.'1 . o3 lnvestigations into Glufosinate Efficacy Against
Raphanus raphanistrum and Lolium rigidum
Submitted by
Anuja Ruwanthi Kumaratilake
B. Ag.Sci. (Hons), The University of Adelaide, Australia
A thesis submitted for the degree of Doctor of Philosophy
ln the Department of Applied and Molecular Ecology
Faculty of Sciences
The University of Adelaide, Australia.
2002 Declaration
This work contains no material which has been accepted for the award of any other degree or diploma in any university or other lertiary institution and to the best of my knowledge and belief, contain no material previously published or written by other person, except where due reference
had been made in the text.
I give my consent that this work may be photocopied or loaned from the library
Anuja Ruwanthi Kumaratilake Acknowledgements
I am greatly indebted to the Bayer CropScience and CRC for Weed Management Systems for offering me a postgraduate scholarship and for continuous financial support which enabled to carry out this project successf ully. I also wish to express my appreciation of the excellent facilities and the happy environment provided by the Department of Applied and Molecular Ecology at the
University of Adelaide.
I owe a special debt of gratitude to Dr Christopher Preston, who as my supervisor was always
available with help and guidance and for continuous interest, encouragement and kindness
through out all phases of this work. I also like to express my sincere thanks to Dr Deborah Colwill
for all the friendly advice and encouragement through out this investrgation. My sincere thanks go
to Mr. Michael Clarke, Bayer CropScience. I always knew that I could count on Mike's help. His
many visits to our laboratory, his interest and enthusiasm continuously motivated me. I would also
like to thank Dr Rob Reid (Depaftment of Plant Science) for his help and guidance in the cell
uptake experiments.
Many thanks to the Council of Australian Weed Science Societies for offering me the 'Young
Scientist Travel Award' (2000) which allowed me to visit and learn from several institutes in USA
and Germany. I also extend my sincere thanks to the CRC for Weed Management Systems for
offering me a Travel Award (2OOO) as well as helping me to attend several national and
international conferences and workshops. This made a significant contribution to my education
and increased confidence. I highly value these opportunities.
My appreciation also extends to Waite Weedies (Rick, Angela, lmam, Dawn, Don, Natalie, Chris,
Mary, Kathy, Janine, Jan, Kelly and Sue) for providing an academically stimulated environment
also rich with fun and laughter. Thanks to Alison for being my good friend and for allthe help and
moral support! Gitta, Terry, Garry, Heather, Sue -thanks for help and support many a times.
I would like to express my sincere thanks to my husband and to my parents to whom this thesis is
dedicated for their love, palience and support through out this study. Abstract
This study addressed an industrial problem related to poor control of major weed species under southern Australian winter conditions by the non selective post emergence herbicide, glufosinate'
The pending introduction of genetically modified canola cultivars (Liberty Link@/ lnVigor@) with glufosinate resistance makes it important to determine the best way to use this herbicide. The study focused on the eff icacy of glufosinate against the Brassicaceae weed
Raphanus raphanistrum (wild radish) and Lotium rigidum (annual ryegrass), which are two important weed species in southern Australian winter cropping regions. ln addition, another common grass species Avena steritis (wild oats) was also included in severalcomparative studies.
The investigations were carried out under artificially simulated winter conditions inside growth chambers as well as under outdoor winter conditions in South Australia.
Several dose response experimenls were performed to assess the efficacy of glufosinate against
L. rigidum and A. steritis under simulated southern Australian winter conditions. The results demonstrated a variable glufosinate efficacy between the two species. Whilst A. sferil¡s was controlled completely (1OO% mortality) with 300 g ai ha-l glufosinate, L. rigidum required 1200 g ai ha-1 to achieve the same mortality. To determine whether the target site sensitivity influenced the above observations, glutamine synthetase activity assays were performed. The results showed no difference between the two weed species in their response to glufosinate. Spray retention experiments were carried out to determine whether the plant size and morphology play a role in the efficacy of glufosinate. The results indicated that glufosinate efficacy was not inf luenced by the
plant size or morphological features, The metabolic studies on glufosinate based on thin Layer
chromatography (TCL) showed that in both plant species only a small proportion of glufosinate
was metabolized. ln addition, there was no difference between the two species in relation to
1aC laO-glufosinate absorption of glufosinate. ln contrast, the basipetaltranslocation of at the shoot
meristematic region was more than twice as high in A. sterilis when compared to L. rigidum under
the same environmental conditions. Glufosinate efficacy was tested under southern Australian winter conditions against several
populations of R. raphanistrum collected f rom different geographical areas. The results showed that, despite the geographical origin, all populations were poorly controlled by glufosinate at the
field rate of 600 g ai ha-1. Similar results were found when a population collected from South
Australia was tested under artificially simulated winter conditions inside a growth chamber.
When dose response experiments were conducted with glufosinate under differential
temperatures (inside growth chambers) herbicide efficacy was enhanced when R. raphanistrum
was grown under warm temperatures. Dose response experiments where plants were grown
under cold conditions and subsequently transferred to warm temperatures showed enhanced
glufosinate efficacy compared to plants that were kept under cold conditions through out the
experiment, suggesting that the temperature following the glufosinate application was crucial to 14C-glufosinate obtaining better herbicide efficacy. Absorption and translocation studies with
showed that under warmer temperatures relatively more glufosinate was translocated to the toC- meristematic regions. Under warm conditions (20l25'C) there was a 5 fold enhancement of
glufosinate translocated to the meristematic regions and to other leaves than the plants kept under
cold conditions continuously.
Experiments conducted with the aim of enhancing glufosinate performance in R. raphanistrum
with the use of co-herbicides, chelating agents, fertilizer salts and hormones. The results showed
that the carotenoid biosynthetic inhibitors did not show a significant enhancement of efficacy.
Amitrole and clomazone were antagonistic in combination with glufosinate on R. raphanistrum.
Diflufenican and benzofenap showed a minor synergistic effect and an additive effect respectively.
Experimental formulations of glufosinate containing different surfactants did not improve efficacy
either. Similarly, when ammonium sulphate was used as a mixture or separately, the efficacy did
not increase. However, when EDTA or kinetin were applied as foliar sprays, 24 hours prior to
glufosinate treatment, it was possible to improve glufosinate efficacy against R. raphanistrum.
Several experiments were conducted to investigate the elfect of pH of the herbicide solution on
cell uptake with R. raphanistrum and Chara australis. These studies showed that the uptake of
IV glufosinate was enhanced by reducing the buffer pH. However, further invesligations on
C. australis showed that only the uptake into the cell wall (apoplast) was increased. ln contrast, the uptake through the plasma lemma remained almost unchanged under low pH suggesting that
by reducing the pH of the herbicide solution, phloem translocation could not be enhanced.
ln summary, the present study showed that the poor efficacy of glufosinate in L. rigidum and
R. raphanistrum was mainly due to poor translocation of the herbicide. ln particular, the lack of
translocation to the meristematic region caused such poor efficacy in the above species. Similarly
the studies wilh Cara australis showed that the uptake through plasma lemma was a major limiting
factor for poor translocation of the herbicide.
It can be recommended through this study that to achieve better control of R. raphanistrum il is
necessary to apply glufosinate under warmer temperature conditions. Table of Contents
Acknowledgements... il
Abstract
Table of Contents......
List of Fi9ures......
List of Tab1es...... , .. xilt
Chapter One: Literature Review ....
1.1 . lntroduction ...... 1.2. Glufosinate and its Mode of Action 1.2.1. Glufosinate 1.2.2. Mode of Action of Glufosinate ......
1 .2.3. Glutamine Synthetase ...... 1.2.4. lnhibition of Photorespiration by Glufosinate 1.3. Herbicide Resistant Crops 1.3.1. Glufosinate Resistant Crops 1.4. lmportant Weeds in Southern Australian Cropping Regions 1.4.1. Ryegrass - Lolium rigidum...... 1 .4.2.Wtd radish -Raphanus raphanistrum
1 .4.2.1. Signif icanc e of Raphanus raphanistrum lo the Canola 1.5. Mechanisms of Herbicide Resistance/ Tolerance 1.6. Absorption and Translocation of Herbicides...... 1.6.1. Foliar Absorption 1.6.2. Absorption Across Membranes.....
1 .6.3. Herbicide Translocation......
1 .6.3. 1. Phloem Translocation of Herbicides ...... 1.6.3. 1.1. Source Sink Relationship 1.6.4. Absorption and Translocation of Glufosinate ...... 1.6.5. Environmental Effects on Absorption and Translocation of Herbicides ....
Chapter Two: Glufosinate Efficacy ln Lolium rigÍdum and Avena sterilis 22
2.1. lntroduction 22
VI 2.2. Malerials and Methods 24 2.2.1. Dose Response Studies. 24 2.2.2. Glulamine Synthetase Assays 25 2.2.3. Spray Retention. 25
2.2.4. Melabolism of G luf osinate. 26 2.2.5. Absorption and Translocation Studies... 27 2.3. Besults 27 2.3.1. Dose Response Studies. 27 2.3.2. Glutamine Synthetase Assays 30 2.3.3. Spray Retention. 31 2.3.4. Metabolism. 31 2.3.5. Absorption and Translocation Studies... 31 2.4. Discussion ...... 34
Chapter Three: Environmental Effects on Gtufosinate Efficacy in Raphanus raphanistrum39
3.1 . lntroduction ...... 39 3.2. Materials and Methods 40 3.2.1. Dose Response Studies: Population Differences...... '.'."'..'.40 3.2.2. Dose Response Studies: Temperature Effect...... -'...'....."41 3.2.2.1. The Effect of Temperature Before and After Treatment on Glufosinate Efficacy4l 3.2.3. Dose Response Studies: Light lntensity Effects.... 42 3.2.4. Glutamine Synthetase Assays ...... -42 3.2.5. Absorption and Translocation: Temperature...... 43 3.3. Results .'.'.."'.."43 3.3.1. Population Differences ...... 43 3.3.2. Dose Response Studies:Temperature Effect...... ""'...".46 3.2.2.1. The Effect of Temperature Before and After Treatment on Glufosinate Efficacy4S 3.3.3. Dose Response Studies: Effect of Light lntensity ...... 52 3.3.4 Glutamine Synthetase Assays...... 54 3.3.5. Absorption and Translocation of Glufosinate 55 3.4. Discussion ...... 59
Chapter Four: lmproving Efficacy of Glufosinate Againsl Raphanus raphanistrum with co- Herbicides, Surfactants, EDTA, Ammonium Sulfate and Kinetin...... 63
4.1 . 1ntroduction ...... 63 4.1.1. Carotenoid Biosynthesis lnhibitors .... 63 4.1.2. Sudactants 65
vil 4.1.3. Chelating Agents 66 4.1.4. Ammonium Sulfate...... 66
4.1 .5. Kinetin,...... 67 4.2. Materials and Methods 68 4.2.1. Herbicide lnteractions: Carotenoid Biosynthetic lnhibitors .... 68 4.2.2. SurÍactant Mixes...... 6B 4.2.3. Elfecl of Ammonium Sulfate...... 6B 4.2.4. Eftecl of EDTA..... 69 1aO-glufosinate 4.2.5. Absorption and Translocation of in EDTA Treated Plants 69 4.2.6. Effecl of Kinetin 69 1aO-glufosinate 4.2.7. Absorption and Translocation of in Kinetin Treated Plants 70 4.2.8. Statistical 4na|ysis...... 70 4.3. Results 70 4.3.1. Herbicide lnteractions: Carotenoid Biosynthetic lnhibitors 70 4.3.1 .1. Amitrole plus Gluf osinate ...... 70
4.3.1 .2. Dif luf enican plus Glufosinate ...... ,.... 73
4.3. 1 .3. Clomazone plus Glufosinate ...... 75 4.3.1 .4. Benzofenap plus Glufosinate ...... 77 4.3.2. Formulations of Glufosinate with Different Surfactants 79 4.3.3. Effect of Ammonium Sulfate. 79 4.3.4. Effect of EDTA..... 82 4.3.5. Effect of Kinetin...... 88
4.4. Discussion .. 92
Chapter Five: pH lnfluence on Glufosinate Absorption in Raphanus raphanistrum and Chara australis... 96
5.1 . lntroduction ..... 5.2. Materials and Method.... 5.2.1. Dose Response Studies...... 5.2.2. Dose Response Pot Experiments ...... 5.2.3. Absorption and Translocation Studies: Effect of pH...... 5.2.4. Uptake into Leaf S|ices...... 5.2.5. Cellular Uptake of Glufosinate...... 5.3. Results 5.3.1 . Dose Response Pot Experiments ...... 5.3.2. Absorption and Translocation : Plants were Treated with Low pH Glufosinate. 5.3.3. Uptake of Glufosinate into Leaf Slices 5.3.4. Uptake of Glufosinate into Ce1|s...... 5.4. Discussion ......
vilt Chapter six: General Discussion...... 117
References..,, 125
X List of Figures
Figure2.1:Survival (A)anddryweights(B) of L. rigidumandA. sterilisinresponsetotreatments
with varying dose rates of glufosinate under controlled growth room conditions...... 29
Figure 2.2: Glutamine synthetase activity in L. rígidum (r) and A. sterilis (r)grown undercontrolled
growth conditions. Data represents the mean+ SE of three replicate experiments...... 30
Figure 2.3: Absorption of radiolabeled glufosinate into leaves of L. rigidum and A. sferilrs under
controlled growth conditions...... '....32
Figure 2.4: Glufosinate translocated from the point of application to lhe upper leaf ol L. rigidum
and,4. steritis under controlled growth conditions...... 33
Figure 2.5: Glufosinate retained in the treated leaf of L. rigidum (r) and A. sterilis (o) under
controlled growth conditions. Values are means t S.E. of 15 replicates...... '33
Figure 2.6: Glufosinate translocated from the point of application to meristematic regions and to
rest of the leaves of L. rigidum and A. sterilis under controlled growth conditions...... 34
Figure 3.1: Survival (A) and dry weights (B) of several populations of F. raphanistrum colleted from
diff erent geographical regions. ... 45
Figure 3.2: Survival (A) and dry weights (B) of H. raphanistrum in response to varying dose rates
of glufosinate under different temperature conditions...... 47
Figure 3.3: Survival (A) and dry matter weights (B) of Æ. raphanistrum grown under cold conditions
of 5/10"C and were transferred to differential growth temperatures ...... 49
Figure 3.4: Survival (A) and dryweights (B) of F. raphanistrum subjected to varying dose rates of
glufosinate when grown under warm temperatures of 20/25"C and were transferred to
differential growth temperatures ....'.'.'...... 51
Figure 3.5: Survival of F. raphanistrum to varying dose rates of glufosinate under differential light
intensities at 5/10'C (A) and 2Ol25"C (B)...... 53
Figure 3.6: Glutamine synthetase activity oÍ R. raphanistrum plants grown under 5/10'C and
20125"C...... 55
Figure 3.7: Absorption of labelled glufosinate into r9. raphanistrum plants grown under 5/10"C and
20125C. 56
X Figure 3.8: Glufosinate distribution within R. raphanistrum plants at 5/10"C or 20125C,...... 58
Figure 4.1:Survival (A) and dryweights (B) of Æ. raphanistrum treated with varying dose rates of
glufosinate and amitrole -'.'.".".72
Figure 4.2: Survival (A) and dryweights (B) of F. raphanistrum treated with varying dose rates of
glufosinate and diflufenican...... '..'.....'74
Figure 4.3. Survival (A) and dryweights (B) of F. raphanistrum treated with varying dose rates of
glufosinate. '.'..'.....76
Figure 4.4: Survival (A) and dryweights (B) of F. raphanistrum treated with varying dose rates of
glufosinate and benzofenap...... "'...'.'78
Figure 4.5: Survival (A) and dry weights (B) of B. raphanistrum treated with different glufosinate
formulations with different surfactants ""....80
Figure 4.6: A Survival (A) and dry weights (B) of F. raphanistrum grown under winter conditions
outside at the Waite Campus treated with varying dose rates of ammonium sulfate and
glufosinate. ".'..'...'...'.....'.81
Figure 4.7: Survival (A) and dry weights (B) of R. raphanistrum grown under winter conditions
outside at the Waite Campus treated with EDTA and glufosinate...... 83
Figure 4.8: Survival (A) and dry weights (B) of R. raphanistrum treated with EDTA and glufosinate
under controlled growth conditions of 5/10'C...... 84 1oC-glufosinate Figure 4.9: Absorption of into leaves ol R. raphanistrum following treatment with
glufosinate or EDTA 24h prior to glufosinate...... 85 laO-glufosinate Figure 4.10: Time course of translocation in EDTA treated and untreated (.) F.
