J. C. Codina

A comparison of microbial bioassays for the detection of metal toxicity

Heavy metal toxicity was studied by assaying six microbiological toxicity tests, both in solution and wastewater. Pseudomonas fluorescens and bakers yeast (Saccharomyces cerevisiae) were used; growth and respirometric determinations were performed. In addition, the Microtox® test was employed as a reference method. The Microtox® test is the most sensitive assay for detecting toxicity of zinc, copper, and mercury but not for cadmium, chromium, and nickel. Wastewater increases the sensitivity threshold (EC20) and EC50 values of the metals in most of the assays, which is correlated to the presence of organic and inorganic compounds that can reduce the bioavailability of the metals, leading to a general loss of sensitivity.All the above-mentioned assays are potentially useful in the detection of chemical toxicity of metals. However, each test shows different sensitivies to each metal, which is related to different sensitivities of the organisms used in the assays, as well as to other factors. Therefore, it would be advisable to use a battery of tests for biological evaluation of metal toxicity.

Copper Resistance in Pseudomonas syringae Strains Isolated from Mango Is Encoded Mainly by Plasmids

ABSTRACT Bacterial apical necrosis of mango, elicited by Pseudomonas syringae pv. syringae, limits fruit production in southern Spain and Portugal. Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 59% were resistant to cupric sulfate. The survey of a mango orchard revealed an increase in frequencies of copper-resistant bacteria after repeated treatments with Bordeaux mixture. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. Most copper-resistant isolates harbored plasmids, although the majority of them contained a 62-kb plasmid that also was present in copper-sensitive strains. The 62-kb plasmids were differentiated by restriction enzyme analysis and hybridization to copABCD DNA. The most frequently found copper-resistant plasmid type (62.1) was transferable by conjugation. Southern blot hybridizations showed that genetic determinants partially homologous to copABCD were present in all the copper-resistant strains examined, and usually were associated with plasmids; these determinants were not detected in copper-sensitive strains. The selective pressure exerted by copper bactericide sprays on the diversity of copper resistance determinants in bacterial populations of mango is discussed.

Comparison of microbial tests for the detection of heavy metal genotoxicity.

Heavy metal genotoxicity was evaluated by using different microbial tests. Four genotoxicity assays were employed: the Ames test, the E. coli WP2 test, the Mutatox™ test detecting mutagenicity, and the SOS assay with E. coli-detecting enzyme induction. All the metals tested (cadmium, chromium, copper, mercury, nickel, and zinc) were detected as genotoxic by the Mutatox™ and the SOS tests. The Ames test and the E. coli WP2 assay only detected chromium as genotoxic, causing a mutagenic effect. The sensitivity to metals of all the assays used was maintained when they were dissolved in sewage, although there was a slight increase in the sensitivity thresholds.

Comparative histochemical analyses of oxidative burst and cell wall reinforcement in compatible and incompatible melon–powdery mildew (Podosphaera fusca) interactions

The spatial-temporal expression patterns of oxidative burst and cell wall reinforcement were analyzed in leaves of resistant and susceptible melon (Cucumis melo L.) cultivars in response to Podosphaera fusca (Fr.) Braun & Shishkoff, the main causal agent of powdery mildew in cucurbits. Extensive development of powdery mildew mycelia and a progressive increase in haustorial count were recorded in the susceptible cultivar after 4d, while in the resistant cultivar powdery mildew failed to grow and small brownish and necrotic leaf areas were frequently observed. Rapid generation of the reactive oxygen intermediates hydrogen peroxide and superoxide radicals 4h after pathogen challenge, but before the fungal haustoria formation, stood upstream in the cascade of events induced during these interactions. This oxidative burst was followed by the accumulation of strengthening polymers of callose and lignin at the cell wall of attacked resistant plant cells. Interestingly, the transcriptional levels of phenylalanine ammonia-lyase (PAL), an important enzyme for phenylpropanoid metabolism, did not significantly change throughout the experiments. Although these physiological changes were observed in both cultivars, their faster kinetics and amplitude in the resistant line compared to the susceptible cultivar governed the differential visual response of these cultivars against P. fusca. These findings, along with data obtained in previous studies, have provided the bases for an integrated model in which the spatial-temporal response patterns of these resistance mechanisms have been arranged, which may ultimately lead to successful protection of melon plants against P. fusca.

