLP analysis and phylogenetic analysis revealed that one of the phytoplasmas identified was representative of novel subgroup. The sequence identity scores between the novel strain and those of previously described ‘Candidatus Phytoplasma fraxini’-related strains was 99 %, while similarity coefficient values were lower than 0.97. These findings provide support to delineate the phytoplasma found in vernonia plants as a reference phytoplasma for a novel subgroup designated 16SrVII-F. This representative of the novel subgroup was denominated VbSP phytoplasma (Vernonia brasiliana Shoot Proliferation; GenBank KX342018). The results of the present study revealed V. brasiliana to be a host of phytoplasmas, evidenced a novel phytoplasma associated with phytoplasmal disease in Brazil and extended the knowledge of the genetic diversity existing within the 16SrVII group.

Vernonia brasiliana is a wild species frequently found in Brazilian savanna areas, also known as “cerrado” areas. In pasture fields, shrubs were observed displaying typical symptoms induced by phytoplasmas, which were characterized by shoot proliferation, mild leaf chlorosis and deformed leaves. Molecular detection revealed the presence of phytoplasmas in approximately 80% of the symptomatic plants. Identification by computer-simulated RFLP allowed the classification of phytoplasma strains as representatives of ‘Candidatus Phytoplasma pruni’, taxonomic subgroups 16SrIII-B and 16SrIII-J. Moreover, phylogenetic analysis evidenced perfect agreement with virtual RFLP. Both phytoplasmas found in diseased vernonia plants have been reported in diverse commercial species cultivated in Brazil, including many vegetables and an extensive number of crops. These findings identify V. brasiliana as a new host of phytoplasmas affiliated with the subgroups 16SrIII-B and 16SrIII-J and suggest this species as a probable reservoir and possible inoculum source of phytoplasmas.

Bougainvillea-potted plants exhibiting typical phytoplasma-induced symptoms, characterized by foliar chlorosis, shoot proliferation, leaf and bract deformations, and decline were observed in commercial nurseries, located in the state of Sao Paulo, Brazil. In this study, PCR assays using group-specific primers revealed that phytoplasmas affiliated with the groups 16SrI and 16SrIII were associated with symptomatic plants. Molecular analysis based on conventional and virtual RFLP patterns and similarity coefficient calculations identified these phytoplasmas as belonging to subgroups 16SrI-B and 16SrIII-B. Phylogenetic analysis confirmed that these phytoplasmas were closely related to representatives of both subgroups. Transmission assays using dodder supported the initial evidence that the symptoms were associated with phytoplasmas.

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.

Papaya apical curl necrosis (PACN) has frequently been observed in several Brazilian states. Affected plants exhibit foliar chlorosis, curvature of the apex, shortening of the internodes leading to bunching of the crown leaves, necrosis of the young apical parts, leaf drop, and dieback. Naturally infected plants were sampled and subjected to PCR assays, which confirmed that a phytoplasma was associated with the disease. Sequencing of the 16S rRNA gene, conventional and computer-simulated RFLP analyses, and phylogenetic analysis allowed the determination of the PACN phytoplasma as a representative of a new subgroup, designated 16SrXIII-E. The phytoplasmas of various 16Sr groups, including 16SrI, 16SrII, 16SrX, 16SrXII, and 16SrXVII, are known to be involved in anomalies in papaya plants in several countries. However, the present study reports, for the first time, the occurrence of a 16SrXIII phytoplasma in association with a papaya disease.

Cassava plants displaying typical symptoms commonly associated with phytoplasmas, such as witches’ broom, general stunt, leaves with chlorosis, deformation, and reduced size, were observed in fields located in the state of Sao Paulo, Brazil. Molecular analysis using specific primers evidenced that the phytoplasma found in all positive samples was affiliated with group 16SrIII (X-disease group). Based on the virtual RFLP patterns and similarity coefficient calculations, the phytoplasma was classified as a member of the group 16SrIII, subgroup B (16SrIII-B). Phylogenetic analysis showed this phytoplasma as closely related to the reference strain for the subgroup 16SrIII-B. Representatives of 16SrIII-B have been described in several cultivated species in Brazil and cassava is an additional host species for this phytoplasma.

