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ABSTRACT Some psyllids transmit 'Candidatus Liberibacter solanacearum' (Lso), the causal agent of devastating plant diseases of cultivated Solanaceae and Apiaceae. The recent detection of Bactericera cockerelli and Lso in Ecuador seriously threatens these crops in South America. There, neither the role of native psyllids in the Lso epidemiology nor the psyllid fauna of vegetables are known. With the aim to identify potential vectors and risk scenarios for the spread of Lso in South America, a survey of the psyllid fauna of Solanaceae and Apiaceae crops and associated weeds was conducted in Brazil. Samples were taken at 29 localities in four states. A total of 2857 specimens were sampled, representing at least 37 species of 23 genera and seven families. The most frequent species on carrot, chilli pepper and potato were Russelliana solanicola, R. capsici and Isogonoceraia divergipennis, respectively. Immatures of R. capsici were found on chilli pepper and of R. solanicola on carrot and potato, confirming these plants as hosts. The two psyllid species have been suspected previously to transmit plant pathogens of unknown identity. Russelliana solanicola is one of the few polyphagous species. Here the species is reported for the first time from carrot. Recent collections in Rio Grande do Sul suggest that Solanum laxum represents the original host of R. capsici, which subsequently shifted to chilli pepper. Both, adaptation to agricultural crops and the possibility of ability to transmit pathogens, make the two Russelliana species dangerous potential vectors of Lso and other plant pathogens in South America.

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Maize striate mosaic virus (MSMV; genus Mastrevirus) was recently reported in maize plants in Brazil and also detected by metagenomic analyses in the corn leafhopper, Dalbulus maidis (DeLong & Wolcott). Although these findings suggested that D. maidis is a potential vector, no transmission studies have been performed. Here, we tested the transmission of MSMV by D. maidis from field-collected infected plants and plants infected with MSMV via leafhopper-mediated transmission in the laboratory; all plants were confirmed positive for MSMV by PCR. In each one of three transmission replicates, aviruliferous D. maidis nymphs and adults were confined together on a source plant during a 4-day acquisition access period (AAP) and subsequently transferred to healthy maize seedlings (10 individuals per test plant) in a series of 4-day inoculation access periods (IAPs). We also tested transmission by the corn aphid, Rhopalosiphum maidis (Fitch) and by mechanical inoculation of healthy maize seedlings. Only D. maidis transmitted MSMV, with overall transmission rates of 29.4 and 39.5% on field-collected infected plants and 18.5% on infected plants in laboratory. D. maidis transmitted MSMV until the third (8 to 12 days after the AAP) or fourth successive IAP (12 to 16 days), with gradual loss in transmission efficiency and rate of viruliferous insects over time, suggesting a persistent but nonpropagative mode of transmission. Infected test plants showed mottling symptoms with mild chlorotic streaks and height reduction. This is the first report of transmission of a mastrevirus by D. maidis, facilitating the completion of Koch's postulate for MSMV.

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In Brazil, the host expansion of Xylella fastidiosa subsp. pauca was recently demonstrated with the report of diseased olive trees (Olea europaea), whose symptoms were associated with olive quick decline syndrome previously described in southern Italy. We employed both polymerase chain reaction-based techniques and culture medium isolation to investigate the geographic distribution of X. fastidiosa as well as the genetic signatures of 21 strains isolated from 11 olive orchards in both São Paulo and Minas Gerais States in Brazil. X. fastidiosa subsp. pauca was detected in 83% of the orchards examined in the region, and was positively diagnosed in 43.7% of all sampled plants with typical scorching symptoms. Of the 21 strains characterized with fast-evolving microsatellite (single sequence repeat [SSR]) markers, 20 different multilocus microsatellite genotypes were observed with the overall allelic diversity of HNei = 0.38. Principal component analysis using the SSR markers clustered all strains, except for three, in one cluster demonstrating a limited range of genetic diversity. Multilocus sequence typing analysis showed the prevalence of a sequence type (ST16) in 75% of the samples; three other novel STs (84, 85, and 86), were detected, all belonging to the X. fastidiosa subsp. pauca cluster. These results show that genetically diverse strains of X. fastidiosa subsp. pauca are widely present in olive orchards in southeastern Brazil, which is consistent with the long history of this bacterium in that region.

