RESUMO Sete acessos selvagens de Coffea arabica da Etiópia prospectados pela FAO Coffee Mission 1964-1965 foram investigados quanto à resistência a 18 linhagens brasileiras e duas linhagens quenianas de Pseudomonas syringae pv. garcae e quatro P. syringae pv. tabaci, agentes etiológicos da mancha-aureolada e da mancha-foliar-bacterina, respectivamente. As cultivares de C. arabica IPR 102, resistente às doenças, e Mundo Novo IAC 376-4, suscetível, foram utilizadas como controle experimental. Nossos resultados indicaram que os acessos etíopes apresentaram altos níveis de resistência a todas as linhagens brasileiras de P. syringae pv. garcae avaliados, mas suscetíveis à infecção causada por linhagens quenianas, que causa diferentes níveis de severidade em acessos selvagens e nos controles experimentais. Os acessos etíopes também foram considerados resistentes às quatro linhagens de P. syringae pv. tabaci, tendo sido observada baixa suscetibilidade, um ponto na escala de severidade, no acesso E-268 em resposta a uma linhagem da bactéria.

ABSTRACT Seven wild accessions of Coffea arabica from Ethiopia prospected by FAO Coffee Mission 1964-1965 were investigated concerning the resistance to 18 Brazilian strains and two Kenyan strains of Pseudomonas syringae pv. garcae and four P. syringae pv. tabaci strains, causal agents of bacterial halo blight and bacterial leaf spot, respectively. The cultivars of C. arabica IPR 102, resistant to the diseases, and Mundo Novo IAC 376-4, susceptible, were used as experimental controls. Our results indicated that the Ethiopian accessions presented high levels of resistance to all Brazilian strains of P. syringae pv. garcae but were susceptible to infection caused by Kenyan strains, which causes different levels of severity in wild accessions and experimental controls. Ethiopian accessions were also considered resistant to the four P. syringae pv. tabaci strains, with low susceptibility observed, one point on the severity scale, in access E-268 in response to a strain of the bacterium.

RESUMO O melhoramento de plantas para resistência genética é um método importante para o manejo de doenças, pelos inúmeros benefícios e baixo custo de implementação. No presente estudo, progênies de 11 espécies de Coffea e 16 acessos selvagens de C. arabica foram testados quanto à resposta a Pseudomonas syringae pv. garcae, agente causal da mancha aureolada, doença disseminada nas principais regiões produtoras de café do Brasil e considerada fator limitante para o cultivo em áreas favoráveis a patógenos; e também para P. syringae pv. tabaci, agente causal da mancha foliar bacteriana, doença altamente agressiva detectada recentemente no Brasil. Experimentos separados para cada doença foram realizados em estufa, por meio da inoculação artificial dos patógenos em condições ideais de umidade para o desenvolvimento das doenças. Os resultados mostraram que as progênies Coffea canephora, C. congensis, C. eugenioides, C. stenophylla e C. salvatrix, além dos acessos selvagens de C. arabica Dilla & Alghe e Palido Viridis e da cultivar IPR 102, possuem níveis satisfatórios de resistência simultânea contra mancha aureolada e mancha foliar bacteriana. Os resultados descritos são úteis em programas de melhoramento para resistência duradoura a múltiplos agentes bióticos, fornecendo novas combinações de alelos de resistência por hibridização, bem como para estudos fitopatológicos, para identificar a variabilidade infraespecífica dos patógenos.