Leonurus sibiricus (honeyweed) plants displaying small, shriveled and mildly chlorotic leaves were observed in commercial cassava fields. Based on nested PCR assays using a group-specific primer pair, computer-simulated RFLP analysis, similarity coefficient calculation and phylogenetic analysis, the phytoplasma identified in these plants was classified as a representative of subgroup 16SrIII-B. This is the first report of honeyweed as a host of phytoplasmas.

Since 2000, a disease has occurred with high levels of incidence in crops of cauliflower grown in the green belt area of the city of Sao Paulo, Brazil. The symptoms are characterized by stunting, malformation of the inflorescence, reddening leaves, and vascular necrosis, suggesting infection by phytoplasma. These symptoms are similar to those described in Brassicas species affected by the aster yellows (16SrI) group of phytoplasma. In the present study, a phytoplasma from the 16SrIII-J subgroup was identified in cauliflower plants based on actual and virtual RFLP patterns and phylogenetic analysis, and was distinct from the phytoplasmas frequently associated with aster yellows disease in Brassicas. Pathogenicity assays using dodder confirmed that the identified phytoplasma is the agent of the observed disease, which is here designated as cauliflower stunt. Consequently, this species of Brassica may be recognized as a new host for subgroup 16SrIII-J, which has frequently been found in diverse species cultivated in Brazil. The spatial pattern of diseased plants was determined in ten cauliflower plots of 300 to 728 plants each. All plants in these plots were evaluated by visual assessments, assigned as diseased or healthy and mapped. The dispersion index and Taylor’s power law were determined for various quadrat sizes and the results showed that the diseased plants were distributed in a random pattern in fields with a low disease incidence and in an aggregated pattern in fields with a disease incidence greater than 25 %. According to an isopath area analysis, diseased plants were predominantly present in the field borders, suggesting that the pathogen is possibly introduced by vector(s) from the external area.

Em um plantio comercial localizado no Vale do Ribeira, no estado de São Paulo, foram observadas plantas de abobrinha-de-moita (Cucurbita pepo L.) apresentando superbrotamento de ramos, malformação da parte aérea e folhas disformes e enrugadas. Através de PCR foi demonstrada a presença de fitoplasma associado aos tecidos doentes. Um fitoplasma do grupo 16SrIII foi identificado por PCR e pela análise de RFLP, conduzida com cinco enzimas de restrição. A presente comunicação se constitui no primeiro relato da presença de um fitoplasma do grupo 16SrIII em abobrinha-de-moita no Brasil. In a commercial field located in the Vale do Ribeira, in the State of São Paulo, Brazil, plants of summer squash (Cucurbita pepo L.) exhibiting witches' broom and leaf deformation were observed. PCR assays demonstrated the presence of phytoplasma associated with diseased tissues. A phytoplasma belonging to the 16SrIII group was identified by PCR and RFLP analysis performed with five restriction enzymes. The present note is the first report of the presence of phytoplasma representative of the 16SrIII group in summer squash in Brazil.

Yellow leaf syndrome was a serious problem in the beginning of the 1990s in Brazil, when yield losses were estimated to be around 50%. The disease is currently endemic, but it is considered potentially important. Previous studies have revealed only the presence of a luteovirus associated with the disease in Brazil. We report that a phytoplasma of 16SrI-B is also associated with this disease. This is the first demonstration of the presence of a group 16SrI-B phytoplasma in association with sugarcane yellow leaf in Brazil.

