Abi Soares dos Anjos Marques

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, garcae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.

Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV)

The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presence of the p35 gene by PCR and/or dot blot hybridization. Transcriptional analysis of regenerated plants showed the presence of specific p35 transcripts in 9 of them. Regenerated plants containing the p35 gene were inoculated with the cowpea aphid-borne mosaic virus (CABMV), the bacterium Xanthomonas axonopodis pv passiflorae, and the herbicide, glufosinate, (Syngenta). None of the plants showed resistance to CABMV. Regenerated plants (p35+) showed less than half of local lesions showed by non-transgenic plants when inoculated with X. axonopodis and some p35+ plants showed increased tolerance to the glufosinate herbicide when compared to non-transgenic plants.

Low genetic diversity among pathogenic strains of Erwinia psidii from Brazil

Erwinia psidii causes bacterial disease of guava in Brazil. Phenotypic and molecular characterization through rep-PCR fingerprinting of 42 strains from different geographical regions showed that E. psidii populations in Brazil have a low level of genetic diversity and suggest that contaminated plant material is the main source for pathogen dissemination in the country.

Sobrevivência e viabilidade de Xanthomonas axonopodis pv. phaseoli em sementes de feijão armazenadas sob condições controladas

The establishment of seed banks for ex situ plant germplasma conservation is widely used. However, seedborne pathogens may affect germplasma integrity when it is regenerated and/or multiplied. The objective of this experiment was to monitor the longevity (survival) and viability (maintenance of infectivity) of Xanthomonas axonopodis pv. phaseoli in a contaminated bean (Phaseolus vulgaris) seed lot (cv. Roxao), stored at -18 and 5 oC, which are, respectively, the normal temperatures for long and average term conservation of plant germplasma. Another sample was maintained at room temperature. Longevity of the bacteria was evaluated by the percentage of infected seeds, scoring typical colonies onto semi-selective medium, and the viability was determined by the capability of the isolates to reproduce the disease. The population of X. axonopodis pv. phaseoli was evaluated in seed stored for five years, by individually isolating the bacteria from seed wash and seedlings. The results showed that the initial contamination rate of 64% dropped to 36-37% for all treatments during the first six months. Final evaluations at 30 and 60 months showed that for seed stored at -18 and 5 oC, the contamination rate was maintained. This differed significantly from seed stored at room temperature. The best temperature for bacteria survival and maintenance was 5 °C. At this temperature, the population level was as high as 1.2 x 108 cfu/seed. Maintenance of infectivity after storage was also demonstrated. We concluded that the optimal conditions for seed conservation are the same as those for maintenance of X. axonopodis pv. phaseoli longevity.

Detecção de Erwinia psidii via enriquecimento em extrato de folhas de goiabeira e imunodifusão radial dupla

One of the most important diseases affecting guava production in Brazil is bacterial blight, caused by the bacterium Erwinia psidii. Pathogen dissemination often occurs through contaminated propagating plant material. The development of more effective diagnostic methods may reduce pathogen dissemination in the country. Considering the need for a reliable and simple method for detecting the pathogen in plant material, the objectives of this study were to produce E. psdii-specific polyclonal antibodies and to develop a detection method using bacterial population enrichment on guava leaf extracts followed by double radial immunodiffusion. The antiserum was produced against the E. psidii type strain IBSBF 435 (ICMP 8426, NCPPB 3555) and its efficiency, specificity and sensitivity threshold were determined. The antiserum As15-1 was tested with strains of several plant-pathogenic bacteria and reacted positively with all strains of E. psidii, although cross reactions were detected with two non-pathogenic isolates from guava flora. Bacterial multiplication on leaf extracts was observed 12 h after incubation from initial populations of 103, 105 and 107 ufc/mL, up to 60 h. After 12 h it was already possible to detect E. psidii in samples with starting populations of 107 cfu/mL. After 36 h, the enrichment technique allowed the detection of E. psidii using double radial immunodiffusion in samples with populations as low as 103 cfu/mL. Considering the possibility of false positives it is desirable to associate other diagnostic methods with the method proposed in this study.