raphanistrum plants exposed to cold conditions of 5/10'C...... 87
Figure 4.11: Survival (A) and dry weights (B) of R. raphanistrum grown under winter conditions
outside at the Waite Campus treated with kinetin and glufosinate...... 89 1oC-glufosinate Figure 4.12: Absorption of into leaves of R. raphanistrum following treatment with
glufosinate or kinetin 24h prior to glufosinate...... 90 1oO-glufosinate Figure 4.13: Time course of translocation in kinetin treated and untreated (.) B.
raphanistrum plants exposed to cold conditions of 5/10'C. 91
Figure 5.1: Survival (A) and dry weights (B) of F. raphanistrum treated with glufosinate solutions
adjusted with phosphoric acid to pH 2.5 , pH 3.5, pH 5 or pH 7...... ,....103
X Figure 5.2: Survival (A) and dry weights (B) of R. raphanistrum treated with glufosinate solutions
adjusted with phosphoric acid to pH 2.5 , pH 3.5 , pH 5 or pH 7 under controlled growlh
conditions of 5/10"C ..104
Figure 5.3: Survival (A) and dry weights (B) of R. raphanistrum treated with the commercial
formulation of glufosinate, or glufosinate adjusted to pH 2.5 with hydrochloric acid, or
phosphoric acid...... 106 laC-glufosinate Figure 5.4: Absorption of in R. raphanistrum lrealed with the commercial
formulation of glufosinate or glufosinate adjusted to pH 2.5 with HCl...... 107 1oC-glufosinate Figure: 5.5: Time course of translocated in plants treated with the commercial
formulation of glufosinate or glufosinate adjusted with HCI to pH 2.5 in R. raphanistrum
grown at 5/'10"C...... 108
Figure 5.6: Concentration dependent uptake of glufosinate into leaf slices ol R. raphanistrum in
Hepes buffer-pH 7.5 and in acetate buffer-pH 5, ...... 109 laO-glufosinate Figure 5.74: Time dependent rate of uptake of into cells of Chara australis...... 1 1 1 1aO-glufosinate Figure 5.78: Time dependant rate of uptake of into cell walls and cytoplasmic
contents ol Chara australís...... 1 11 1aC-glufosinate Figure 5.8: Concentration dependent uptake of into cell walls and cell contents of
Chara australis...... 112
Figure 5.94: Total rate of uptake of glufosinate into cells of Chara australis at different pH...... 112 1aC-glufosinate Figure 5.98: Rate of uptake of into cell walls and cytoplasmic contents ol Chara
australis at different pH...... 1 13
xil List of Tables
Table 2.1: LDuo values for glufosinate (0 to 1200 g ai ha-1) in L. rigidum and A. sterilis, as
determined through three dose response experiments conducted under controlled growth
conditions and for experiments conducted outdoors in winter...... 28
Table 3.1: LD5q values for glufosinate of R. raphanistrum, as determined through three dose
response experiments conducted under controlled growth conditions. Plants were originally
grown under cold temperatures of 5i10'C...... 50
Table 3.2: LD5e values for glufosinate of F. raphanistrum, as determined through dose response
experiments conducted under controlled growth conditions. Plants were originally grown
under warm temperatures ol20l25C ...... 52
Table 3.3: LD5e values for glufosinate for F. raphanistrum, as determined through dose response
experiments conducted in controlled growth chambers under different light intensities...... 54
Table 5.1: LD5e values for glufosinate for R. raphanistrum. Herbicide solutions were adjusted with
phosphoric acid to pH 2.5,3.5 5 or 7. Plants were grown and treated outdoors in winter or
under controlled growth conditions of 5/10'C...... 105
xilt Chopter 1: Literolure review
Chapter One: Literature Review
1,1 . lntroduction
Glufosinate is a non-selective foliar-active post emergence herbicide used successfully for weed controlsince mid 1980s over 50 different countries including Australia (Rasche 1995). lt has been used as a non-specific herbicide in minimum tillage systems, orchards, vineyards, chemicalfallow and as a pre-harvest desiccant in cropping systems (Mersey et al. 1990). However, it was only after the introduction of glufosinate resistant varieties (Liberty Link@/ lnVigor@) that glufosinate is available as an effective post emergence herbicide in broad acre crops. lt is been used successfully in conjunction with resistant crops such as canola, wheat, maize and soybean that have been genetically modified to be resistant to glufosinate in Canada and USA (Rasche 1997).
Currently it is in the process of been introduced to Australian cropping systems as the sole herbicide to control weeds in particular, Brassicaceae weeds in lnVigor@ canola crops.
Despite it's non-selective nature, not all weeds are controlled equally well by glufosinate. Up to 70 fold differences in sensitivity to glufosinate has been observed in seven annual weed species
(Ridley and McNally 1 985). lt has been reported that differential sensitivity observed was a result of differential uptake and translocation of glufosinate, and that metabolic degradation did not play a role (Mersey et al. 1990, Steckel et al. 1997, Pline et al. 1999). According to Carlson and
Burnside (1984) efficacy of glufosinate depended on various environmental conditions and other factors such as weed species and application rates. Anderson et al. (1993) demonstrated that both temperature and relative humidity have a considerable effect on the activity of glufosinate in barley and in green foxtail. lt has been shown that glufosinate is not translocated extensively from the site of application (Hass and Muller 1987, Bromilow et al. 1993, Steckel et al. 1997). To date the reasons for this limited translocation are unknown (Beriault et al. 1999), According to Kleier
(1988) the physicochemical properties of glufosinate (pK" <2,2.9 and 9.8, log Ko* estimated at -
1 Chopler 1: Literoture review
3.9) have the requisite characteristics for phloem mob¡lity. Therefore, it has been assumed that the rapid action of glufosinate at the site of application causes localized phytotoxicity in leaves limiting translocation (Beriault et al. 1999). Even though several scientists have commented on poor absorption and translocation of glufosinate, to date, no detailed study has been conducted to determine the actual mechanisms involved in these processes in major weeds such as Lolium rigidum and Raphanus raphanistrum. Similarly, no evidence is available on the influence of environmental factors on absorption and translocation of glufosinate under Australian conditions,
Field studies conducted in Canada and in Europe showed successfulweed controlwith glufosinate
(Rasche 1995). However, when field tested under Australian conditions, glufosinate had variable performance against several major weed species (Michael Clark, pers. comm.). The reason for this is unknown. Before the impending release of lnVigor@ canola in Australia it is important to investigate the efficacy of glufosinate against the major weed species. Studies on absorption and translocation are necessary to understand the physiological reasons for poor control of weeds under Australian conditions.
1.2. Glufosinate and its Mode of Action
1.2.1. Glufosinate
Glufosinate is the synthetic version of phosphinothricine (PPT), a breakdown product of bialaphos
(phosphinothricinyl-L-alanine-L-alanine) produced by the bacterial species Streptomyces viridochromogenes and S. hygroscopiscus (Hoagland 1999, Lydon and Duke 1999). lt is available as a broad-spectrum foliar spray under the trade name of Basta@. Research on toxicity of glufosinate in mammals, show that under the conditions of recommended use, any detrimental effect on the health of both users and consumers is extremely unlikely (Hack et al. 1993).
According to Hack et al. (1993) glufosinate dose not interfere with various neurotransmitter receptors in vitro or does not lead to a breakdown of ammonium metabolism in mammalian tissues. Glufosinate has been used in Australia for several years in horticultural and industrial weed control quite successfully. Like glyphosate, glufosinate is also a non-selective herbicide with no crop or weed species demonstrating high levels of tolerance. Due to it's non-selective activity,
2 Chopter 1: Lileroture review
glufosinate has not been used in broad acre crops. However, with the introduction of glufosinate resistant (lnVigor@) crops, glufosinate will be used as a novel herbicide in broad acre crops for weed control.
A major advantage of glufosinate is that there is no significant metabolism in plants that are not
genetically transformed for glufosinate resistance (Mersey et al. 1990). ln studies using thin layer
chromatography detection systems, Mersey et al. (1990) found that more than 85% of radioactivity 1aC-glufosinate g6 extracted f rom plants treated with for 24, 48,72 and hours co-chromatographed
withl4C labelled glufosinate standards. Hass and Muller (1987) reported that metabolism of
glufosinate in shoots proceeded extremely slowly such that after 3 days of exposure to glufosinate
84% glufosinate was still present in S. halepense, 821o in T. subterranum and B2/" in C. arvensis.
Similarly, various plants have been analysed for glufosinate metabolism by the above authors and
reported that only very small quantities of 3-methyl-phosphonico-propanoic-acid and other
compounds were found as metabolites (Dröge et al. 1992, Dröge-Laser et al. 1994)
Several studies on glufosinate degradation in soil showed that glufosinate is degraded rapidly (half
life between 4 and 7 days) in soil due to soil microbial activities (Smith 1989). Rasche (1995)
reported that glufosinale has never been detected as a residue in harvested crops. This rapid
degradation of glufosinate in soil makes it one of the most environmentally friendly herbicides in
the market. Studies conducted by Tebbe and Reber (1991) showed that several strains of soil
bacteria are able to use glufosinate as a nitrogen source. Behrendt et al. (1990) stated that
glufosinate ammonia degradation yielded two major metabolites in soil. These were: 3-(methyl
phosphinyl) propanoic acid (MPPA) and 2(methyl phosphinyl) acetic acid (MPAA-2).
1.2.2. Mode of Action of Glufosinate
Glufosinate is the only commercial herbicide that targets glutamine synthetase (GS) in higher
plants to induce phytotoxicity (Devine and Shukla 2000). This herbicide is considered to be the
most effective form of natural inhibitors of GS that includes tabtoxinine-p-lactam, oxetin and
methionine sulfoximine (Uchytil and Durbin 1980, Omura et al. 1984, Jeannoda et al. 1985). The
3 Chopter 1: Literoture review
inhibition of GS causes rapid accumulation of intracellular ammonia, cessat¡on of photorespiration and photosynthesis (Tachibana and Kaneko 1986). Even though accumulation of ammonia may cause the death of the plant, the primary cause of phytotoxicity is believed to be as a result of inhibition of photorespiration (Devine et al. 1993).
1 .2.3. Gl utamine Synthetase
The enzyme glutamine synthetase (GS) of higher plants catalyses the ATP dependent conversion of glutamate into glutamine using ammonia as a substrate (as shown below) and plays an
important role in nitrogen metabolism (Rastogi et al. 1998).
GS Glutamate + NH3 + ATP------Glutamine+ H2O +ADP + Pi
GS is an octameric protein, which has a native molecular mass of 350-400KDa (Lea 1993). ln
size and quaternary structure, higher plant GS strongly resembles the mammalian GS but is
structurally different from the bacterial GS (Lea 1993). However, studies on X-ray crystalography
of GS have demonstrated that GS lrom Salmonella typhimurium (baclerium) and eukaryotic
octameric GS were similar in spite of structural differences (Yamashita et al. 1989). Liaw and
Eisenberg (1994) and Liaw et al. (1995) investigated the reaction of glutamate to glutamine
catalysed by GS. According to these authors, ATP binds first to the active site of the bacterial GS.
Then glutamate reacts with the bound ATP to produce y-glutamyl phosphate and ADP. An
ammonium ion is then bound to the active site and loses a proton to form ammonia which then
attacks y-glutamyl phosphatase to produce glutamine and phosphate.
Studies using ion exchange chromatography of GS have shown that there are two GS isoenzymes
in leaves of higher plant (McNally et al. 1983). These two isoenzymes have been referred to as
G51 and GS2. Whilst GS1 has been found in the cytoplasm of phloem elements and root cells
(Edwards et al. 1990), GS2 has been found in the chloroplast (Lea 1993). lt has been shown that
the proportions of GS1 and GS2 vary with the plant species and the developmental age (Lea
1993). For example, in spinach, only GS2 is present (Hirel and Gadal 1982) whereas other C3
4 Chopter 1: Literoture review
ptants such as rice (Hireland Gadal 1981), pea (H¡rel and Gadal 1981) and barley (Mann, Fentem et al. 197g) contain 20% of GS1. ln sorghum, 70% oÍ total GS activity found in leaves is due to
GS1 isoenzyme (Hirel and Gadal 1982). The major isoform (GS2) of GS in the leaves is the
cholroplastic form (Leegood 1999). The primary role of GS2 is the reassimilation of ammonia
released by photorespiration (Leegood 1999). Woodall et al. (1996) demonstrated that in barley, a
change in temperature caused alterations in the activity of chloroplastic GS. When barley plants
were grown at 25'C (day temp) and transferred to 15'C (day temp), 75"/" ol the activity of the
chloroplastic GS was lost after 3 days (Woodall et al. 1996).
Recent studies on the cytoplasmic GS isoform have shown that the cytoplasmic GS is localized in
the vascular system of tobacco plants (Carvalho et al. 1992) and in the phloem companion cells of
potatoes (Pereira et al. 1992). ln rice, cytoplasmic GS was found to be in large and small vascular
bundles (Kamachi et al. 1992). A study conducted on cytoplasmic GS showed that relatively high
amounts of cytoplasmic GS remained during senescence but the chloroplastic GS decreased by
B0% in line with Rubisco activity (Kamachi et al. 1992). lt is generally thought that the cytoplasmic
GS plays a major role in the assimilation of ammonium derived from nitrate reduction in roots
(Edwards et al. 1990). The role of GS1 in vascular tissues is not well defined (Edwards et al.
l ggo). However, one possibility is that GS may be involved in vascular nitrogen transfer (Edwards
et al. 1990). To study the cytoplasmic GS in vascular tissues Brugiere et al. (1999) conducted a
study involving antisence RNA in tobacco. They concluded that the cytoplasmic GS in the phloem
plays a major role in regulating proline synthesis.
Glufosinate, an analogue of glutamate, binds to the active site of GS irreversibly inhibiting GS
activity. Glufosinate appears to have two actions (Mersey et al. 1990). ln the presence of ATP, a
time dependent irreversible inhibition of GS occurs (Logusch et al. 1989), whilst under initial rate
conditions, it behaves as a reversible inhibitor of GS by competing with glutamate (Mersey et al.
1990). ln higher plants, the GS phosphorylated glufosinate complex may not be so labile
(Mandersceid and Wild 1986; Hopfner et al. 1988). To date it is not clear to what extent GS can
be inhibited with glufosinate under differential environment factors such as temperature and light
5 Chopter 1: Lileroture review
intensity, or whether there is a difference in GS inhibition in weeds such as R. raphanistrum and L. rigidum when compared to more susceptible species such as Avena sterilis and Sisymbrium orientale.
1.2.4. lnhibition of Photorespiration by Glufosinate
The inhibition of the GS enzyme by glufosinate and other known GS inhibitors, such as tabtoxin-B-
lactam, oxetin and methionine sulfoximine in a range of plants, results in an accumulation of
ammonia (Kocher 1989, Wild and Ziegler 1989) and a strong decline in the amino acids,
glutamine, glutamate, aspartate, alanine, serine and glycine (Wendler and Wild 1990). This
increase of intracellular ammonia causes disruptions of ion transfer and other membrane functions
following GS inhibition (Trogisch et al. 1989). However, studies conducted by Ziegler and Wild
(1989) suggested that accumulation of ammonium was not the primary cause for inhibition of
photosynthesis by glufosinate. The inhibition of photosynthesis caused by glufosinate was based
on inhibition of carbon recycling following photoinhibition. lnvestigations conducted by Wild and
Wendler (1991) on the effect of GS inhibition with glufosinate and bialaphos on photorespiration
and photosynthesis demonstrated that under non-photorespiratory conditions (0.1%v/v CO2,2%
O2), the photosynthetic rate of Srnapsrs alba remained constant at 80% of the controlled rate.
However, when plants were transferred to normal at (0.04% vlv CO2, 21% o2) there was a rapid
fall in the photosynthetic rate. This rapid inhibition of photosynthesis observed under normal air
was reversed by the addition of glutamine (Suauer et al. 1987). ln contrast, ammonia
accumulation continued to increase even when the inhibition of photosynthesis was reversed.
Studies conducted by Walton (1983) and Yu et al. (1984) demonstrated that when
photorespiratory glyoxylate is aminated to glycine in the peroxisome by glutamate-glyoxylate
amino transferase or via serine-glyoxylate amino transferase, glutamine and serine were the
amino donors to glyoxylate. GS inhibition with glufosinate caused a lack of these amino acids
required for transamination of glyoxylate to glycine. This in turn resulted in inhibition of the
photorespiratory carbon oxidation cycle (Wendler and Wild 1990). Ziegler and Wild (1989)
6 Chopter 1: Lileroture review
demonstrated that the subsequent accumulation of high concentrations of glyoxylate or phosphoglycolate inhibits Rubisco leading to a reduction in photosynthetic CO2 fixation in plants
(Hall and Roa 1999).