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Homologous recombination affects the evolution of bacteria such as Xylella fastidiosa, a naturally competent plant pathogen that requires insect vectors for dispersal. This bacterial species is taxonomically divided into subspecies, with phylogenetic clusters within subspecies that are host specific. One subspecies, pauca, is primarily limited to South America, with the exception of recently reported strains in Europe and Costa Rica. Despite the economic importance of X. fastidiosa subsp. pauca in South America, little is known about its genetic diversity. Multilocus sequence typing (MLST) has previously identified six sequence types (ST) among plant samples collected in Brazil (both subsp. pauca and multiplex). Here, we report on a survey of X. fastidiosa genetic diversity (MLST based) performed in six regions in Brazil and two in Argentina, by sampling five different plant species. In addition to the six previously reported ST, seven new subsp. pauca and two new subsp. multiplex ST were identified. The presence of subsp. multiplex in South America is considered to be the consequence of a single introduction from its native range in North America more than 80 years ago. Different phylogenetic approaches clustered the South American ST into four groups, with strains infecting citrus (subsp. pauca); coffee and olive (subsp. pauca); coffee, hibiscus, and plum (subsp. pauca); and plum (subsp. multiplex). In areas where these different genetic clusters occurred sympatrically, we found evidence of homologous recombination in the form of bidirectional allelic exchange between subspp. pauca and multiplex. In fact, the only strain of subsp. pauca isolated from a plum host had an allele that originated from subsp. multiplex. These signatures of bidirectional homologous recombination between endemic and introduced ST indicate that gene flow occurs in short evolutionary time frames in X. fastidiosa, despite the ecological isolation (i.e., host plant species) of genotypes.

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Two haplotypes of Xylella fastidiosa subsp. pauca (Xfp) that correlated with their host of origin were identified in a collection of 90 isolates infecting citrus and coffee plants in Brazil, based on a single-nucleotide polymorphism in the gyrB sequence. A new single-nucleotide primer extension (SNuPE) protocol was designed for rapid identification of Xfp according to the host source. The protocol proved to be robust for the prediction of the Xfp host source in blind tests using DNA from cultures of the bacterium, infected plants, and insect vectors allowed to feed on Xfp-infected citrus plants. AMOVA and STRUCTURE analyses of microsatellite data separated most Xfp populations on the basis of their host source, indicating that they were genetically distinct. The combined use of the SNaPshot protocol and three previously developed multilocus SSR markers showed that two haplotypes and distinct isolates of Xfp infect citrus and coffee in Brazil and that multiple, genetically different isolates can be present in a single orchard or infect a single tree. This combined approach will be very useful in studies of the epidemiology of Xfp-induced diseases, host specificity of bacterial genotypes, the occurrence of Xfp host jumping, vector feeding habits, etc., in economically important cultivated plants or weed host reservoirs of Xfp in Brazil and elsewhere.

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Aphids are the most important vectors of viruses infecting potato (Solanum tuberosum). We focused on the response of the aphid vector Myzus persicae (Sulzer) to five commercial potatocultivars: Ágata, Jaette Bintje, Mondial, Monalisa and Santè, by traditional antibiosis and antixenosis tests and by the EPG (Electrical Penetration Graph) technique, as a step forward to the design of effective management practices. Our aim was to identify plant factors involved in resistance of these cultivars against M. persicae, both at the surface and in deeper plant tissues. Results from the antixenosis test confirmed a strong preference of M. persicae for the Mondial cultivar. The antibiosis study indicated a lower population development of the aphid in 'Monalisa' when compared to 'Ágata' and 'Jaette Bintje'. EPG assays indicated that 'Santè' inhibited the initial feeding process of M. persicae, whereas 'Monalisa' showed a physical-type of resistance as demonstrated by a very high number of short probes. The cultivar Mondial showed average values for all EPG variables analyzed. The behavior in 'Jaette Bintje' indicated this cultivar was an ideal host for aphid feeding and reproduction. Together, the EPG data revealed the existence of pre and post-phloematics factors in the cultivars under study, which have important implications on the efficiency of transmission and spread of virus in potato by M. persicae.

