Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 2/2/2005
Publication Date: 6/15/2006
Citation: Stanford, R.L., Medrano, E.G., Thompson, T.E., Ellis, E.A., Grauke, L.J. 2005. Recognition of uncultured gram positive cocci associated with Fusicladosporium effusum [abstract]. American Society for Microbiology. 105:187.
Technical Abstract: Background: Pecan Scab is a significant yield limiting disease that is caused by Fusicladosporium effusum. Research focused on studying F. effusum pathogenic mechanisms has been hampered by poor in vitro fungal growth. Using light microscopy, we observed what appeared to be bacterial cocci sparsely distributed along the surface of F. effusum hyphae. These cocci exhibited a Gram positive staining reaction, yet were non-culturable on standard bacteriological media (e.g. Luria Burtanni, Trypticase Soy, or Brain Heart Infusion) under either aerobic or anaerobic conditions. Therefore, our objective was to determine whether or not the observed structures were bacteria in otherwise pure F. effusum cultures. Methods: Three week old potato dextrose broth cultures of F. effusum were vortexed in an attempt to separate the cocci from the hyphae. The mixture was centrifuged and then the supernatant was separated from the mycelial pellet by pipet transfer. A bacterial genomic DNA extraction procedure was conducted on the supernatant. A universal degenerate primer set designed to target 16s ribosomal DNA using PCR was employed to test for an amplified product. A 1.4 kb PCR product was cloned, sequenced and used in a BLAST inquiry. Preparation of F. effusum samples for viewing using scanning electron microscopy (SEM) consisted of fixation, critical point drying, and then coating with gold palladium. For transmission electron microscopy (TEM) visualization, samples were infiltrated with resin then thinly sectioned. Results: The cloned and sequenced PCR fragment had a 99% nucleotide identity with Propionibacterium spp. 16s ribosomal DNA gene sequences. Cocci with a diameter ranging from 1.5-2.5um were apparent on SEM images. The TEM images revealed morphological traits consistent with bacteria. Conclusion: These results demonstrated the presence of uncultured bacteria associated with F. effusum. Studies to assess potential biological interactions and to determine if F. effusum isolates from diverse geographical locations also harbor bacteria are in progress.