O lenho mole da macieira é uma doença relevante em diversas partes do mundo. Sintomas típicos desta doença têm sido observados em pomares instalados em estados do sul do território brasileiro desde a década de oitenta. Enxertia tem revelado a natureza infecciosa da doença e a observação de corpúsculos filamentosos no floema tem evidenciado possível associação com fitoplasma. No presente trabalho plantas com sintomas de lenho mole foram coletadas em pomar comercial, visando demonstrar a presença de fitoplasma em tecido doente, bem como identificar molecularmente este fitoplasma. Através do emprego de duplo PCR com iniciadores universais R16mF2/R1 e R16F2n/R2, fitoplasma foi consistentemente detectado em plantas sintomáticas. A identificação conduzida com duplo PCR usando-se iniciadores específicos R16(III)F2/R demonstrou que o fitoplasma detectado pertencia ao grupo 16SrIII. Análises de RFLP conduzidas com as endonucleases AluI, KpnI, HinfI, HpaII, MseI, RsaI e SauIIIA confirmaram que o fitoplasma era um representante típico do grupo 16SrIII. A detecção e identificação molecular se constitui numa forte evidência que um fitoplasma está associado ao lenho mole da macieira no Brasil, complementando os trabalhos realizados anteriormente com transmissão por enxertia e observação por microscopia eletrônica . Apple rubbery wood is an important disease occurring worldwide. Typical symptoms have been observed since 80' decade in orchards located in the South part of Brazil. In previous studies, grafting has evidenciated that the disease had infeccious etiology and visualization of filamentous bodies inside phloem had indicated that a phytoplasma could be associated with the disease. In the present study, plants with symptoms of rubbery wood were sampled in a commercial orchard in order to demonstrate the presence of phytoplasma in infected tissue and to identify molecularly that the organism. Using nested PCR with universal primers pairs R16mF2/R1 and R16F2n/R2, phytoplasma was consistently detected in symptomatic plants. Identification conducted with specific primer pair R16(III)F2/R1 in nested PCR demonstrated that the phytoplasma was a member of group 16SrIII. RFLP analyses with endonucleases AluI, KpnI, HinfI, HpaII, MseI, RsaI e SauIIIA confirmed that the phytoplasma was a representative of the group 16SrIII. The detection and molecular identification are strong evidences that a phytoplasma is associated with apple rubbery wood in Brazil and agrees with previous evidences using grafting and electron microscopy.

Erigeron sp. plants showing symptoms of witches' broom and stunting were found near orchards of passion fruit in São Paulo state, Brazil. These symptoms were indicative of infection by phytoplasmas. Thus, the aim of this study was to detect and identify possible phytoplasmas associated with diseased plants. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR conducted with the primer pairs P1/Tint and R16F2n/16R2. Amplification of genomic fragments of 1.2 kb from the 16S rRNA gene confirmed the presence of phytoplasma in all symptomatic samples. The sequence identity scores between the 16S rRNA gene of the phytoplasma strain identified in the current study and those of previously reported ‘Candidatus Phytoplasma fraxini’-related strains ranged from 98 % to 99 % indicating the phytoplasma to be a strain affiliated with ‘Candidatus Phytoplasma fraxini’. The results from a phylogenetic analysis and virtual RFLP analysis of the 16S rRNA gene sequence with 17 restriction enzymes revealed that the phytoplasma strain belongs to the ash yellows phytoplasma group (16SrVII); the similarity coefficient of RFLP patterns further suggested that the phytoplasma represents a novel subgroup, designated 16SrVII-D. The representative of this new subgroup was named EboWB phytoplasma (Erigeron bonariensis Witches' Broom).

