The fungus was isolated from about 50 days outdated seedlings. For this purpose the megagametophyte and also the cotyledons had been removed, superficially disinfected in 96% ethanol and submersed in 1% sodium hypochlorite for 10 minutes. The material was desiccated within a laminar flow bench plus the megagametophyte was separated through the cotyledons. Infected tissues were transferred to tubes with PDA medium working with a sterile platinum loop. Tubes had been incubated at 26 C for 7 days and ex amined for fungal development. The emerged fungus was transferred to fresh PDA medium. Constant culture was on ISP 2 agar, The microorganism was identified in all plants showing symptoms of infection. The pathogenicity check was carried out by using healthier seeds excised from mature cones collected in 2011.
Seeds have been disinfected as previ ously described and scarified by getting rid of the integuments from the seed tip, exposing the megagametophyte. Scarified seeds had been incubated at 25 C in darkness with all the fungus. For this objective, selleckchem seeds had been placed within a tray and partially covered with sterile water containing myce lium. Mycelial plugs of 14 day previous cul tures within the isolate had been in ten ml sterile water. Controls consisted of sterile water, supplemented with an agar plug not having fungus. Trays have been maintained on an orbital shaker for 48 h. After this period, seeds had been extra, as well as resulting seedlings were trans ferred to polyethylene jars, as described over. Each ex periment consisted of two replicates with 33 seeds every single. When seeds were incubated while in the presence of your fungus, 42% of germinated plants developed the illness and died up to 70 days immediately after inoculation, presenting precisely the same symp toms previously observed.
Isolation and culture of bacteria Mainly because bacteria from bulk soil is often unique from these attached for the root surface, they have been extracted full article from each roots and sandy soil under Araucaria cunnighamii trees. The area was Wild Cattle Creek State Forest, Megan NSW, Australia, Soil samples had been taken in February from your respective rhizosphere, which was defined because the root containing natural layer soon after removal of your uppermost undigested litter layer. Rhizosphere sampling was be tween three to eight cm through the surface and at a distance of approximate two m from your tree trunk. 3 randomly taken samples were mixed and dried at 60 C. About 500 mg of dried soil have been extracted with sterile 50 ml HNC medium, deciding on particularly for Actinomycetes, The medium contained glass beads, and also the samples have been kept on a rotatory shaker at 200 rpm and 42 C. The resulting suspension was filtered by way of cotton. Filtrates were diluted ten or one hundred fold with water, and 50 ul plated on Petri dishes with ISP 2 agar, twenty g dissolved in one l tap water.