FUNGI ISOLATED FROM COPTOTERMES FORMOSANUS SHIRAKI CONTROL THE BROWN ROT FUNGUS, GLOEOPHYLLUM TRABEUM Poornima Jayasimha and Gregg Henderson Department of Entomology, LSU AgCenter, Baton Rouge, LA Wood decayed by brown rot fungus, Gloeophyllum trabeum, has been shown to be attractive and more nutritious to Formosan subterranean termites, Coptotermes formosanus Shiraki. To evaluate this association, Formosan termite workers were released into Petri dishes containing wood chips inoculated with G. trabeum. Controls received all components as above but without termites. Unexpectedly we observed significant suppression in the growth of fungus in treatments with termites. Wood chips from the termite treatments were placed on potato dextrose yeast agar (PDYA) medium to determine G. trabeum survivorship. G. trabeum did not grow but, green spored fungi were predominant in all the cultures. We hypothesized that these fungi may be carried by termites and might play a role in suppressing the growth of G. trabeum. Dual culture tests of fungi isolated from the external surface of Formosan termites showed that several isolates were antagonists and effectively controlled the growth of G. trabeum. The isolated parasitic fungi were identified as Aspergillus flavus, Trichoderma harzianum, T. virens, T. asperillum and T. ghanense. Our study is the first to show that fungi present on the external body surface of Formosan termites control the growth of a brown rot fungus. Literature is replete with evidence showing a mutual beneficial relationship between termites and fungi. Wood decayed by certain brown rot fungi increase its nutritional value to termites (1, 5). In turn, some termites may carry and spread the fungal spores. This was demonstrated by the isolation of fungi from the exterior and from the gut of termites (2), and by the observations of hyphae, conidiophores, and conidia clinging to the bodies and appendages of termites (3). The objectives of our experiment were to see if Formosan termites had any effect on growth of G. trabeum Madison 617 and to test whether fungi present on Formosan termites had an effect on growth of G. trabeum. Fig 2: G. trabeum is suppressed when termites are present © 2005 Michael A. Seymour Fig 1: G. trabeum with dense mycelial growth in controls © 2005 Michael A. Seymour Seven termite workers from each colony were allowed to walk in Petri dishes containing PDYA medium with streptomycin (25 mg/l) for 1 minute. Termites were then removed. Petri dishes were sealed with parafilm ® and were incubated at 25 0 C for 4 to 7 days. Controls were handled in the same way only termites were not added to the Petri dishes. Any fungi growing were visually differentiated based on their color and colony growth characteristics. These fungi were then subcultured 2 or 3 times to obtain pure cultures. Subcultures were made thereafter every week to maintain the pure cultures. The experiment was repeated 6 times with 3 different lab maintained colonies and 3 or 5 times with 6 different field collected colonies. Selected fungi were sent for identification to Dr. Steven Carpenter, Abbey Lane Laboratory LLC, Philomath, Oregon. Dual culture techniques to test the effects of selected fungi against G. trabeum: To determine if selected fungi inhibited G. trabeum growth, a disc of G. trabeum was placed at one end of the Petri dish containing PDYA medium. After two days a disc of the selected fungi isolated from the termites was placed on the other end. Controls contained only G. trabeum. This procedure was replicated 5 times with each of the selected fungi. The total growth of the G. trabeum and the isolated fungi were measured. Abstract Introduction Materials & Methods Growth of G. trabeum on wood chips in presence and absence of termites: An autoclaved wet wood chip was placed in each of 14 Petri dishes (replicated on 4 colonies) and a loop full of G. trabeum was inoculated on to the wood chip. Fifty worker termites were added to each of the 7 Petri dishes. No termites were added to controls. Petri dishes were then sealed with parafilm ® and were incubated at 25 0 C. Length and breadth of the fungal growth were measured to obtain total growth of the fungus. Wood chips from the treatments were then placed on PDYA medium to determine the survival of the inoculated fungus. Fig 4: Growth of G. trabeum in controls © 2005 Michael A. Seymour Fig 5: Aspergillus flavus against G. trabeum © 2005 Michael A. Seymour Fig 6: Trichoderma virens against G. trabeum © 2005 Michael A. Seymour Fig 7: Trichoderma harzianum against G. trabeum © 2005 Michael A. Seymour Fig 8: Trichoderma ghanense against G. trabeum Fig 9: Trichoderma asperillum against G. trabeum Results & Discussion Growth of G. trabeum on wood chips in presence and absence of termites: Growth of G. trabeum was significantly (P<0.0001) suppressed in treatments when compared to controls (Fig 1-3). Suppressed G. trabeum did not grow when placed on PDYA, but several potentially parasitic fungi were observed. Fig 3: Comparison of mean growth of G. trabeum with and without termites Fig 10: Growth of G. trabeum compared with growth of parasitic fungi Conclusion Though G. trabeum makes wood attractive and more nutritious, Formosan termites suppress the growth of the fungus. Our study shows that Aspergillus flavus, Trichoderma harzianum, T. virens, T. asperillum, T. ghanense are present on the external surface of Formosan termites and suppress the growth of G. trabeum. Although these results were unexpected the competitive exclusion principle suggests that two species cannot coexist when they have identical needs of a limited resource. References 1) Becker, G., Termites and Fungi. Material und Organismen 3: ) Hendee, E. C., The association of the termites, Kalotermes minor, Reticulitermes hesperus, and Zootermopsis angusticollis with fungi. University of California publications in Zoology 39, No. 5: ) Hendee, E. C., The association of termites and fungi: In: Termites and termite control (Kofoid, C.A Ed). University of California press, Berkeley. 4)Mankowski, M. E., Schowalter, T. D., Morrell, J. J., and Lyons, B., Feeding habits and gut fauna of Zootermopsis angusticollis (Isoptera: Termopsidae) in response to wood species and fungal associates. Community and Ecosystem Ecology 27 (6): ) Ruyooka, D. B. A., (1979). Associations of Nasutitermes exitiosus (Hill) (Termitidae) and wood rotting fungi in Eucalyptus regnans F. Muell, and Eucalyptus grandis W. Hill ex Maiden: choice feeding, laboratory study. Zeit. Angew. Entomol. 87: Acknowledgements We would like to thank Karen Nix and Dr. Huxin Fei for collecting termites. We would also like to thank Suresh Babu Kadaru for helping us with SAS programs. A special thanks to Dr. Steven Carpenter for identifying the fungi. Regardless of the colony origin (field or laboratory) most of the isolated fungi tested in dual cultures did not significantly differ in their effect on G. trabeum (P>0.05). A. flavus isolated from one colony significantly varied in its effect on G. trabeum from A. flavus isolates from 3 other colonies (P<0.0084). Interestingly, some of these fungi, in addition to helping termites by suppressing a competing cellulose consumer, may directly benefit termite fitness. Trichoderma viride inoculated wood increased the number of gut protozoa in the Pacific dampwood termite, Zootermopsis angusticollis (4). Whether Trichoderma spp. directly benefits C. formosanus needs to be investigated. Identification of the isolated fungi: Selected fungi were identified by Dr. Steven Carpenter as Aspergillus flavus, Trichoderma harzianum, T. virens, T. asperillum and T. ghanense Front view Flipped back Isolation and identification of fungi from termites: Worker termites were collected from 9 different colonies. Dual culture techniques to test the effects of selected fungi against G. trabeum: All the isolated fungi suppressed G. trabeum in dual cultures (Fig 4-9). There was a significant difference in the growth of G. trabeum in dual cultures compared to controls (P<0.0001) (Fig 10).