1.3. Herbicide Resistant GroPs
After the discovery of atrazine resistance in weeds, research was under taken to develop atrazine resistant crops. At first such herbicide resistant crops were developed using traditional breeding techniques. To produce triazine resistant canola, Brassica napus (canola) was crossed with triazine resistant B. campestris in successive generations (Grant and Beversdotf 1985). However, there were limitations of using such breeding techniques to transfer resistant traits, as this was
possible in a limited number of species. Therefore, other approaches have been used to create
herbicide resistance in crop plants. Techniques such as somatic hybridisation of protoplasts (from
resistant to susceptible individuals), induced mutations and recombinant DNA technology have
been used.
1.3.1. Glufosinate Resistant Grops
Glufosinate tolerance has been conferred to at least 36 plant species including canola, corn,
soybean, sugarbeet and maize through genetic engineering for more rapid glufosinate metabolism
(Devine and Shukla 2000). Glufosinate tolerant canola was first planted commercially in Canada
in 1995 and glufosinate herbicide has been used as the sole herbicide for weed control in these
crops (Rasche and Gadsby 1997). lt was reported that an application of 300 to 500 g ai ha-1 of
glufosinate had more than 90% efficacy in controlling weeds in glufosinate tolerant canola fields
(Rasche and Gadsby 1997). Studies conducted on glufosinate tolerant canola varieties treated
with glufosinate or which received standard herbicides, showed that glufosinate consistently gave
better results than the standard herbicide treatments (Rasche and Gadsby 1997). ln Europe,
when glufosinate was field tested in glufosinate tolerant canola crops in 1993 and 1994,85-97%
control of weeds was achieved with dose rates of 300 to 600 g ai ha-1 (Rasche and Gadsby 1997).
Therefore, it was concluded that the level of weed control with glufosinate in resistant crops in
7 Chopler l: Literolure review
most cases was superior to standard herbicide treatments (Rasche and Gadsby 1997). ln contrast to the successful use of glufosinate in Canada and Europe, field studies conducted with glufosinate showed variable performance against several weed species in cropping regions of southern Australia (Michael Clarke, pers. comm.).
1.4. lmportant Weeds in Southern Australian Cropping
Regions
Controlling weeds in broad acre cropping systems is a major expenditure for Australian growers costing $400 million on herbicides alone annually. Althought many different plant species are considered as weeds, a few species dominate cropping regions of Australia. Lolium rigidum
(ryegrass) is considered by far the most important weed in Australian cropping systems, due to it's abundance through out the cropping regions in southern Australia. Other major grass weed species in this region are Avena fatua (wild oats) and A. ludoviciana, Bromus spp (bromes),
Hordeum glaucum (northern barley grass), H. leporinum (barley grass) and Vulpia spp
(silvergrasses). There are also several major dicotyledonous plant species that are weeds in southern Australia. Haphanus raphanistrum (wild radish) is classified as the principal broad leaf weed through out the cropping zone. Other major dicotyledon weeds include Arctotheca calendula
(capeweed), various Brassicaceae spp., including Sisymbrium orientale, (lndian Hedge mustard), and Brassicaceae tourneforiii (wild turnip), and Emex spp.
1.4.1. Ryegrass - Lolium rigidum
The genus Lolium originated in temperate Europe, North Africa and the Eastern Mediterranean.
Several species have been recorded as widespread and naturalized in southern regions of
Australia. L. rigidum is an easily established pasture species with good seedling vigour and rapid tillering enabling it to compete with other plant species. ln the past, there have been many deliberate introductions of this species across southern Australia since it was productive and palatable to livestock (Mullett 1919). The distribution and persistence ol L. rigidum and it's
8 Chopler l: Literoture review
success as a pasture plant in southern regions of Australia have made this species a major annual grass weed in cereal and other crops (Forcella 1984). Reeves (1976) showed that the percent yield loss in wheat is directly proportional to the square root of lhe L. rigidum density per unit area.
Therefore, 100 plants m-2 could result in a 10% yield loss. Control of L. rigidum in cropping
regions is achieved by either herbicide applications or cultural methods. Millions of dollars are spent annually on herbicides for L. rigidum control. An estimate in 1989 indicated that in excess of
$33 million was spent on herbicides for L. rigidum control in Western Australia alone (Gill 1996).
Such heavy reliance on herbicides for L. rigidum control during the last twenty years with both
selective and non-selective herbicides has accentuated the development of herbicide resistance
(Mathews et al. 1996). ln Australia, herbicide resistance first appeared in L. rigidum in 1980s
(Heap and Knight 1982) and is now widespread across the regions of southern Australia (Preston
et al. 1999). According to Nietschke et al. (1996) in some regions of southern Australia morethan
40% oÍ cropping fields are infested with herbicide resistant L. rigidum. Multiple herbicide resistant
populations of L. rigídum are resistant to more than 12 herbicide chemistries of seven modes of
action (Preston et al. 1999). lt was observed that in southern Australia there has been a rapid
evolution of herbicide resistance in this species. Resistance to both ACCase and ALS inhibiting
herbicides has occurred following only a few applications of these herbicides.
1.4.2. W¡td radish -Raphanus raphanistrum
Raphanus raphanistrum (wild radish) is a major weed species of winter grain crops of Australia.
The weed is a member of the Brassicaceae (mustard) family, which comprises over 3,500 species
of plants. The genus Raphanus consists of eight species, which are native to Western and
Central Europe, through the Mediterranean to Central Asia. ln Australia, three species of
Raphanus have been recorded, H. raphanistrum, R. meritium and R. sativus (Anonymous. 1982).
R. raphanistrum was first introduced to Australia in the 19th century as a contaminant of
agriculture products (Donaldson 198ô). H. raphanistrumis a weed of cultivation, often found in
winter grain crops, along roadsides, in wastelands and other disturbed habitats in Australia, ln
southern Australia, R. raphanistrum is one of the most widespread and troublesome weeds of
I Chopter 1: Lileroture review
cereal and grain legume crops, in a range of soil types. lt competes vigorously with the crop and can become the dominant species in cultivated land. The plants have the ability to reach up to one meter in height and, due to allelopathic effects, can rapidly dominate dwarf varieties of modern crop cultivars.
Damage caused by R. raphanistrum to the Australian agriculture industry has ranked this species as the most damaging weed of cereal crops (Pool and Gill 1987). ln Victoria, nearly 50% yield loss of wheat has been recorded with a R. raphanisfrum density of 200 plants m-2. According to
Moore (1979) in Western Australia, 7-11% yield loss occurred with 25 R. raphanistrum plants m-2 and 50 plants m-'had resulted in a yield loss of 15-20%. ln New South Wales, 160 plants m-2
reduced wheat yields by 7O-BO% (Dellow and Milne 1987). ln addition, the contamination of grain with ¡9. raphanistrum pod segments causes quality and price reductions (Cheam and Code 1998).
ln Western Australia, grain with more than 6% of H. raphanistrum contamination is rejected
(Cheam and Code 1998). lt is almost impossible to remove the pod segments ol R. raphanistrum
from wheat grain due to their similar size (Cheam and Code 1998). Previous findings showed
R. raphanislrum causes toxic effects when eaten by livestock. Trouche. (1936) reported that
grazing of flowering H. raphanisfrum caused jaundice, haemoglobinuria, liver damage and
increased mortality in lambs. Seeds ol R. raphanistrum are regarded as the most dangerous part
of the plant (Connor 1977). Cooper and Johnson (198a) identified that the toxic compound in
R. raphanistrum is allylisothiocyanate, derived f rom glucosinolates. R. raphanistrum also acts as
an alternate host to a number of pests and diseases such as thrips (Thrips spp), flee beetles
(Phytlotreta spp) and club root disease of Brassicas (Plasmodiophora brassicae Woron) (Cheam
and Code 1998). Cupertino et al. (1984) reported thal in California active tobacco streak virus was
isolated from F. raphanistrum collected in tomato fields near Sacramento. Dikova (1989) reported
that Æ. raphanistrum also act as an alternative host to cucumber mosaic virus.
1.4.2.1. Significance of Raphanus raphanistrum to the Canola lndustry
R. raphanistrum infestation is a major problem for the booming canola industry in Australia. ln
1996,283,000 tons of canola were exported from Australia with a value of over $100 million
(ABARE Australian Commodity Statistics 1997). ln many parts of Australia, canola is grown over
10 Chopler 1: Literoture review
thousands of hectares of agricultural land and ¡t is predicted that this trend will increase in the future. ln southern Australia, the area under canola comprised 1.6 million hectares in 1999 and seems to be doubling every three years (ABARE Australian commodity Statistics, 1999).
R. raphanistrum not only reduces the canola yield, but also lowers the quality and quantity of canola oil. However, herbicides cannot be applied to control Brassicae weeds such as
R. raphanistrumin canola fields since both the weed and the crop are Brassicae species, sharing similar genetical characteristics including susceptibility to herbicides. Therefore, there are no
herbicides available that can eradicate R. raphanistrum without damaging the standard varieties of
canola.
ln order to overcome the problems associated with weed control in canola, herbicide resistant
canola cultivars have been introduced. Transgenic, herbicide resistant varieties have been
commercialised in America, and are being developed in Australia. Non-transgenic varielies are
also available in the market. Triazine tolerant canola (TT) has been successfully introduced during
the 1990s and represented over 50% of the canola crop sown in Australia in 1999 (Trent Potter,
pers. comm.). The triazine tolerant canola varieties are not an ideal solution to this problem since
these varieties have a substantial (20%) yield loss and lower oilcontent and oilquality (Beversdotl
et al. 19BB). ln addition, extensive use of triazine herbicides in North America has resulted in
ground water contamination with herbicide residues (Ribaudo 1993). Another non-transgenic
canola variety with imidazolinone tolerance was released in 2000 in Australia. A major
disadvantage with these varieties is that many weed species have already developed resistance to
trazine and imidazolinone herbicides (Heap and LeBaron 2001). ln addition, canolavarieties with
resistance to herbicides glyphosate, glufosinate and bromoxymil are also proposed for release in
Australia in the next few years (Preston and Rleger 2000). Glufosinate resistant canola cultivars
are available under the trade names of Liberty link@ and lnVigor@. This new variety has been
marketed in Canada since 1995 (Rasche and Gadsby 1997) and has been readily adopted by the
Canadian growers. ln 1997 about 830,000 hectares of Liberty link canola was grown in Canada
(Rasche and Gadsby 1997). With the introduction of the Liberty link@ canola, the herbicide
11 Chopler 1: Literotur¿ review
glufosinate-ammonia was also introduced to Canada and has been successfully used for controlling weeds.
Field studies have been conducted with many weed species to investigate the suitability of
glufosinate under Australian conditions in Western Australia. The results indicated that there is a
variable performance of glufosinate on F. raphanistrum (Michael Clarke, pers. comm.). However,
contrary to this finding, recent investigations have shown that R. raphanistrum can be easily
controlled by glufosinate in Europe (Michael Clarke, pers. comm.). Furthermore, when the
Australian populatrons ol R. raphanistrum were grown in Europe they were effectively controlled by
glufosinate (Michael Clarke, pers. comm.). Therefore, it can be hypothesised that the poor control
of R. raphanistrum by glufosinate in Australia is most likely to be associaled with the
envlronmental conditions in Australia. Further investigations are necessary to understand the
physiological reasons for poor control ol R. raphanistrum in Australia
1.5. Mechanisms of Herbicide Resistance/ Tolerance
Shortly after the introduction of herbicides, researches predicted the possibility of spreading
herbicide resistant weeds as such evolved resistance was observed in insects, fungi and bacteria
(Heap and Le Baron 2001). The very first herbicides used in broad acre crops were the auxin
herbicides 2,4-D and MCPA in 1947 (Heap and LeBaron 2001). Since then many herbicides have
been introduced to eradicate weeds in broad acre systems. First report of resistance appeared to
trazine herbicides in the early 1970s (Ryan 1970, LeBaron and Gressel 1982). Atthe sametime
resistance lo 2,4-D and MCPA was observed in several weed species. Since then many weeds
have developed resistance to many of the commercially available herbicides making herbicide
resistance in weeds a worldwide problem. According to Preston, et al. (1999) it is most severe in
the southern Australian grain belt. The above authors also reported that based on a survey
conducted in 1997, approximately 17% of cereal growers in southern Australia believed that they
have herbicide resistant weed populations. Development of resistance in weeds is not confined to
cereal cropping systems but is also known from orchards, pastures, roadsides, railways, perennial
12 Chopter 1: Lilerqture review
lucerne fields and other sites. To date, 22 weed spec¡es with resistant populations are known in
Australia (Preston et al. 1999).
Several mechanisms have been postulated for the development of herbicide resistance or tolerance. The most commonly observed resistance mechanism is the target site insensitivity where due to a modification of the target enzyme, binding of the herbicide to the enzyme is reduced or eliminated. Cross resistance, where a single resistant mechanism confers resistance to several herbicides, is also known in several weed species. For example, resistance to two chemically dissimilar classes of herbicides, the aryloxyphenoxypropanoates and cyclohexianediones that inhibit the enzyme acetyl coenzyme A carboxylase within the same plant is known (Heap and LeBaron 2001). Resistance is also conferred by mechanisms other than target site modification. Non target site resistance can be due to mechanisms such as enhanced metabolism, reduced rates of herbicide translocation or herbicide sequestration (Heap and
LeBaron 2OO1). Resistance in several populations of L. rigidum has occurred as a result of
increased herbicide detoxification of Group A, B or C herbicides. ln addition populations ol Avena
sterilis, Digitaria sanguinalis and Hodeum leporinum to group A herbicides show increased
detoxification of herbicides. Other potential resistance mechanisms include reduced uptake into
cells or tissues, and avoidance due to physical or temporal separation of the herbicide. ln cases
such as reduced uptake into cells and physical and temporal separations, resistance is described
to be due to sum of complex morphological and physical factors (Devine and Shukla 2000)'
Tolerance to paraquat and diquat in H. glaucum, H.leporinum, A. calendula and resistance due to
glyphosate in L. rigidum are considered to be due to altered transport of herbicide into or out of the
cell cytoplasm (Devine and Shukla 2000). According to studies in these populations, considerably
less herbicide reached the site of action in the chloroplast. However, the aspects of absorption,
translocation and entry of herbicides into cells have not been thoroughly investigated to
understand the reasons for poor translocation of the above mentioned herbicides to the sites of
action.
13 Chopter 1: Lileroture revi¿w
1.6. Absorption and Translocat¡on of Herbicides
Herbicides are taken onto the plant through two major routes. Pre-emergence (soil active)
herbicides are absorbed from the soil through the root system of the plant while the post-
emergence (foliar active) herbicides enter the plant directly through the leaves. With soil active
herbicides, the herbicide molecules enter the roots via bulk flow of water along with dissolved ions.
Once inside the root, the herbicide is transported to the shoots in the transpiration stream. For a
foliar active herbicide to reach it's active site, the herbicide must pass through a series of barriers
of different chemical and structural compositions (Gunawan and Donald 1989). These include the
cuticular layer, cell wall plasma membrane, and sometimes the organelle membranes (Bayer and
Lumb 1973).
1.6.1. Foliar Absorption
Foliar absorption of herbicides via the leaves depends upon the thickness, chemical composition,
and permeability of the cuticle of the plant species, leaf age and the environmental conditions
under which the leaf has developed. Martin and Juniper (1 970) studied the cuticular thickness of
various plant species and reported that the cuticular thickness varied between 0.7 ¡rm to 13'5 pm.
They concluded that cuticular thickness varies widely depending on the plant species and the
environmental conditions. Tribe et al. (19ô8) showed that the quality of wax produced in oat and
barley leaves were directly proportional to light intensity but inversely proportional to relative
humidity. Norris (1974) indicafed that herbicide absorption is not necessarily related to cuticular
thickness or weight but more to the cuticular lipids and the degree to which they block the passage
of solute. ln order to prove this, Norris (1974) removed the cuticular wax of various species and
allowed 2,4-D lo penetrate the leaves. He found that the 2,4-D penetration was increased by a
factor of 2 lo 25 in the absence of cuticular waxes depending on the species. Similarly, water
permeability of the cuticle increased by a factor of 200 to 500, when the soluble lipids were
removed (Schmidt et al. 1981). There is evidence that herbicide penetration decreases as the leaf
ages (Kirkwood et al. 1982). Schmidt et al. (1981) found that young Kaffer lily (Clivia miniata
14 Chopter 1: Lileroture review
regte) leaves were more permeable to water than old leaves. He attributed this to greater polarity of the cutin of the young leaves. Also the penetration of 1-naphtylactic acid was greatest when leaves were expanding most rapidly, due to thin or discontinuous wax coverage on such tissues
(Barker and Hunt 1981).