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In some American countries, grapevines are affected by Pierce's disease (PD), which is caused by a particular strain of Xylella fastidiosa not yet reported in Brazil. In order to investigate the potential for PD spread in Brazil in case of pathogen introduction, we conducted a faunistic analysis of leafhoppers (Hemiptera: Cicadellidae) associated to vineyards in the State of Rio Grande do Sul, with emphasis in the subfamily Cicadellinae (sharpshooters), which includes the main potential vectors of X. fastidiosa. Leafhopper samplings were carried out fortnightly during two years (9/2004-9/2006) in four Vitis vinifera vineyards in the municipalities of Bento Gonçalves and Farroupilha (RS). Thirtyfour leafhopper and six spittlebug species were collected, but most (98.4%) of the 3,893 specimens trapped were leafhoppers, distributed in the subfamilies Cicadellinae (60.2%), Gyponinae (34.1%), Deltocephalinae (3.8%) and Coelidinae (0.3%). The sharpshooter specimens were divided in the tribes Cicadellini (68.5%; 12 species) and Proconiini (31.5%; 11 species). Based on the faunistic indices, five species of Cicadellini, Bucephalogonia xanthophis (Berg), Dilobopterus dispar (Germar), Macugonalia cavifrons Stal, Sibovia sagata (Signoret) and Spinagonalia rubrovittata Cavichioli, and three of Proconiini, Molomea consolida (Schöder), Oncometopia facialis (Signoret) and Oncometopia fusca Melichar were prevalent in the vineyards. The high diversity of native sharpshooters in Rio Grande do Sul indicates the existence of a high risk of PD spread if the pathogen is introduced in grapevines.

Assuntos

RESUMO

In some American countries, grapevines are affected by Pierce's disease (PD), which is caused by a particular strain of Xylella fastidiosa not yet reported in Brazil. In order to investigate the potential for PD spread in Brazil in case of pathogen introduction, we conducted a faunistic analysis of leafhoppers (Hemiptera: Cicadellidae) associated to vineyards in the State of Rio Grande do Sul, with emphasis in the subfamily Cicadellinae (sharpshooters), which includes the main potential vectors of X. fastidiosa. Leafhopper samplings were carried out fortnightly during two years (9/2004-9/2006) in four Vitis vinifera vineyards in the municipalities of Bento Gonçalves and Farroupilha (RS). Thirtyfour leafhopper and six spittlebug species were collected, but most (98.4 percent) of the 3,893 specimens trapped were leafhoppers, distributed in the subfamilies Cicadellinae (60.2 percent), Gyponinae (34.1 percent), Deltocephalinae (3.8 percent) and Coelidinae (0.3 percent). The sharpshooter specimens were divided in the tribes Cicadellini (68.5 percent; 12 species) and Proconiini (31.5 percent; 11 species). Based on the faunistic indices, five species of Cicadellini, Bucephalogonia xanthophis (Berg), Dilobopterus dispar (Germar), Macugonalia cavifrons Stal, Sibovia sagata (Signoret) and Spinagonalia rubrovittata Cavichioli, and three of Proconiini, Molomea consolida (Schõder), Oncometopia facialis (Signoret) and Oncometopia fusca Melichar were prevalent in the vineyards. The high diversity of native sharpshooters in Rio Grande do Sul indicates the existence of a high risk of PD spread if the pathogen is introduced in grapevines.

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Aphids are the most important vectors of viruses infecting potato (Solanum tuberosum). We focused on the response of the aphid vector Myzus persicae (Sulzer) to five commercial potatocultivars: Ágata, Jaette Bintje, Mondial, Monalisa and Santè, by traditional antibiosis and antixenosis tests and by the EPG (Electrical Penetration Graph) technique, as a step forward to the design of effective management practices. Our aim was to identify plant factors involved in resistance of these cultivars against M. persicae, both at the surface and in deeper plant tissues. Results from the antixenosis test confirmed a strong preference of M. persicae for the Mondial cultivar. The antibiosis study indicated a lower population development of the aphid in 'Monalisa' when compared to 'Ágata' and 'Jaette Bintje'. EPG assays indicated that 'Santè' inhibited the initial feeding process of M. persicae, whereas 'Monalisa' showed a physical-type of resistance as demonstrated by a very high number of short probes. The cultivar Mondial showed average values for all EPG variables analyzed. The behavior in 'Jaette Bintje' indicated this cultivar was an ideal host for aphid feeding and reproduction. Together, the EPG data revealed the existence of pre and post-phloematics factors in the cultivars under study, which have important implications on the efficiency of transmission and spread of virus in potato by M. persicae.

