Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/17/2017
Publication Date: 6/10/2017
Citation: Medrano, E.G., Grauke, L.J., Thompson, T.E., Stanford, R. 2017. Evidence for the presence of an endosymbiont in the pecan scab pathogen Venturia effusa (basyonym: Fusicladium effusum). Journal of Applied Microbiology. 123:491-497.
Interpretive Summary: Pecans are a worldwide crop of particular importance to US agriculture. The most important disease of the plant is called Pecan Scab and it is caused by the fungus Venturia effusa (formerly named Fusicladium effusum). Infections result in leaf and shuck damage that can reduce the amount and quality of harvested nuts. Fungicide is the primary method to control the fungus. Unfortunately, constant applications will eventually result in the development of resistance by V. effusa. The pursuit of alternative means to control infections is hindered by the difficulty in culturing the fungus in the laboratory for study. During microscopic examination of V. effusa structures we observed what appeared to be bacterial cells. In this report, we provide high powered microscopic images and molecular biology data that showed bacteria within the fungus. Bacterial structures were found to mainly occur just under the fungal cell wall. Further, the fungal cell wall illuminated upon exposing to fluorescent DNA probes that target a bacterial gene. The findings from this work provide a novel perspective in studying biological and infectious processes of this pecan pathogen.
Technical Abstract: Aims: To determine whether Venturia effusa (basyonym: Fusicladium effusum) the causative fungal agent of Pecan Scab harbors a bacterial symbiont. Methods and Results: Beginning with monoconidial isolates, V. effusa was maintained on potato dextrose agar amended with antibiotics (chloramphenicol 100 µg/ml; tetracycline 100 µg/ml). Genomic DNA extracted from V. effusa mycelia was used to target eubacterial 16S rDNA. A 1.4 PCR amplified product using 16S rDNA degenerate primers was cloned, sequenced and found to have 99% identities with Actinobacteria representatives. Attempts to culture the genetically detected bacteria apart from the fungus following agitation and fungal cell lysis were unsuccessful using standard bacteriological media under either aerobic or anaerobic conditions. Fungal structures were visualized using scanning electron microscopy (SEM). The SEM technique revealed putative bacterial formations associated with the fungal mycelia. The fluorescence in situ hybridization (FISH) cytogenetic technique using 16S rDNA oligonucleotides illuminated spores and portions of the hyphae. Conclusions: This is the first report that provided both molecular microbiological and microscopic evidence in support of the hypothesis that V. effusa harbors endosymbiotic bacteria. Bacteria were observed within the fungal cell wall based on FISH illumination upon exposing to fluorescent DNA probes that targeted a bacterial gene. Significance and Impact of the Study: Pecan Scab is the major yield limiting disease of the plant. Findings from this research contribute fundamental information regarding the biology of the fungus that may ultimately lead to identifying a target of the infection process for use in management and/or avoidance strategies.