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[SANET-MG] kentucky field tests of GM fungus

August 14, 2005

Prof. Joe Cummins

“Field testing genetically modified fungal endophytes of ryegrass”

The University of Kentucky has prepared Environmental Assessment for Field Tests of Genetically Engineered Neotyphodium. USDA/APHIS has prepared a docket containing the documentation on the environmental field test of the modified fungus Agency Docket Number: 05-062-1 which is available for comment Comment Period Ends on 12 September 2005 (1). Those concerned about the release of genetically modified (GM) fungi should provide comments.

Fungal endophytes are fungi that form symbiotic relationships with plants. The fungi grow in or around plant cells in a cozy relationship in which the plant feeds the fungus which takes what it is given and no more to avoid becoming a parasite on the plant. The fungus Neotyphhodium provides rye grass with alkaloids which protect the grass from animal predators. The grass bearing the endophyte have a clear advantage over grass lacking the fungus. However, alkaloids such as the ergot family adversely effect grazing mammals. The fungus reproduces asexually in the grass and does not produce sexual spores. However, fungi have active mitotic recombination and somatic gene conversion in what is called a para sexual cycle. The fungus is passed through maternal tissue to the seed and inoculation of a plant lacking the fungus is difficult.

The proposed release has two different strains modified in different gene for alkaloid formation. One of the transgenic endophytes has a gene for dimethylallytryptophan synthase (dmaW) disrupted, the other transgenic endophyte has a gene for lysergyl peptide synthetase (dmaW) disrupted. The disrupted dmaW eliminated production of the alkaloid ergovaline and its precursors including lysergic acid. Disrupted lpsA eliminated ergovaline but not lysergic acid. In that strain lysergic acid increases markedly about twenty five times. Both transgenic strains were disrupted by introducing the hygomycin B phosphotransferase (hph) gene from a bacterium into the dmaW and lpsA genes. The hph confers antibiotic resistance, the gene is driven by a promoter gene from the fungus Neurospora and a transcription terminator from the fungus Aspergillus. The hph genes are targeted specifically to disrupt the dmaW and lpsA genes by the insertion of short DNA sequences from the target gene at both ends of the hph gene insertion(2). Details of the gene disruptions used to modify Neotyphodium endophyte are provided in peer reviewed publications (3,4).

The inserted antibiotic resistance gene was assumed, in the proposal, to have no impact on the growth of the endophyte in the absence antibiotic treatment, but evidence confirming that assumption was not provided. Furthermore , gene disruption leads to “pop out” in some fungi , intra-chromosomal homologous recombination splices out the inserted gene mobilizing the excised insert as a circular DNA unit and restoring the disrupted gene to full activity. This consideration seems not to have been contemplated in the proposal.

The proposal believes that the fungal endophyte and its antibiotic resistance gene will be stable and not be transmitted horizontally. There was little or no discussion of the potential impact of the protein produced by the antibiotic resistance gene, hygomycin B phosphotransferase, other than to note the United States Environmental Protection agency had granted it an exemption from tolerance. The toxicity and allergenicity of the protein does not seem to have been considered in the proposal. The proposal argues that there will be little or no horizontal transfer of the resistance marker but did not provide evidence about the breakdown of the grass bearing the transgenic endophyte. Certainly, the endophyte will break down releasing antibiotic resistance gene to the soil environment where it may transform soil bacteria or during digestion of the grass in the pasture animals. It seem unwise to ignore logical possibilities or to deny that they exist.

GM endophytes have previously been tested, a bacterial endophyte of yellow lupine was modified with genes for degrading an organic pollutant along with genes for antibiotic and nickel resistance. The bacterial enodphyte turned out to be a pathogen for humans! As in the proposal above little concern was given to the spread of antibiotic genes in the environment (5).

The proposal to field test GM endophyte modified pasture grass should have been preceded by a mouse or rat feeding experiment with the modified grass. The animals should be examined by a full necropsy to study the impact of the grass. The fate of the introduced antibiotic resistance gene should be studied in decaying and fed modified grass. Those experiments should be undertaken before the grass is released in an open field test.


1. University of Kentucky; Availability of Environmental Assessment for Field Tests of Genetically Engineered Neotyphodium Docket ID: APHIS-2005-0067 Comment Period End Date September 12,2005 http://docket.epa.gov/edkfed/do/EDKStaffCollectionDetailView?objectId=0b0007d4809475d5

2. USDA/APHIS Environmental Assessment in response to permit application (05-152-01r) received from the University of Kentucky for field testing of two genetically engineered fungal endophyte Neotyphodium sp. isolate Lp1 strains introduced in perennial ryegrass (Lolium perenne). 2005 http://docket.epa.gov/edkfed/do/EDKStaffAttachDownloadPDF?objectId=090007d480947684

3. Panaccione DG, Johnson RD, Wang J, Young CA, Damrongkool P, Scott and Schardl CL. Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte. Proc Natl Acad Sci U S A. 2001 Oct 23;98(22):12820-5

4. Wang J, Machado C, Panaccione DG, Tsai HFand Schardl CL. The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass. Fungal Genet Biol. 2004 Feb;41(2):189-98

5. Cummins,J. and Ho,MW Bio-remediation without caution ISIS press Release 2004 http://www.i-sis.org.uk/

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