Tomato plants displaying foliar chlorosis, shoot proliferation, calyx deformation and reduced leaves, flowers and fruits all symptoms of tomato big bud, were found in a culture at Braganca Paulista region, State of Sao Paulo, Brazil. Samples were collected and analyzed by electron microscopy, PCR and RFLP to detect and identify the possible phytoplasma associated with diseased plants. Pleomorphic and round corpuscles were observed in the phloem vessels of diseased plants; the presence of phytoplasma was confirmed by PCR using universal primer pairs. PCR assays primed by group-specific primer pairs enabled the identification of the detected phytoplasma as a member of the 16SrIII group. Molecular identification by RFLP using various endonucleases confirmed the classification of this phytoplasma as a representative of the 16SrIII group. Phytoplasmas that are associated with big bud have been classified in different groups. The big bud phytoplasma identified in Brazil belongs to the same group of phy...

Phytoplasmas are associated with several plant diseases occurring in Brazil. A phytoplasma of group 16SrIII found in tomato plants with symptoms of big bud was identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA. RFLP patterns using HhaI and RsaI endonucleases were distinct from those exhibited by phytoplasmas representatives of diverse subgroups of group 16SrIII. Nucleotide sequence analyses demonstrated sequence heterogeneity expressed through a few base positions and restriction site among cloned fragments, revealing lineages different from members of currently known subgroups. The detection of lineages within tomato big bud phytoplasma present in Brazil revealed the diversity of representatives of group 16SrIII in tropical ecosystem and confirmed the genetic diversity of phytoplasmas of that group around the world.

Since 2007, a new disease in broccoli (Brassica oleracea var. italica Plenck) has been observed in the Sao Paulo state, Brazil. The characteristic symptoms of the disease are plant stunting, inflorescence malformation, reddening of the leaves and phloem necrosis. Nested polymerase chain reaction with P1/Tint and F2n/R2 primer pairs revealed the presence of phytoplasmas in diseased broccoli plants. Restriction fragment length polymorphism and phylogenetic analysis of the 16S rDNA gene showed that phytoplasmas belonging to 16SrI, III and XIII groups were associated with the plants. To the best of our knowledge, this is the first report of phytoplasmas in this Brassica species in Brazil, as well the first time phytoplasmas of 16SrIII and XIII groups have been associated with broccoli plants.

The objective of the present work was to study biochemical alterations in corn plants infected by the maize bushy stunt phytoplasma. A resistant and a susceptible corn hybrid were used and changes in protein, phenolic compounds, chlorophyll and reducing sugars in infected plants were evaluated. The experiments were carried out inside greenhouse, and the corn plants were inoculated by using infective Dalbulus maidis leafhoppers 10 days after seeding. When symptoms started to appear, leaf samples were harvested at different times for the biochemical analyses. The results showed an increase in all biochemical parameters evaluated for both hybrids, with exception for the chlorophyll content that decreased. In a general way, the values observed for the level of the different compounds were higher in the susceptible hybrid when compared to the resistant one. The increases in protein, reducing sugar and phenolic compound contents in inoculated plants point out changes in host metabolism due to the phytoplasma, and the reduction in chlorophyll content shows that the phytoplasma can interfere in photosynthesis and perhaps speedy senescence in the leaf tissue.

Ornamental plants of Celosia argentea L. and Celosia spicata L. displaying typical phytoplasma-induced symptoms were observed in Piracicaba, State of Sao Paulo, south-eastern Brazil. Our aim was to identify the possible phytoplasma involved. PCR revealed the association of phytoplasma with diseased plants of both species. Based on actual and virtual RFLP analysis and phylogenetic analysis, the phytoplasma was characterized as a member of the 16SrIII-J subgroup. Transmission of the pathogen by dodder supported the evidence that the symptoms observed in naturally diseased plants were induced by a phytoplasma. Our results show that C. spicata is a new host for phytoplasma and that this is the first report of a 16SrXIII-J phytoplasma infecting plants of C. argentea and C. spicata in Brazil.

