Clara Pliego

Screening for candidate bacterial biocontrol agents against soilborne fungal plant pathogens

Over the years, many bacterial isolates have been evaluated as potential biocontrol agents against soilborne fungal phytopathogens. However, few of them were ultimately successful after evaluation in field trials. One of the major reasons for this failure is the lack of appropriate screening procedures to select the most suitable microorganisms for disease control in diverse soil environments. For this reason, the study of bacterial screening has a future that is characterised by many technical and conceptual challenges. In this review, we summarise and discuss the convenience of use of the main screening methods currently applied to select bacterial candidates for biocontrol of fungal and oomycete soilborne phytopathogens. Also, a comparative case study of the application of different screening methods applied to an experimental pathosystem is shown, revealing the success of bacterial candidates selected by different strategies for biocontrol of the phytopathogenic fungus Rosellinia necatrix in avocado plants. Screening for antagonism against this fungal pathogen, one of the more straightforward methods used for the selection of bacterial biocontrol agents, was proven to be a valid strategy for this experimental system.

GFP sheds light on the infection process of avocado roots by Rosellinia necatrix

In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron microscopy (SEM) images allowed detailed visualisation of the hyphal network generated by invasion of R. necatrix through the epidermal, cortical and vascular cells, including hyphal anastomosis and branching points. To our knowledge, this is the first report describing the construction of GFP-tagged strains belonging to the genus Rosellinia for monitoring white root rot using CLSM and SEM.

Plant Growth-Promoting Bacteria: Fundamentals and Exploitation

Many plant-beneficial rhizobacteria have been described in the literature. These have been isolated from the plant root, where they usually live under conditions of nutrient starvation and at a low pH. In order to be beneficial, they usually need to colonize the root efficiently. Moreover, they have to multiply fast in order to be competitive with other organisms. To this end, traits such as chemotaxis to, and fast utilization of, the nutrients secreted by the root are required. These nutrients mainly consist of organic acids and sugars. Some plant-beneficial bacteria promote plant growth directly, e.g., by making nutrients available to the plant or by stimulating the growth of plants by production of hormones. Other plant-beneficial bacteria stimulate plant growth indirectly, e.g., by degrading environmental pollutants which inhibit plant growth or by controlling the growth of pathogens.

Developing tools to unravel the biological secrets of Rosellinia necatrix, an emergent threat to woody crops.

UNLABELLED White root rot caused by Rosellinia necatrix is one of the most destructive diseases of many woody plants in the temperate regions of the world, particularly in Europe and Asia. Recent outbreaks of R. necatrix around the globe have increased the interest in this pathogen. Although the ecology of the disease has been poorly studied, recent genetic and molecular advances have opened the way for future detailed studies of this fungus. TAXONOMY Rosellinia necatrix Prilleux. Kingdom Fungi; subdivision Ascomycotina; class Euascomycetes; subclass Pyrenomycetes; order Sphaeriales, syn. Xylariales; family Xylariaceae; genus Rosellinia. IDENTIFICATION Fungal mycelium is present on root surfaces and under the bark, forming mycelium fans, strands or cords. A typical presence of pear-shaped or pyriform swellings can be found above the hyphal septum (with diameters of up to 13 µm). Sclerotia are black, hard and spherical nodules, several millimetres in diameter. Black sclerotia crusts may also form on roots. On synthetic media, it forms microsclerotia: irregular rough bodies composed of a compact mass of melanized, interwoven hyphae with no differentiated cells. Chlamydospores are almost spherical (15 µm in diameter). Synnemata, also named coremia (0.5-1.5 mm in length), can be formed from sclerotia or from mycelial masses. Conidia (3-5 µm in length and 2.5-3 µm in width) are very difficult to germinate in vitro. Ascospores are monostichous, situated inside a cylindrical, long-stalked ascus. They are ellipsoidal and cymbiform (36-46 µm in length and 5.5-6.3 µm in width). HOST RANGE This fungus can attack above 170 different plant hosts from 63 genera and 30 different families, including vascular plants and algae. Some are of significant economic importance, such as Coffea spp., Malus spp., Olea europaea L., Persea americana Mill., Prunus spp. and Vitis vinifera L. DISEASE SYMPTOMS Rosellinia necatrix causes white (or Dematophora) root rot, which, by aerial symptoms, shows a progressive weakening of the plant, accompanied by a decline in vigour. The leaves wilt and dry, and the tree can eventually die. White cottony mycelium and mycelial strands can be observed in the crown and on the root surface. On woody plant roots, the fungus can be located between the bark and the wood, developing typical mycelium fans, invading the whole root and causing general rotting. DISEASE CONTROL Some approaches have been attempted involving the use of tolerant plants and physical control (solarization). Chemical control in the field and biological control methods are still under development.

Two similar enhanced root-colonizing Pseudomonas strains differ largely in their colonization strategies of avocado roots and Rosellinia necatrix hyphae

Pseudomonas alcaligenes AVO73 and Pseudomonas pseudoalcaligenes AVO110 were selected previously as efficient avocado root tip colonizers, displaying in vitro antagonism towards Rosellinia necatrix, causal agent of avocado white root rot. Despite the higher number of antagonistic properties shown in vitro by AVO73, only AVO110 demonstrated significant protection against avocado white root rot. As both strains are enhanced root colonizers, and as colonization is crucial for the most likely biocontrol mechanisms used by these strains, namely production of non-antibiotic antifungal compounds and competition for nutrients and niches, we decided to compare the interactions of the bacterial strains with avocado roots as well as with R. necatrix hyphae. The results indicate that strain AVO110 is superior in biocontrol trait swimming motility and establishes on the root tip of avocado plants faster than AVO73. Visualization studies, using Gfp-labelled derivatives of these strains, showed that AVO110, in contrast to AVO73, colonizes intercellular crevices between neighbouring plant root epidermal cells, a microhabitat of enhanced exudation. Moreover, AVO110, but not AVO73, also colonizes root wounds, described to be preferential penetration sites for R. necatrix infection. This result strongly suggests that AVO110 meets, and can attack, the pathogen on the root. Finally, when co-inoculated with the pathogen, AVO110 utilizes hyphal exudates more efficiently for proliferation than AVO73 does, and colonizes the hyphae more abundantly than AVO73. We conclude that the differences between the strains in colonization levels and strategies are likely to contribute to, and even can explain, the difference in disease-controlling abilities between the strains. This is the first report that shows that two similar bacterial strains, selected by their ability to colonize avocado root, use strongly different root colonization strategies and suggests that in addition to the total bacterial root colonization level, the sites occupied on the root are important for biocontrol.

Biocontrol bacteria selected by a direct plant protection strategy against avocado white root rot show antagonism as a prevalent trait.

Aim:  This study was undertaken to study bacterial strains obtained directly for their efficient direct control of the avocado white root rot, thus avoiding prescreening by any other possible mechanism of biocontrol which could bias the selection.

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae.