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Title:   Author  item
                                            James, Rosalinditem
                                            BUCKNER, J. - BIOSCIENS RES.LAB., ND
  Submitted to: Mycopathologia
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 6/25/2004
                                    Publication  Date: 12/15/2004
                                    Citation: James, R.R., Buckner, J.S. 2004. Lipids stimulate spore germination in the entomopathogenic ascomycete Ascosphaera aggregata. Mycopathologia. 158:293-302Interpretive Summary: The alfalfa leafcutting is used extensively in the U.S. for pollination of alfalfa seed crops.  These bees nest in holes drilled in wood or polystyrene blocks, and their brood are highly prone to a disease called chalkbrood caused by a fungus called Ascosphaera aggregata.  It is difficult to test whether or not the spores are alive because none of the growth media currently used are adequate to test for spore germination.  We found that vegetable oils can enhance germination, a highly unusual property for a fungus.  We compared the effects of oils extracted from the bee host with those that come from various plants.  Both plant and bee oils increased spore germination from 50% (without oil) to 75-85% (with oil). Percent germination was greater with oils, but it did not matter which oil was used.  Chalk brood spores attached to oils in an interesting way, lining up along the oil/water interface in the broth.  The spores swelled and germinated only on the broth-side of the spore, with the other half of the spore staying attach to a lipid droplet (looking like a root into the oil drop), where it remained, without swelling.   It is possible that chalkbrood has developed a specialty of using fats and oils as a nutrient because their host, larvae of this bee, are unusually fat for an insect, storing at least twice as much fat as most other insects.
                                Technical Abstract: The alfalfa leafcutting bee (Megachile rotundata (Fabr.)) is a solitary bee that is managed on a large scale for pollination of alfalfa seed crops.  The bees nest in holes drilled in wood or polystyrene blocks, and their larvae are highly prone to a fungal disease called chalkbrood.  The most prevalent form of chalkbrood is caused by Ascosphaera aggregata, an Ascomycete that is difficult to culture.  Hyphae will grow on standard fungal media, but spore germination is difficult to achieve and highly variable.  We found that lipids enhance germination, and compared the effects of internal lipids derived from the bee host with oils from various plants.  Plant and bee lipids increased germination from 50% (without oil) to 75-85% (with oil). Percent germination was significantly greater in the presences of lipids, but germination was not significantly different when different oils were used.  A. aggregata spores oriented along the lipid/aqueous interface in the broth in a polar fashion, with swelling and germ tube formation always occurring into the aqueous portion of the broth. The other half of the spore tended to attach to a lipid droplet, where it remained, without swelling, during germ tube formation.  A. aggregata is a host-specific, obligate pathogen.  It is possible that it has developed a specialization of utilizing lipids as a carbon source because larvae of this bee have an unusually high lipid content for an insect, storing at least twice as much lipid as most other insects.  Further research is needed to determine where the spores are encountering lipids when germinating in the host gut, where germination normally occurs.


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