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The causal agent of citrus variegated clorosis, Xylella fastidiosa, is transmitted by leafhoppers of the subfamily Cicadellinae, whose species vary regionally. The goal of this study was to identify potential vectors of this pathogen in citrus groves of Bahia North Coast, Brazil. The survey was done from March/2002 to February/2003 in three seven- to nine-year-old sweet orange (Citrus sinensis, Pêra variety) groves located in Rio Real, BA. Fifteen yellow sticky cards (8.5x11.5 cm) were installed 40 m apart in each grove, hanged at 1.5 m high on the upper north side of citrus canopies, and replaced fortnightly. A sweep net was periodically used to sample leafhoppers on herbaceous weeds inside the groves, by selecting five points at random and performing 30 sweeps in each point. A total of 1,360 specimens of 49 Auchenorrhyncha species were collected in this study, mostly in the family Cicadellidae (90.2 percent). The subfamily Cicadellinae, which includes the sharpshooter vectors of X. fastidiosa, showed the largest number of species (14) and specimens (84.8 percent). Acrogonia flagellata Young, A. citrina Marucci & Cavichioli, Homalodisca spottii Takiya, Cavichioli & McKamey and an unidentified Cicadellini (species 1) were the dominant species trapped on citrus canopies, while Hortensia similis (Walker) and Erythrogonia dubia (Medler) were dominant in the weedy vegetation. Among the Cicadellinae species already known as vectors of X. fastidiosa in citrus, only A. citrina, Bucephalogonia xanthophis (Berg) e Ferrariana trivittata (Signoret) were found. The two latter species were accidentally trapped by sweep net in the weedy vegetation.

O agente causal da clorose variegada dos citros, Xylella fastidiosa, é transmitido por cigarrinhas da subfamília Cicadellinae, cujas espécies variam regionalmente. O objetivo deste trabalho foi identificar potenciais vetores do patógeno em pomares cítricos do Litoral Norte da Bahia. Os levantamentos foram conduzidos de mar/2002 a fev/2003, em três propriedades no município de Rio Real, em pomares de laranjeira doce (Citrus sinensis) variedade Pêra, com sete a nove anos. Em cada pomar, 15 cartões adesivos amarelos foram dispostos a 40 m entre si, instalados a 1,5 m de altura em ramos da face norte, na periferia da copa, sendo substituídos quinzenalmente. Também foram realizadas amostragens com rede de varredura na vegetação rasteira dos pomares, em cinco pontos escolhidos aleatoriamente, efetuando-se 30 redadas por ponto. No total, foram coletados 1360 espécimes pertencentes a 49 espécies de Auchenorrhyncha, a maioria da família Cicadellidae (90,2 por cento). A subfamília Cicadellinae, que inclui os vetores de X. fastidiosa, apresentou maior número de espécies (14) e de indivíduos coletados (84,8 por cento). Entre as espécies de Cicadellinae, Acrogonia flagellata Young, A citrina Marucci & Cavichioli, Homalodisca spottii Takiya, Cavichioli & McKamey e uma espécie não-identificada de Cicadellini (espécie 1) foram dominantes na copa das plantas cítricas, enquanto que Hortensia similis (Walker) e Erythrogonia dubia (Medler) foram dominantes na vegetação rasteira. Entre os cicadelíneos já conhecidos como vetores de X. fastidiosa em citros, apenas A. citrina, Bucephalogonia xanthophis (Berg) e Ferrariana trivittata (Signoret) foram observados, sendo que as duas últimas espécies tiveram ocorrência acidental na vegetação rasteira.

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The population of Hemiptera Auchenorrhyncha was studied in sweet citrus groves (Citrus sinensis), coffee plantations (Coffea arabica) and a semi-deciduous seasonal forest with shrub physiognomy in Bebedouro, SP, to evaluate the influence of the natural ecosystem on the species composition of the agroecosystems. Monitoring was carried out by using yellow stick cards, which were replaced every 15 days and all Auchenorrhyncha collected were counted and identified. Seven families, 11 subfamilies and 98 species were collected, with Cicadellidae being the most abundant. The native forest presented larger wealth, diversity and equitability of Auchenorrhyncha species, demonstrating to be more stable than the other habitats. The high values of similarities obtained between the agroecosystems and the forest demonstrated that great part of Auchenorrhyncha species occurring in the agricultural habitats was also occurring at the forest, indicating that the last may serve as reservoir of species. The abundance of the taxonomic groups of Auchenorrhyncha collected varied with the evaluated habitats, with Proconiini being the most abundant in the coffee plantation next to the forest, Athysanini, Scaphytopiini, Neocoelidiinae and Coelidiinae in the orange orchard and coffee plantation distant from the forest; Cicadellinae and Agalliinae were not related to any of the habitats. The presence of vector insects and possible vectors of plant diseases in the appraised habitats indicate the need of the implementation of strategies for landscape management.