Recently, disease in broccoli plants (Brassica oleracea var. italica) was associated with three distinct phytoplasmas in Brazil. The disease named broccoli stunt (BS) has caused significant economic losses in Sao Paulo State. Group 16SrIII phytoplasmas is the most common group that have been associated with BS. The BS disease is still poorly understood, and the lack of information about the vectors further impairs its management. In this study, leafhoppers belonging to 18 different species were collected from weeds thriving near broccoli fields that were affected by group 16SrIII phytoplasmas. Specific primers revealed the presence of group 16SrIII phytoplasmas in five leafhoppers: Empoasca spp., Agallia albidula Uhler, Agalliana sticticollis (Stal), Planicephalus flavicosta (Stal), and Atanus nitidus (Linnavuori). The identity of the phytoplasmas was confirmed through DNA sequencing analysis. The leafhoppers were infected by phytoplasmas of the 16SrIII group (proposed “Candidatus Phytoplasma pruni” species) and are phylogenetically related to the broccoli stunt phytoplasma (BSP) strains detected in the study area and, thus, are considered potential vectors of group 16SrIII phytoplasmas to broccoli plants.

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provid...

Plantas de hibisco com superbrotamento e definhamento seguido de morte têm sido observadas nos municípios de São Paulo, Campinas e Piracicaba. Como os sintomas são sugestivos daqueles induzidos por fitoplasmas, o presente trabalho buscou identificar o possível fitoplasma associado com a doença. Assim, 14 plantas sintomáticas de hibisco foram coletadas em Piracicaba (SP) e submetidas ao PCR duplo com os primers P1/Tint-R16F2n/R2 e ao exame em microscópio eletrônico de transmissão. A identificação foi realizada por análise de RFLP com as enzimas de restrição BfaI, DraI, HaeIII, HhaI, HpaII, MboI, MseI, RsaI e TaqI. Testes de transmissão foram conduzidos com enxertia de ramos e uso de Cuscuta subinclusa. Os resultados de nested-PCR revelaram a presença consistente de fitoplasmas em todas as plantas sintomáticas e foram confirmados pela observação de corpúsculos pleomórficos no floema, através da microscopia eletrônica. A análise de RFLP mostrou que o fitoplasma encontrado em hibisco pertence ao grupo 16SrXV, o mesmo grupo do Candidatus Phytoplasma brasiliense. O fitoplasma foi transmitido de planta doente para sadia, tanto pela enxertia como pela C. subinclusa, demonstrando ser o agente do superbrotamento do hibisco.

Begonia is a very appreciated genus of ornamental plants, of economic relevancy, having species of flowers and foliage. In commercial croppings, plants exhibiting characteristic symptoms of phytoplasma infection have been observed, such as shoot proliferation, reduced plant, size small leaves and flowers, and phyllody. Leaves were sampled and total DNA was extracted to be used in nested Polymerase Chain Reaction (PCR), in order to detect and identify an expected phytoplasma. The results confirmed consistently the presence of a phytoplasma associated with symptomatic plants through the amplification of a typical genomic fragment of 1.2 kb by using the universal primers R16mF2/mR1 and R16F2n/R2. The use of specific primers R16(III)F2/R1 allowed to identify the phytoplasma detected as a representative of the group 16SrIII. This information is very expressive, because different diseases caused by fungus, bacteria, virus and nematodes have been reported for begonia, however, reports have not been found for begonia diseases associated with phytoplasmas.

Maize rayado fino virus (MRFV), present in various countries in Latin America, has shown similarities to corn streak virus that occurs in Brazil, regarding pathogenic, serological and histological characteristics. In the current report both virus were molecularly compared to confirm the similarities between them. MRFV was identified by nucleic acid hybridization in samples of maize tissues exhibiting symptoms of "corn stunt" disease, collected from two Brazilian States - São Paulo and Minas Gerais. The coat protein gene and 3'non-translated region of MRFV were amplified from infected tissues by reverse transcription-polymerase chain reaction (RT-PCR) using MRFV-specific primers, and were characterized by nucleotide sequence and phylogenetic analysis of the cloned PCR products. Phylogenetic analysis of the relationships between the Brazilian isolates and isolates obtained from Latin America and the United States reveals a close relationship to isolates from Brazil, Peru and Bolivia. Results support the proposal that the Brazilian corn streak virus be regarded as an isolate of MRFV and provide evidence for the presence of MRFV in "corn stunt' disease in Brazil.

