Subject: [SANET-MG] kentucky field tests of GM fungus

From: jcummins <[email-masked]>
Date: Mon, 15 Aug 2005 12:10:05 -0400

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.

References

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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