A população de Hemiptera Auchenorrhyncha foi estudada em pomares de laranja doce (Citrus sinensis), cafezais (Coffea arabica) e floresta estacional semidecidual com fisionomia arbustiva, em Bebedouro (SP), com o objetivo de avaliar a influencia do ecossistema natural na composição de espécies do agroecossistemas. O monitoramento foi feito com cartões adesivos amarelos, os quais foram trocados a cada 15 dias, efetuando-se a contagem e identificação dos Auchenorrhyncha coletados. Capturou-se o total de sete famílias, 11 subfamílias e 98 espécies, sendo Cicadellidae o grupo mais abundante. A floresta nativa apresentou a maior riqueza, diversidade e equitabilidade de espécies de Auchenorrhyncha, demonstrando ser mais estável que os demais habitats. Os altos valores de similaridades obtidos entre os agroecossistemas e a floresta demonstram que grande parte das espécies de cigarrinhas que está ocorrendo nos habitats agrícolas também está ocorrendo na floresta, indicando que esta pode estar funcionando como reservatório de espécies. A abundância de grupos taxonômicos de Auchenorrhyncha coletados variou nos habitats avaliados, sendo Proconiini o mais abundante no cafezal próximo à floresta, Athysanini, Scaphytopiini, Neocoelidiinae e Coelidiinae no pomar de laranja e cafezal distante da floresta; Cicadellinae e Agalliinae não se relacionaram a nenhum dos habitats. A presença de insetos vetores e possíveis vetores de doenças de plantas cultivadas nos habitats avaliados indicam a necessidade da implantação do manejo de pragas nessa área.

Assuntos

RESUMO

The population of Hemiptera Auchenorrhyncha was studied in sweet citrus groves (Citrus sinensis), coffee plantations (Coffea arabica) and a semi-deciduous seasonal forest with shrub physiognomy in Bebedouro, SP, to evaluate the influence of the natural ecosystem on the species composition of the agroecosystems. Monitoring was carried out by using yellow stick cards, which were replaced every 15 days and all Auchenorrhyncha collected were counted and identified. Seven families, 11 subfamilies and 98 species were collected, with Cicadellidae being the most abundant. The native forest presented larger wealth, diversity and equitability of Auchenorrhyncha species, demonstrating to be more stable than the other habitats. The high values of similarities obtained between the agroecosystems and the forest demonstrated that great part of Auchenorrhyncha species occurring in the agricultural habitats was also occurring at the forest, indicating that the last may serve as reservoir of species. The abundance of the taxonomic groups of Auchenorrhyncha collected varied with the evaluated habitats, with Proconiini being the most abundant in the coffee plantation next to the forest, Athysanini, Scaphytopiini, Neocoelidiinae and Coelidiinae in the orange orchard and coffee plantation distant from the forest; Cicadellinae and Agalliinae were not related to any of the habitats. The presence of vector insects and possible vectors of plant diseases in the appraised habitats indicate the need of the implementation of strategies for landscape management.

Assuntos

RESUMO

The causal agent of citrus variegated clorosis, Xylella fastidiosa, is transmitted by leafhoppers of the subfamily Cicadellinae, whose species vary regionally. The goal of this study was to identify potential vectors of this pathogen in citrus groves of Bahia North Coast, Brazil. The survey was done from March/2002 to February/2003 in three seven- to nine-year-old sweet orange (Citrus sinensis, Pêra variety) groves located in Rio Real, BA. Fifteen yellow sticky cards (8.5x11.5 cm) were installed 40 m apart in each grove, hanged at 1.5 m high on the upper north side of citrus canopies, and replaced fortnightly. A sweep net was periodically used to sample leafhoppers on herbaceous weeds inside the groves, by selecting five points at random and performing 30 sweeps in each point. A total of 1,360 specimens of 49 Auchenorrhyncha species were collected in this study, mostly in the family Cicadellidae (90.2%). The subfamily Cicadellinae, which includes the sharpshooter vectors of X. fastidiosa, showed the largest number of species (14) and specimens (84.8%). Acrogonia flagellata Young, A. citrina Marucci & Cavichioli, Homalodisca spottii Takiya, Cavichioli & McKamey and an unidentified Cicadellini (species 1) were the dominant species trapped on citrus canopies, while Hortensia similis (Walker) and Erythrogonia dubia (Medler) were dominant in the weedy vegetation. Among the Cicadellinae species already known as vectors of X. fastidiosa in citrus, only A. citrina, Bucephalogonia xanthophis (Berg) e Ferrariana trivittata (Signoret) were found. The two latter species were accidentally trapped by sweep net in the weedy vegetation.