Periwinkle (Catharanthus roseus), a tropical perennial plant, was found to be infected by a phytoplasma. Plants exhibiting virescence, phyllody and variegation symptoms were collected in the states of Minas Gerais and Sao Paulo, Brazil. The phytoplasma was transmitted by grafting from an infected periwinkle plant to healthy plants and by dodder to a citrus plant. Phytoplasma isolates from periwinkle plants from Brazil had the 16S rDNA gene sequenced and were classified in the 16SrIX group, subgroup A, belonging to the 'Candidatus P. phoenicium' species.

In the present work we studied the effect of inoculating corn plants with the maize bushy stunt phytoplasma on the activity of the enzymes peroxidase, β-1,3 glucanase and chitinase. The experiments were carried out inside a greenhouse. Plants of a resistant and a susceptible corn hybrid were inoculated by using infective Dalbulus maidis leafhoppers 10 days after sowing. When symptoms started to appear, leaf samples were collected at different periods to quantify enzyme activity. The results showed an increase in the activity of the three enzymes in inoculated plants of both hybrids. In general, the values observed for the level of the different enzymes were higher in the susceptible hybrid when compared to the resistant one. Thus, the increases in peroxidase, β-1,3 glucanase and chitinase levels in inoculated plants are evidence of changes in the host metabolism caused by the phytoplasma. On the other hand, since the increases could not be correlated with plant resistance further studies are needed to explain such changes. No presente trabalho foi estudado o efeito da inoculação de plantas de milho com o fitoplasma, agente do enfezamento vermelho, sobre a atividade das enzimas peroxidase, β-1,3 glucanase, quitinase. Os experimentos foram conduzidos em casa de vegetação. Plantas de um híbrido de milho resistente e de um suscetível foram inoculadas através de cigarrinhas infectivas de Dalbulus maidis, 10 dias após a semeadura. Quando os sintomas começaram a aparecer, amostras de folhas foram coletadas em diferentes períodos de tempo para quantificar a atividade das enzimas. Os resultados mostraram um aumento na atividade das três enzimas em plantas inoculadas de ambos os híbridos. De maneira geral, os valores observados para o teor das diferentes enzimas foram mais elevados no híbrido suscetível quando comparado com ao resistente. Assim, os aumentos nos níveis de peroxidade, β-1,3 glucanase, e quitinase em plantas inoculadas se constituíram em evidência de alterações no metabolismo do hospedeiro causadas pelo fitoplasma. Por outro lado, uma vez que os aumentos não puderam ser correlacionados com a resistência de planta, há necessidade de novos estudos para poder interpretar estas alterações.

Symptoms resembling giant calyx, a graft-transmissible disease, were observed on 1–5 % of eggplant (aubergine; Solanum melongena L.) plants in production fields in Sao Paulo state, Brazil. Phytoplasmas were detected in 12 of 12 samples from symptomatic plants that were analysed by a nested PCR assay employing 16S rRNA gene primers R16mF2/R16mR1 followed by R16F2n/R16R2. RFLP analysis of the resulting rRNA gene products (1.2 kb) indicated that all plants contained similar phytoplasmas, each closely resembling strains previously classified as members of RFLP group 16SrIII (X-disease group). Virtual RFLP and phylogenetic analyses of sequences derived from PCR products identified phytoplasmas infecting eggplant crops grown in Piracicaba as a lineage of the subgroup 16SrIII-J, whereas phytoplasmas detected in plants grown in Bragança Paulista were tentatively classified as members of a novel subgroup 16SrIII-U. These findings confirm eggplant as a new host of group 16SrIII-J phytoplasmas and extend the known diversity of strains belonging to this group in Brazil.