Effects of leaf wetness duration and temperature on infection of Prunus by Xanthomonas arboricola pv. pruni

Xanthomonas arboricola pv. pruni is the causal agent of bacterial spot disease of stone fruits and almond. The bacterium is distributed throughout the major stone-fruit-producing areas of the World and is considered a quarantine organism in the European Union according to the Council Directive 2000/29/EC, and by the European and Mediterranean Plant Protection Organization. The effect of leaf wetness duration and temperature on infection of Prunus by X. arboricola pv. pruni was determined in controlled environment experiments. Potted plants of the peach-almond hybrid GF-677 were inoculated with bacterial suspensions and exposed to combinations of six leaf wetness durations (from 0 to 24 h) and seven fixed temperatures (from 5 to 35°C) during the infection period. Then, plants were transferred to a biosafety greenhouse, removed from bags, and incubated at optimal conditions for disease development. Although leaf wetness was required for infection of Prunus by X. arboricola pv. pruni, temperature had a greater effect than leaf wetness duration on disease severity. The combined effect of wetness duration and temperature on disease severity was quantified using a modification of the Weibull equation proposed by Duthie. The reduced-form of Duthie's model obtained by nonlinear regression analysis fitted well to data (R = 0.87 and R2adj = 0.85), and all parameters were significantly different from 0. The estimated optimal temperature for infection by X. arboricola pv. pruni was 28.9°C. Wetness periods longer than 10 h at temperatures close to 20°C, or 5 h at temperatures between 25 and 35°C were necessary to cause high disease severity. The predictive capacity of the model was evaluated using an additional set of data obtained from new wetness duration-temperature combinations. In 92% of the events the observed severity agreed with the predicted level of infection risk. The risk chart derived from the reduced form of Duthie's model can be used to estimate the potential risk for infection of Prunus by X. arboricola pv. pruni based on observed or forecasted temperature and wetness durationMinisterio de Educación, Ciencia y Deporte (AGL2013-41405-R, FPU13/04123) of Spain (https://www.mecd.gob.es/). University of Girona (SING12/13, MPCUdG2016/085) (www. udg.edu). European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement number 613678 (DROPSA

Basis for a predictive model of Xanthomonas arboricola pv pruni growth and infections in host plants

Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot disease of stone fruits and almond. The bacterium is considered a quarantine pathogen in Europe and it has become a new and emerging threat for European crops. As the disease is strongly influenced by the weather, a forecasting model that predicts Xap infections based on climatic conditions could be implemented in stone fruit integrated pest management. The objective of this work was to constrain the basis for the development of a predictive model of Xap growth and infections, determining the effects of pathogen, host and climatic parameters on infection and disease development. A non-pathogenic specialization of Xap and cross-infection among host species was observed, although strains isolated from peach were the most virulent in peach leaves. Xap was able to infect unwounded leaves and it was observed that the presence of wounds on the leave surface did not favour Xap penetration in peach leaves. Otherwise, the water condition of plants played an important role in Xap infections and disease development in peach. The presence of water congestion and leaf wetness 48 h before inoculation favoured Xap infections and the duration of leaf wetness after inoculation was directly correlated to disease severity. Temperature and leaf age had a significant effect on Xap infections. Temperatures above 20°C favoured Xap infections, which were basically produced in young leaves; whereas severity was significantly lower at temperatures below 15°C and in mature leavesSupported by research grants BR 2013/31 from University of Girona and FPU13/04123 from Spain MECD, and the projects AGL2013-41405-R from Spain MINECO and the European Union Seventh Framework (FP7 / 2007-2013) under the agreement n°613678 (DROPSA

Pathogenic plant-microbe interactions. What we know and how we benefit

Plants, like humans and other animals, also get sick, exhibit disease symptoms, and die. Plant diseases are caused by environmental stress, genetic or physiological disorders and infectious agents including viroids, viruses, bacteria and fungi. Plant pathology originated from the convergence of microbiology, botany and agronomy; its ultimate goal is the control of plant disease. Microbiologists have been attracted to this field of research because of the need for identification of the agents causing infectious diseases in economically important crops. In 1878—only two years after Pasteur and Koch had shown for the first time that anthrax in animals was caused by a bacteria—Burril, in the USA, discovered that the fire blight disease of apple and pear was also caused by a bacterium (nowadays known as Erwinia amylovora). In 1898, Beijerinck concluded that tobacco mosaic was caused by a “contagium vivum fluidum” which he called a virus. In 1971, Diener proved that a potato disease named potato spindle tuber was caused by infectious RNA which he called viroi

Plant-associated microorganisms: a view from the scope of microbiology

Microorganisms interact with plants because plants offer a wide diversity of habitats including the phyllosphere (aerial plant part), the rhizosphere (zone of influence of the root system), and the endosphere (internal transport system). Interactions of epiphytes, rhizophytes or endophytes may be detrimental or beneficial for either the microorganism or the plant and may be classified as neutralism, commensalism, synergism, mutualism, amensalism, competition or parasitis

Lysozyme enhances the bactericidal effect of BP100 peptide against Erwinia amylovora, the causal agent of fire blight of rosaceous plants