THE INHIBITION OF METHANOGENIC ACTIVITY FROM ANAEROBIC DOMESTIC SLUDGES AS A SIMPLE TOXICITY BIOASSAY

Abstract A specific toxicity assay to detect heavy metal toxicity to the anaerobic digestion process was evaluated. The relative toxicity of metals, obtained by using the inhibition of methanogenic activity assay, was Zn>Cr>Cu>Cd>Ni>Pb. The sensitivity of this assay to metal toxicity was compared with four microbiological toxicity tests. In general, Microtox ® , the Pseudomonas fluorescens growth inhibition (PFNEN) and the inhibition of methane production tests showed the highest sensitivity for the detection of metal toxicity from wastewaters and sludges.

The Mangotoxin Biosynthetic Operon (mbo) Is Specifically Distributed within Pseudomonas syringae Genomospecies 1 and Was Acquired Only Once during Evolution

ABSTRACT Mangotoxin production was first described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for the mbo operon to examine its distribution within the P. syringae complex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of the mbo operon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.

62-kb Plasmids Harboring rulAB Homologues Confer UV-tolerance and Epiphytic Fitness to Pseudomonas syringae pv. syringae Mango Isolates

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.

Effects of nickel and lead and a support material on the methanogenesis from sewage sludge

The effects of the addition of two heavy metals (nickel and lead) and a support material (purified sepiolite) on the methanogenesis have been evaluated in two types of domestic sewage sludges. A higher toxic effect of the metals was observed on the production of methane from loading sludge than from the anaerobic digester sludge, nickel being more toxic than lead in all cases studied. Antagonistic effects between both heavy metals were obtained when the loading sludge was supplemented with several concentrations of these metals. The addition of sepiolite to the loading sludge reduced toxic effects of both metals, which contrasts with the results obtained using sludges from anaerobic digester.

Serotypes and pyocin types of Pseudomonas aeruginosa isolated from natural waters.

Two important epidemiological characteristics (serotype and pyocin type) of Pseudomonas aeruginosa strains isolated from freshwater and seawater were investigated. Serotype I predominated, followed by serotype 6, whereas serotypes 11 and 9 were only occasionally isolated. The most frequent pyocin types from seawater and freshwater were 13B and 16B, respectively. The distribution of the different types in relation to the degree of faecal pollution was also studied. Pyocin types 12A and 13B were the best adapted to the marine environment, and serotype 10 and the pyocin type 17B were worst adapted to these aquatic environments

Evaluation of Phenotypic and Genetic Techniques to Analyze Diversity of Pseudomonas syringae pv. syringae Strains Isolates from Mango Trees

Bacterial apical necrosis of mango, produced by Pseudomonas syrin- gae pv. syringae (Pss), is the main disease affecting mango production in the Mediterranean area. Surveys carried out in the main areas of cultivation ascertained the presence of Pss strains and resulted in a collection of Pss strains from different seasons and locations (including mainland Spain and Canary Islands, Portugal, Italy and Israel). To study the diversity relationships among these Pss strains, different phenotypic and genetic techniques were evaluated by using a selection of repre- sentative Pss strains isolated from mango tissues. The use of physiological tests were based on conventional identification techniques (API tests), toxins production based on biological tests, and analysis of copper resistance. The genetic diversity studies were mainly based on repetitive PCR fingerprinting using ERIC, BOX and REP primers set with UPGMA analysis, and 16S rDNA gene sequencing and ARDRA analysis. Additionally, the native plasmid profiles of these representative strains were determined, and the presence of some genes of interest were detected by hybridization analysis in the most abundant plasmid (62 kb). Preliminary results indicate a considerable phenotypic diversity. Analysis of genetic techniques resulted in repetitive PCR fingerprintings using some primers sets, showing higher diversity than the other techniques used.