ABSTRACT Breeding for genetic resistance is an important method of crop disease management, due to the numerous benefits and low cost of establishment. In this study, progenies of 11 Coffea species and 16 wild C. arabica accessions were tested for their response to Pseudomonas syringae pv. garcae, the causal agent of bacterial halo blight, a widespread disease in the main coffee-producing regions of Brazil and considered a limiting factor for cultivation in pathogen-favorable areas; and also to P. syringae pv. tabaci, causal agent of bacterial leaf spot, a highly aggressive disease recently detected in Brazil. Separate experiments for each disease were carried out in a greenhouse, with artificial pathogen inoculations and ideal moisture conditions for disease development. The results showed that C. canephora, C. congensis, C. eugenioides, C. stenophylla, and C. salvatrix progenies, the wild C. arabica accessions Dilla & Alghe and Palido Viridis, and cultivar IPR 102 contain satisfactory levels of simultaneous resistance against bacterial halo blight and bacterial leaf spot. These results are useful in breeding programs for durable resistance to multiple biotic agents, providing new combinations of resistance alleles by hybridization, as well as for phytopathological studies, to identify infraspecific variability of the pathogens.

ABSTRACT Bacterial halo blight, caused by Pseudomonas syringae pv. garcae, is an important disease of coffee crop occurring in Brazil and other countries. In recent years, outbreaks of this disease have damaged several coffee crops in Brazil. Aggressiveness and genetic diversity of 25 strains of P. s. pv. garcae, obtained between the years 1958 and 2011, in 23 cities of São Paulo and Minas Gerais states, as well as three strains from Kenya were evaluated in this study. The strains were inoculated on coffee seedlings cultivar Mundo Novo, and their genetic diversity was evaluated by ERIC-PCR, REP-PCR, and their combination. All the strains were pathogenic to the coffee seedlings; the results of pathogenicity tests, in both experiments, could be divided in four aggressiviness classes (highly aggressive; aggressive; moderately aggressive and less aggressive). The Kenyan strains grouped separately from the Brazilian strains with ERIC-PCR and the combination of ERIC- and REP-PCR. The Brazilian strains could be grouped in two sub-clusters, the first including the older strains, obtained from 1958 to 1978, and the other comprising the remaining strains. With a few exceptions, strains isolated from 1997 to 2011, grouped mainly by their region of origin, were predominantly isolated from higher altitude regions, above 800 m. This probably occurred because the climatic conditions that prevail in these regions, characterized by milder temperatures and regular rainfall, are favorable for the coffee crop and for the production of high quality coffee beverage, but can be also favorable to bacterial halo blight.

RESUMO: Bactérias da espécie Pseudomonas syringae van Hall, 1902, causam perdas na produção em inúmeras culturas de importância econômica. Em cafeeiros, P. syringae pv. garcae provoca a mancha-aureolada e P. syringae pv. tabaci ocasiona a mancha-foliar-bacteriana, doenças cuja ocorrência e agressividade têm aumentado nos últimos anos no Brasil. Embora a idade das folhas influencie na expressão da severidade de mancha-aureolada, não se sabe ainda se essa influência se mantém em plantas infectadas por estirpes altamente virulentas da bactéria. Desse modo, o presente estudo foi realizado com a finalidade de examinar diferenças na severidade de mancha-aureolada em folhas de cafeeiro com diferentes idades, bem como estudar comparativamente tais aspectos na interação entre cafeeiro e mancha-foliar-bacteriana, empregando-se isolados altamente virulentos. Os resultados evidenciaram que, assim como a mancha-aureolada, a severidade da mancha-bacteriana também é maior em folhas jovens do primeiro internódio, entretanto, as estirpes mais agressivas de P. syringae pv. garcae e P. syringae pv. tabaci provocaram danos de magnitude semelhantes em folhas de diferentes idades, do primeiro ao quinto internódio.

ABSTRACT: Pseudomonas syringae van Hall, 1902, causes yield losses in innumerous economic important crops. On coffee trees, P. syringae pv. garcae causes the bacterial-halo-blight (BHB) and P. syringae pv. tabaci the bacterial-leaf-spot (BLS). Recently, these diseases incidence has increase in occurrence areas and aggressiveness in Brazil. Although leaf age plays a role in the severity response of BHB, it is not known yet if this phenomenon also occurs in coffee-BLS interaction, and with highly virulent strains. So, we examined differences in the diseases severity by inoculation of P. syringae pv. garcae and P. syringae pv. tabaci strains on coffee leaves with different ages, to compare this aspect with coffee-BLS interaction. Our results showed that, for both pathovars, the severity was greater at the first internodes leaves, although for the most aggressive strains it was quite similar on any leaf age.