Background: Fire blight is an important disease affecting rosaceous plants. The causal agent is the bacteria Erwinia amylovora which is poorly controlled with the use of conventional bactericides and biopesticides. Antimicrobial peptides (AMPs) have been proposed as a new compounds suitable for plant disease control. BP100, a synthetic linear undecapeptide (KKLFKKILKYL-NH2), has been reported to be effective against E. amylovora infections. Moreover, BP100 showed bacteriolytic activity, moderate susceptibility to protease degradation and low toxicity. However, the peptide concentration required for an effective control of infections in planta is too high due to some inactivation by tissue components. This is a limitation beause of the high cost of synthesis of this compound. We expected that the combination of BP100 with lysozyme may produce a synergistic effect, enhancing its activity and reducing the effective concentration needed for fire blight control. Results: The combination of a synhetic multifunctional undecapeptide (BP100) with lysozyme produces a synergistic effect. We showed a significant increase of the antimicrobial activity against E. amylovora that was associated to the increase of cell membrane damage and to the reduction of cell metabolism. Combination of BP100 with lysozyme reduced the time required to achieve cell death and the minimal inhibitory concentration (MIC), and increased the activity of BP100 in the presence of leaf extracts even when the peptide was applied at low doses. The results obtained in vitro were confirmed in leaf infection bioassays. Conclusions: The combination of BP100 with lysozyme showed synergism on the bactericidal activity against E. amylovora and provide the basis for developing better formulations of antibacterial peptides for plant protectionThis work has been supported by the project AGL2009-13255-C02-01/AGR from MICINN of Spain. The research group has been supported in part by CIRIT of the Catalonian Government (Ref. 2009SGR812) and by the University of Giron

Mechanisms of antagonism of Pseudomonas fluorescens EPS62e against Erwinia amylovora, the causal agent of fire blight

Pseudomonas fluorescens EPS62e was selected during a screening procedure for its high efficacy in controlling infections by Erwinia amylovora, the causal agent of fire blight disease, on different plant materials. In field trials carried out in pear trees during bloom, EPS62e colonized flowers until the carrying capacity, providing a moderate efficacy of fire-blight control. The putative mechanisms of EPS62e antagonism against E. amylovora were studied. EPS62e did not produce antimicrobial compounds described in P. fluorescens species and only developed antagonism in King’s B medium, where it produced siderophores. Interaction experiments in culture plate wells including a membrane filter, which physically separated the cultures, confirmed that inhibition of E. amylovora requires cell-to-cell contact. The spectrum of nutrient assimilation indicated that EPS62e used significantly more or different carbon sources than the pathogen. The maximum growth rate and affinity for nutrients in immature fruit extract were higher in EPS62e than in E. amylovora, but the cell yield was similar. The fitness of EPS62e and E. amylovora was studied upon inoculation in immature pear fruit wounds and hypanthia of intact flowers under controlled-environment conditions. When inoculated separately, EPS62e grew faster in flowers, whereas E. amylovora grew faster in fruit wounds because of its rapid spread to adjacent tissues. However, in preventive inoculations of EPS62e, subsequent growth of EPS101 was significantly inhibited. It is concluded that cell-to-cell interference as well as differences in growth potential and the spectrum and efficiency of nutrient use are mechanisms of antagonism of EPS62e against E. amylovor

Cyclic Lipopeptide Biosynthetic Genes and Products, and Inhibitory Activity of Plant- Associated Bacillus against Phytopathogenic Bacteria

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, srfAAand 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. subtilisand 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. cereusand B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillusagainst 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. subtilisand 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 cLPsFunding was provided by Programa Nacional de Agricultura y Alimentación (CICYT) (Ref. AGL-2009-13255-C02-01) of the MINECO of Spain, and FP7 KBBE.2013.1.2-04. Ref. 613678. DROPSA project of the European Union. The research group is accredited and supported in part by SGR 2009-0812, 2014 SGR 697 and TECNIO net from Cataloni

Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions

Four methods were tested to assess the fire-blight disease response on grafted pear plants. The leaves of the plants were inoculated with Erwinia amylovora suspensions by pricking with clamps, cutting with scissors, local infiltration, and painting a bacterial suspension onto the leaves with a paintbrush. The effects of the inoculation methods were studied in dose-time-response experiments carried out in climate chambers under quarantine conditions. A modified Gompertz model was used to analyze the disease-time relatiobbnships and provided information on the rate of infection progression (rg) and time delay to the start of symptoms (t0). The disease-pathogen-dose relationships were analyzed according to a hyperbolic saturation model in which the median effective dose (ED50) of the pathogen and maximum disease level (ymax) were determined. Localized infiltration into the leaf mesophile resulted in the early (short t0) but slow (low rg) development of infection whereas in leaves pricked with clamps disease symptoms developed late (long t0) but rapidly (high rg). Paintbrush inoculation of the plants resulted in an incubation period of medium length, a moderate rate of infection progression, and low ymax values. In leaves inoculated with scissors, fire-blight symptoms developed early (short t0) and rapidly (high rg), and with the lowest ED50 and the highest yma

Diversity of plant defense elicitor peptides within the Rosaceae

Plant elicitor peptides (Peps) are endogenous molecules that induce and amplify the first line of inducible plant defense, known as pattern-triggered immunity, contributing to protect plants against attack by bacteria, fungi and herbivores. Pep topic application and transgenic expression have been found to enhance disease resistance in a small number of model plant-pathogen systems. The action of Peps relies on perception by specific receptors, so displaying a family-specific activity. Recently, the presence and activity of Peps within the Rosaceae has been demonstrated. Here we characterized the population of Pep sequences within the economically important plant family of Rosaceae, with special emphasis on the Amygdaleae and Pyreae tribes, which include the most relevant edible species such as apple, pear and peach, and numerous ornamental and wild species (e.g. photinia, firethorn and hawthorn)This work was supported by the Spanish MINECO AGL2013–41405-R and the FP7 KBBE.2013.1.2–04 with reference 613678 (DROPSA

Diversity of the bacterial community in the surface soil of a pear orchard based on 16S rRNA gene analysis

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environment