Agrobacterium rhizogenes, what’s in a name!

status: publishe

Gekke wortels vormen nieuwe uitdaging

status: publishe

Enquête bewijst blijvende nood aan onderzoek rond gekke wortels

status: publishe

Efficacy of hydrogen peroxide treatment for control of hairy roots disease caused by rhizogenic agrobacteria

Aim: Rhizogenic Agrobacterium biovar 1 strains are the causative agent of hairy root disease (HRD), an increasing problem in the hydroponic cultivation of tomato and cucumber in Europe. A previous study has demonstrated that different lineages of rhizogenic agrobacteria are able to form biofilms. Although hydrogen peroxide (H2O2) is a frequently used biocide in hydroponic systems, until now its effectiveness to remove rhizogenic agrobacteria has not yet been unequivocally demonstrated. Therefore, the main objective of this study was to assess the efficacy of H2O2 in controlling Agrobacterium-containing biofilms. Methods and Results:. Using lab-scale experiments we found a huge variation between different rhizogenic Agrobacterium biovar 1 strains in EC50 values, ranging from 18.8 to 600 ppm H2O2, representing the lowest and highest concentration tested, respectively. Using pilot-scale experiments in which different H2O2 concentrations were tested, treatment with 25 ppm H2O2 was found to be ineffective. In contrast, treatment with 50 ppm significantly affected a catalase-negative Agrobacterium population, while a catalase-positive population was only marginally affected. For the catalase-positive Agrobacterium population, a treatment of 100 ppm H2O2 was required to be effective. Finally, H2O2 treatment of HRD in two commercial greenhouses was monitored, and showed that the H2O2 concentration decreased considerably towards the end of the irrigation circuits. Further, a clear correlation was found between the actual concentration of H2O2 and the incidence of HRD. Conclusion: We showed that H2O2 may be effective to reduce biofilm formation by rhizogenic bacteria. Nevertheless, it was clear from our results that the required H2O2 concentration depends on the particular Agrobacterium strain(s) present in the greenhouse. Significance and Impact of Study: This is the first study that examined the effectiveness of H2O2 to control HRD in hydroponic systems, and the effect of catalase activity on H2O2 effectiveness. Our study has direct relevance for the highly-intensive horticultural sector.status: publishe

Development of a qPCR assay for detection and quantification of rhizogenic agrobacterium biovar 1 strains

Agrobacterium biovar 1 strains harbouring a Ri-plasmid cause extensive root proliferation (so-called “hairy roots”) on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in severe economic losses. The aim of this study was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay based on the rol locus of the Ri-plasmid for detection and quantification of these rhizogenic agrobacteria in hydroponic systems. The assay is designed based on all rolB sequences currently available in GenBank and developed using a collection of both target and non-target strains, and was specific for rhizogenic Agrobacterium biovar 1 strains. Based on a calibration with artificially contaminated water samples mimicking hydroponic conditions, unknown bacterial concentrations could be accurately quantified in water samples from surveys carried out in different greenhouses. Further, a detection limit was obtained of less than one cell per mL water, following filtration of a volume of 500 mL. Altogether, our results illustrate the power of this qPCR assay for routine detection of rhizogenic Agrobacterium biovar 1 strains in aqueous samples. Importantly, the assay can be used for pathogen assessment at the presymptomatic stage of infection, enabling adequate control of the disease in hydroponics before plants become infected. Keywords Agrobacterium Quantitative real-time PCR Ri-plasmid rolB SYBR greenstatus: publishe

Assessing the usefulness of hydrogen peroxide to control Agrobacterium biovar 1 containing biofilms in a pilot-scale system