ABSTRACT The bacterial-halo-blight (Pseudomonas syringae pv. garcae) is disseminated by the main coffee areas in the producing states of Brazil. On the other hand, the disease bacterial-leaf-spot (Pseudomonas syringae pv. tabaci) was reported only once in coffee seedlings in a sample collected in the State of São Paulo. In mid-2015, samples of coffee leaves with symptoms of foliar lesions surrounded by yellow halo, were collected in coffee plantations in the State of Paraná and fluorescent bacteria producing or not brown pigment in culture medium were isolated and determined as belonging to the Group I of P. syringae. Through biochemical, serological and pathogenicity tests, the pathogens were identified as P. syringae pv. garcae and P. syringae pv. tabaci, with prevalence of isolates belonging to pathovar tabaci and, as well as in certain samples, it was identified simultaneous infection by both etiological agents. Then, this is the first report of associated occurrence of garcae and tabaci pathovars of P. syringae and of the incidence of “bacterial-leaf-spot” under field conditions and in the State of Paraná.

ABSTRACT Coffee resistance to Pseudomonas syringae pv. garcae has been associated to pleiotropic effect of SH1 allele, present in coffee plants resistant to certain races of Hemileia vastatrix, the causal agent of leaf rust, or genetic linkage between resistance alleles to both pathogens. To validate this hypothesis, 63 coffee plants in F2 generation were evaluated for resistance to 2 isolates of H. vastatrix carriers of alleles, respectively, v2, v5 (isolate I/2015) and v1; v2; v5 (isolate II/2015) with the objective to confirm presence of SH1 allele in resistant plants to isolate I/2015. The same coffee plants were evaluated for resistance to a mixture of P. syringae pv. garcae strains highly pathogenic to coffee. Results showed that, among F2 coffee allele SH1 carriers, resistant to isolate I/2015, resistant and susceptible plants to bacterial halo blight were found; the same segregation occurs between F2 homozygous for SH1 allele, susceptible to the same isolate (I/2015) of H. vastatrix. Results also indicate that there is no pleiotropic effect of gene or allele SH1 connection between genes conferring resistance to leaf rust caused by H. vastatrix and bacterial halo blight caused by P. syringae pv. garcae.

RESUMO: Há várias bactérias que causam problemas para o cafeeiro, incluindo Pseudomonas cichorii, P. syringae pv. garcae, P. syringae pv. tabaci, Burkholderia andropogonis e Xylella fastidiosa, todas elas já descritas no Brasil. Tentativas de diferenciar essas bactérias por testes serológicos de dupla difusão em ágar (dda), com antissoros produzidos contra células íntegras de P. s. pv. garcae, mostraram reações cruzadas, principalmente entre P. s. pv. garcae e P. s. pv. tabaci. Dessa forma, foram produzidos antissoros contra P. s. pv. garcae (linhagem patotipo IBSBF-248 - Coleção de Culturas de Fitobactérias do Instituto Biológico - IBSBF), obtidos por meio de imunizações de coelhos com antígenos de proteínas do complexo proteico da membrana (CPM). Esses antissoros foram testados por dupla difusão em agarose (dda) contra diversas formas de antígenos extraídos de P. cichorii, P. s. pv. garcae e P. s. pv. tabaci [células autoclavadas, células tratadas com formol, exopolissacarídeos (EPS), glicoproteínas (GP) da cápsula bacteriana, proteínas de membrana e suspensão bacteriana (SB) em NaCl 0,85%]. Os resultados mostraram que, dependendo do antígeno e do meio suporte da dupla difusão (com ou sem MgCl2 e/ou azul de tripano), os antissoros reagem somente com P. s. pv. garcae. Desse modo, esses antígenos podem ser usados para a rápida diagnose da mancha aureolada do cafeeiro nos testes de dda.