1.6.2. Absorption Across Membranes
The effectiveness of a herbicide can be influenced by herbicide absorption across membranes.
To date, little work has been conducted in general on the mechanisms of herbicide absorption
across membranes (Sterling 1994) and there is very little information regarding glufosinate
absorption. Almost all studies in this area have been conducted on excised tissues such as root
slices, cotyledons or leaf tissues, protoplasts, plasma membrane vesicles, or algal cells.
Most herbicides have been shown to move across plant membranes via non-facilitated diffusion
(Hess 1985). Such absorption of herbicide molecules can be divided into two groups, depending
on physicochemicalcharacteristics of the molecule (Sterling 1994). They are:the lipophilic-neutral
herbicides, and lipophilic-ionic herbicides. Bromillow and Chamberlian (1991) pointed out that the
most important physicochemical properties that a herbicide molecule must posses in order to get
absorbed by the cell are lipophilicity (non polarity) and acidity. Neutral, lipophilic molecules are
thought to move into cells via passive diffusion with the lipophillic nature allowing them to diffuse
rapidly across the lipid bilayer of plant membranes down their concentration gradient to reach
equilibrium between external and internal concentrations (Sterling 1994). Several studies
conducted in this area supply evidence to support this hypothesis. lnvestigations with amitrole
(Lichtner 1983) and atrazine (Price 1982) have shown there is a linear relationship between
herbicide absorption and external herbicide concentrations. Similarly, amitrole, atrazine, diclofop-
methyl, metribuzin, and simazine absorption are not saturated at high concentrations and the
absorption is not carrier mediated (Sterling 1994). lt has been shown that weak acid herbicides
are absorbed by simple diffusion and transport is via nonJacilitated diffusion (Sterling 1994).
However, many weak acid herbicides reach cellular concentrations greater than external
concentrations as a result of ion trapping. According to Sterling (1994), the features controlling
'15 Chopler 1: Literoture review
transport of weak acid herbicides across membranes are their ability to solubilize in lipids as the neutralform, and their weak acidic nature.
There are some herbicides that need active transport to cross the plant membranes. To date, the evidence of active transport is limited to dalapon (Prasad and Blackman 1965),2,4-D (Kasai and
Bayer 1991), glyphosate (Denis and Delrot 1993) and paraquat (Hart et al. 1992). Prasad and
Blackman (1965) observed dalapon absorption inlo Lemma minor occurred in two phases. ln
phase 1, absorption occurred for 30 mins and was related linearly to external dalapon concentration. ln phase 2 the rate of absorption was curvilinearly related to dalapon
concentrations, indicating absorption via active transport. Glyphosate is absorbed into plants
through nonfacillitated diffusion and also through active transport (Burton and Balke 1987, and
Denis and Delrot 1993). Glyphosate uptake in Bermuda grass leaf tips, broad bean leaf
protoplasts and in suspension cultures of carrots had a nonlinear rapid initial uptake followed by a
slower continual uptake (Burton and Balke 1987).
Several studies have investigated the possible role of a transporter for glyphosate in plant cell
membranes. The results show that the saturable component of glyphosate transport was inhibited
by phosphate, supporting an involvement of a phosphate carrier which recognises the phosphate
group on the glyphosate molecule (Denis and Delrot 1993). Brecke and Duke (1980) observed
glyphosate inhibited absorption of phosphate by isolated bean cells. Therefore, it was suggested
that glyphosate uptake by plant cells may be mediated by a phosphate transport system located
on the plasma membrane (Brecke and Duke 1980). Glufosinate has a phosphinate group that
behaves differently to a phosphate group. Therefore, the absorption of glufosinate is likely to
occur differently to glyphosate. However, to date no studies have been conduced on glufosinate
absorption into plant cells. Further studies in this area are warranted to determine the movement
of glufosinate into the plant cell and then to the active site.
16 Chopter l: Literoture review
1.6.3. Herbicide Translocat¡on
Herbicides can be transported in plants from the site of absorption via both the xylem and phloem.
Whilst pre-emergence herbicides are translocated from roots to the shoots via the xylem, most post emergence herbicides are applied to the shoot system and are translocated into leaves and stems in plant via the phloem (Hess 1985). ln addition, there are many herbicides that get translocated in both xylem and phloem.
1.6.3. 1. Phloem Translocation of Herbicides
Most pesticides can enter the phloem sieve tubes freely but due to far greater water flow in the
xylem, only those compounds that are well retained in the phloem sieve tubes are able to be
transported effectively via the phtoem (Bromillow and Chamberlian 1991). Tyree et al. (1979)
developed the concept that many compounds are able to enter the symplast but only those having
limited permeation rates through membranes could be retained sufficiently well for long distant
transport via phloem. The distinction between compounds that are mobile in the xylem and the
phloem is not always clear and even the compounds that are mobile in the xylem enter the
symplast freely. Similarly all phloem mobile compounds can also move in xylem (Monaco et al.
2002).
Phloem mobile herbicides exhibit a pattern of movement similar to sugars. They are translocated
from source to sink. Therefore, movement occurs from the leaves (source) to any energy
requiring regions (sink) such as the apical meristems, axillary buds, developing shoots and roots
(Devine et al. 1993). The mechanism of phloem translocation is driven by osmotic and pressure
gradients (Devine et al. 1993). ln perennial plants and in grass weeds efficient phloem
translocation of herbicides is important for effective control.
17 Chopter 1: Literoture review
1.6.3. 1,1. Source Sink Relationship
A number of processes in source and sink regions help maintain export of assimilated carbon from source organs and it's distribution among sinks (Geiger and Bestman 1990). Carbon fixed in the chloroplast is used for respiration, and synthesis of compounds that remain in the leaf or is converted to sucrose which is then available for export. Sucrose can be directly stored in the cytosol and vacuole or may enter the sieve elements to be transported to the sinks. Sucrose imported from the source regions is partitioned among various sinks such as roots, fruits, apical meristems and among developing leaves. At the sink regions, the solutes exit the sieve element by phloem unloading processes depending on the nature of the sink organ. For example, in developing bean seeds sucrose and amino acids are taken up through membranes into cells of the developing tissue (Thorne 1986). The flux of sucrose importing and unloading is maintained by the rate of sucrose degradation by enzymes such as acid invertase, alkaline invertase or sucrose synthase and subsequent utilization in these organs for respiration and synthesis of various products.
The phytotoxic action of a phloem mobile herbicide may affect one or more of the processes that maintain or regulate translocation and distribution of carbon among sinks. ln source leaves, toxic effects can affect the photosynthetic carbon fixation, allocation of both newly fixed and stored carbon, short distance transfer and membrane transport by sieve elements. Therefore, herbicide toxicity can interfere with export of both assimilated carbon and the herbicide. Throughout the translocation path, processes can be affected due to damage to the conducting tissues that may result in leakage of solutes and loss of pressure from the phloem. Long distance transport through the sieve elements of the translocation path appears to result primarily from osmotically driven mass flow (Geiger and Sovonick 1975). Physiological properties of the phloem mobile herbicides in lransit determine the extent of their entry and leakage from the sieve elements and their phloem mobility (Gougler and Geiger 1981 and Kleier 1988). The current model of phloem tranlocation of xenobiotics considers that their phloem mobility also depends on properties of the plant such as translocation velocity and length of translocation path (Kleier 1988, Tyree et al.
1979). Herbicide induced slowing of translocation may cause a greater leakage of a herbicide that
'18 Chopter 1: Literoture review
is at the liipophilic end of the range of phloem mobility (Kleier 1988) and as a result prevents establishment of an effective phytotoxic level in the sink organs.
1.6.4. Absorption and Translocation of Glufosinate
Glufosinate, a foliar active non-selective herbicide, is known to be absorbed through the foliage of
plants and is translocated to the sink tissues via the phloem (Steckel et al. 1997). Several authors
have reported that glufosinate does not translocate extensively well from the site of application, in
several species of plants (Hass and Muller 1987; Bromilow et al. 1993, Steckel et al. 1997). To
date, the reasons for this limited translocation are unknown (Beriault et al. 1999) and it is assumed
that the phytotoxicity induced by rapid action of glufosinate at the site of application causes
localized phytotoxicity in leaves limiting phloem translocation (Beriault et al. 1999). Other
herbicides such as chlorosulfuron and glyphosate are also known to limit phloem translocation
through rapid action (Beriault et al. 1999). Holloway and Stock (1990) pointed out that differential
herbicide absorption, translocation and metabolism can determine the degree of plant sensitivity or
tolerance to certain herbicides. Although glufosinate is a non-selective broad spectrum herbicide,
efficacy has been shown to be influenced by environmental factors, plant species and application
rates (Carlson and Burnside 1984). According to Anderson et al. (1993) both temperature and
relative humidity were found to affect glufosinate activity in barley (tolerant) and green foxtail
(sensitive). The differences in activity of glufosinate on barley and green foxtail were explained by
differences in foliar absorption and translocation but not by a metabolic degradation (Mersey et al.
1 990).
A limited amount of information is available on translocation of glufosinate in glufosinate resistant
and susceptible plants (Droge et al. 1992). Studies in combination with acetyl L-glufosinate, the
initial detoxification product and glufosinate have shown both compounds possess similar
distributions in the plants. For both forms of glufosinate, most of the herbicide is retained in the
treated leaf with a small amount transported to the stem and upper leaves (Droge et al. 1992).
Beriault et al. (1999) observed that acetyl-L-glufosinate was much more phloem mobile and
substantial amounts were transported to the upper leaves, flowers, and to the roots whilst
19 Chopler 1: Lileroture review
translocation of L-glufosinate (the active form of glufosinate) was limited and this was explained as, a result of the combined effects of ammonia accumulation, reduced carbon accumulation and depletion of glutamine.
1.6.5. Environmental Effects on Absorpt¡on and Translocation of
Herbicides
According to Shaner (1994) environmentalfactors such as temperature, light intensityand relative humidity can have a profound effect on the activity of a herbicide as these factors can influence the rate of absorption, translocation or metabolism of herbicides directly. ln addition, indirect effects on plant metabolic processes can influence herbicide efficacy. Shaner (1994) pointed out that for phloem mobile herbicides, any environmental factor that disrupts photosynthesis and photosynthate transport (such as low temperature and water stress) will reduce the movement of that herbicide. Several studies have been conducted to determine the effect of different temperatures, light intensities and relative humidities on absorption and translocation of many herbicides in different weed species. Xie et al. (1996) investigated the effects of high (20l30"C) and low temperature (5/10'C) with different light intensity on absorption of fenoxaprop-ethyl and imazamethabenz-methyl in Avena fatua. Absorption and translocation of both herbicides increased under high temperature. Low temperalure resulted in decreased translocation and translocation efficiency for both herbicides. Xie et al. (1996) further observed an enhanced phytotoxic effect of both herbicides under low light (70% shading) intensity. Despite the differences between high and low temperatures on absorption and translocation of fenoxaprop- ethyl and imazamethabenz-methyl in Avena fatua, herbidde efficacy was only slightly affected. ln a similar study, Coupland (1989) reported that the performance of fluazifop-butyl againsl Elymus repens was significantly influenced by the environmental conditions in which the plants had grown prior to treatment. This investigation showed the effect of soil moisture deficit had reduced herbicide performance (Coupland 19Bg). This study also reported that there was a significant environmental effect on spray retention, foliar uptake and the amount of herbicide translocated to roots and rhizomes (Coupland 1989).
20 Chopter 1: Lil¿roture review
As mentioned earlier, glufosinale performance against weeds such as L. rigidum and
R. raphanistrumhas been variable in field trials in Australia and often poor control of these weeds has been observed. As F. raphanistrum is a major weed of canola crops in Australia it is important that herbicide efficacy is maintained. Control of R. raphanistrum by glufosinate is apparently not a problem in Europe. Canola growing conditions can be very different across
Australia and between Australia and Europe. As glufosinate has generally poor translocation within plants any decrease in absorption and translocation due to prevailing climatic conditions can have a large impact on efficacy (Bromilow et al. 1993). Therefore, it is important to study the environmental effects on absorption and translocation of glufosinate in weeds such as
R. raphanistrum lo understand the reasons for variable performance observed under Australian
conditions.
21 Chopfer 2: Glufosinote Efficocy in L. rigídumond A. sterilis
Chapter Two: Glufosinate Efficacy in Lolium rigidum and Avena sterilis
2.1- lntroduction
Even though glufosinate is considered to be a non-selective herbicide its absorption and the translocation are known to be species dependent. Pline et al. (1999) after studying the absorption of 1oO-glufosinate found variations in absorption in five weed species. ln this study the highest uptake was found after 72 hours in S. carolinense (73% of the applied radioactivity), which was followed by S. faberi(54%), C. obtusifolia (44%), C. album (41%) and A. syriaca (37%). Steckel et lac-glufosinate al. (1997) showed that the absorption of was 677o, 53o/",42yo and 16% in Setaria faberi, Echinochtoa crus-galli, Abutilon theophrastiand Chenopodium album. ln these species, the 1aO-glufosinate subsequent translocation from the absorbed was only 15%, 14%,5% and <1"/" following 24 hours of treatment (Steckelet al. 1997). Mersey et al (1990) and Steckelet al. (1997) concluded that the differential sensitivity observed between weed species as a result of differential uptake and translocation of glufosinate. ln this chapler it is intended to study the absorption and translocation of glufosinate in two closely related grass weeds, Lolium rigidum and Avena sterilis.
Lotium rigidum Gaudin, the most important ryegrass species in Australia, is found in allstates and in off shore islands (Kloot 1983). L. rigidum is a major weed of crops and pastures, as well as along roadsides and other non agricultural situations (Mullett 1919). L. rigidum competes vigorously for light and nutrients with the crop causing significant crop yield losses. ln 1989, in
Western Australia alone $33 million was spent on herbicides for L. rigidum control (Gill 1996). To control this weed Australian farming systems rely heavily on selective and non selective herbicides
(Gill 1996). However, the intensive use of herbicides has caused the evolution of herbicide
resistance in this species. The first report of resistance appeared in L. rigidum in 1982 to diclofop
22 Chopter 2: Glufosinote Efficocy in L. rigidunond A. slerilis
methyl (Heap and Knight 1982). Since then many populations of this species have developed resistance to a large number of herbicides (Preston et al. 1999).
Avena spp. (wild oats) are also considered as weeds of great economic importance (Medd 1996),
They occur throughout the Australian grain belt (Medd 1996). According to Harden (1994), there are four species present in Southern Australia. These are A. fatua L., A. ludoviciana Durieu, A. barbata and A. sterilis. ln 1990, the estimated losses to southern Australian agriculture due to
Avena spp., was $42 million (Medd and Pandey 1990). Avena spp. are renowned for their competitive ability, reducing crop yields and quality (Combellack 1992). The nationalyield loss is estimated to be 102, 330 tones (Medd and Pandey 1990). Many different techniques are used to control Avena spp in Australia. However, herbicides are a major tool for the control of these species. A range of selective herbicides are used for the control of Avena spp. Of these, Group A herbicides, the aryloxyphenoxypropionates and cyclohexanediones, are the most widely used post emergence herbicides and triallate and simazine are the key pre emergence herbicides used in controlling Avena spp. However, the continuous use of lhese herbicides has caused development of herbicide resistance in this species.
For populations of L. rigidum or A. sterilis that have developed resistance to a herbicide or to many herbicides the prospect of using existing herbicides can be limited. Therefore, the introduction of new herbicide chemistries to control these weeds is a necessity. Glufosinate, a novel herbicide in the broad acre crops can be used effectively to control such weeds in glufosinate resistant crops, as the weeds have not yet developed resistance to this herbicide. The impending release of glufosinate tolerant crop cultivars in Australia makes it important to assess the efficacy of glufosinate on major weed species such as L. rigidum and ,4. sterilis. lt is intended through this chapter to study glufosinate efficacy in these two economically important grass weed species.