“Crazy roots” (also known as “hairy root disease”) caused by rhizogenic Agrobacterium biovar 1 strains has become a major problem in hydroponics greenhouses, especially in the cultivation of tomato and cucumber (Weller et al., 2006). Hairy root disease is characterized by excessive root development, leading to a strong vegetative growth and severe losses in marketable yield. Importantly, infections are often persistent as many Agrobacterium strains are able to form biofilms in the irrigation system. Prevention and removal of biofilms are considered to be crucial in management of the disease. To this end, hydrogen peroxide (H2O2) is frequently used in hydroponics systems as a disinfecting agent. However, in a recent study it was demonstrated that many rhizogenic Agrobacterium biovar 1 strains show catalase activity, enabling them to survive exposure to H2O2 (Bosmans et al., 2015). Therefore, the effectiveness of H2O2 to control Agrobacterium containing biofilms can be questioned. The objective of this study was to evaluate the effectiveness of H2O2 to control biofilms that harbour rhizogenic agrobacteria using a pilot-scale system. Material and Methods Experiments were performed using the pilot-scale system described by Vankerckhoven et al., (2011). Briefly, the system consists of four subsystems (allowing to test different H2O2 concentrations at once), each containing a water container. From each container water was pumped through a piping system harboring a biofilm sampling device consisting of polycarbonate rings. Following inoculation with Agrobacterium (106 cells per ml), biofilms were allowed to develop on the polycarbonate rings for 65 h. Subsequently, H2O2 was applied at different concentrations, including 25 ppm, 50 ppm and 100 ppm. A treatment in which no H2O2 was applied was used as negative control. Next, every 24 h bulk water and biofilm samples were taken to quantify the number of Agrobacterium cells as well as the total number of culturable bacteria. Quantification was performed by quantitative PCR (rhizogenic agrobacteria; Bosmans et al., 2016), and traditional culture-plate enumeration on Plate Count Agar, respectively. The experiment was performed for a catalase-positive and catalase-negative Agrobacterium biovar 1 strain. Results and Conclusions While application of 25 ppm H2O2 was ineffective for both the catalase-positive and catalase-negative strain, substantial differences between both strains were observed for the other concentrations tested. More particularly, while treatment of the catalase-negative strain with 50 ppm resulted in a significant reduction in Agrobacterium cells, the same treatment was ineffective for the catalase-positive Agrobacterium strain. When 100 ppm H2O2 was applied both the catalase positive and catalase negative strain were affected. These results clearly indicate that the efficacy of the H2O2 treatment is strongly dependent on the particular Agrobacterium strain present in the greenhouse. Moreover, the presence of a catalase-positive Agrobacterium strain is able to protect other bacteria against H2O2 treatment as well. Altogether, our data suggest that the treatment against hairy roots disease should be attuned to the specific Agrobacterium strain present in the greenhouse.status: publishe

Assessment of the genetic and phenotypic diversity among rhizogenic Agrobacterium biovar 1 strains infecting solanaceous and cucurbit crops

Rhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium.status: publishe

Didymella bryoniae in cucumber: an integrated control strategy

Didymella bryoniae (synonym Stagonosporopsis cucurbitacearum) is a necrotrophic fungus which can cause internal and external fruit rot in cucurbits. Infection of living material occurs by mycelium growth starting from dying material or by infection via the flower. This results in important production losses due to rotten and malformed fruits. A set of control measures has been evaluated, which resulted in an advice to the growers based on a combination of both preventative and curative measures. Out of 12 fungicides and six biocontrol organisms tested for their effectiveness against D. bryoniae in a semi-commercial crop, four chemical crop protection products showed a good result. These treatments are, however, a temporary solution. Differences in plant infections depend mainly on cultivation methods, plant morphology, fruit charge and climate. Moisture plays an important role. Climate and infection data of 6 growers show that problems occur when the humidity deficit is low. This finding was confirmed in a greenhouse trial. In addition, irrigation should be reduced in the evening and the night. It was demonstrated that air circulation has a limited but positive influence in preventing infections and inhibiting fungal growth. The results of these trials were combined in an integrated control strategy for D. bryoniae in cucumber.status: publishe

Differential Tomato Transcriptomic Responses Induced by Pepino Mosaic Virus Isolates with Differential Aggressiveness1[W][OA]

Pepino mosaic virus (PepMV) is a highly infectious potexvirus and a major disease of greenhouse tomato (Solanum lycopersicum) crops worldwide. Damage and economic losses caused by PepMV vary greatly and can be attributed to differential symptomatology caused by different PepMV isolates. Here, we used a custom-designed Affymetrix tomato GeneChip array with probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation of tomato seedlings with a mild and an aggressive PepMV isolate that share 99.4% nucleotide sequence identity. The two isolates induced a different transcriptomic response, despite accumulating to similar viral titers. PepMV inoculation resulted in repression of photosynthesis. In addition, defense responses were stronger upon inoculation with the aggressive isolate, in both cases mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for a PepMV-encoded silencing suppressor. Finally, perturbation of pigment biosynthesis, as shown by differential regulation of the flavonoid and lycopene biosynthesis pathways, was monitored. Metabolite analyses on mature fruits of PepMV-infected tomato plants, which showed typical fruit marbling, revealed a decrease in carotenoids, likely responsible for the marbled phenotype, and an increase in alkaloids and phenylpropanoids that are associated with pathogen defense in the yellow sectors of the fruit

Potential for Biocontrol of Hairy Root Disease by a Paenibacillus Clade

status: publishe