A reliable qPCR technique for detecting viable Xanthomonas arboricola pv. pruni cells

Xanthomonas arboricola pv. pruni (Xap) is the causal agent of bacterial spot of stone fruits and almond (Prunus spp). Detection of Xap is typically carried out using quantitative real-time PCR (qPCR) combined with culture-based isolation. However, qPCR does not differentiate between viable and dead cells, potentially leading to an overestimation of the infective population in a sample. Such overestimation could result in unnecessary phytosanitary measures. The present study aims to develop a specific protocol ideally targeting to detection of only live Xap bacterial cells. To address this challenge, the viable quantitative PCR (v-qPCR) method was evaluated using three nucleic acid-binding dyes: propidium monoazide (PMA), a combination of PMA and ethidium monoazide (EMA), and PMAxx™, an improved version of PMA. PMAxx™ proved to be the most suitable dye for the detection and quantification of living bacterial cells. This methodology was also evaluated in infected plant material over time and can be considered a rapid and reliable alternative to PCR methods for detecting only those putative infective Xap that may pose a risk for Prunus crops.Este trabajo fue financiado por el proyecto PID2021-123600ORC44, con el apoyo de MICIU/AEI/https://doi.org/10.13039/501100011033 y por el FEDER, UECut-off CtLODXanthomonas arboricola pv. pruniV-qPCRIntercalating dyeViable cell detectionPublishe

Interacción de lipasas con superficies sólidas hidrofóbicas aplicacioes biotecnológicas

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 06-07-199

Interaccion de lipasas con superficies solidas hidrofobicas Aplicaciones biotecnologicas

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

Biofilm formation in xanthomonas arboricola pv. Pruni: Structure and development

Centro de Investigación Forestal (CIFOR)Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of stone fruit and almond, an important plant disease with a high economic impact. Biofilm formation is one of the mechanisms that microbial communities use to adapt to environmental changes and to survive and colonize plants. Herein, biofilm formation by Xap was analyzed on abiotic and biotic surfaces using different microscopy techniques which allowed characterization of the different biofilm stages compared to the planktonic condition. All Xap strains assayed were able to form real biofilms creating organized structures comprised by viable cells. Xap in biofilms differentiated from free-living bacteria forming complex matrix-encased multicellular structures which become surrounded by a network of extracellular polymeric substances (EPS). Moreover, nutrient content of the environment and bacterial growth have been shown as key factors for biofilm formation and its development. Besides, this is the first work where different cell structures involved in bacterial attachment and aggregation have been identified during Xap biofilm progression. Our findings provide insights regarding different aspects of the biofilm formation of Xap which improve our understanding of the bacterial infection process occurred in Prunus spp and that may help in future disease control approaches.This work was supported financially by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Ministerio de Ciencia Innovación y Universidades and Agencia Estatal de Investigación (AEI) from Spain, projects RTA2014-00018-C02-01 and RTI2018-096018-R-C31 cofinanced by FEDER.Peer reviewed18 Pág. (This article belongs to the Special Issue Effect of Plant–Microbe Interaction on Biotic Resistance in Crops

Biocontrol of tomato wilt by Penicillium oxalicum formulations in different crop conditions

Eight formulations of Penicillium oxalicum (FOR1 to FOR8) were obtained by the addition of various ingredients, in two separate steps of the production and drying of P. oxalicum conidia. These formulations were then evaluated against tomato wilt in three glasshouse (G1 to G3) and two field (F1 and F2) experiments. All formulations were applied to seedlings in seedbeds 7 days before transplanting at a rate of 107 spores g-1 seedbed substrate. The conidial viability of each formulation was estimated by measuring germination just after fluid bed-drying, before seedbed application and after 1 and 2 years of storage at 4 °C under vacuum. The densities of P. oxalicum were estimated in the seedbed substrate and in the rhizosphere of three plants per treatment just before transplanting. Initial conidial viability of formulations just after fluid bed-drying was approx. 80%, except for FOR1, FOR4, and FOR7 which were 60%. The initial viability was maintained up to 40-50% for 2 years of storage at 4 °C under vacuum, except for FOR1. All formulations had {greater than or slanted equal to}50% viability at application time. Populations of P. oxalicum in the seedbed substrate just before transplanting were >106 cfu g-1 soil in G3 and F2; populations in rhizosphere were also >106 cfu g-1 fresh root, except for FOR3, FOR5, and FOR6 in G2. A range of 22-64% of disease reduction was observed with all formulations, although these reductions were not significant (p = 0.05) for FOR1, FOR4, and FOR5 in any experiment. Contrast analysis showed significant differences between biological treatments and untreated control (p = 0.05) in all experiments, but no significant differences between biological and chemical treatments. Initial conidial viability of P. oxalicum in formulations and populations of P. oxalicum in the seedbed substrate explained 78.26% of the variability in P. oxalicum populations in tomato rhizosphere before transplanting. Disease incidence in untreated plants was negatively correlated (r = -0.54) with the percentage of disease control. The relationship between the viability of formulations, the populations of P. oxalicum in seedbed and rhizosphere, and the control of tomato wilt is discussed