23 Chopler 2: Glufosinole Efficocy ín L. rigidunond A. sterilis
2.2. Materials and Methods
2.2.1. Dose Response Studies
Dose response pot experiments were performed using single populations of L. rigidum (VLR 1) and
A. sterilis (SAS 2). These two populations were originally collected from areas with no history of herbicide use and are susceptible to all herbicides used for their control, Seeds of both species were germinated on 0.6% (w/v) agar in a germination cabinet with 12 h 19"C (30 pmol m't s-t¡ day and 12 h, 19'C night. Wild oat seed trays were kept at 4'C for 24 h before placing them in the germination cabinet. Germinated seedlings were transplanted into 15 cm square pots filled with
potting soil. Each pot contained nine L. rigidum or four A. sterilis seedlings. The plants were
grown in a growth chamber set at 12 h, 2OoC, (480 pmol m-t s-t) day and a 12 h,15"C night. The
relative humidity ranged from 60 lo 70%. The same dose response pot experiments were
performed outside during autumn and winter, the normal growing season for these weed species in
southern Australia.
At the 2 to 3 leaf stage, seedlings were sprayed with Basta@ (2OO g ai glufosinate L-1). Herbicide
was applied with a laboratory moving boom sprayer equipped with T-jet flat fan nozzles at a speed
of 1 m s-t. The output of the sprayer was calibrated a|125 L ha-1 at a pressure of 250 Kpa. Dose
rates ranged from O to12O0 g ai ha'1 where the field rate of glufosinate is likelyto be 600 g aiha-1.
Plants were returned to the growth chamber after spraying and assessed for survival 21 days after
treatment (DAT). Plants were harvested and shoot dry weights were determined after drying the
shoots at 6O'C for 48 h. There were three replicates for each herbicide rate and the experiment
was repeated three times at different times under the same controlled growth environment for the
growth room sludies. The outdoor experiments were peñormed three times with each containing
six replicates for each herbicide treatment outside at the Waite Campus during the winter months
of 1999 and 2000. Data from all three repeats were pooled, as the results were not different.
Probit analysis was used on the pooled data to determine the LD5e values for each species kept in
the growth room and outside (GENSTAT, 2000).
24 Chopter 2: GlulosinoteEfficocy in L. rigidunond A. sterilis
2.2.2. Gl utami ne Synthetase Assays
GS assays were performed using seedlings of both species. The seedlings were grown as for the dose response experiments in the same growth room. When the seedlings were at the 2 to 3 leaf stage, fully expanded leaves were harvested and the GS assay was performed as described by
Mandersceid (1993), with a few modifications. Fresh leaf tissue (2 g) was ground with acid- washed sand at 4'C in pre-cooled Buffer A (50 mM Tricine, 0.5 mM EDTA, 1 mM MgSOa, 10 mM
DTT) and the homogenate was centrifuged (20,000 g) for 10 min. The supernatant was taken and the proteins precipitated by drop-wise addition of an equal volume of saturated (NH4)2SO4. The solution was stirred at 4'C for t h and then was centrifuged (27,000 g) for 30 min. The pellet was collected and resuspended in Buffer B (100 mM Hepes,50 mM MgSOa, 10 mM EDTA,'l mM
DTT). The enzyme was desalted on a SephdexrM G-2.5 column. The enzyme was eluted with
Buffer B and assayed for GS activity immediately. The reaction was started by addition o1200 ¡tL of partially purified protein to 2 mL of assay buffer (100 mM Hepes, 50 mM MgSOa, 10 mM EDTA,
10 mM NH2OH, 15 mM ATP and 12.5 mM glutamate) containing glufosinate (0.05-'100trM)). The reaction was allowed to proceed for 15 min at 37"C and was stopped by adding 1 mL of 0.37 M
FeCl3, 0.67 M HCl, and 0.2 M trichloroacetic acid. Formation of y-glutamylhydroxamate was measured spectrophotometrically at 540 nm. Experiments were repeated on three separate enzyme extracts. The mean inhibition of GS was calculated across the three experiments,
Proteins in the enzyme extracts were measured by the method of (Bradford 1976). The l5e value for GS was determined by plotting a sigmoidal curve to the data of the formula Y=D+(C-
D)/(1+10((logl50-logx).8)). Where D is the enzyme activity at the lowest glufosinate concentration,
C is the enzyme activity at the highest concentration, l5e is the concentration lor 50o/" inhibition, X is the logarithm of glufosinate concentration and B is the slope of the line at l5e.
2.2.3. Spray Retention
To determine the amount of glufosinate retained by L. rigidum and A. sterilis, spray retention experiments were performed. Seedlings of both species were germinated and grown as described above. When the plants reached the 3-leaf stage, they were sprayed with a commercial glufosinate solution (equivalent to 150 g ai ha'l) containing Chicago Blue (0.5% w/v). lmmediately
25 Chopter 2: Glufosinote Efficocy in L. rigidunond A. sterilis
after spraying, plants were harvested at soil leveland the shoots were washed in 0.1% (viv)Triton
X-100 in water (50 mL). Absorbance of this solution was measured spectrophotometrically at
620nm. The shoots were dried in an oven at 60'C for 48 hours. The amount of glufosinate retained was determined as ¡imol per gram dry weight. The experiment was repeated twice with six replicates. Each replicate contained 9 seedlings ol L. rigidum and 5 seedlings of A. sterilis.
2.2.4. Metabolism of Glufosinate
Experiments were conducted to determine the metabolism of glufosinate in both weed species.
Seedlings were germinated and grown as for the dose response experiments. When the plants had reached the 3-leaf sfage, they were sprayed with Basta@ (150 g ai ha-1) and immediately afterwards 2 ¡tL1aC-glufosinate (250 Bq ¡.rL-1 made up in a commercial formulation of glufosinate at
1.2 g ai L-l) was applied to the center of the 2nd leaf . The specific activity of the labeled glufosinate was 2.33 GBq g-t. Pots were kept in the same growth room after treatment. Plants were harvested at soil level from each replicate after 8,24, 48 and72 hour intervals and were washed in
0.1% (v/v) Triton X-100 in water (50 mL). The fresh weights were recorded and the shools were
wrapped in aluminium foil. The foil wrapped parcels were frozen in liquid nitrogen and were kept at
-2OC until extracted. The frozen plant materials were ground in 2 mL ol 10% methanol as
described by Muller et al. (2001). The ground plant materialwas centrifuged (15,000 g)for 10 min
and the supernatant collected. The pellet was extracted with further 1 mL of 10% methanol and
centrifuged (15,000 g) for 1O min. Supernatants were pooled, mixed with 3 mL ice-cold acetone
and centrifuged to remove proteins and lipids, The supernatant was decanted, dried under
vacuum and resuspended in 100 pL of 10% methanol. The components of this solution (25 pL)
were separated by TLC on cellulose plates (10 cm X 10 cm X 0.1 mm). The plates were
developed twice in a solvent containing pyridine, n-butanol, acetic acid and water (10:15:3:12 by
vol) as described by Strauch et al. (1988). Radiolabeled materials were visualized by
phosphorimaging and the radiolabeled compounds were isolated from the TLC plates by scraping
the radiolabelled spots. The amount of radioactivity per sample was determined by LSS. The
experiment was replicated four times.
26 Chopter 2: Glufosinole Efficocy in L. rígidunond A. sterilis
2.2.5. Absorption and Translocation Studies
Experiments to determine absorption and translocation of glufosinate were performed on seedlings of both weed species. Seedlings were germinated and grown as described above except five seedlings were grown in each pot. When the plants had reached the 3-leaf stage, they were 1aC-glufosinate sprayed with glufosinate (150 g ai ha-1) and immediately afterwards 2 ¡.rL (250 Bq pL-1 made up in a commercial formulation of glufosinate at 1 .2 g ai L-1) was applied to the center of the 2nd leaf and the area of application clearly marked. Pots were kept in the same growth room after treatment. Five plants were harvested from each replicate after 5, 10, 24,48 and 72 hour intervals. Each plant was divided into three segments, the treated area (treated leaf), area above the treated section (leaf tip) and rest of the shoots. The treated leaf was washed in 5 mL 0.1%
(v/v) Triton X-1OO in water. The amount of radioactivity present in the wash was determined using
LSS following the addition of scintillation fluid. The harvested shoots were dried in an oven at 60'C for 48 hours and combusted in a biological sample oxidizer. The COz released was trapped in a carbon trapscintillation fluid mix. The amount of radioactivity per sample was determined by LSS.
Experiments contained five replicates and were repeated three times. All data were pooled and the mean and the standard error were calculated.
2.3. Results
2.3.1. Dose Response Studies
Application of glufosinate to A. steritis and L. rigidum plants grown under controlled growth environments showed A. sterilis was better controlled with glufosinate when compared to
L. rigidum. A rate of 300 g ai ha-1 was sufficient for full mortality ol A. sterilis when nearly 60%
survival was observed for L. rigidum at this rate (Figure 2.1A). The rate required to reduce the
growth by 50% (LDso) was 156.4 and 41 1.6 g ai ha-l lor A. steritis and L. rigidum respectively
(Table 2.1). This indicated that almost three times as much glufosinate was required to control 50%
of the population of L. rigídum when compared to A. sterilis under controlled growth conditions of
15/2OC. L. rigidum seedlings showed signs of damage after application of 150 g ai ha-1
27 Chopter 2: Glufosinote Efficocy in L. rigidunond A. sîerilis
glufosinate, particularly tip burn, however all plants grew back and the dry weights were reduced by only 45/" (Figure 2.18). ln contrast A. sterilis plants were severely affected by this rate of glufosinate with a dry weight reduction of over B0%. Experiments conducted under field conditions in winter, outside the Waite Campus, South Australia also showed that A. sterilis was better controlled by glufosinate than L. rigidum. The LD5e lor L. rigidum was approximately twice that of
A. sterilis (Table 2.1). Experiments conducted under growth room conditions as wellas underfield conditions demonstrated that glufosinate provided better control ol A. sterilis than L. rigidum.
Table 2.1: LD5s values for glufosinate (O to 1200 g ai ha-1) in L. rigidum and A. sterilis, as determined through three dose response experiments conducted under controlled growth conditions and for experiments conducted outdoors in winter.
Species Growth Room (15l2OC) Field Experiments
L. rigidum 411.6 t32.2 647.0 r 58.1
A. sterilis 156.4 t26.9 390 + 61.6
28 Chopter 2: Glulosínale Efficocy in L. rígidunond A. sterilis
A 100
80 \oo\ tgou .¿ ã40
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
B
3
o)
.9 2 o = o 1
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 2.1: Survival (A) and dry weights (B) of L. rigidum (r) and A. sterilis 1o) in response to treatments with varying dose rates of glufosinate under controlled growth room conditions. Values are means t S.E. of 15 replicates
29 Chopter 2: Glufosinole Efficocy in L. rigídunond A. sterilis
2.3.2. GI utamine Synthetase Assays
Glufosinate readily inhibited GS activity in vitro in both species (Figure 2.2). Ihe l5e valuê was 5.4 pM for L. rigidum and 8.9 pM for A. sterilis. This difference in GS sensitivity to glufosinate observed would not be sufficient to account for the greater tolerance of L. rigidum to glufosinate.
à := 100 o(u gõE^ 80 9È Ë5 60 =oØ-o gÐ ¿o .E (ft Ë20 6 0 0.01 0.1 1 10 100 G lufosinate concentration (mM)
Figure 2.2: Glutamine synthetase activity in L. rigidum (r) and A. steritis 1r) grown under controlled growth conditions. Data represents the mean+ SE of three replicate experiments. l5e wâs 5.4 pM
for L. rigidum and 8.9 ¡rM for A. steritis. Enzyme activity of the control was 4.95 f 0.36 pmol mg-1 protein min-1for L. rigidumand 3.78 !0.77 ¡rmol mg'1 protein min-1for A. sterilis. The equations of (ross 4-loex)L1'23)¡ (roo8's-roox)11 26)¡ the curves are: !=!./a 95.9/(1+10 for L. rigidum and y=3.7a 93.3/(1+19 lor A. sterilis.
30 Chopler 2: Glufosinote Efficocy in L. rigidumand A. sterilis
2.3.3. Spray Retention
When plants were treated with a mixture of glufosinate (Basta@) and Chicago Blue, it was found that L. rigidum retained more glufosinate on dry weight basis (1.3 t 0.2 pmol g dw-1 1n = 12)) than
A.steritis (O.B t 0.1 pmol g dw-t (n = 12)). Therefore, increased susceptibility to glufosinate in
A. sterilis was not due to greater retention of the herbicide by the plants.
2.3.4. Metabolism
1aO-glufosinate ln these experiments , 92o/o oÍ the radioactivity of the applied ran as a single spot.
Al 72 HA-l 92% of the radioactivity extracted from L. rigidum co-chromatographed with the glufosinate standard and 80% of the extracted radioactivity from A. sterilis co-chromatographed with the glufosinate standard. This indicates no significant metabolism of glufosinate in L. rigidum and only a small amount in A. sterilis. Therefore, differential detoxification of glufosinate could not account for the increased tolerance ol L. rigidum.
2.3.5. Absorption and Translocation Studies
Studies on absorption and translocation of the radiolabeled glufosinate showed an identical pattern of absorption into both species. Absorption was 44 and 43"/" lor L. rigidum and A. sterilis 5 HAT, toC-glufosinate respectively. Within 24 HAT 98 to 99% of the applied was absorbed by leaves of 1oC-glufosinate both species (Figure 2.3). Of the absorbed 55% was translocated to the tip of the treated leaf of A. sterilis,whenTS'/" was observed in the leaf tips ol L. rigidum24HAT (Figure 2.4).
The herbicide continued to move to the leaf tip throughout the course of the experiment. A
difference of 18 to 20% translocation to the leaf tips until 72 HAT was maintained in L. rigidum over toO-glufosinate A. steritis (Figure 2.4). The retained in thetreated leaf decreased with time in both
species but was higher in A. sterilis (Figure 2.5). After 24h only 14/" ol the labeled glufosinate was
retained in the treated area of L. rigidum, whereas 28/" was retained in A. sterilis. The amount of
glufosinate found in the rest of the shoots increased with time over the course of the experiment.
31 Chopler 2: Glufosinote Efficocy in L. rigídunond A. slerílís
The amount translocated to the meristematic reg¡ons and to other leaves (basipetal) 24 HAT was 1aO-glufosinate 1 1 and 22% ol the applied in L. rigidum and A. sterilis, respectively (Figure 2.6).
100
IG .= 80 oâø 5E cDo J)6t\vv ^n :obO ôL F IJ 40 õ: -äE .ct820
0 0 20 40 60 80 Time (HAT)
Figure 2.3: Absorption of radiolabeled glufosinate into leaves of L. rigidum (r) and A. steritis (o)
under controlled growth conditions. Values are means t S.E. of 15 replicates
32 Chopter 2: Glufosinole Efficacy in L. rigidumond A. sterilis
100 It f;A Bo tE Egg Ê 60 OG E=. 40 gE (,E20
0 0 20 40 60 80 Time (HAT)
Figure 2.4: Glufosinate translocated from the point of application to the upper leaf of L. rigidum (t)
and A. steritis (o) under controlled growth conditions. Values are means t S.E. of 15 replicates
100 ït Oa .=(úX ç80
Ë.Euo(úG 'ã *. ¿o .-3s 620
0 0 20 40 60 80 Time (HAT)
Figure 2.5: Glufosinate retained in the treated leaf of L. rigidum (r) and A. sterilis 1o) under controlled growth conditions. Values are means t S.E. of 15 replicates.
33 Chapler 2: Glufosinate Efficocy in L. rigidumond A. sferilis
30 f, q) 25 (ú o f, o (¡) Ø t¡ 20 c o G to -o 1 5 (¡) G (ú o Ë, rt '6 -o 1 0 o ¡¡\ f 5 o 0 0 20 40 60 80 Time (HAT)
Figure 2.6: Glufosinate translocated from the point of application to meristematic regions and to rest of the leaves ol L. rigidum (r) and A. steritis 1o) under controlled growth conditions. Values are means + s.E. of 15 replicates.
2.4. Discussion
The dose response curves of L. rigidum and A. sterilis demonstrated that glufosinate could control
A. sterilis easily whereas L. rigidum was poorly controlled. The LD5e value for A. sterilis was 156.4 g ai ha-1 and f or L rigidum was 41 1 .6 g ai ha-1. Therefore, to achieve 50% mortality with glufosinate it requires more than twice as much herbicide for L. rigidum when compared to A. sterilis. Such variation in efficacy of glufosinate has been observed in previous studies. Mersey et al. (1990) found the rate of glufosinate required to reduce growth by 50% was 65 and 500 g ai ha-1 lor Setaria viridis and lor Hordeum vulgare respectively. ln a similar study Steckel et al. (1997) have found the rate of glufosinate required to reduce shoot dry weight by 50% was 69 and 235 g ai ha-1 Íor Setaria faberiand Chenopodium album. Our results on whole plant dose response to glufosinate showed that such variation exists between closely related grass species such as A. sterilrs and L. rigidum.