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RESUMO

Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) is a bacterial pathogen transmitted by several sharpshooters in two tribes of Cicadellinae (Proconiini and Cicadellini). Here, we compared the transmission efficiency of X. fastidiosa in coffee (Coffea arabica L.) and citrus [Citrus sinensis (L.) Osbeck] by Cicadellini [Bucephalogonia xanthophis (Berg) and Dilobopterus costalimai Young] and Proconiini [Homalodisca ignorata Melichar and Oncometopia facialis (Signoret)] sharpshooters that occur in both crops. At different seasons, healthy adults of each species were submitted to a 48-h acquisition access period on citrus or coffee source plants infected with X. fastidiosa isolates that cause Citrus variegated chlorosis (CVC) and Coffee leaf scorch (CLS), respectively, and then confined on healthy seedlings of the corresponding host plant for a 48-h inoculation access period. No significant effect of inoculation season was observed when comparing infection rates of citrus or coffee plants inoculated by vectors at different times of the year. In citrus, the transmission rate by single insects was significantly higher for H. ignorata (30%) in relation to B. xanthophis (5%) and O. facialis (1.1%), but there was no difference among vector species in coffee, whose transmission rates ranged from 1.2 to 7.2%. Comparing host plants, H. ignorata was more effective in transmitting X. fastidiosa to citrus (30%) in relation to coffee (2.2%), whereas the other vectors transmitted the bacterium to both hosts with similar efficiencies. Despite these variations, vector efficiency in coffee and citrus is lower than that reported in other hosts.

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Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

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Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis (CVC), Pierce's disease of grapevine, and leaf scald of coffee and plum and many other plant species. This pathogen is vectored by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and resides in the insect foregut. Scanning electron microscopy was used to determine the retention sites of X. fastidiosa for the most common vector species in Brazilian citrus groves, Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, and Oncometopia facialis. After a 48-h acquisition access period on infected citrus or plum, adult sharpshooters were kept on healthy citrus seedlings for an incubation period of 2 weeks to allow for bacterial multiplication. Then the vector heads were incubated for 24 h in a fixative and transferred into a cryoprotector liquid. Bacterial rod cells exhibiting similar X. fastidiosa morphology were found laterally attached to different regions inside the cibarial pump chamber (longitudinal groove, lateral surface, cibarial diaphragm and apodemal groove) of A. citrina, O. facialis, and D. costalimai, and polarly attached to the precibarium channel of O. facialis. Polymerase chain reactions of vector's heads were positive for the presence of X. fastidiosa. No X. fastidiosa-like cells were detected in B. xanthophis. A different type of rod-shaped bacterium was found on B. xanthophis cibarium chamber and images suggest that the cibarium wall was degraded/digested by these bacteria. Colonization patterns of X. fastidiosa in their vectors are fundamental aspects to be explored toward understanding acquisition, adhesion, and transmission mechanisms for development of X. fastidiosa control strategies.

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Xylella fastidiosa is a plant pathogen that threatens a US$ 4.6 billion worldwide wine and citrus industry. Monitoring its presence and distribution in plants and vectors is crucial for designing control strategies, as well as for understanding its ecological role and fate. We developed two fluorescent oligonucleotide probes complementary to different regions of the 16S rRNA gene of X. fastidiosa. The specificity of the newly designed probes S-S-X.fas-0067-a-A-18 and S-S-X.fas-1439-a-A-18 was demonstrated using fluorescence in situ hybridization (FISH) for 12 Xylella isolates, 15 closely related microorganisms and three plant endophytes. These probes were used to detect and quantify X. fastidiosa in plant sap (average value of 2.9 +/- 0.3 x 10(6) cells ml(-1)) from three different citrus orchards. In a second experiment, cells were quantified in honeydew (2.2 +/- 0.2 x 10(4) cells ml(-1)) collected from the insect vector Bucephalogonia xanthophis during the acquisition access period on an infected plant. The number of pathogen cells retained or digested by the insect is 10,000 times greater than the estimated minimum value to ensure an efficient transmission. Polymerase chain reaction (PCR) amplification using specific primers with plant sap and honeydew samples, followed by sequencing, confirmed the presence of the plant pathogen. This is the first demonstration of FISH being used for environmental samples, such as plant sap and insect honeydew, to estimate the abundance of a plant pathogen during infection.

