First Report of bacterial speck caused by Pseudomonas syringae pv. tomato Race 1 affecting tomato in different Regions of Chile.

In Chile, tomato is one of the most widely cultivated vegetables, with around 5,000 ha for fresh market and 8,000 ha for processing industry. During recent years, symptoms of bacterial speck caused by Pseudomonas syringae pv. tomato, have been observed more frequently in tomato plants in different regions of Chile. This pathogen was first identified in Chile in 1987 (Latorre & Lolas, 1988) and the presence of an apparent new variant was reported in 2004 (Besoain et al. 2004). To characterize the pathogen that was affecting this crop, samples of diseased tomato plants were taken in three regions of Chile. The samples were collected in 2016 in Northern Chile in Lluta Valley from the Arica y Parinacota Region, and in Central Chile, in 2014 in Limache from Valparaíso Region and in 2015 in Pichidegua from O´Higgins Region. Affected tomato plants exhibited dark brown to black lesions surrounded by yellow halos in the leaves, and dark brown to black lesions in the stems, pedicels, and peduncles. Plants tissues were macerated, and the suspension was spread on King's B medium, resulting in fluorescent colonies visualized under 366 nm UV light. LOPAT tests results of three selected isolates from different Regions, were: levan production (+), oxidase reaction (-), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+) (Lelliot et al. 1966). Molecular identification was carried out by amplification and sequence analysis of housekeeping genes cts, encoding citrate synthase, gyrB, encoding DNA gyrase B, and rpoD, encoding sigma factor 70 (Hwang et al. 2005; Sarkar & Guttmann 2004) (GenBank Accessions No. OK001658-OK001666). BLAST analysis of cts and rpoD genes of the three isolates resulted in a match with a 100% identity (919 bp and 491 bp respectively) with Pseudomonas syringae pv. tomato strain B13-200 (GenBank: CP019871.1). BLAST analysis of gyrB gene of two isolates resulted in a match with a 100% identity (684 bp) and one isolate with 99.85% (683 bp) with Pseudomonas syringae pv. tomato strain B13-200. To identify the race 1, each strain was inoculated in five tomato plants cv. San Pedro, susceptible to both races of P. syringae pv. tomato, and cv. Rio Grande, resistant to race 0. The tomato plants were slightly wounded with a metal sponge and then sprayed with the bacterial suspension (108 CFU mL-1) of each isolate, including the reference strain DC3000 (race 0). Negative controls were sprayed with water. The plants inoculated with Chilean strains in both cv. San Pedro and cv. Rio Grande, showed symptoms of bacterial speck after 7 days. Plants inoculated with DC3000 strain showed symptoms only in cv. San Pedro, whereas control plants remained asymptomatic. Strains were re-isolated from symptomatic plants and identified by gene sequence analyses as Pseudomonas syryngae pv. tomato. This is the first report of Pseudomonas syryngae pv. tomato race 1 in Chile. Race 1 was previously reported in Canada (Lawton and MacNeill. 1986), in Italy (Buonaurio et al. 1996), in California (Arredondo and Davis 2000), in Portugal (Cruz et al. 2010), and in other states in the USA and countries in South America, Europe, Africa, and Australia, becoming the most commonly isolated race today (Cai et al 2011). These results will be the base for future studies of epidemiology, characterization, and virulence in order to explain the outbreak of this disease and the severity of symptoms observed.

WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana.