ABSTRACT: Some bacterial diseases have been described causing problems in coffee, whose causal agents are Pseudomonas cichorii, P. syringae pv. garcae, P. s. pv. tabaci and Burkholderia andropogonis, all of them also occurring in Brazil. Attempts to differentiate these bacteria by double diffusion agar (dda) technique with antisera produced against whole cells of P. s. pv. garcae showed cross-reactions, especially among P. s. pv. garcae and P. s. pv. tabaci. Thus, antisera were produced against P. s. pv. garcae (pathotype strain IBSBF-248 - Phytobacteria Culture Collection of Instituto Biológico - IBSBF), using rabbit immunizations with antigens of the protein complex of membranes. These antisera were tested against different kind of antigens (autoclaved cells, cells treated with formaldehyde, capsule polysaccharides, glycoproteins, bacterial membrane proteins and bacterial suspension in 0.85% NaCl) extracted from P. cichorii, P. s. pv. garcae and P. s. pv. tabaci. The results showed that depending on the kind of antigen and the medium used in the double diffusion test (with or without MgCl2 and/or trypan blue), the antiserum reacted only with P. s. pv. garcae. Thus, these antigens may be used as an auxiliary tool in the rapid diagnosis of bacterial halo blight of coffee in the double diffusion test.

RESUMOApesar de pouco estudada, a mancha-aureolada é uma importante doença nos principais Estados produtores de café do Brasil. Os danos externos e as alterações anatômicas em folhas foram mensurados em mudas de Coffea arabica cv. Mundo Novo, suscetível a Pseudomonas syringae pv. garcae a partir de cortes histológicos realizados aos 10 e 20 dias após a inoculação (DAI). As alterações na epiderme foram menores do que as lesões mensuradas no mesofilo, independente do período de colonização avaliado, mostrando que os danos internos causados pela bactéria representam o dobro do observado externamente. A partir do ponto de inoculação, foi verificado lise das células epidérmicas e dos parênquimas paliçádico e esponjoso, com forte coloração de seus conteúdos celulares, espaços intercelulares anormais no parênquima paliçádico, hipertrofia e hiperplasia das células do mesofilo e destruição parcial de cloroplastos. Além disso, este estudo revelou a presença de corpos de inclusão em células da epiderme e do mesofilo. Massas bacterianas foram encontradas no apoplasto entre e dentro de células mesofílicas. Bactérias também foram observadas na bainha do feixe e em feixes vasculares, sendo a ocorrência bacteriana, mais pronunciada aos 20 DAI, não apenas próximo ao ponto de inoculação, mas também, em regiões distantes, sugerindo seu deslocamento pelo sistema vascular. Estes resultados podem ser úteis na compreensão da interação planta-patógeno.

ABSTRACTAlthough poorly studied, the bacterial halo blight is an important disease in the major coffee-producing states of Brazil. External damage and anatomical changes on leaves were measured in seedlings of Coffea arabica cv. Mundo Novo, susceptible to Pseudomonas syringae pv. garcae, by using histological sections obtained at 10 and 20 days after inoculation (DAI). The changes on the epidermis were smaller than the lesions measured in the mesophyll, irrespective of the evaluated colonization period, showing that the internal damage caused by the bacterium represent twice the damage observed externally. From the inoculation site, lysis occurred on the epidermal cells and on the palisade and spongy parenchyma cells, with strong staining of their cellular contents, as well as abnormal intercellular spaces in the palisade parenchyma, hypertrophy and hyperplasia of mesophyll cells and partial destruction of chloroplasts. Additionally, this study revealed the presence of inclusion bodies in epidermal and mesophyll cells. Bacterial masses were found in the apoplast between and within mesophyll cells. Bacteria were also observed in the bundle sheath and vascular bundles and were more pronounced at 20 DAI, not only near the inoculation site but also in distant areas, suggesting displacement through the vascular system. These results can be useful to understand this plant-pathogen interaction.