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Several diseases caused by viruses, bacteria and fungi affect plant crops, resulting in losses and decreasing the quality and safety of agricultural products. Plant disease control relies mainly on chemical pesticides that are currently subject to strong restrictions and regulatory requirements. Antimicrobial peptides are interesting compounds in plant health because there is a need for new products in plant protection that fit into the new regulations. Living organisms secrete a wide range of antimicrobial peptides produced through ribosomal (defensins and small bacteriocins) or non-ribosomal synthesis (peptaibols, cyclopeptides and pseudopeptides). Several antimicrobial peptides are the basis for the design of new synthetic analogues, have been expressed in transgenic plants to confer disease protection or are secreted by microorganisms that are active ingredients of commercial biopesticides.

The anaerobic zones of Lakes Cis6 and Vilar (Banyoles karstic area, NE Spain) had mass developments of purple sulfur bacteria during summer 1982. In Lake Vilar, Chromatium spp. was dominant (up to 92% of the microbial biovolume). In Lake Ciso, the predominant microorganisms were Chromatium spp. (up to 7 1%) and another purple sulfur bacterium forming aggregates (20%).The bacterial layer could be divided according to the physiological state of the cells into a top part of maximal specific activity, a peak of maximal abundance, and a bottom part of inactive cells. The bacteria in the peak were predominantly limited by light; sulfide, phosphate, and acetate were not limiting in the middle of the day. The light limitation started at the depth having the maximal concentration of cells; the top of the layer appeared to be sulfide-limited. Specific contents of photopigments, elemental sulfur, and reserve polymers decreased from the top to the bottom of the bacterial layer. These phenomena point to the crucial role of light in the development of layers of phototrophic bacteria in stratified lakes.

Plant protection against pathogens, pests and weeds has been progressively reoriented from a therapeutic approach to a rational use of pesticide chemicals in which consumer health and environmental preservation prevail over any other productive or economic considerations. Microbial pesticides are being introduced in this new scenario of crop protection and currently several beneficial microorganisms are the active ingredients of a new generation of microbial pesticides or the basis for many natural products of microbial origin. The development of a microbial pesticide requires several steps addressed to its isolation in pure culture and screening by means of efficacy bioassays performed in vitro, ex vivo, in vivo, or in pilot trials under real conditions of application (field, greenhouse, post-harvest). For the commercial delivery of a microbial pesticide, the biocontrol agent must be produced at an industrial scale (fermentation), preserved for storage and formulated by means of biocompatible additives to increase survival and to improve the application and stability of the final product. Despite the relative high number of patents for biopesticides, only a few of them have materialized in a register for agricultural use. The excessive specificity in most cases and biosafety or environmental concerns in others are major limiting factors. Non-target effects may be possible in particular cases, such as displacement of beneficial microorganisms, allergenicity, toxinogencity (production of secondary metabolites toxic to plants, animals, or humans), pathogenicity (to plants or animals) by the agent itself or due to contaminants, or horizontal gene transfer of these characteristics to non-target microorganisms. However, these non-target effects should not be evaluated in an absolute manner, but relative to chemical control or the absence of any control of the target disease (for example, toxins derived from the pathogen). Consumer concerns about live microbes due to emerging food-borne diseases and bioterrorism do not help to create a socially receptive environment to microbial pesticides. The future of microbial pesticides is not only in developing new active ingredients based on microorganisms beneficial to plants, but in producing self-protected plants (so-called plant-incorporated pesticides) by transforming agronomically high-value crop plants with genes from biological control agents.

BackgroundPantoea agglomerans strains are among the most promising biocontrol agents for a variety of bacterial and fungal plant diseases, particularly fire blight of apple and pear. However, commercial registration of P. agglomerans biocontrol products is hampered because this species is currently listed as a biosafety level 2 (BL2) organism due to clinical reports as an opportunistic human pathogen. This study compares plant-origin and clinical strains in a search for discriminating genotypic/phenotypic markers using multi-locus phylogenetic analysis and fluorescent amplified fragment length polymorphisms (fAFLP) fingerprinting.ResultsMajority of the clinical isolates from culture collections were found to be improperly designated as P. agglomerans after sequence analysis. The frequent taxonomic rearrangements underwent by the Enterobacter agglomerans/Erwinia herbicola complex may be a major problem in assessing clinical associations within P. agglomerans. In the P. agglomerans sensu stricto (in the stricter sense) group, there was no discrete clustering of clinical/biocontrol strains and no marker was identified that was uniquely associated to clinical strains. A putative biocontrol-specific fAFLP marker was identified only in biocontrol strains. The partial ORF located in this band corresponded to an ABC transporter that was found in all P. agglomerans strains.ConclusionTaxonomic mischaracterization was identified as a major problem with P. agglomerans, and current techniques removed a majority of clinical strains from this species. Although clear discrimination between P. agglomerans plant and clinical strains was not obtained with phylogenetic analysis, a single marker characteristic of biocontrol strains was identified which may be of use in strain biosafety determinations. In addition, the lack of Koch's postulate fulfilment, rare retention of clinical strains for subsequent confirmation, and the polymicrobial nature of P. agglomerans clinical reports should be considered in biosafety assessment of beneficial strains in this species.

Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH 2 ). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED 50 ) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation were also determined. Pep3 and several analogues inhibited growth of the three pathogens and had a bactericidal effect at low micromolar concentrations (ED 50 of 1.3 to 7.3 M). One of the analogues consisting of a replacement of both Trp and Val with Lys and Phe, respectively, resulted in a peptide with improved bactericidal activity and minimized cytotoxicity and susceptibility to protease degradation compared to Pep3. The best analogues can be considered as potential lead compounds for the development of new antimicrobial agents for use in plant protection either as components of pesticides or expressed in transgenic plants.

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The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of antimicrobial cLPs.