KEY MESSAGE: NbWRKY22 and NbWRKY25 are required for full activation of bacteria-associated pattern- and effector-triggered immunity as well as for the response to other non-bacterial defense elicitors. Plants defend themselves against pathogens using a two-layered immune system. Pattern-triggered immunity (PTI) can be activated upon recognition of epitopes from flagellin including flg22. Pseudomonas syringae pv. tomato (Pst) delivers effector proteins into the plant cell to promote host susceptibility. However, some plants express resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomato lines such as Rio Grande-PtoR (RG-PtoR) recognize two Pst effectors, AvrPto and AvrPtoB, and activate ETI through the Pto/Prf protein complex. Using RNA-seq, we identified two tomato WRKY transcription factor genes, SlWRKY22 and SlWRKY25, whose expression is increased during Pst-induced ETI. Silencing of the WRKY25/22 orthologous genes in Nicotiana benthamiana led to a delay in programmed cell death normally associated with AvrPto recognition or several non-bacterial effector/R protein pairs. An increase in disease symptoms was observed in silenced plants infiltrated with Pseudomonas syringae pv. tabaci expressing AvrPto or HopQ1-1. Expression of both tomato WRKY genes is also induced upon treatment with flg22 and callose deposition and cell death suppression assays in WRKY25/22-silenced N. benthamiana plants supported their involvement in PTI. Our results reveal an important role for two WRKYs as positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens.

Imidazolium salts as alternative compounds to control diseases caused by plant pathogenic bacteria.

AIMS: To evaluate the inhibitory effect of five structurally different imidazolium salts on the in vitro growth of plant pathogenic bacteria that belong to divergent taxonomic genera as well as their ability to reduce the severity of common bacterial blight of common bean caused by Xanthomonas axonopodis pv. phaseoli and bacterial speck of tomato caused by Pseudomonas syringae pv. tomato. METHODS AND RESULTS: Growth inhibition of Xanthomonas, Pseudomonas, Erwinia, Pectobacterium and Dickeya strains by imidazolium salts was assessed in vitro by radial diffusion on agar medium and by ressazurin reduction in liquid medium. The reduction of common bacterial blight and bacterial speck symptoms and the area under de disease progress curves were determined by spraying two selected imidazolium salts on healthy plants 48 h prior to inoculation with virulent strains of the bacterial pathogens. All imidazolium salts inhibited the growth of all plant pathogenic bacteria when tested by radial diffusion on agar medium. The strength of inhibition differed among imidazolium salts when tested on the same bacterial strain and among bacterial strains when tested with the same imidazolium salt. In liquid medium, most imidazolium salts presented the same minimum inhibitory concentration (MIC) and minimum bactericidal concentration values (200 µmol l-1 ), the most notable exception of which was the MIC (at least 1000 µmol l-1 ) for the dicationic MImC10 MImBr2 . The imidazolium salts C16 MImBr and C16 MImCl caused significant reductions in the severity of common bacterial blight symptoms when compared with nontreated plants. CONCLUSION: Imidazolium salts inhibit the in vitro growth of plant pathogenic bacteria and reduce plant disease symptoms to levels comparable to an authorized commercial antibiotic product. SIGNIFICANCE AND IMPACT OF THE STUDY: New compounds exhibiting broad-spectrum antibacterial activity with potential use in agriculture were identified.

Seleção de bactérias endofíticas de tomateiro como potenciais agentes de biocontrole e de promoção de crescimento / Screening of endophytic bacteria isolated from tomato plants as potencial biocontrol agents and growth promotion

Quarenta isolados bacterianos endofíticos de plantas sadias de tomateiro foram avaliados quanto à sua potencialidade como agentes de biocontrole de doenças do tomateiro. Foi realizada, em casa de vegetação, uma seleção massal utilizando-se Pseudomonas syringae pv. tomato e Alternaria solani, como patógenos desafiantes. Com base na média do número de lesões por planta, quatro isolados foram selecionados como potenciais agentes de biocontrole dessas enfermidades fúngica e bacteriana do tomateiro. Esses isolados foram identificados, por meio do sequenciamento do gene 16S do DNA ribossômico, como Acinetobacter johnsonii (UFV-E05), Serratia marcescens (UFV-E13), Sinorhizobium sp. (UFV-E25) e Bacillus megaterium (UFV-E26). Os mesmos isolados selecionados para o biocontrole também foram avaliados quanto à sua capacidade de promover o crescimento em plantas e somente S. marcescens (UFV-E13) proporcionou aumento na altura das plantas.

