TY - THES
T1 - Identification of biochemical and molecular mechanisms of resistance to glufosinate and glyphosate in Eleusine indica
AU - Jalaludin, Adam
PY - 2015
Y1 - 2015
N2 - Herbicides are important tools in agriculture. They allow for a simple and
effective method of weed control for growers in order to meet the global food
demand. Unfortunately, intensive herbicide usage with diminishing diversity of
weed control methods has resulted in weeds evolving resistance to herbicides.
Herbicide resistance is now a major issue and challenge for growers globally. One
of the problematic weed species, especially in the tropical and warm climate
regions is Eleusine indica. Eleusine indica is a pernicious weed that is prone to
evolve resistance to herbicides. Currently, global incidences of evolved resistance
in E. indica include eight different herbicide sites of action, including glufosinate.
Working with a glufosinate-resistant E. indica population from Malaysia,
the resistance profile was further characterised and assessed for multiple resistance.
Glufosinate resistance was confirmed in the E. indica population, with the GR50
(rate required to reduce the growth by 50%) and LD50 (rate required to kill 50% of
the population) R/S ratios being 5- and 14-fold, respectively. More importantly,
multiple resistance was observed, with the selected glufosinate-resistant subpopulation
(R*) exhibiting a very high level of glyphosate resistance. The GR50 and
LD50 R/S ratios obtained were 12- and 144-fold, respectively for glyphosate. This
population had also evolved resistance to paraquat, albeit at a low level (GR50 and
LD50 R/S ratios 2 to 3-fold, respectively). This species is the first to be reported to
have evolved resistance to all three non-selective herbicides. Additionally,
resistance to several ACCase-inhibiting herbicides, namely fluazifop-p-butyl,
haloxyfop-p-methyl and butroxydim, was caused by a Trp-2027-Cys substitution in
the ACCase protein sequence.
In order to investigate the glufosinate resistance mechanism(s) in the R*
population, activity of glutamine synthetase (GS) (the target -site of glufosinate)
was compared in the S and R* populations. No difference in enzyme sensit ivity
towards glufosinate was observed. Specific GS activity was also similar between S and
R*. Differences in foliar uptake and translocation of [14C]-glufosinate were not
significant between the two populations. HPLC analysis of glufosinate metabolism did
not detect any metabolites in S or R* plants. Consequently, the resistance mechanism to
glufosinate is not due to an insensitive target-site, target-site over production, differential glufosinate uptake and translocation, nor enhanced glufosinate metabolism,
and remains to be determined.
Sequencing of the glyphosate target gene, EPSPS, in the highly glyphosate
resistant E. indica population revealed that a double mutation in the EPSPS gene, i.e.
Thr-102-Ile and Pro-106-Ser (TIPS), was responsible for the high level glyphosate
resistance. Importantly, this double mutation is similar to the first commercialised
transgenic, glyphosate-tolerant EPSPS in maize, but has never been reported to occur
naturally. Dose-response experiments showed that the naturally evolved TIPS mutants
are 180-fold (LD50 based) more resistant to glyphosate than the wild type (WT) E.
indica plants, and 32-fold more resistant than the Pro-106-Ser (P106S) (LD50 based)
mutants. EPSPS inhibition assays also revealed similar results, with the TIPS EPSPS
enzyme activity showing very high glyphosate resistance relative to wild type (WT)
EPSPS (2600-fold) and P106S EPSPS (600-fold). Interestingly, the highly resistant
TIPS mutant exhibited a resistance cost in terms of vegetative growth and seed
production, while no resistance cost was observed for plants with the P106S mutation.
Plants with the TIPS mutation had a higher basal shikimic acid (the substrate for
EPSPS) level and lower tryptophan (a downstream product) levels than WT and P106S
plants. The evolution of the TIPS double mutation is likely a sequential event, with the
P106S mutation being selected first, followed by the T102I mutation, creating the
highly glyphosate resistant TIPS EPSPS.