34 Chopler 2: Glufosínote Efficocy in L. rígidumond A. sterilis
These stud¡es confirmed that the rate of 300 g ai ha-1 of glufosinate would easily control Avena sterilis when less than 50% ol L. rigidum was controlled at this rate.
GS activity was similar in the two species and was inhibited by glufosinate (Figure 2.2). The l5s value was approximately 6 pM lor L. rigidum and about B pM for A. sterilis indicating that GS from
L. rigidum was slightly more sensitive to glufosinate than that from A. sterilis. Therefore, a difference in GS sensitivity to glufosinate does not account for the greater tolerance ol L. rigidumlo glufosinate. Ridley and McNally ('1985), after studying GS activity in seven species of plants reported that the inhibition of GS in these plants were similar. They concluded that the variations in whole plant susceptibility to glufosinate may not be related to differences in the inhibition of GS enzyme.
The spray retention experiments conducted with glufosinate suggest that the leaf size, plant architecture and the size of the plant do not contribute for greater or lesser glufosinate efficacy.
Studies conducted on glufosinate metabolism in the two species showed no significant metabolism of glufosinate in L. rigidum and only a small amount in A. sterilis. Therefore, differential detoxification of glufosinate could not account for the increased tolerance oÍ L. rigidum. Previous studies on glufosinate metabolism showed low or minimal metabolism of glufosinate in
untransformed plants. Mersey et al. (1990) showed no significant metabolism of glufosinate in
H. vulgare and in S. viridis when the extracts were analysed by TLC. According to these authors
more than 85% of the radioactivity was found at the R1 value of the glufosinate standard and a
small quantity of radioactivity was detected in the samples at R¡ values of known nontoxic
metabolites. Hence it was concluded that the detoxification of glufosinate is not an important
mechanism conferring tolerance to glufosinate in the above species. Similarly, Hass and Muller 1aO-glufosinate (1987) reported that in a TLC study only 11 to 13% of the absorbed was
metabolized in the shoots and 0.2 to 0.6% in the roots of three species. After studying twenty plant
species for glufosinate metabolism, Jansen et al (2000) concluded that even though the metabolic
capacity to transform and detoxify glufosinate varied noticeably between species, none of the plant
species tested had a metabolic capacity sufficient for complete tolerance of glufosinate. Dröge-
35 Chopter 2: Glufosinote Efficocy in L. rigidunond A. sferilís
Laser et al. (1994) after studying the metabolites of glufosinate in a similar cellulose TLC method to the present study showed that in unmodified plants small quantity of glufosinate was metabolized and these metabolites were clearly different f rom the parent compound.
Neto et al. (2000) proposed lhal Xanthium strumariurn was more susceptible to glufosinate than
L pomoea purpureaand Commelina diffusa because it absorbs three times more glufosinate than l. purpurea and six times more than C. diffusa. However, in the present study an identical pattern of absorption of glufosinate in L. rigidum and A. sterilis implied that differential sensitivity could not be due to differences in glufosinate absorption in the two species. The presence of relattvely more laO-glufosinate in the leaf tips of L. rigidum and more in the shoot bases of A. sterilis indicated increased acropetaltranslocation and reduced basipetaltranslocation to meristematic regions in L. rigidum when compared to A. sterilis. Mersey et al. (1990) also found that in the more tolerant H. vulgare, two-fold more herbicide was translocated to the tip of the treated leaf than in the more susceptible S. viridis. Herbicide translocation can be vitally important for efficacy. The pattern of translocation is known to differ between herbicides and between species. ln a study with glyphosate, Martin and Edington (1981) showed that H. vulgare had a similar distribution of the herbicide to that observed for glufosinate in this study. Preston et al. (1992) and Purba et al.
(1995) showed that paraquat resistant Hordeum gluaucum and H. leporinum had reduced basipital translocation of paraquat compared to susceptible plants. The mechanism of this is not known but suggested to be reduced absorption of paraquat into cells (Preston et al. 1992). ln the present study, less than 25% of the absorbed glufosinate was translocated to the meristematic region in both species. However, more than twice the amount of the radiolabeled glufosinate was translocated in a basipetal direction 24 HAT in A. sterilis when compared to L. rígidum. Glufosinate that accumulates in the leaf tip has moved apoplastically in the transpiration stream. Glufosinate that accumulates in other leaves and the rest of the shoots has moved symplastically via the
phloem from the treated leaf. lt would seem that there is reduced symplastic movement of
glufosinate in L. rigidum.
Reduced symplastic movement of the herbicide in L. rigidum is correlated with decreased
sensitivity to glufosinate in this species and is coupled with increased translocation of the herbicide
36 Chopter 2: Glufosinote Eflicacy in L. rigidumond A. sterílis to the tip of the treated leaf. Dröge-Laser et al. (1994) after studying acetyl L-glufosinate and L- glufosinate showed both compounds have similar distribution patterns in plants. For both forms of glufosinate most of the herbicide was retained in the treated leaf with a small amount transported to the stem and to upper leaves (Dröge-Laser et al. 1994). ln a similar study Beriault (1999) observed that acetyl-L-glufosinate was much more phloem mobile and substantial amounts were transported to the upper leaves, flowers, and to the roots whilst the translocation of L-glufosinate was limited. This limited translocation of L-glufosinate was explained as a result of the combined effects of ammonia accumulation, reduced carbon accumulation and depletion of glutamine
(Beriault, 1999).
Several other post emergence herbicides have demonstrated similar phloem translocation in other species. ln our study, both species showed phytotoxic effects caused by glufosinate. However, severe damage was observed in ,4. sterilis from which only the plants that were exposed to the lowest dose rate were able to recover. ln contrast, some L. rigidum plants were able to recover from all but the highest rate of application. Both species showed the appearance of the symptoms in the tip of the treated leaf. However, a rapid development of chlorosis and necrosis was apparent in A. sterilis when compared to L. rigidum. Mersey et al. (1990) also found that the injury symptoms in H. vulgare progressed from the leaf tips towards the base of the leaves, whereas in
S. viridis, symptoms developed over the entire leaf . Neto (2000) found the same pattern of injury symptoms in C. diffusa, l. purpurea and in X. strumarium.
The equal susceptibility of GS of L. rigidum and A. sterilis (Figure 2.2) means that the different patterns of symptoms displayed by the two species is reflective of the ability of leaf applied glufosinate to react the target enzyme. This would lead to a greater extent of toxic impacts, such as ammonium accumulation and inhibition of photosynthesis, in the leaves of A. sterilis compared lo L. rigidum. , Therefore, this different pattern of symptom development was likely a result of more basipetal translocation of glufosinate in A. sterilis. lt is possible that the reduced basipetal translocation in L. rigidum may be the result of glufosinate being less readily absorbed into cells compared lo A. sterílis. A herbicide that is less readily absorbed by cells would be expected to accumulate in the leaf tips as was observed for L. rigídum.
37 Chopter 2: Glufosinole EÍficacy in L. rþidun ond A. sterílis
ln conclusion the current study demonstrates that A. sterilis should be controlled readily by glufosinate but control of L. rigidum by this herbicide would be poor and the ability of glufosinate to translocate to the meristematic zone is crucialfor effective control of grass species.
38 Chopter 3: Glufosinole Efficocy in P. rophanislrum
Chapter Three: Environmental Effects on Glufosinate Efficacy ¡n Raphanus raphanistrum
3.1. lntroduction
Although glufosinate is considered to be a non-selective herbicide, several workers have shown that its efficiency depends on environmental factors such as temperature, relative humidity (RH), and light intensity (Carlson, 1984). Anderson et al. (1993) demonstrated that both temperature and relative humidity have a considerable effect on the activity of glufosinale in Hordeum vulgare and
Setaria viridis. This study demonstrated that low temperatures prior to glufosinate application reduced its efficacy against H. vulgare and S. viridis. Coetzer et al. (2000) studied glufosinate efficacy in Amaranthus retroflexus, A. palmeriand A. rudis. fhey reported that all three species were controlled better at 90% RH than at 35% RH. They concluded that RH did not alter glufosinate absorption but increased RH increased the subsequent translocation out of the treated leaves. Studies conducted on glufosinate efficacy under differential light intensities in Galium aparine showed that low light intensity reduced the accumulation of ammonia in the plants and subsequently reduced glufosinate efficacy (Peterson and Hurle 2001).
Raphanus raphanistrum is one of the most widespread and troublesome broad leaf weeds in the
cropping regions of southern Australia. ln particular it presents a major problem to the canola
industry. To date there are no herbicides available that can eradicate H. raphanisfrum without
damaging standard varieties of canola as both canola and F. raphanistrum are genetically related.
The only possibility is to use herbicide resistant canola cultivars. Such varieties would allow the
use of non-selective herbicides such as glyphosate and glufosinate to be used in the crop to
control all weeds including R. raphanistrum. As cited earlier, field studies conducted with
glufosinate against R. raphanistrum and other major weed species in Western Australia showed
variable performance of the herbicide in contrast to the field studies conducted in Europe showed
39 Chopter 3: Glufosinote Efficocy in P. raphanisfrun
R. raphanistrum was easily controlled with glufosinate (Michael Clarke, pers. comm.). Similarly, when several Australian populations of Æ. raphanistrum were tested in Europe they were easily controlled with glufosinate (Michael Clarke, pers. comm.) suggesting that the poor control of F. raphanistrum by Glufosinate in Australia may be related to local environmental conditions. Studies on herbicidal activity against R. raphanistrum are limited and to date no study has investigated the influence of environmental factors on absorption and translocation of glufosinate in this weed under
Australian conditions.
The objective of this study was to determine the effect of environmental factors such as temperature and light intensity on glufosinate efficacy in H. raphanistrum in order to develop an understanding of why this weed is poorly controlled under Australian winter conditions.
3.2. Materials and Methods
3.2.1. Dose Response Studies: Population Differences
Several dose response pot experiments were performed outside at the Waite Campus in winter
(May to August 2001). Three populations of seeds were collected f rom farms in Victoria, New
South Wales and South Australia and one population was obtained from Berkshire, England
(Curtsey of Herbiseeds Ltd). Dehulled R. raphanisfrum seeds were soaked in 1:1 sodium hypochlorite for 30 minutes followed by a 30 minute soak in water. The seeds were germinated on
0.6% (w/v) agar in a germination cabinet with 12h, 19"C day (30 pmol.m't s-t¡ 12h, 19'C night after incubating at 4'C lor 24h. Germinated seedlings were transplanted into 1Scm square pots filled with potting soil. Each pot contained four F. raphanistrum seedlings. At the 2-4 leaf stage, seedlings were sprayed with glufosinate (Basta@), using a laboratory moving boom sprayer as described in Chapter 2. Plants were returned outside after spraying and were assessed for survival 30 DAT. Plants were considered as dead provided the meristematic area was completely burnt out. According to our experience plants with green meristems regrew 30 DAT. Therefore,
40 Chopter 3: Glufosinote Efficacy ín P. rophanísfrun
any plant that had green meristematic tissue was classified as a survivor. Plants were harvested and the shoot dry weights were determined after drying the shoots at 60'C tor 72h.
3.2.2. Dose Response Studies: Temperature Effect
Several dose response pot experiments were performed in controlled growth chambers using the population of R. raphanistrum collected from South Australia. Seedlings were germinated as mentioned above. The plants were grown under different temperatures (5/10"C, 15/20'C and
2Ol25"C). The night temperatures were kept 5'C less than the day temperatures to simulate field conditions. For example under cold conditions of 5/10'C, the night temperature was 5"C when the day temperature was 10'C. However, in the growth rooms the relative humidity and light intensities were kept constant throughout the experiment. Relative humidity was maintained between 60 to
70% whilst the light intensity was kept at 553 p mol m'ts'2. At the 2-4leaf stage seedlings were sprayed with glufosinate (Basta@) as described above. Plants were returned to the growth chamber after spraying and were assessed for survival 30 DAT. Plants were harvested and the shoot dry matter weights were determined after drying the shoots at 60qC for 48h.
3.2.2.1.The Effect of Temperature Before and After Treatment on Glufosinate
Efficacy
Dose response experiments were conducted to observe the effect of post application on temperature on glufosinate efficacy in R. raphanistrum. Plants were grown in two growth chambers where the temperatures were 5/10"C and 2Ol25"C (nighV day temperatures) with the same light intensity and RH. Plants were sprayed with glufosinate (Basta@) allhe2-4leaf stage
as described above. Plants kept in each growth room were divided into four replicates (treatments)
prior to the spray treatment. Each replicate was a full dose response experiment provided with a
different temperature treatment after the spray application. ln each growth room one replicate
(Treatment 1) was kept under the same growth temperature through out the experiment.
Treatment 2 was transferred to the other growth room for three days and was returned back to the
original growth room for the duration of the experiment. Treatment 3 was kept in the original
41 Chopler 3: Glufosinote ElÍicocy in P. raphonisfrun
growth room for three days and was transferred to the other growth room for the duration of the experiment and Treatment 4 was moved to the other growlh room directly after the spray treatment and was kept there. Survival was assessed after 30 days. The plants were harvested and the shoot dry weights determined after drying the shoots at 60"C for 48h.
3.2.3. Dose Response Studies: Light lntensity Effects
Dose response pot experiments were performed in controlled growth chambers under different light intensities. Seedlings of F. raphanistrum were germinated as described above. Temperature and relative humidity were kept constant while the light intensity was varied between pots by covering plants with shade cloth that reduced the light intensity by 60%. The pots kept under high light intensity received 553 pmol m-2s-1 whilst the pots under the shade cloth received only 217 pmol m-2s-1. The relative humidity was kept between 60-70% throughout the experiment. Plants at
The 2-4leaf stage were sprayed as described earlier. After 30 days plants were harvested and shoots were dried in an oven at 60oC for 72 hours before dry weights were taken.
3.2.4 Glutamine Synthetase Assays
GS assays were performed using seedlings of wild radish grown under different temperature
regimes. When the seedlings were at the 2-4 Þaf stage, fully expanded leaves were harvested and GS assay was performed as described by Mandersceid (1993). Fresh leaf tissue (59) was
ground with acid-washed sand at 4oC in pre-cooled Buffer A (50mM Tricine,0.5mM EDTA, 1mM
MgSOa, lOmM DTT) and the homogenate was centrifuged for 10 mins at 20,000x g. The reaction
was started by addition ol2OO¡IL of the supernatant to 2mL of assay buffer (100mM Hepes, 50mM
MgSOa, 10mM EDTA, 1OmM NH2OH, 15mM ATP, 12.5mM glutamate) containing glufosinate
(0.05-1OpM). The reaction proceeded for 15 mins at 37'C and was stopped by adding 1mL of
0.37M FeOls, 0.67M HCI and 0.2M trichloroacetic acid. Formation of y-glutamylhydroxamate was
measured spectrophotometrically at 540nm.
42 Chopter 3: Glufosinqle EfÍicocy in P. raphanislrun
3.2.5. Absorption and Translocation: Temperature
Studies on absorption and translocation of glufosinate were performed using 2-4 leaf stage seedlings. They were germinated and grown as described above at either 5/10'C or 20125"C except five seedlings were grown in each pot. When the plants had just reached the 3-leaf stage 1aC-glufosinate they were sprayed with glufosinate (300 g ai ha-1) and immediately afterwards 2¡tL at 250 Bq-1 (made up in commercial formulation at 300 g ai ha-1) was applied to the center of a f ully expanded leaf and the area of application clearly marked. The pots were kept in the same growth room after treatment. Five plants were harvested f rom each replicate after 5, 10, 24, 48 and 72h intervals. Each plant was divided into five segments, the treated area (Treated Leaf), area above the treated section (Leaf tip), rest of the treated leaf, apical meristem and rest of the shoots (Rest of the plant). The treated leaf was washed in SmL 0.1% Triton X-100 in water. The amount of radioactivity present in the wash was determined as described in Chapter 2.The harvested shoots were dried in an oven at 60'C lor 24 hours and combusted in a biological sample oxidizer as described in Chapter 2.
3.3. Results
3.3.1. Population Differences
During germination and growth, the four R. raphanistrum populations derived from different
locations showed variation in relevance to leaf size, intensity of leaf hair and flower colour.
