Isolation of potential biocontrol agents of 'Candidatus Phytoplasma mali'

Background and objectives Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’, is one of the most important phytoplasma diseases in Europe. So far, due to the absence of AP resistant varieties, the management of AP mainly consists in insecticide treatment against the insect vectors and in the eradication of diseased plants. These treatments have a strong economic and environmental effect, representing a risk for both operators and final customers. One of the most innovative solutions to develop sustainable approaches is the use of endophytes as inducers of the natural plant defense responses. Bacterial endophytes are plant-associated bacteria that affect the plant life cycles in different manners such as nitrogen fixation or the biocontrol of plant pathogens (Lugtemberg and Kamilova 2009). A basic point for the success of sustainable management of plant diseases based on biocontrol agents is the study of endophytic bacterial community associated with plants. In this work, the endophytic bacterial community associated with healthy and AP phytoplasma-infected apple roots was described and characterized in order to find potential biocontrol agents. Materials and Methods Apple roots were collected from five asymptomatic and five AP-symptomatic plants during field survey conducted in 2010 in North-western Italy. Roots were opportunely sterilized and the total DNA was extracted with the method describe by Doyle and Doyle (1990), with some modifications. Extracted DNA was used as template for ‘Ca. Phytoplasma mali’ identification by (PCR)-based amplification of ribosomal RNA genes. In detail, ‘Ca. Phytoplasma mali’ was detected by the use of primer pairs fAT/rAS specific for 16SrX phytoplasmal group (Smart et al., 1996). Endophytic bacterial community associated with healthy and ‘Ca. Phytoplasma mali’-infected apple roots was described by cultivation dependent and independent methods (16S rRNA gene library analyses and sequencing). Endophytic bacteria were isolated by cultivation on Tryptic Soy Agar (TSA) and Luria-Bertani (LB). Total DNA from bacterial colonies was extracted and the 16S rRNA genes were amplified with bacterial universal primers and sequenced. The endophytic bacteria isolated on culture media were characterized for five beneficial traits related to mineral nutrition (phosphate solubilization, siderophores, nitrogen fixation), development (indolacetic acid synthesis), stress relief (catalase activity), disease control (siderophores). Results and Discussion The diversity of microbiota associated with healthy and phytoplasma-infected apple roots was investigated both with cultivation-independent and -dependent methods in order to increase the range of diversity explored in a sample. In this study, 16S rRNA gene libraries from infected and uninfected apple roots were analyzed to describe the endophytic bacterial community. A total of 120 clones were sequenced, 76 clones from the healthy roots and 44 from the infected ones. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 Operational Taxonomic Units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Also in previous work, clone library analysis of plant-associated bacteria in ‘Ca. Liberibacter asiaticus’-infected and uninfected citrus roots evidenced differences in the composition of their bacterial community (Trivedi et al., 2010). The influence of pathogen infection on endophytic bacterial community was also reported in healthy and phytoplasma-infected grapevine leaves (Bulgari et al., 2011). Cultivation dependent methods allowed to isolate twelve colonies with different morphology from healthy roots and six colonies from infected roots. Sequences of the 16S rRNA gene identified Firmicutes of the genus Bacillus, Lysinibacillus and Paenibacillus; Gammaproteobacteria of the genus Pseudomonas. Six different Bacillus species were isolated from healthy apple trees and, among these, Bacillus amyloliquefaciens and Bacillus gibsonii were found also in infected plants. Interestingly, bacterial strains, here isolated, belong to genera widely studied for developing biocontrol strategies to contain plant pathogens. The endophytic bacteria isolated on culture media were characterized for five beneficial traits related to mineral nutrition (phosphate solubilization, siderophores, nitrogen fixation), development (indolacetic acid synthesis), stress relief (catalase activity), disease control (siderophores) in order to select some strains for in vivo biocontrol tests. These assays allow to select different strains belonging to the genus Pseudomonas and Lysinibacillus that showed biocontrol and plant-growth promotion abilities. 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