Diferenças em colonização do hospedeiro por isolados de Xylella fastidiosa de citros e cafeeiro em inoculações recíprocas

Clorose variegada dos citros (CVC) e atrofia dos ramos do cafeeiro (ARC) são doenças importantes no Brasil, associadas a estirpes de Xylella fastidiosa que são geneticamente próximas. Entretanto, pouco se sabe a respeito de plantas hospedeiras em comum e da importância de citros e cafeeiro como fontes de inóculo dessas estirpes. Neste estudo, realizaram-se experimentos de inoculação recíproca para determinar se isolados de X. fastidiosa de CVC e de ARC são biologicamente semelhantes em plantas de citros e café. Estes dois hospedeiros foram mecanicamente inoculados com um isolado de CVC e um isolado de ARC, em quatro concentrações que variaram de 10³ a 10(9) unidades formadoras de colônias UFC mL-1. Aos dois, quatro e oito meses após a inoculação, a eficiência de infecção e a população bacteriana dos isolados em cada hospedeiro foram determinadas por cultura. O isolado de CVC infectou tanto plantas de citros quanto de café, mas desenvolveu populações mais baixas em cafeeiro. O isolado de ARC não colonizou citros. A inoculação de plantas de café com o isolado de CVC resultou em baixas taxas de infecção e exigiu uma concentração de inóculo dez vezes mais alta que a necessária para obter uma taxa de infecção semelhante (25%) em citros. A reduzida taxa de infecção e a baixa população bacteriana do isolado de CVC em cafeeiro em relação a citros sugerem que cafeeiro não é um hospedeiro adequado para atuar como fonte de inóculo da estirpe de CVC para disseminação deste patógeno para pomares de laranja ou dentro de cafezais.Citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) are important diseases in Brazil associated with closely-related strains of Xylella fastidiosa, but little is know about host aoverlappingnd importance of citrus and coffee as inoculum sources of these strains. In this study, reciprocal-inoculation experiments were performed to determine if CVC and CSA isolates are biologically similar within citrus and coffee plants. These two hosts were mechanically inoculated with a CVC and a CSA isolate of X. fastidiosa at four concentrations ranging between10³ and 10(9) colony forming units CFU mL-1. At two, four and eight months after inoculation, the infection efficiency and bacterial populations of the isolates in each host were determined by culturing. The CVC isolate infected both citrus and coffee plants, but developed lower populations in coffee. The CSA isolate did not colonize citrus. Inoculation of coffee plants with the CVC isolate resulted in low rates of infection and required an inoculum concentration ten-fold higher than that necessary to obtain a similar (25%) rate of infection in citrus. The relatively low infection rates and bacterial numbers of the CVC isolate in coffee plants compared with those observed in citrus suggest that coffee is not a suitable host to serve as a source of inoculum of the CVC strain for primary spread to citrus or within coffee plantations

Multiplication and movement of Xylella fastidiosa within Citrus sinensis and its efficiency of acquisition and inoculation by sharpshooter vectors