AB - Herbicides are important tools in agriculture. They allow for a simple and
effective method of weed control for growers in order to meet the global food
demand. Unfortunately, intensive herbicide usage with diminishing diversity of
weed control methods has resulted in weeds evolving resistance to herbicides.
Herbicide resistance is now a major issue and challenge for growers globally. One
of the problematic weed species, especially in the tropical and warm climate
regions is Eleusine indica. Eleusine indica is a pernicious weed that is prone to
evolve resistance to herbicides. Currently, global incidences of evolved resistance
in E. indica include eight different herbicide sites of action, including glufosinate.
Working with a glufosinate-resistant E. indica population from Malaysia,
the resistance profile was further characterised and assessed for multiple resistance.
Glufosinate resistance was confirmed in the E. indica population, with the GR50
(rate required to reduce the growth by 50%) and LD50 (rate required to kill 50% of
the population) R/S ratios being 5- and 14-fold, respectively. More importantly,
multiple resistance was observed, with the selected glufosinate-resistant subpopulation
(R*) exhibiting a very high level of glyphosate resistance. The GR50 and
LD50 R/S ratios obtained were 12- and 144-fold, respectively for glyphosate. This
population had also evolved resistance to paraquat, albeit at a low level (GR50 and
LD50 R/S ratios 2 to 3-fold, respectively). This species is the first to be reported to
have evolved resistance to all three non-selective herbicides. Additionally,
resistance to several ACCase-inhibiting herbicides, namely fluazifop-p-butyl,
haloxyfop-p-methyl and butroxydim, was caused by a Trp-2027-Cys substitution in
the ACCase protein sequence.
In order to investigate the glufosinate resistance mechanism(s) in the R*
population, activity of glutamine synthetase (GS) (the target -site of glufosinate)
was compared in the S and R* populations. No difference in enzyme sensit ivity
towards glufosinate was observed. Specific GS activity was also similar between S and
R*. Differences in foliar uptake and translocation of [14C]-glufosinate were not
significant between the two populations. HPLC analysis of glufosinate metabolism did
not detect any metabolites in S or R* plants. Consequently, the resistance mechanism to
glufosinate is not due to an insensitive target-site, target-site over production, differential glufosinate uptake and translocation, nor enhanced glufosinate metabolism,
and remains to be determined.
Sequencing of the glyphosate target gene, EPSPS, in the highly glyphosate
resistant E. indica population revealed that a double mutation in the EPSPS gene, i.e.
Thr-102-Ile and Pro-106-Ser (TIPS), was responsible for the high level glyphosate
resistance. Importantly, this double mutation is similar to the first commercialised
transgenic, glyphosate-tolerant EPSPS in maize, but has never been reported to occur
naturally. Dose-response experiments showed that the naturally evolved TIPS mutants
are 180-fold (LD50 based) more resistant to glyphosate than the wild type (WT) E.
indica plants, and 32-fold more resistant than the Pro-106-Ser (P106S) (LD50 based)
mutants. EPSPS inhibition assays also revealed similar results, with the TIPS EPSPS
enzyme activity showing very high glyphosate resistance relative to wild type (WT)
EPSPS (2600-fold) and P106S EPSPS (600-fold). Interestingly, the highly resistant
TIPS mutant exhibited a resistance cost in terms of vegetative growth and seed
production, while no resistance cost was observed for plants with the P106S mutation.
Plants with the TIPS mutation had a higher basal shikimic acid (the substrate for
EPSPS) level and lower tryptophan (a downstream product) levels than WT and P106S
plants. The evolution of the TIPS double mutation is likely a sequential event, with the
P106S mutation being selected first, followed by the T102I mutation, creating the
highly glyphosate resistant TIPS EPSPS.
KW - Glufosinate
KW - Glyphosate
KW - Multiple resistance
KW - TIPS
KW - EPSPS
M3 - Doctoral Thesis
ER -