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RESUMO

The efficiency of Xylella fastidiosa transmission by sharpshooters on citrus plants is low and varies with the vector species. The feeding behavior of the sharpshooter vector related to X. fastidiosa acquisition and inoculation is one of the probable conditioning factors in transmission efficiency. The objective of this study was to assess the behavior of two sharpshooter species, Dilobopterus costalimai Young and Oncometopia facialis (Signoret), in selection of citrus nursery trees and feeding places and to ascertain the influence of sprouting on plant attractiveness to the vectors. Free choice tests were performed with the two species in observation chambers (63 x 63 x 120 cm), releasing 40 individuals per chamber. The number of dead insects were assessed as well as those that chose, or not, one of the treatments, at 3, 15, 21, 24, 39, 45 and 48h after the release. For D. costalimai the leaves were important at the start of host selection, and were substituted over time by the secondary branch. D. costalimai preferred to stay on the secondary leaf nervures followed by the secondary branch. O. facialis showed no difference in preference for staying on the leaves (central and secondary nervures) and secondary branch on citrus plants, and this varied with the time of day. The presence of sprouting in the nursery trees increased the attractiveness of the plant to the two sharpshooter species raising the chances of X. fastidiosa acquisition or inoculation. So, control measures should be adopted against these insects in periods of greater vegetation in orange orchards.

A eficiência de transmissão de Xylella fastidiosa por cigarrinhas em citros é baixa e variável com a espécie vetora. O comportamento alimentar do vetor relacionado à aquisição e inoculação de X. fastidiosa é um dos prováveis fatores condicionantes da eficiência de transmissão. Assim, os objetivos desse trabalho foram avaliar o comportamento de duas espécies de cigarrinhas, Dilobopterus costalimai Young e Oncometopia facialis (Signoret), na seleção de mudas cítricas e de locais de alimentação e verificar a influência das brotações na atratividade aos vetores. Realizaram-se testes de livre escolha em câmaras de observação (63 x 63 x 120 cm) com as duas espécies, liberando-se 40 indivíduos por câmara. Avaliou-se o número de insetos mortos e que escolheram ou não um dos tratamentos, após 3, 15, 21, 24, 39, 45 e 48h da liberação. Para D. costalimai, as folhas foram importantes no início da seleção hospedeira, sendo substituídas com o passar do tempo pelo ramo secundário. D. costalimai preferiu pousar na nervura secundária da folha, seguida pelo ramo secundário. Para O. facialis não houve diferença na preferência para pouso entre folhas (nervura central e secundária) e ramos secundários das mudas cítricas, sendo esta variável com o horário do dia. Comprovou-se que a presença de brotação nas mudas aumenta a atratividade das plantas para as duas espécies de cigarrinhas, elevando as chances de aquisição ou inoculação de X. fastidiosa pelos vetores. Assim, sugere-se adotar medidas de controle desses insetos em períodos de maior vegetação dos pomares de laranja.

RESUMO

A distribuição espacial das espécies de cigarrinhas (Dilobopterus costalimai Young, Acrogonia sp. e Oncometopia facialis Signoret), vetoras da Xylella fastidiosa, agente causal da Clorose Variegada dos Citros, foi estudada com o uso da geoestatística. As avaliações foram feitas em um pomar comercial de laranja 'Pêra' (Citrus sinensis [L.] Osb.), objetivando estabelecer meios para melhor controle dos vetores e da doença. O monitoramento da ocorrência das cigarrinhas no pomar foi feito através de amostragens mensais, utilizando-se armadilhas adesivas amarelas de 3" x 5", distribuídas uniformemente em 50 pontos na área, dispostas em laranjeiras à altura de 1,5 m do solo e substituídas mensalmente. Acrogonia sp. foi a espécie prevalente nas amostragens. Os resultados possibilitaram ajustar modelos aos semivariogramas da distribuição espacial das três espécies no pomar estudado. Durante os três anos consecutivos de amostragem, as populações de Acrogonia sp., D. costalimai e O. facialis apresentaram modelos de distribuição agregada somente nos meses de verão, inverno e primavera, respectivamente, mostrando a necessidade de monitoramento constante desses vetores para reduzir a sua população em épocas favoráveis ao seu desenvolvimento. Através de parâmetros geoestatísticos foi possível calcular a área de agregação das cigarrinhas no pomar. A espécie Acrogonia sp. apresentou área média de agregação de 15.760 m², enquanto para O. facialis e D. costalimai foi possível constatar áreas médias de agregação de 11.555 m² e 10.980 m², respectivamente. Esses resultados indicaram que para um levantamento seguro de cigarrinhas é necessário pelo menos dispor de uma armadilha por hectare.

The spatial distribution of the three principal species of sharpshooter (Dilobopterus costalimai Young, Acrogonia sp. e Oncometopia facialis Signoret), vectors of Xylella fastidiosa causal agent of Citrus Variegated Chlorosis, was studied by using geostatistics. The evaluations were carried out in a commercial 'Pera' sweet orange (Citrus sinensis [L.] Osb.) grove budded on Rangpur lime (Citrus limonia Osb.) located at Bebedouro, São Paulo state, aiming to establish better means for vector and disease control. The sharpshooters were monitored using with 50 uniformly distributed 3" x 5" yellow sticky traps hanging down in branches at 1.5 m up to the ground, which were replaced monthly. Acrogonia sp. was the prevalent species sampled. The results were used to adjust population models to semivariograms of the spatial distribution of the three sharpshooters species in the grove. During the three consecutive years of sampling, the population of Acrogonia sp., D. costalimai and O. facialis showed an aggregated distribution only during summer, winter and spring, respectively, showing that intensive monitoring of these vectors is necessary to control their population in periods favorable to their development. The aggregation area of the sharpshooters within the orange grove was determined using geostatistic parameters. Acrogonia sp. showed a mean aggregation area of 15,760 m², whereas for O. facialis and D. costalimai, the corresponding areas were 11,555 m² and 10,980 m², respectively. Therefore, in order to obtain a reliable estimate of sharpshooter populations, at least one trap should be used per hectare.

RESUMO

The appropriate preservation of the DNA of a certain organism is important for successful application of molecular techniques like RAPD-PCR. This study was designed to compare simple methods of preservation of specimens of Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) with respect to quantity and quality of DNA for use in RAPD-PCR, after different storage periods. Eight methods were tested: freezing (-20°C); ethyl alcohol 70 percent (-20°C and room temperature); absolute ethyl alcohol (-20°C and room temperature); air-dry; and preservation in extraction buffer (whole and homogenized insect). At intervals of 10 to 30 days, insect DNA was extracted, quantified and amplified through RAPD-PCR using primer OPA-04. The quality of extracted DNA was observed on 0.8 percent agarose gel. After 210 days of preservation, freezing (-20°C) showed to be the best method. Satisfactory quantities of DNA were also obtained from insects conserved in absolute ethyl alcohol (-20°C), ethyl alcohol 70 percent (-20°C) and extraction buffer (whole and homogenized insect). Insects conserved in absolute ethyl alcohol (room temperature), ethyl alcohol 70 percent (room temperature) and air-dry conditions were inappropriate for RAPD-PCR studies after 120, 60 and 10 days of storage, respectively.

A preservação adequada do DNA de um determinado organismo é fundamental para o sucesso no uso de técnicas moleculares como RAPD-PCR. O objetivo deste trabalho foi comparar métodos simples de preservação de espécimes de Dalbulus maidis (DeLong & Wolcott) (Hemiptera: Cicadellidae) quanto à quantidade e qualidade do DNA para uso em RAPD-PCR, após períodos sucessivos de armazenamento. Avaliaram-se oito métodos: congelamento (-20°C); álcool etílico 70 por cento (-20°C e temperatura ambiente); álcool etílico absoluto (-20°C e temperatura ambiente); secagem ao ar; e preservação em tampão de extração (inseto inteiro e macerado). A intervalos de 10-30 dias, o DNA foi extraído, quantificado e amplificado via RAPD-PCR pelo oligonucleotídeo OPA-04. A qualidade do DNA extraído foi observada em gel de agarose 0,8 por cento. Após 210 dias de armazenamento, o congelamento (-20°C) mostrou ser a melhor técnica. Quantidades satisfatórias de DNA também foram obtidas dos insetos conservados em álcool etílico absoluto (-20°C), álcool etílico 70 por cento (-20°C) e tampão de extração (inseto inteiro e macerado). Insetos conservados em álcool etílico absoluto (temperatura ambiente), em álcool etílico 70 por cento (temperatura ambiente) e secos ao ar mostraram-se impróprios para estudos com RAPD-PCR após 120, 60 e 10 dias de armazenamento, respectivamente.