A pesquisa teve por objetivo investigar possíveis fatores determinando a baixa eficiência de transmissão da bactéria Xylella fastidiosa por cigarrinhas (Hemiptera, Cicadellidae) em citros. Inicialmente, foram adaptadas técnicas para isolamento primário e quantificação de X. fastidiosa em citros e no vetores, e para inoculação mecânica do patógeno em mudas cítricas. A seguir, foram conduzidos estudos para avaliar as eficiências de aquisição e inoculação de X. fastidiosa por cigarrinhas, bem como a multiplicação e movimentação deste patógeno em mudas cítricas após sua inoculação mecânica. A metodologia de isolamento primário, envolvendo maceração de tecidos infectados em homogeneizador mecânico e plaqueamento da suspensão resultante em meio de cultura, foi eficiente para detecção e quantificação de X. fastidiosa em citros. Os meios de cultura PW e PWG, específicos para o crescimento de X. fastidiosa, propiciaram diâmetro de colônias de aproximadamente 1 mm aos 14 dias após a inoculação. Outro meio específico testado, BCYE, foi cerca de 60-70% menos eficiente para recuperação do patógeno em isolamento primário que os dois anteriores, gerando colônias menores e de crescimento mais lento. A adição de macerado cítrico à suspensão bacteriana não inibiu o crescimento de X. fastidiosa em meio sólido, mas o favoreceu quando comparado a suspensões puras da bactéria. Nenhum dos indivíduos sadios de Dilobopterus costalimai e Bucephalogonia xanthophis, submetidos a um período de acesso à aquisição de 96 horas em planta-fonte de X. fastidiosa, foi positivo para a bactéria pelos testes de cultura e ELISA, indicando baixa eficiência de aquisição dos vetores. No estudo de eficiência de inoculação, nenhum dos 19 indivíduos infectivos de Oncometopia facialis transmitiu X. fastidiosa para plantas-teste durante período de acesso à inoculação de 96 horas, sugerindo que a inoculação em citros por vetores também é um processo ineficiente. A multiplicação e movimentação de X. fastidiosa, em mudas cítricas foi avaliada em períodos sucessivos (1, 2, 4, 8 e 16 semanas) após sua inoculação em caule ou folhas. O método de inoculação mecânica com alfinete entomológico teve eficiência variável de 45 a 100 %. Com apenas 1 semana da inoculação, a bactéria foi isolada de áreas do caule localizadas 4 cm acima e 4 cm abaixo do ponto de inoculação, assim como na primeira folha acima deste; demonstrando o caráter sistêmico de sua infecção em citros. A porcentagem de plantas positivas detectadas com 1 semana após a inoculação não aumentou significativamente nas semanas subsequentes (até 16 semanas); desta forma o sucesso da infecção pode ser diagnosticado precocemente pelo método de isolamento. A população bacteriana nas duas primeiras semanas após a inoculação variou de 103 a 105 unidades formadoras de colônias (UFC) por grama de tecido, sendo que no período de 4 a 16 semanas este valor manteve-se entre 104 e 106 UFC/g. Sintomas foliares de CVC foram observados 2 meses após a inoculação. Com base em informações oriundas de outro patossistema (doença de Pierce em videira), postula-se que um fator limitante da eficiência de transmissão de X. fastidiosa em citros possa ser a baixa eficiência de aquisição do patógeno pelos vetores, decorrente da pequena população bacteriana encontrada em plantas cítricas infectadas.The main goal of this research was to investigate possible factors determining the low transmission efficiency of Xylella fastidiosa by sharpshooter leafhoppers (Hemiptera, Cicadellidae) in citrus. Initially, to techniques to isolate and quantify X. fastidiosa population in citrus and vectors, and to needle inoculate the pathogen in the host plant. Studies were then carried out to evaluate the acquisition and inoculation efficiencies of X. fastidiosa by leafhoppers, as well as the multiplication and movement of this bacterium in citrus. The isolation and quantification procedure was successfully adapted, being efficient to detect the bacterium in citrus. ln the specific media for X. fastidiosa growth, PWG and PW, colonies were observed 7-8 days after plating, with a diameter achieving 1 mm after 14 days of incubation, these results are superior to those obtained with BCYE medium, in which colonies were observe 14-15 days after plating with smaller size, this medium also was 60-70 % less efficient in bacterial recovery than the previous two. Homogenized citrus tissue did not inhibit X. fastidiosa bacterial growth on solid medium, in fact, it stimulated more colony recovery when compared to pure suspensions of the pathogen. None of the assayed leafhoppers by the diagnostic tests used acquired X. fastidiosa in citrus, therefore the acquisition might be an inefficient step of the transmission. About 10 % of the tested insects in the inoculation experiment had X. fastidiosa isolated in medium from their heads, but no inoculation was observed, also suggesting inefficient inoculation. The multiplication and movement of X. fastidiosa in citrus observations were done after needle inoculation of the pathogen in stems and leaves of test plants, this method had a 45 - 100 % efficiency. Only 1 week after inoculation the bacterium was recovered in areas above and below the inoculation points, as wells as in the first leaf above it. The amount of positive plants 1 week after inoculation remained constant until the end of the assays (16 weeks), therefore, the infection success rate can be early identified. The bacterial population in the first weeks after inoculation varied from 103 to 106 colony forming units (CFU) per gram of tissue, after 4 weeks this value remained between 105 and 106 CFU/g. Symptoms were observed 2 months after inoculation in all tests done. Based on the Pierces disease of grapevines pathosystem, possibly the limiting factor to X. fastidiosa transmission in CVC is the low acquisition efficiency by vectors, due to the low bacterial population found in symptomatic plants

XadA2 Adhesin Decreases Biofilm Formation and Transmission of Xylella fastidiosa subsp. pauca.

Xylella fastidiosa is a vector-borne bacterium that causes diseases in many plants of economic interest. The bacterium-vector initial interactions involve bacterial membrane-bound adhesins that mediate cell attachment to the foregut of insect vectors. We investigated the role of the afimbrial adhesin XadA2 in the binding and biofilm formation of X. fastidiosa subsp. pauca to vector surfaces in vitro, as well as its potential to disrupt pathogen transmission. We showed that XadA2 has binding affinity for polysaccharides on sharpshooter hindwings, used as a proxy for the interactions between X. fastidiosa and vectors. When in a medium without carbon sources, the bacterium used wing components, likely chitin, as a source of nutrients and formed a biofilm on the wing surface. There was a significant reduction in X. fastidiosa biofilm formation and cell aggregation on vector wings in competition assays with XadA2 or its specific antibody (anti-XadA2). Finally, pathogen acquisition and transmission to plant were significantly reduced when the vectors acquired X. fastidiosa from an artificial diet supplemented with anti-XadA2. These results show that XadA2 is important in mediating bacterial colonization in the insect and that it could be used as a target for blocking X. fastidiosa transmission

XadA2 Adhesin Decreases Biofilm Formation and Transmission of Xylella fastidiosa subsp. pauca.

Xylella fastidiosa is a vector-borne bacterium that causes diseases in many plants of economic interest. The bacterium-vector initial interactions involve bacterial membrane-bound adhesins that mediate cell attachment to the foregut of insect vectors. We investigated the role of the afimbrial adhesin XadA2 in the binding and biofilm formation of X. fastidiosa subsp. pauca to vector surfaces in vitro, as well as its potential to disrupt pathogen transmission. We showed that XadA2 has binding affinity for polysaccharides on sharpshooter hindwings, used as a proxy for the interactions between X. fastidiosa and vectors. When in a medium without carbon sources, the bacterium used wing components, likely chitin, as a source of nutrients and formed a biofilm on the wing surface. There was a significant reduction in X. fastidiosa biofilm formation and cell aggregation on vector wings in competition assays with XadA2 or its specific antibody (anti-XadA2). Finally, pathogen acquisition and transmission to plant were significantly reduced when the vectors acquired X. fastidiosa from an artificial diet supplemented with anti-XadA2. These results show that XadA2 is important in mediating bacterial colonization in the insect and that it could be used as a target for blocking X. fastidiosa transmission

Panretinal photocoagulation versus intravitreal injection retreatment pain in high-risk proliferative diabetic retinopathy

PURPOSE: To compare pain related to intravitreal injection and panretinal photocoagulation in the management of patients with high-risk proliferative diabetic retinopathy. METHODS: Prospective study including patients with high-risk proliferative diabetic retinopathy and no prior laser treatment randomly assigned to receive panretinal photocoagulation (PRP group) or panretinal photocoagulation plus intravitreal ranibizumab (PRPplus group). In all patients, panretinal photocoagulation was administered in two sessions (weeks 0 and 2), and intravitreal ranibizumab was administered at the end of the first laser session in the PRPplus group. Retreatment was performed at weeks 16 and 32 if active new vessels were detected at fluorescein angiography. Patients in the PRPplus group received intravitreal ranibizumab and patients in the PRP group received 500-µm additional spots per quadrant of active new vessels. After the end of retreatment, a 100-degree Visual Analog Scale was used for pain score estimation. The patient was asked about the intensity of pain during the whole procedure (retinal photocoagulation session or intravitreal ranibizumab injection). Statistics for pain score comparison were performed using a non-parametric test (Wilcoxon rank sums). RESULTS: Seventeen patients from PRPplus and 14 from PRP group were evaluated for pain scores. There were no significant differences between both groups regarding gender, glycosylated hemoglobin and disease duration. Mean intravitreal injection pain (±SEM) was 4.7 ± 2.1 and was significantly lower (p<0.0001) than mean panretinal photocoagulation pain (60.8 ± 7.8). Twelve out of 17 patients from the PRPplus group referred intensity pain score of zero, while the minimal score found in PRP group was found in one patient with 10.5. CONCLUSION: In patients with high-risk proliferative diabetic retinopathy who needed retreatment for persistent new vessels, there was more comfort for the patient when retreatment was performed with an intravitreal injection in comparison with retinal photocoagulation. Further larger studies are necessary to confirm our preliminary findings

Host colonization differences between citrus and coffee isolates of Xylella fastidiosa in reciprocal inoculation Diferenças em colonização do hospedeiro por isolados de Xylella fastidiosa de citros e cafeeiro em inoculações recíprocas

Citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) are important diseases in Brazil associated with closely-related strains of Xylella fastidiosa, but little is know about host aoverlappingnd importance of citrus and coffee as inoculum sources of these strains. In this study, reciprocal-inoculation experiments were performed to determine if CVC and CSA isolates are biologically similar within citrus and coffee plants. These two hosts were mechanically inoculated with a CVC and a CSA isolate of X. fastidiosa at four concentrations ranging between10³ and 10(9) colony forming units CFU mL-1. At two, four and eight months after inoculation, the infection efficiency and bacterial populations of the isolates in each host were determined by culturing. The CVC isolate infected both citrus and coffee plants, but developed lower populations in coffee. The CSA isolate did not colonize citrus. Inoculation of coffee plants with the CVC isolate resulted in low rates of infection and required an inoculum concentration ten-fold higher than that necessary to obtain a similar (25%) rate of infection in citrus. The relatively low infection rates and bacterial numbers of the CVC isolate in coffee plants compared with those observed in citrus suggest that coffee is not a suitable host to serve as a source of inoculum of the CVC strain for primary spread to citrus or within coffee plantations.<br>Clorose variegada dos citros (CVC) e atrofia dos ramos do cafeeiro (ARC) são doenças importantes no Brasil, associadas a estirpes de Xylella fastidiosa que são geneticamente próximas. Entretanto, pouco se sabe a respeito de plantas hospedeiras em comum e da importância de citros e cafeeiro como fontes de inóculo dessas estirpes. Neste estudo, realizaram-se experimentos de inoculação recíproca para determinar se isolados de X. fastidiosa de CVC e de ARC são biologicamente semelhantes em plantas de citros e café. Estes dois hospedeiros foram mecanicamente inoculados com um isolado de CVC e um isolado de ARC, em quatro concentrações que variaram de 10³ a 10(9) unidades formadoras de colônias UFC mL-1. Aos dois, quatro e oito meses após a inoculação, a eficiência de infecção e a população bacteriana dos isolados em cada hospedeiro foram determinadas por cultura. O isolado de CVC infectou tanto plantas de citros quanto de café, mas desenvolveu populações mais baixas em cafeeiro. O isolado de ARC não colonizou citros. A inoculação de plantas de café com o isolado de CVC resultou em baixas taxas de infecção e exigiu uma concentração de inóculo dez vezes mais alta que a necessária para obter uma taxa de infecção semelhante (25%) em citros. A reduzida taxa de infecção e a baixa população bacteriana do isolado de CVC em cafeeiro em relação a citros sugerem que cafeeiro não é um hospedeiro adequado para atuar como fonte de inóculo da estirpe de CVC para disseminação deste patógeno para pomares de laranja ou dentro de cafezais

Identification of a non-host plant of Xylella fastidiosa to rear healthy sharpshooter vectors Identificação de uma planta não-hospedeira de Xylella fastidiosa para criação de insetos vetores sadios

Rearing leafhopper (Hemiptera: Cicadellidae) vectors free of Xylella fastidiosa is a requirement for studies of various aspects of vector-pathogen interactions. The selection of a plant that allows vector development but not bacterial multiplication is desirable to produce healthy vectors. In this study, two leafhopper hosts, Vernonia condensata ('boldo') and Aloysia virgata ('lixeira') were needle inoculated with citrus and coffee strains of X. fastidiosa to evaluate if these plants support pathogen colonization. The inoculated plants did not present symptoms and the pathogen was not detected by culture and PCR tests, neither soon after inoculation (7-14 days) nor later, at 1, 4, 6 and 12 months after inoculation. To obtain healthy adults of the leafhopper vectors Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, Homalodisca ignorata and Oncometopia facialis, early-instar nymphs were reared on V. condensata. X. fastidiosa was not detected in any of 175 adults obtained. V. condensata and A. virgata are nonpropagative hosts of X. fastidiosa and enable the production of healthy leafhoppers for vector studies.<br>A obtenção de cigarrinhas (Hemiptera: Cicadellidae) livres de Xylella fastidiosa é importante para estudos de interação entre essa bactéria e seus vetores, sendo desejável a seleção de uma planta que permita a criação desses insetos, mas não a multiplicação da bactéria. Neste estudo, duas plantas hospedeiras de cigarrinhas, Vernonia condensata (boldo) e Aloysia virgata (lixeira), foram inoculadas por agulha com as estirpes de citros e de cafeeiro de X. fastidiosa, para avaliar a possibilidade deste patógeno colonizá-las. Não foram observados sintomas, nem se detectou a bactéria por isolamento em meio de cultura e/ou PCR em períodos curtos (7 e 14 dias) ou longos (1, 4, 6 e 12 meses) após a inoculação. Para obtenção de adultos sadios das cigarrinhas vetoras, Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, Homalodisca ignorata e Oncometopia facialis, ninfas de primeiros ínstares foram criadas em plantas de boldo. Não foi detectada X. fastidiosa em nenhum de 175 adultos obtidos da criação. V. condensata e A. virgata não permitem a colonização de X. fastidiosa, possibilitando assim a obtenção de cigarrinhas sadias para estudos com vetores