Comparing Acidovorax citrulli strains from melon and watermelon: Phenotypic characteristics, pathogenicity and genetic diversity

Melon and watermelon bacterial fruit blotch, incited by Acidovorax citrulli, is limited to some areas in Brazil but causes important losses, mainly in melon-producing regions. Although genetic diversity has been observed among strains belonging to the species, they are considered a homogeneous group based on the fact that they show only slight physiological or nutritional differences. The objective of this study was to compare Brazilian strains from melon and watermelon by means of biochemical, pathogenicity, serological and molecular assays. Fifteen biochemical tests, cross inoculation between strains and hosts, ELISA and repetitive sequence analysis (rep-PCR) with the primers REP, ERIC and BOX were conducted. No differences were revealed by nutritional characterization or serology, but cross inoculation showed different pathogenicity groups, which could explain high aggressiveness of the bacteria to melon crops in some regions. Molecular analysis by BOX-PCR clustered strains according to their geographical origin, while ERIC- and REP-PCR, analyzed together, indicated genetic diversity, but without geographical or host origin relationships. One test that could be used to verify the pathogenicity of strains by inoculating detached leaf petioles, showing results in 36 h, is proposed here.

Recovering optimization of Pseudomonas syringae pv. tabaci by use of two modified semiselective medium

RESUMEN Los medios de cultivo, selectivos y semiselectivos, favorecen al aislamiento de las bacterias fitopatogenas, mediante la supresion de microorganismos no deseados. Para optimizar la recuperacion de Pseudomonas syringae pv. tabaci, a partir de semillas de tabaco se emplearon dos medios de cultivo: KB (medio B de King et al.) y MSP (“modified sucrose peptone”, medio de Mohan y Schaad), a los que fueron adicionados los antibioticos ciclohexamida y cefalexina. La eficacia de los medios se comprobo mediante siembra de una suspension bacteriana y de semillas de tabaco infectadas, utilizando como testigo el medio KB sin drogas. Ninguno de los medios estudiados suprimio el crecimiento de la bacteria, mientras que los contaminantes presentes en las muestras, fueron reducidos en los medios con antibioticos dos o mas veces, en comparacion con el testigo. Las colonias de P. syringae pv. tabaci en el medio MSP modificado pudieron ser evaluadas a las 48 horas y mostraron una morfologia facilmente distinguible de las colonias contaminantes.

Population dynamics of Pseudomonas savastanoi pv. phaseolicola in bean, throughout the epiphytic and pathogenic phases

The objective of this work was to monitor traits of the life cycle of Pseudomonas savastanoi pv. phaseolicola , in order to better understand the outbreak of bean halo blight, originating from a bacterial population in asymptomatic plants. Five experiments were conducted in the field, in greenhouses, and in humidity chambers. Changes in population size were evaluated in three field plantings, by introducing the bacteria in contamination focal points and observing the weather conditions favoring an outbreak. The dispersion of the bacteria in the field was followed by isolation and Bio‑PCR analysis. Two assays were conducted in greenhouses and humidity chambers to evaluate the effect of leaf age on disease expression and the relationship between population level and number of leaf spots. The bacteria multiply intensively when in contact with a compatible host and reach high population sizes, with or without symptoms. The most favorable factor for bacterial multiplication and symptom triggering was water, and its role in the changeover from the epiphytic to the pathogenic phase might be linked to rainfall volume and intensity. Bacterial asymptomatic dispersion in the field is greater than disease emergence. In Brazil, the bacteria should be categorized as a present quarantine pest.

Fundamentos biológicos, ferramentas operacionais e inovação em quarentena vegetal

The demand toward food and nutritional security delimitates models for agricultural intensification, in which yield loss prevention is essential. The globalized agriculture scenario, the increase of trade routes, and the displacement of people and products maximize the potential of nondeliberate pest introductions in undamaged areas, endangering the production systems. Plant quarantine is presented as a measure to control plant product movement, in order to limit the dispersal of agricultural pests. Surveillance advocates the anticipation and recognizes the threat, focusing on the accuracy and efficiency of the diagnosis, on the consolidation of methods of pest risk analysis, and on the evolution of operational tools. Responses to the challenges posed to agriculture security are expected, considering the appropriation of new technologies, enhancement of inspectorate structures, and a strong, innovative capacity. This work approaches the historical aspects related to quarantine actions, legal framework, influence of international trade, analytical tools, innovation perspectives, and infrastructure qualification. The objective of this work is to contextualize the risk importance of introducing new pests, faced with a thriving agriculture and the intensification of commercial interchange. It also analyses the challenges to plant quarantine actions, while it aligns the biological grounding on which the phytosanitary regulation should be sustained, in order to subside the formulation of legal security measures.