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.

Selection for biocontrol bacteria antagonistic toward Rosellinia necatrix by enrichment of competitive avocado root tip colonizers.

Biological control of soil-borne pathogens is frequently based on the application of antagonistic microorganisms selected solely for their ability to produce in vitro antifungal factors. The aim of this work was to select bacteria that efficiently colonize the roots of avocado plants and display antagonism towards Rosellinia necatrix, the causal agent of avocado white root rot. A high frequency of antagonistic strains (ten isolates, 24.4%) was obtained using a novel procedure based on the selection of competitive avocado root tip colonizers. Amplification and sequencing of the 16S rRNA gene, in combination with biochemical characterization, showed that eight and two of the selected isolates belonged to the genera Pseudomonas and Stenotrophomonas, respectively. Characterization of antifungal compounds produced by the antagonistic strains showed variable production of exoenzymes and HCN. Only one of these strains, Pseudomonas sp. AVO94, produced a compound that could be related to antifungal antibiotics. All of the ten selected strains showed twitching motility, a cell movement involved in competitive colonization of root tips. Production of N-acyl-homoserine lactones and indole-3-acetic acid was also reported for some of these isolates. Resistance to several bacterial antibiotics was tested, and three strains showing resistance to only one of them were selected for biocontrol assays. The three selected strains persisted in the rhizosphere of avocado plants at levels considered crucial for efficient biocontrol, 10(5)-10(6) colony forming units/g of root; two of them, Pseudomonas putida AVO102 and Pseudomonas pseudoalcaligenes AVO110, demonstrated significant protection of avocado plants against white root rot.