Copper formulations in bacterial blight control and toxic effects on coffee seedlings

Bacterial blight of coffee (Pseudomonas syringae pv. garcae) is an important coffee disease and can be controlled using antibiotics and copper-based compounds. However, copper-based compounds raise doubts among coffee growers regarding bacterial blight control efficiency and phytotoxic potential. In this work, coffee plants were sprayed with different copper molecules in order to study their efficiency on bacterial blight control and the phytotoxic potential. Seven copper formulations, cuprous oxide, copper oxychloride, copper nitrate, copper hydroxide 1 (water-dispersible granules) and 2 (concentrated suspension), copper sulfate 1 (complexed with gluconic acid) and 2 (Bordeaux mixture) were studied. The copper formulations efficiency was compared with the antibiotic kasugamycin, saline solution, and control. In controlled environmental conditions of temperature, relative humidity, and photoperiod, coffee seedlings were sprayed with the treatments and after 24 hours they were inoculated with Pseudomonas syringae pv. garcae suspension. Disease incidence and severity assessments were performed in a 2-day interval during a 16-day period. Phytotoxicity incidence and severity, mapping, and quantification of copper on the leaf tissue surface, dried leaves weight, and total copper leaf content were assessed 16 days after pathogen inoculation. Data were submitted to the Scott-Knott test (p < 0.05). Cuprous oxide and copper sulfate 2 proved most efficient to bacterial blight control, causing lower phytotoxicity effect, best covering, and persistence on leaf tissues. Copper nitrate and copper sulfate complexed with gluconic acid were more phytotoxicity compared to other copper formulations.

Multiple resistance to bacterial halo blight and bacterial leaf spot in Coffea spp / Resistência múltipla à mancha aureolada e à mancha foliar bacteriana em Coffea spp

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.(AU)

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.(AU)

A 14-20 kDa protein binds to the upstream region of the phtM operon involved in the synthesis of phaseolotoxin in Pseudomonas syringae pv. phaseolicola NPS3121 / Una proteína de 14-20 kDa se une a la región río arriba del operón phtM involucrado en la síntesis de faseolotoxina en Pseudomonas syringae pv. phaseolicola NPS3121

Pseudomonas syringae pv. phaseolicola is a phytopathogenic bacterium in beans that produces a phytotoxin called phaseolotoxin, in whose synthesis a group of genes that belong to the "Pht cluster" are involved. This cluster comprises 23 genes arranged in 5 transcriptional units, two monocistronic (argK, phtL) and three polycistronic (phtA, phtD, phtM) operons, whose expression is increased at 18°C, correlating with the production of phaseolotoxin by the bacterium. So far, the regulatory mechanisms involved in phaseolotoxin synthesis are poorly understood and only the requirement of low temperatures for its synthesis has been demon strated. Therefore, in this study we searched for regulatory proteins that could be involved in the phaseolotoxin synthesis, focusing on the regulation of the phtM operon. Gel shift assays showed that the promoter region of the phtM operon contains binding sites for putative regulatory proteins, which are encoded outside the Pht cluster and are independent of the GacS-GacA two-component system. Deletion assays with the promoter region of the phtM operon show that the binding site for a putative transcription factor is located within a 58 bp region. The putative transcription factor of the phtM operon has an apparent molecular mass in the 14-20 kDa range. Furthermore, the results demonstrate that the transcription factor recognizes and binds the upstream phtM region as monomer o multimer of a single polypeptide. Our findings provide new insights into the regulatory mechanisms involved in phaseolotoxin production, and suggest that the Pht cluster was integrated into the global regulatory mechanism of P. syringae pv. phaseolicola.

Can the leaf age influence the susceptibility to bacterial-leaf-spot and bacterial-halo-blight on coffee seedlings? / A idade das folhas pode influenciar na suscetibilidade de cafeeiros à macha-foliar-bacteriana e à mancha-aureolada?

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.(AU)

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.(AU)

Diferenciação de bactérias do gênero Pseudomonas patogênicas ao cafeeiro por técnicas serológicas / Differentiation of bacteria of the genus Pseudomonas pathogenic to coffee by serological techniques

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.(AU)

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.(AU)

Draft genome sequences of five Pseudomonas syringae pv. actinidifoliorum strains isolated in France

ABSTRACT Pseudomonas syringae pv. actinidifoliorum causes necrotic spots on the leaves of Actinidia deliciosa and Actinidia chinensis. P. syringae pv. actinidifoliorum has been detected in New Zealand, Australia, France and Spain. Four lineages were previously identified within the P. syringae pv. actinidifoliorum species group. Here, we report the draft genome sequences of five strains of P. syringae pv. actinidifoliorum representative of lineages 1, 2 and 4, isolated in France. The whole genomes of strains isolated in New Zealand, representative of P. syringae pv. actinidifoliorum lineages 1 and 3, were previously sequenced. The availability of supplementary P. syringae pv. actinidifoliorum genome sequences will be useful for developing molecular tools for pathogen detection and for performing comparative genomic analyses to study the relationship between P. syringae pv. actinidifoliorum and other kiwifruit pathogens, such as P. syringae pv. actinidiae.

Identification of plant defence regulators through transcriptional profiling of Arabidopsis thaliana cdd1 mutant.

A sustainable balance between defence and growth is essential for optimal fitness under pathogen stress. Plants activate immune response at the cost of normal metabolic requirements. Thus, plants that constitutively activate defence are deprived of growth. Arabidopsis thaliana mutant constitutive defence without defect in growth and development1 (cdd1) is an exception. The cdd1 mutant is constitutive for salicylic acid accumulation, signalling, and defence against biotrophic and hemibiotrophic pathogens, without having much impact on growth. Thus, cdd1 offers an ideal genetic background to identify novel regulators of plant defence. Here we report the differential gene expression profile between cdd1 and wild-type plants as obtained by microarray hybridization. Expression of several defence-related genes also supports constitutive activation of defence in cdd1. We screened T-DNA insertion mutant lines of selected genes, for resistance against virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Through bacterial resistance, callose deposition and pathogenesis-associated expression analyses, we identified four novel regulators of plant defence. Resistance levels in the mutants suggest that At2g19810 and [rom] At5g05790 are positive regulators, whereas At1g61370 and At3g42790 are negative regulators of plant defence against bacterial pathogens.

BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae: P. viridiflava group

The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garçae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.