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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
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
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
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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