However, as shown in Figure 3.1, the response to glufosinate in all the populations showed a similar response up to 600 g.ai.ha-1. Some variation was evident between populations at the higher
rates of glufosinate, however, none of the populations were adequately controlled under the
conditions used (Figure 3.14). The LDso values obtained varied between populations. The
population from NSW had the lowest LD5e at 1 157 g ai ha-l which was followed by the populations
from South Australia (LD5e to be 1249 g ai ha-1), Europe (LD5q at 1486 g ai ha-1) and that collected
from Victoria (LD5s al 231S g ai ha-1). These results showed that even though there were
differences in the LD5e values of the populations tested, under southern Australian winter
43 Chopler 3: Glufosinole Efficacy in R. rophanislrum
conditions glufosinate had poor control ol H. raphanistrum regardless of the populations tested. All populations tested showed reduction of dry weights as the concentration of glufosinale increased.
A 4 fold decrease in dryweights at 1200 g ai ha-1 in the SA, N.S.W and European populations was observed whilst the Victorian population showed 1.4 fold decrease (Figure 3.18).
44 Chopter 3: Glufosinote Efficocy in P. raphanistrum
A 100
80
o\rO r60 .l ãqo
20
0 0 300 600 900 1200 1 500 Glufosinate (g ai ha-1)
6 B
5
cn 4 Ø c 'õct) 3 = o 2
1
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 3.1: Survival (A) and dry weights (B) of several populations of F. raphanistrum collected from different geographical regions, grown and treated under South Australian winter conditions outside at the Waite lnstitute. Populations sourced from New South Wales (o), South Australia (o), Victoria (r) and Europe (o). Values are means + S.E. of 12 replicates.
45 Chopter 3: Glufosinofe Efficacy in P. raphoníslrun
3.3.2. Dose Response Studies: Temperature Effect
The results of the dose response experiments under controlled growth room conditions demonstrated that glufosinale efficacy was enhanced by increasing temperature (Figure 3.24). At a rate of 6009 ai ha-1 glufosinate, B0% survival occurred under 5/10'C when the same rate of herbicide was sufficient to reduce the survival to 30% under 1 5120"C and 10% under 20l25"C in the same population of R. raphanistrum. Under cold conditions (5/10'C) even the highest rate of glufosinate (1200 g ai ha-1) only controlled 50% of the population in contrast to warmer temperatures (15/20'C and20l25C) where 100% mortality was achieved. The LDso vales were significantly different when grown and treated under the three temperature regimes. Under cold conditions of 5/10'C the LDso was 1160 g ai ha-1 whilst under 15l2OC it was only 402 g ai ha-l which was further reduced lo 287 g ai ha-1 under warm temperatures ol2Ol25C. Dry weights of the above experiment showed only slight differences belween temperatures after 30 days (Figure
3.2 B). All treatments showed a reduction of dry weights. However, the plants kept under cold conditions of 5/10'C grew back approximately two weeks after the treatment whereas the ones that were exposed to warmer conditions had no regeneration.
46 Chapter 3: Glufosinafe Efficacy in R. raphanísfrum
100
80
Eoo (ú .¿
Ø=40 20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
25 B
20 ol U' 1 5 'õct)
= 1 0 o 5
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-l)
Figure 3.2: Survival (A) and dry weights (B) of ,9. raphanistrum in response to varying dose rates of glufosinate under different temperature conditions 5/10"C (o), 15l2OC (u) and 20125'C (t). Values are means t S.E. of 12 replicates.
47 Chopter 3: Glufosinote Efficacy in R. rophoníslrun
3.2.2.1. The Effect of Temperature Before and After Treatment on Glufosinate
Efficacy
The results of the dose response experiments on the effect of temperature after glufosinate treatment showed that when cold grown plants were transferred to warm conditions, glufosinate efficacy was enhanced (Figure 3.3 A). Plants that were transferred into the warmer growth room immediately after the glufosinate treatment for the duration of the experiment showed a significant increase in glufosinate efficacy when compared to plants kept under cold conditions through out the experiment. With 3OO g ai ha-1 of glufosinate, the survival of R. raphanistrum was reduced by half under warm temperatures when compared to the plants kept under cold conditions. At the field rate of 600 g ai ha-1 glufosinate, more than five fold reduction of survival was observed in plants that were transferred to warm conditions when compared to the plants that were kept under cold conditions. Transferring plants to the warmer conditions for 3 days also resulted in better control than the plants kept under cold conditions throughout the experiment. The LD5e values of this experiment confirmed the observations made on survival ol R. raphanistrum. As shown in
Table 3.1 the highest LD5qwas 1130 g ai ha-1 for plants kept under cold conditions throughout the experiment and the lowest was for those plants that were transferred to the warmer growth room for the duration of the experiment (LDs' 502 g ai ha-1). The other two lreatments showed that the plants kept under warmer growth conditions for three days were better controlled compared to the plants kept for 3 days under cold conditions. Despite variations in survival, dry weights of plants were similar for alltreatments (Figure 3.3 B).
48 Chopter 3: Glufosinote Efficacy in P. rophanistrum
A 100
BO 10o\ õ60 .¿ õ40
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-l)
14 B
12
CD, 0 U' E .9 8 o 6 = o 4
2
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 3.3: Survival (A) and dry matter weights (B) of F. raphanistrum grown under cold conditions, treated with varying dose rates of glufosinate and were exposed to different temperature conditions after the spray treatment, 5/10'C constantly (r), 5/10'C for 3 days lhen 20125C (¡), 20125C lor 3 days then 5/10'C 1o) and 2Ol25"C constantly (o). Values are means t S.E. of 9 replicates.
49 Chopler 3: Glufosinote EÍficocy in R. raphdntslrum
Table 3.1: LDso values for glufosinate of R. raphanistrum, as determined through three dose response experiments conducted under controlled growth conditions. Plants were originally grown under cold temperatures of 5/1OqC.
Temperature after Spraying LDso (9 ai ha-1)
5/10"C constantly 1 130
5/10'C for 3 days lhen 20125'C 824
20125"C for 3 days then 5/10'C 806
20125C constantly 502
As shown in Figure 3.4, when the same experiment was repeated with plants grown under warm temperatures (20125'C) plants that were kept under warm temperatures (20/25"C) through out the experiment were well controlled than the ones transferred from the warm (20125'C) to the cold temperatures after the spray treatment (Figure 3.4A). Under warm temperatures 100% mortality was achieved with 300 g ai ha'1. However, plants that were transferred to the cold growth room directly after the spray treatment were not controlled totally even with 1200 g ai ha-1 glufosinate.
The LD5e value was 116 g ai ha-1for those plants that were kept under warm temperatures through
out the experiment when the plants that were transferred to the cold growth room after the spray treatment showed this was 305 g ai ha-l lTable 3.2). Plants that were kept under warm conditions
for 3 days or the plants that were transferred into the cold growth room for 3 days did not show
much difference in survival. The LD5q values showed a slight difference. The LDso was 141 g ai
ha-1 and 150 g ai ha-1 for the plants that were kept under warm temperatures for 3 days and the
ones that kept under cold conditions for 3 days respectively. The dry weights were similar between
treatments except plants transferred to cold conditions maintained a greater dry weight (Figure
3.48).
50 Chopter 3: Glufosinote Efficacy in R. rophanisîrun
100 A
80 :O o\ 860 .t õ+o
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-r)
14 B
12
UT 1 0 Ø .9 8 o = 6 o 4
2
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-l)
Figure 3.4: Survival (A) and dry weights (B) of F. raphanistrum subjected to varying dose rates of glufosinate when grown under warm temperatures of 20l25"C and were transferred to differential growth temperatures,2Ol25"C constantly (o),5/10'C for 3 days lhen20l25"C (o), 20l25"Cfor 3 days then 5/10'C (À) and 5/10'C constantly (A). Values are means t S.E. of 9 replicates.
51 Chopter 3: 6lufosinole Efficocy in P. rophonisfrum
Table 3.2: LD56 values for glufosinate of F. raphanistrum, as determined through dose response experiments conducted under controlled growth conditlons. Plants were originally grown under warm temperatures of 20l25eC
Growth Temperature LDso (g ai ha-1)
2Ol25C constantly 116
5/10"C for 3 days then 20l25"C 150
2Ol25C for 3 days then 5/10"C 141
5/10'C constantly 305
3.3.3. Dose Response Studies: Effect of Light lntensity
Figures 3.5 A, and B, show the effect of light intensily (217 and 553 pmol m-2s-t¡ under cold
(5/10'C) and warm (20125"C) temperatures. The results of the pot experiments conducted in growth chambers showed that light intensity did not greatly influence efficacy of glufosinate when kept at cold temperatures of 5/10"C (Figure 3.5 A). There were no mortality up to 600 g ai ha'1 at either light intensity and the highest rate of glufosinate gave 40-50% control ol R. raphanistrum.
Under warmer temperatures (20l25'C), the plants kept under both light intensities were well controlled. Those plants that were kept under low light intensity showed 100% mortality at 300 g ai ha-1. Under high light intensity 92T" morlality was observed at this rate (Figure 3.5 B). The LDue values did not show a significant difference between the two light intensities under each temperature (Table 3.3). However, a significant difference in LD56 was found between temperatures. Under cold conditions of 5/10'C the LD5e for high light intensity was 1231 g ai ha'1
and under low light intensity this was 1200 g ai ha-1. Under warm conditions with high light
'17 intensity, the LD5s was 166 g ai ha-1 and under low light was reduced to 1 g ai ha-1.
52 Chopter 3: Glufosinote Efficocy in P. raphanístrum
A 100
BO o\-o õ60 .t ã40
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
B 100
80
òq 660 .l ãqo
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-l)
Figure 3.5: Survival of F. raphanistrum to varying dose rates of glufosinate under high (r) and low (n) light intensities at 5/10'C (A) and 20125"C (B). Values are means t S.E. of 9 replicates.
53 Chapter 3: Glufosinole Efficocy in R. raphonisfrum
Table 3.3: LD5e values for glufosinate for R. raphanistrum, as determined through three dose response experiments conducted in controlled growth chambers under different light intensities.
Growth Temperature Cold conditions (5/1 0"C) Warm conditions (20125"C)
LDso (9 ai ha-1) LDso (g ai ha-1)
High light 1231 166
Low light 1 200 117
3.3.4. Glutamine Synthetase Assays.
Glufosinate readily inhibited GS activity in vitro for plants grown under both cold and warm
temperature regimes (Figure 3.6). The l5e value was approximately 5,9M. Therefore, enhanced
efficacy with increasing temperatures does not seem to be an effect of enhanced GS sensitivity to
glufosinate in R. raphanistrum.
54 Chopter 3: Glufosinole Efficacy in P. raphanistrun
--a .E o G o 80 (EãaL^ 0È F6 60 ;o Ul -o coÐ E (ú 6= 0 0.01 0.1 1 10 G lufosinate concentration (pM)
Figure 3.6: Glutamine synthetase activ¡ty of R. raphanistrum grown under 5/10'C (.) and 20125'C (r). Values are means + S.E. of 6 replicates. l5e was 1.85+0.11pM for plants kept under 5/10'C and 1 .99t0.17pM for plants kept under 20125"C. The equations of the curves are: y=5.6+g2.4/(1+1g((rosr.asrosx)-1.38)) for 5/10"c and y=6.5+g2.g/(1+1O((rosr.gg-rosx)-138)) br 2ol25c.
3.3.5. Absorption and Translocation of Glufosinate
Absorption and translocation studies conducted in growth chambers under different temperatures
indicate that glufosinate is absorbed rapidly into leaves of R. raphanistrum regardless of the
temperature (Figure 3.6). Absorption of glufosinate was 60-70% 5 HAT. Within 24 HAT 80-90% of
the labeled glufosinate was absorbed and by 72 HAT over 90% of glufosinate was absorbed into
the treated plants. Up to 24 HAT, the percentage glufosinate absorbed under 5/10"C was lower
compared to absorption under warmer temperatures of 20125"C. However lrom 48-72 HAF
absorption was same for both temperatures.
55 Chopler 3: Glufosinole Efficocy in P. raphonisfrum
10 1 00 o G tr 80 '6 o
(5 60
\to o 40 c o o- 20 o tt, l¡ 0 0 20 40 60 80 Time (HAT)
Figure 3.7: Absorption of labelled glufosinate into B. raphanistrum plants grown under 5/10"C (o) and20l25"C (r). Values are means t S.E. of 15 replicates.
A significantly higher proportion of labeled glufosinate translocated to the tip of the treated leaf (leaf tip) under cold conditions of 5/10'C when compared to warmer temperatures ot 20125C (Figure
3.74). ln the period up to 1O HAT, translocation to the leaf tip increased to 35% of the absorbed
under both temperature regimes. However from 24 lo 72 HAT the amount of glufosinate
translocated to the leaf tip increased under cool conditions, bul remained unchanged under warm
conditions. The amount of glufosinate retained in the treated leaf varied between 40-50% of that
absorbed throughout the experiment (Figure 3.78). Translocation to the rest of the plant from the
treated leaf was poor with a slight increase over time. There were no differences in herbicide
retained at the site of application between temperatures. However, more basipital translocation
was observed under warmer conditions than under cold conditions (Figure 3.7C) and a significant
difference in translocation of the labeled glufosinate to meristematic regions and to the other
leaves was observed under warm temperatures. As shown in figure 3.7D, under cold conditions,
translocation to the meristematic regions was >17o throughout the experiment whilst under warm
temperatures labeled glufosinate continued to accumulate in the meristem. By 72 HAT 4.5% of the
56 Chopter 3: Glufosinole Efficocy in R. rophanistrum labeled herbicide accumulated in the meristem showing a 5-fold difference between the two treatments (Figure 3.7D). A similar pattern was evident in the amount of labeled glufosinate translocated to other leaves (Figure 3.7E). ln the untreated leaves radiolabeled glufosinate amounted up to 1% under cold conditions but this was increased to 5.5% in untreated leaves of plants kept under warm conditions.
57 Chopter 3: Glufosinote Efficocy in R. raphanislrun
60
6
20
0 B 60
40
20
E' to c ¡¡8oo IE Ðas-o o o tr 'õ 20 o e o (Ju
4
2
0 10 E I 6
4 2
0 o206080 Tirne (HAT)
Figure 3.8: Glufosinate distribution within R. raphanistrum plants at 5/10'C (o) or 20125C (r), (A) recovered from the tip of the treated leaf (B) recovered from the treated zone (C) recovered from the base of the treated leaf (D) recovered from the meristematic zone and (E) recovered from the untreated leaves. Values are means 1S.E. of 15 replicates.
58 Chopler 3: Glufosinole Efficocy in R. raphonistrum
3.4, Discussion
Preliminary data derived from field studies conducted by the Bayer Crop Sciences Ltd, showed that glufosinate had variable performance against R. raphanistrum under Western Australian winter conditions (Michael Clark, pers. comm.). ln the present study the efficacy of glufosinate against different populations of R. raphanistrum collected from diverse geographical areas was tested under southern Australian winter conditions. ln addition, efficacy of glufosinate was tested against
R. raphanisfrurn grown inside growth rooms under simulated winter conditions (5/'10'C, RH 0-70% and light intensity 553 pmol m-'s-t). The results of outside pot experiments showed that when the herbicide was used at the field rate (600 g ai ha-1), plant mortality reached a maximum ol 20%.
Results of the growth room experiments also confirmed the results showing only 20% plant mortality. Therefore, it can be concluded that Australian winter conditions are critical in determining glufosínate efficacy against B. raphanístrum.
ln the subsequent experiments, the effect of temperature on glufosinate against R. raphanistrum was studied in detail by growing the plants inside a growth room at different day/night temperatures
(5/10'C, 15l2O"C and 2Ol25"C) with constant RH and light intensity. Glufosinate efficacy was markedly enhanced at higher temperatures. For example,90-100% mortality was achieved at
20125C at the field rate of glufosinate. This was fufiher demonstrated by experiments where plants were moved from cold (5/10'C) to warm (2Ol25C') temperatures. ln this experiment when the plants were grown, sprayed and maintained at temperature 20125'C 100% plant mortality was achieved with 300 g ai ha-1 of glufosinate (Figure 3.3). However, lhe plants grown, sprayed and maintained at 5/10'C showed 100% survival with 300 g ai ha-1 of glufosinate clearly demonstrating the role played by the temperature. The results of the same experiment showed that by transferring the plants immediately after the spray treatment from warm (2Ql25 C) to cold conditions (5/'10'C) reduced glufosinate efficacy, whilst those plants transferred from the cold to warm temperatures enhanced the herbicide efficacy (Figures 3.3 and 3.4). These results also demonstrated that more than the temperature in which the plants were grown, the temperature following the spray treatment is crucialto the efficacy of glufosinate in controlling R. raphanistrum.
59 Chopter 3: Glufosinole Efficocy in P. raphanistrum
The importance of temperature on the efficacy of glufosinate was prev¡ously reported by Anderson et al. (1993), in Hordeum vulgare and Setaria viridis. The results showed that exposure to low temperatures prior to glufosinate application markedly reduced herbicide efficacy. The same study demonstrated that as the temperature decreased, less ammonia was produced in treated plants.
