Plant microbiome interactions: Growth of the facultative pathogen Lasiodiplodia theobromae is altered by endophytic bacteria Ivelisse Irizarry, Mónica S. Torres, James F. White, Jr. Rutgers University, School of Environmental and Biological Sciences (SEBS). Department of Plant Biology and Pathology ABC Cocultures of Endophytes Pigment Production Aerial Hyphae Pycnidia Present Lasiodiplodia theobromae++ + Lasiodiplodia theobromae + Pantoea agglomerans-+ + Lasiodiplodia theobromae + Pseudomonas sp.-- - Lasiodiplodia theobromae is a fungal endophyte and facultative pathogen that is commonly found in a diverse group of hosts, particularly in subtropical and tropical regions. This fungus has been documented as a causal agent of blue- stain disease of wood and dieback of economically important crops. We explored how the endophytic bacteria Pantoea agglomerans and Pseudomonas sp. affect the growth and pigment production of the fungus Lasiodiplodia theobromae. The effects were observed by setting up cocultures of Lasiodiplodia with each bacterial endophyte and microscopically assessing their adhesion to hyphae. Colony diameter and hyphal width of L. theobromae in cocultures was measured. Cocultures were observed for pigment and pycnidia production. Both bacterial endophytes, Pantoea agglomerans and Pseudomonas, were able to adhere to Lasiodiplodia hypha. Pantoea agglomerans was additionally observed moving within a vesicle inside the fungus. This is the first report of endohyphal bacteria actively moving within L. theobromae. Both bacterial endophytes reduced colony diameter and hyphal width of L. theobromae, with Pseudomonas sp. being the most effective at reducing growth. After 14 days, only cultures of L. theobromae produced the blue stain pigment while cocultures with bacterial endophytes did not produce any fungal derived pigment. Pseudomonas appeared to have a much more aggressive antagonism against L. theobromae, as evidenced by a strong inhibition of growth and pigment production, less aerial mycelia, and no production of pycnidia as compared to cocultures with Pantoea agglomerans or controls. This study demonstrates that endophytic bacteria from plant microbiomes have the capacity of altering the physiology of a facultative fungal phytopathogen and possibly decrease the aggressiveness of certain virulence factors. Abstract Introduction Isolations: L. theobromae was isolated from seeds of the coastal tree Thespesia populnea (Malvaceae), commonly known as Portia tree. Seed samples were obtained from the US Virgin Islands, surface sterilized, and inoculated on potato dextrose agar (PDA). Pantoea agglomerans and Pseudomonas sp. were found as seed transmitted endophytes of Festuca arundinacea in a previous study 5. Fungal and bacterial identification: The fungus was identified by microscopic examination of fungal spores, characteristics of fungal cultures, and sequencing of the ITS1-ITS4 region. The bacteria were previously identified by sequencing the 16S rRNA gene 5. Fungal and bacterial endophyte cocultures: Bacterial endophytes were spread throughout the entire surface of Petri dishes containing PDA using an inoculating loop. A very small piece of mycelia from the same culture of Lasiodiplodia theobromae was then inoculated in the center of the plate. The plates were incubated at room temperature. Measurements: Colony diameter was measured (n=3). Cocultures were observed at 1000x and 50 hyphal cells per sample were measured and averaged. Results Discussion and Conclusions References Acknowledgements Figure 1. Attachment of bacterial cells to hyphae of Lasiodiplodia theobromae. A) L. theobromae (control), B) L. theobromae and Pantoea agglomerans, C) L. theobromae and Pseudomonas sp. Wet mounts stained with aniline blue were observed at 1000x. Lasiodiplodia theobromae interacts with other endophytic bacteria that are able to decrease fungal growth, pigment production, and reproduction by pycnidia. Beneficial bacteria from plant microbiomes are able to alter the growth of phytopathogenic fungi and possibly decrease their virulence. Pseudomonas is a more effective antagonist than Pantoea agglomerans by decreasing blue pigment production and pycnidia production in L. theobromae. Bacteria can affect the virulence of pathogenic fungi by decreasing pigment and aerial hyphae production. Production of aerial hyphae is important for dispersal. Could be of great importance for management practices in agriculture and crop production – certain bacteria may be protecting crops from fungal diseases. 1. 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Ecol. 76: White JF, Crawford H, Torres M, Mattera R, Irizarry I, Bergen M A proposed mechanism for nitrogen acquisition of grass seedlings through oxidation of symbiotic bacteria. Symbiosis. 57(3): The authors acknowledge financial support from the Rutgers Center for Turfgrass Science Figure 4. Cocultures of L. theobromae after 14 days. A) L. theobromae (control), B) L. theobromae and P. agglomerans, C) L. theobromae and Pseudomonas sp. Table 1. Aerial hyphae, pigment production, and presence of pycnidia in cocultures of Lasiodiplodia theobromae as observed after 14 days. +, observed; -, not observed Figure 3. Average hyphal width of L. theobromae cocultured with Pantoea agglomerans and Pseudomonas. 50 hyphal cells were measured per sample (n = 3). Figure 2. Average colony diameter of L. theobromae cocultures with P. agglomerans and Pseudomonas (n=3). Pseudomonas decreased the growth of the fungus most effectively. The facultative pathogen Lasiodiplodia theobromae interacts with the endophytic bacteria Pantoea agglomerans and Pseudomonas sp. (Figure 1). These endophytic bacteria are able to decrease colony growth and hyphal width of the fungus (Figures 2 and 3). Both bacteria were able to inhibit pigment production and decrease production of aerial hyphae in culture after 14 days (Table 1). Pseudomonas was additionally able to inhibit the production of pycnidia Materials and Methods Bacteria have been documented to affect growth of phytopathogens and potentially improve plant growth 4. Bacterial endophytes have also been identified as possible endosymbionts of fungi 1,2,3. L. theobromae is a cosmopolitan fungus in tropical regions that causes blue staining of wood and die back disease. It has also been reported as an endophytic fungus, therefore making it a facultative pathogen. The way that plant microbiome bacteria interact with potential pathogens could affect the outcome of the interaction between pathogen and plant. This study could have an effect on methods of disease management in crop production. Our objectives are: To assess the interaction of the fungus L. theobromae with endophytic bacteria. To evaluate the effects of endophytic bacteria on the physiology and growth patterns of the fungus. This study represents the first attempt to elucidate the interactions that this tropical pathogen engages in with the endophytic bacteria Pantoea agglomerans and Pseudomonas sp. C B A