Dispersal improvement of a powder formulation of Penicillium oxalicum, a biocontrol agent of tomato wilt

Sugars, polyalcohols, inorganic salts, and detergents were added to conidia of Penicillium oxalicum at three different points of the production-formulation process to improve water dispersal. Effects also were tested on conidial germination and production. Conidial germination without additives ranged from 51 to 79%. Additives did not reduce conidial germination except for 50% polyethylene glycol (PEG) 300 and 10% CaCl2. Sunflower oil and sodium alginate, sucrose (0.5, 15, 30, and 60%), D-sorbitol (30 and 60%), glycerol (2, 5, 20, and 30%), 30% PEG 300, CaCl2 (0.01 to 1%), Tween 20 (0.01, 0.02, 0.5, and 1%), and Tween 80 (0.01 to 1%) enhanced conidial germination. Production without additives ranged from 0.57 to 4.58 conidia x 108 g-1 substrate. Additives did not affect conidial production except for reduction by 60% D-sorbitol, 60% fructose, and 10% CaCl2. Conidial dispersal in water improved when 1.5% sodium alginate was added to substrate in bags before production, and when 1.5% sodium alginate, 60% sucrose, 60% D-sorbitol, 60% fructose, 5 to 20% PEG 8000, or 20% glycerol were added to conidia before drying. Dispersal of dried conidia was enhanced with 1% Tween 20, 1% Tween 80, 1% Trition X-100, 10% Agral, and 1.5% sunflower oil. Two P. oxalicum formulations (conidial suspensions maintained with 60% sucrose or 1.5% sodium alginate for 10 min before drying) significantly reduced tomato wilt caused by Fusarium spp. under greenhouse conditions and, in a preliminary trial, by Verticillium spp. in a field assay. © 2005 The American Phytopathological Society

Development of a dried Penicillium oxalicum conidial formulation for use as a biological agent against Fusarium wilt of tomato Selection of optimal additives and storage conditions for maintaining conidial viability

The fungus, Penicillium oxalicum Thom.;has been proposed as a biocontrol agent against vascular wilts caused by Verticillium spp.;Fusarium oxysporum. In this paper, we report our findings on the effects of different additives and storage conditions of varying durations, temperatures, and types on the viability and biocontrol efficacy of three different types of P. oxalicum conidial formulations. The viability of the three different P. oxalicum conidial formulations with various solvent stabilizers and moisture contents between <5% and 21% was determined after their preparation and storage with or without vacuum for as long as one year at temperatures that ranged from 4 to 50°C. The results were compared to those obtained using a dried conidial formulation that was prepared from fresh conidia without any solvent stabilizers. After conducting a series of laboratory assays, and several glasshouse and field experiments over two growing seasons with the different types of conidial formulations, we found that a P. oxalicum conidial formulation with biocontrol efficacy and a long shelf life for controlling Fusarium tomato wilts can be a non-vacuum-packed or vacuum-packed formulation that contains 1.5% sodium alginate, 20% glycerol, 5% sucrose and 5% sorbitol and has <15% moisture content. Although the results on the efficacy of this biocontrol product are promising when the disease pressure of Fusarium wilt is moderate, this P. oxalicum formulation will still need to be improved in order to develop a commercial product that will be efficacious when the disease pressure of Fusarium wilt is high. © 2010 Elsevier Inc

The use of stable and unstable green fluorescent proteins for studies in two bacterial models: Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris

Fluorescent proteins have been used to track plant pathogens to understand their host interactions. To be useful, the transgenic pathogens must present similar behaviour than the wild-type isolates. Herein, a GFP marker was used to transform two plant pathogenic bacteria, Agrobacterium and Xanthomonas, to localize and track the bacteria during infection. The transgenic bacteria were evaluated to determine whether they showed the same fitness than the wild-type strains or whether the expression of the GFP protein interfered in the bacterial activity. In Agrobacterium, the plasmid used for transformation was stable in the bacteria and the strain kept the virulence, while Xanthomonas was not able to conserve the plasmid and transformed strains showed virulence variations compared to wild-type strains. Although marking bacteria with GFP to track infection in plants is a common issue, works to validate the transgenic strains and corroborate their fitness are not usual. Results, presented here, confirm the importance of proper fitness tests on the marked strains before performing localization assays, to avoid underestimation of the microbe population or possible artificial effects in its interaction with the plant.The research was supported by the Florida Citrus Production Research Advisory Council, FDOC contract 00079694, and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) project RTA2006-00149.Peer reviewe

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A "dried conidia without stickers" with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a "dried conidia without stickers" conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers. © 2008 Elsevier Inc. All rights reserved