L. O. S. Beriam

Structural and Functional Characterization of Basic PLA2 Isolated from Crotalus durissus terrificus Venom

The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA2 also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2 showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2 isoform, determined by automatic sequencing, was HLLQFNKMLKFETRKNAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2 isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2 cited in the literature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2 as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity.

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

BackgroundAn interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex.ResultsThis PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity.The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm.PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound.ConclusionThe unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules.

Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor.

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by sPLA2r + EOCC was less than that induced by sPLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native sPLA2r. Native sPLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r + EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native sPLA2r from 14,299.34 Da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of sPLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the sPLA2 in Crotalus durissus sp. venom as well as other sPLA2s.

Induced resistance against Xanthomonas axonopodis pv. passiflorae in passion fruit plants

Control of bacterial leaf spot of yellow passion fruit using the abiotic resistance inducer, acibenzolar-S-methyl (ASM), and the biotic agents, harpin protein and glycoproteins extracted from two Xanthomonas species, was evaluated. The inducers were applied by spraying the leaves 72 h before the inoculation with Xanthomonas axonopodis pv. passiflorae. The inducers were also applied by seed immersion and the inoculation was performed when the seedlings had four true leaves. The results showed that ASM conferred a protection up to 70% at the concentration of 12.5 µg a.i. mL-1, while harpin led to an increase in bacterial symptoms. The glycoproteins from Xanthomonas spp. conferred up to 72% protection in plants against the bacterium. ASM or harpin provided up to 90% and 47% protection, respectively, in yellow passion fruit seedlings raised from treated seeds. Thus, leaf treatment with ASM or the glycoproteins from Xanthomonas spp. and seed treatment with ASM or harpin are potent inducers of resistance in passion fruit plants against X. axonopodis pv. passiflorae.

Pseudomonas syringae pv. tabaci in papaya seedlings

The natural occurrence of Pseudomonas syringae pv. tabaci causing leaf spot symptoms in papaya seedlings is reported. The pathogen was identified through biochemical, physiological, serological, and molecular assays and artificial inoculations in papaya plants. It was also shown that the strains were pathogenic to bean and tobacco plants. The restriction patterns obtained with Afa I, Alu I, Dde I, Hae III, Hpa II, Hinf I, Sau 3A I and Taq I of the PCR-RFLP of 16S-23S DNAr were identical to the P. s. pv. tabaci patterns. Primers corresponding to hrpL gene of P. syringae were also tested and the results grouped the papaya strains with P s. pv. tabaci. Bacterial strains were deposited at Colecao de Culturas IBSBF, Instituto Biologico, Campinas, Brazil, under access numbers 1687 and 1822.

Mancha bacteriana em Ruscus sp. causada por Burkholderia andropogonis no Brasil

Plants of ruscus (Ruscus sp.) showing symptoms of leaf spot were received for analysis in April 2008 from a field located in the region of Santo Antonio de Posse, state of Sao Paulo, Brazil. These spots were small, rounded, with 5 to 8 mm in diameter, dark brown in color, showing necrotic center and surrounded by chlorotic haloes. Slow-growing, cream-colored bacterial colonies were consistently obtained. The bacterium was Gram-negative, oxidative and not fluorescent. Artificial inoculations on healthy ruscus seedlings reproduced the symptoms observed in natural infections; the pathogen was re-isolated from the lesions. Biochemical, cultural, physiological and serological tests of the isolates identified the causal agent as Burkholderia andropogonis (former Pseudomonas andropogonis). This is the first report of this pathogen in ruscus in Brazil. Bacterial strains were deposited in the Phytobacteria Culture Collection of the Instituto Biologico (IBSBF) under numbers 2594 and 2595.

Avaliação da solarização do solo para o controle de Ralstonia solanacearum

The use of soil solarization for the control of bacterial wilt, caused by Ralstonia solanacearum, was evaluated by burrowing nylon bags containing soil infested with the bacteria in plots solarized or not. Two experiments were carried out in Campinas (SP), from February to April/2001, and Piracicaba (SP), from December/2001 to February/2002. The experiments were set up in a completely randomized factorial design, with four replications, in 4 x 4 meter plots. The factors evaluated were soil solarization with a transparent plastic film 100 µm thick, period of treatment (30 and 60 days and 37 and 60 days for the first and second experiments, respectively) and, only for the second experiment, soil depth (10 and 20 cm). The soil of each nylon bag collected after the previously established solarization periods, was placed in pots where tomato (Lycopersicon esculentum) seedlings were transplanted. In the non-solarized soil, in both experiments, 43 to 100% of the tomato plants wilted. In the second experiment, 6 to 22% of the plants grown on the soil solarized for 37 days wilted. No wilted tomato plants were observed in the solarized plots of the first experiment nor in the soil solarized for 60 days, in both soil depths, of the second experiment. The results obtained indicate that soil solarization has potential for the control of R. solanacearum.

Bacterial halo blight of coffee crop: aggressiveness and genetic diversity of strains

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 PLANT PROTECTION Article Bacterial halo blight of coffee crop: aggressiveness and genetic diversity of strains Karen Wolf Maciel1*, Suzete Aparecida Lanza Destefano1, Luis Otavio Saggion Beriam1, Irene Maria Gatti de Almeida1, Flavia Rodrigues Alves Patricio2, Lucas Mateus Rivero Rodrigues3, Oliveiro Guerreiro Filho3 1.Instituto Biológico Laboratório de Bacteriologia Vegetal Campinas (SP), Brazil. 2.Instituto Biológico Laboratório de Fitopatologia Campinas (SP), Brazil. 3.Instituto Agronômico Centro de Café Campinas (SP), Brazil. *Corresponding author: karenwmaciel@hotmail.com Received: Jul 6, 2016 – Accepted: Feb. 28, 2017 strains grouped separately from the Brazilian strains with ERIC-PCR and the combination of ERICand 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.

Ocorrência de Serratia marcescens bizio sobre lagartas de Heliothis virescens (Fabr.)

A large quantity of dead worms was observed in rearing of Heliothis virescens. A bacteria, later identified as Serratia marcescens Bizio, was isolated from the dead worms. The present work registers the occurrence and confirms the pathogenicity of S. marcescens on H. virescens.

First report of mixed infection by Pseudomonas syringae pathovars garcae and tabaci on coffee plantations

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 Sao 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 Parana and fluorescent bacteria producing or not brown pigment in culture medium were isolated and determined as belonging to the Group I PLANT PROTECTION - Article First report of mixed infection by Pseudomonas syringae pathovars garcae and tabaci on coffee plantations Lucas Mateus Rivero Rodrigues 1 *, Gustavo Hiroshi Sera 2 , Oliveiro Guerreiro Filho 1 , Luis Otavio Saggion Beriam 3 , Irene Maria Gatti de Almeida 1.Instituto Agronomico - Centro de Cafe - Campinas (SP), Brazil.2.Instituto Agronomico do Parana - Area de Melhoramento e Genetica Vegetal - Londrina (PR), Brazil.3.Instituto Biologico - Laboratorio de Bacteriologia Vegetal - Campinas (SP), Brazil.