Forty isolates of endophytic bacteria obtained from healthy tomato plants were tested for their potential as biocontrol agents of tomato diseases. A massal screening was performed at greenhouse using Pseudomonas syringae pv. tomato and Alternaria solani as challenging pathogens. Based on the average number of lesions per plant, four isolates were selected as potential agents of biocontrol of these tomato diseases caused by fungi and bacteria. These isolates were identified by 16S ribosomal DNA sequence analysis as Acinetobacter johnsonii (UFV-E05), Serratia marcescens (UFV-E13), Sinorhizobium sp. (UFV-E25) and Bacillus megaterium (UFV-E26). The four endophytes selected for biocontrol were also evaluated for their ability of promoting plant growth and only S. marcescens (UFV-E13) presented increase in the height of the plants.

Bactérias endofíticas no controle e inibição in vitro de Pseudomonas syringae pv tomato, agente da pinta bacteriana do tomateiro / Control with endophytic bacteria and in vitro inhibition of Pseudomonas syringae pv tomato, agent of bacterial speck of tomato

Para avaliar o potencial de 53 isolados de bactérias endofíticas no controle da pinta bacteriana do tomateiro (Lycopersicum esculentum Mill.), realizaram-se seleções massais em casa-de-vegetação e a seguir foi avaliado, in vitro, o antagonismo desses isolados sobre a bactéria desafiante Pseudomonas syringae pv. tomato (Pst). A inoculação das bactérias endofíticas foi feita por microbiolização das sementes de tomate cv. Santa Clara e da desafiante (Pst) por pulverização. Aos 7, 14 e 21 dias após a inoculação da Pst, foram realizadas as avaliações da severidade da pinta bacteriana, bem como da altura das plantas. As espécies e os isolados bacterianos mais eficazes na redução da severidade da pinta bacteriana foram: Acinetobacter johnsonii (isolado 10), Bacillus pumilus (isolados 3, 12, 20, 39, 51), Paenibacillus macerans (isolados 37 e 47), PIM 11, Bacillus sphaericus (isolado 45), B. amyloliquefaciens (isolado 50), TOM 2, TOM 24 e Staphylococcus aureus (isolado 18). Mais de 50 por cento dos isolados eficazes na redução da severidade foram da espécie Bacillus pumilus. Das espécies endofíticas mais eficazes na redução da severidade da pinta bacteriana, Bacillus pumilus e B. amyloliquefaciens inibiram também o crescimento da Pst in vitro.Vários dos isolados promoveram também o crescimento das plantas.

To asses the potential of fifty three isolates of endophytic bacteria on the control of Pseudomonas syringae pv. tomato (Pst) in tomato (Lycopersicum esculentum Mill.), several screening were done in greenhouse followed by in vitro studies on antagonism of those isolates to Pst. The inoculation of endophytic bacteria was done by microbiolization of tomato cv Santa Clara seeds. The challenging bacterium (Pst) inoculation was done by spraying. At 7, 14 and 21 days after Pst inoculation the assessment of bacterial speck severity was done, and height of plants was also measured. The most efficient endophytic species and isolates in reducing disease severity were: Acinetobacter johnsonii (isolate 10), Bacillus pumilus (isolates, 3, 12, 20, 39, 51), Paenibacillus macerans (isolates, 37, 47), PIM 11, Bacillus sphaericus (isolate 45), B. amyloliquefaciens (isolate 50), TOM 2 , TOM 24 and Staphylococcus aureus (isolate 18). More than 50 percent of the endophytic isolates efficient in reducing disease severity belonged to Bacillus pumilus. From the most efficient endophytic species group, the species Bacillus pumilus and B. amyloliquefaciens inhibited the Pst growth in vitro. Several bacterial isolates promoted growth of tomato.