Several reports suggest that temperature plays an impodant role in determining the efficacy of herbicides, other than glufosinate. Purba et al. (1995) demonstrated that paraquat resistant
Hordeum leporínum and H. glaucum were highly resistant to the herbicide when grown and treated under winter conditions. ln contrast, the same weed species were more susceptible to paraquat under summer conditions. These authors reported that under warm conditions of 30"C more basipital translocation of paraquat occurred in these populations. McWhorter and Ouzls (1994) reported that in Sorghum hatepense absorption of glyphosate was approximately doubled and the translocation slightly increased as the air temperature increased from 24'lo 35'C. Duke and Hunt
(1977) reported that cool conditions were responsible for the lack of glyphosate translocation in
Elytrigia repens at temperatures below 7'C. lt was found that E. repens control with sethoxydim was enhanced by increasing both the humidity and temperature.
The relationship between the light intensity and glufosinate efficacy has been previously studied
(Köcher 1983, Peterson and Hurle 2001). Köcher (1983) showed that high light intensity at and after glufosinate application increased the herbicidal efficacy. According to Peterson and Hurle
(2001), low light intensity reduced the accumulation of ammonia and the performance of glufosinate in the plant. The dose response experiments of the present study showed similar
results to the previous studies conducted with light intensity. However, under cold conditions light
intensity had no significant effect on efficacy of glufosinate against H. raphanistrum. ln contrast,
under warm conditions F. raphanístrum was better controlled at low light than at high light intensity
(Figure 3.5). lt is possible under low light at warmer temperatures less ammonia was produced
causing delayed appearance of phytotoxicity allowing more translocalion of the herbicide in the
plant. Such enhanced efficacy with low light has been observed with other herbicides. Xie et al.
(1996) after studying the influence of temperature and light intensity on absorption, translocation
60 Chopler 3: Glufosinqte Efficacy in R. raphanisfrun
and phytotoxicity of fenoxaprop-ethyl and imazamethabenz-methyl in Avena fatua reporled that the absorption of these herbicides was affected by low light intensily (70% shading) under warm temperatures of 20l30'C.
ln the present study, when glufosinate inhibition of glutamine synthetase was tested in vitro, the l5e value was approximately 5 pM for F. raphanistrum plants grown under either cold or warm temperatures. Therefore, enhanced efficacy under warm temperatures was not due to increased growing temperatures having an effect on enhancing glutamine synthetase sensitivity in F. raphanistrum. Previous research demonstrated that variable sensitivity against different weeds is not due to variable performance of glutamine synthetase in higher plants. Ridley and McNally
(1985) after studying GS activity in seven species of plants showed that inhibition of GS from these plants by glufosinate was similar, and concluded the variations in whole plant susceptibility to glufosinate were not caused by differences in GS.
The mechanisms behind the variable efficacy of glufosinate in F. raphanistrum under cold and warm temperatures was examined by studying the absorption and translocation of radiolabelled 1aO-glufosinate glufosinate. The results showed that the absorption of was not greatly affected by 1aO-glufosinate the growth temperature (Figure 3.7). However, the subsequent translocation of was greatly influenced by temperature (Figure 3.8). Under cold conditions a relatively large proportion of the labelled glufosinate was translocated to the tip of the treated leaf indicating significant apoplastic translocation with almost no labelled glufosinate reaching the meristematic laO-glufosinate regions. However, under warm temperatures, in addition to the large proportion of loO-glufosinate translocated to the tip of the treated leaf, 5 fold more translocated to the meristematic regions within 72 hours after the treatment when compared to the plants kept under 1aO-glufosinate cold conditions. These differences in translocation of may explain the relatively
poor activity against R. raphanistrum under cool conditions.
Translocation experiments conducted with two grass weed species L. rigidum and A. sterilis
(described in Chapter 2), also suggested that translocation of glufosinate was predominantly to the
shoot tips and that differences in translocation to the mereistematic regions resulted in differences
61 Chopter 3: Glufosinole Efficocy in P. rophanistrum
in efficacy. Steckel et al. (1997) showed that glufosinate is absorbed read¡ly by leaves oÍ Setaria faberi, Echinochloa crus-galli, Abutilon theophrasti, and Chenopodíum album and suggested that variable sensit¡vity between the species was caused by differential translocation of glufosinate in these species. The same can be true for other herbicides. Xie et al. (1996) showed that basipital translocation of imazamethabenz-methyl was increased in Avena fatuaby high temperatures whilst low temperatures ol 5110'C decreased total translocation and translocation efficiency of this herbicide. Harker and Dekker (1988) showed strong evidence that temperature increased basipital translocation, as increasing temperatures from 5/10"C to 15120C to 25l30"C resulted more translocation to shoots in a range of herbicides including glyphosate, sethoxydim, cycloxydim, and fluazifop-bulylin Agropyron repens. Purba et al. (1995) showed increased basipitaltranslocation of paraquat in paraquat resistant H. leporinum at higher temperatures.
Taken together these studies demonstrate that translocation of herbicides to the meristematic zone is vital to efficacy, such that increased translocation to the meristematic zone results in increase efficacy. Where environmental variables such as temperature, significantly reduce translocation, a reduction in efficacycan be expected. Such is the case with R. raphanistrum where poorefficacy of glufosinate is achieved under the cool, wet conditions prevalent during winter in southern
Australia.
62 Chopler 4: fmproving Glufosinote Efficocy ogoinst P. raphanístrun
Chapter Four: lmprov¡ng Efficacy of Glufosinate Against Raphanus raphanistrumwith co-Herbicides, Surfactants, EDTA, Ammonium Sulfate and Kinetin
4.1. lntroduction
14C-glufosinate Experiments conducted with on R. raphanistrum in Chapter 3 showed that
translocation of the labeled glufosinate from the treated leaf to other parts of the plant was poor and
the basipetal translocation was influenced by temperature. Southern Australian winter conditions
further aggravated this poor translocation of glufosinate to the shoot meristematic region. Bromilow
et al. (1993) and Steckel et al. (1997) also suggested that the low efficacy of glufosinate in weed
species maybe due to poor translocation of the herbicide to the meristematic regions.
The work presented in this chapter aimed at improving the efficacy of glufosinate against
R. raphanistrum under southern Australian winter conditions. One strategy was the pre{reatment of
plants with several co-herbicides and plant growth regulators prior to glufosinate application. These
agents included carotenoid biosynthesis inhibitors (amitrole, clomazone, diflufenican and
bezofenap), the chelating agent EDTA and the growth regulator kinetin. Ammonium sulfate and
EDTA were also applied simultaneously with glufosinate as a mixture. ln addition, two experimental
glufosinate formulations that contained different surfactants were also compared with the
commercial form ulation of glufosinate (Basta).
4.1.1. Caroteno¡d Biosynthes¡s lnhibitors
Carotenoids (carotenes and xanthophylls) are isoprenoids that are responsible for yellow to red
colours in plants and are present in all green tissues as components of the chloroplasts (Bramley
and Pallett 1993). Carotenoids playa useful role in photosynthesis byprotecting chlorophyllagainst
photooxidative destruction by singlet oxygen molecules (Sandmann et al. 1991). Singlet oxygen
63 Chopter 4: fmproving 6lufosinote Efficocy ogoinst R. raphonístrun
radicals are formed in excess through excited chlorophyll when the photosynthes¡s occurs too slowly to accommodate strong light (Sandmann et al. 1991). lt is known that molecules such as lycopene,
B-carotene or xanthophylls can quench singlet oxygen molecules and dissipate the generated energy as heat (Foote 1976). ln the absence of carotenoids, singlet oxygen and thriplet chlorophyll tend to initiate degrading reactions resulting in chlorophyll and membrane destruction (Bramley
1gg3). Carotenoids are also important as agents that act as accessory light harvesting pigments.
They absorb light and pass on the excitation energy by singlet-singlet energy transfer, via antenna
chlorophyll to the reaction centres. They also have a role as a precursor of the plant hormone
abscisc acid (Bramley 1993).
Carotenoid biosynthesis inhibitors (CBl) have been used as herbicides and are characterised as
bleaching herbicides that produce white foliage following the treatment. This occurs as a result of a
primary inhibition of carotenoid biosynthesis coupled to a secondary inhibition of chlorophyll
synthesis and the destruction of existing chlorophyll (Bramley 1 993). The symptoms appear on the
young developing tissues at the meristematic regions. CBI predominantly target the meristematic
regions for action and are considered to be potent molecules requiring relatively low dose rates
(Bramley and Pallett 1993). Since one of the actions of glufosinate is to slow photosynthesis it
might be expected that CBI would synergise the toxic effects of glufosinate.
Amitrole is one of the CBI that is commonly used on annual and perennial broad leaf and grass
weeds (Vecchia et al.2001). lt is an inhibitor of carotenogenesis at phytoene desaturation and
lycopene cyclization in plants (Vecchia et al.2001). When applied, amitrole is absorbed directly
through the foliage and translocates through the phloem in plants. The translocation of this
herbicide is affected by light (Vecchia et a|.2001). According to Rocca et al (2000), amitrole
produces dramatic photo-oxidation of organelles in Hordeum vulgare and the treated plants have
significantly altered membranes and possess very smallquantities of chlorophyll.
Diflufenican is another CBI used as a pre and early post emergence herbicide for the selective
control of broadleaf andgrassweedsinwintercereals(Crampetal, 1985,Kyndtetal. 1985). The
primary mode of action of diflufenican is on inhibiting phytoene desaturase in the carotenoid
64 Chopter 4: fmproving Glufosinote Efficacy ogoinst P. raphanislrun
biosynthesis pathway (Ashton et al. 1994). According to Britton et al. (1987) diflufenican treated radish plants (Raphanus sativus) had greatly reduced carotenoids and chlorophylls, but possessed a substantial amount of the biosynthetic intermediate, phytoene. These results confirmed that diflufenican caused the inhibition of carotenoid biosynthesis by blocking the phytoene desaturase act¡vity. Ashton et al. (1994) reported that diflufenican also inhibits plant fatty acid biosynthesis.
Clomazone is a broad spectrum herbicide that is absorbed through roots, emerging shoots and to a
lesser extent through leaf tissues (Devine et al. 1993). ln addition to inhibition of carotenoid
biosynthesis, it also inhibits the synthesis of gibberellins, plastoquinone, and chlorophyll in
susceptible plants (Devine et al. 1993). Application of clomazone causes bleached foliage through
reducing or stopping the accumulation of plastid pigments in susceptible species. Clomazone is
known as a plastoquinone inhibitor. Plastoquinone is an essential co-factor for caroteinoid
biosynthesis.
Benzofenap is a pre and early post emergence herbicide used mainly in rice fields (lkeda and Goh
1991). lt is effective against a range of broad leaf weeds. Benzofenap has been used in rice fields
in Japan, the far east and southern Europe since 1981. Benzofenap is classified as a pyrazole
derivative that is absorbed through the roots and the base of the target weeds (lkeda and Goh
1991). The death of the plants is through bleaching and yellowing. ln studies conducted with rice
plants under different temperatures, the phytotoxicity caused by this herbicide was nol affected by
temperature fluctuations (lkeda and Goh 1991).
4.1.2. Surfactants
The activity of foliar applied herbicides is influenced by the efficacy of cuticle retention and
penetration, tissue absorption and translocation (Kirkwood 1993). Surfactants are used as
spreaders or stickers, in herbicide formulations. Factors such as spray retention and penetration of
formulated herbicides are influenced by the oil/water partition coefficient, physicochemical
properties and concentration of organic solvents and surfactants present in the mixture (Scher
1988). According to Kirkwood (1993), surfactants have a major effect in reducing the surface
65 Chopler 4: fmproving Glufosinote Efficocy ogoinsl P. raphanistrum
tension of the spray droplets at the air-water interface and on the conlact angle of the water plant
interface. Surfactants also have an influence on spray droplet spectrum, spray drift, efficacy of
delivery to the leaf suÍace, adhesion, spreading, wetting and coverage. De Ruiter et al. (1990) and
Kirkwood et al. (1992) have suggested that at higher concentrations, surfactants have the ability to
solubilise the cuticle waxes, which would reduced surface tension, improve spray retention and
enhance cuticular penetration. Overall, surfactants are expected to increase penetration of
herbicides and potentially increase translocation.
4.1.3. Ghelating Agents
Chelating agents such as EDTA posses the ability to bind to cations. The presence of ions such as
calcium, iron, zinc, aluminium and magnesium in the water used as spray carriers have an
antagonistic affect on the herbicidal activity of glyphosate (Shillevg and Waller 19Bg). The same is
probably true for glufosinate and other acid herbicides. Calcium ions have the ability to bind to any
negatively charged herbicide molecule reducing the chance of entry. The presence of cations in the
cytoplasm may also prevent negatively charged herbicide molecules reaching the target site. ln
addition to the physical effects of EDTA in reducing binding of herbicides by calcium ions and other
divalent cations, EDTA may also influence phloem movement in plants (Knoblauch et al.2001).
Calcium has the ability to stop phloem mobility by dispersing P protein bodies, which are present in
the phloem sive elements. The exact function of these large protein bodies seems to be unknown.
It has been observed that rapid and reversible conformational switches in response to wounding
(sap sucking insects such as aphids attack), change in turgor or disturbances of membrane integrity
in the phloem can cause these P protein bodies to disperse in the presence of calcium and to block
the sieve elements as a self defence mechanism to prevent the continuous flow of phloem exudates
(Knoblauch et al. 2001). However, EDTA stops this process and the P protein bodies remain intact
leaving phloem movement unaltered.
4.1 .4. Ammonium Sulfate
Addition of ammonium sulfate to the spray solution has been shown to improve the efficacy of
gluf osinate against many weed species. For example eff icacy was enhanc ed in Galium aparine and
Brassica rapa when ammonium sulfate was used with glufosinate (Peterson and Hurle 2001).
66 Chopter 4: fmproving Glufosinote Efficocy agoinsl R. raphaníslrun
Langeluddeke et al. (1988) and Mathiassen and Kudsk (1993) also found an increase in the efficacy of glufosinate with ammonium sulfate and suggested ammonium sulfate increased glufosinate uptake. Roggenbuck and Penner (1997) reported that the efficacy was enhanced when ammonium sulfate was used with glufosinate against Abutilon theophorasti, Chenopodium album and Setaria
faberi. ln a more recent study Maschhoff et al. (2000) reported that adding ammonium sulfate at
2Og L-1 increased the efficacy of glufosinale on Echinochloa crus-galli, Setaria faberiand Abutilon 1aC-glufosinate theophorastl. ln the above study, ammonium sulfate increased foliar absorption of to variable levels. Ammonium sulfate also increased translocation of 'oC-glufosinate out of the treated leal 24 hours after treatment in A. theophorasti and S. faberi, but not in C. album.
Ammonium sulfate is also known to enhance efficacy of glyphosate (Donald 1988, Ruiter and
Meinen 1996, Salisbury et al. 1991, Wills and Whoder 1985), picloram (Moxness and Lym 1989,
Wilson and Nishimoto 1975), imazerthapyr (Kent et al. 1991, Wills and Mc Whorter'1987) and
sethoxydim (Jordan et al. 1989). The exact mechanism responsible for an increased uptake of
these herbicides by ammonium sulfate is unclear. However, Kirkwood (1993) and Wade et al.
(1993) suggested that ammonium sulfate changes the pH in the apoplast enhancing herbicide
uptake due to a H* co-transportor, which carries the herbicide through the plasma-membrane.
4.1.5. Kinetin
Kinetin (6{urfurylaminopurine) is a synthetic cytokinin first isolated from herring sperm DNA (Moor
1989). Cytokinins are an important class of plant growth regulators that are able to promote cell
division, cell differentiation, chlorophyll senescence and apical dominance (Mok 1994). According
to Mok (1994) apart from influencing growth and development of plants, cytokinins also affect
flowering and many other plant processors. lt might be expected that cytokinins such as kinetin that
enhance cell growth might indirectly enhance glufosinate translocation to the newly forming cells at
the meristematic region in the plant.
67 Chopler 4: frnproving Glufosinote Efficocy ogoinst R. rophanisfrun
4.2. Materials and Methods
4.2.1. Herbicide I nteractions: Carotenoid Biosynthetic lnhi bitors
Raphanus raphanistrum seedlings were grown outside at the Waite Campus as described in
Chapter 3. When the plants were at 2-4 leaf slage, plants were sprayed with amitrole
(Weedazole@) at concentrations of 0, 96, 192, gB4 and 768 g ai ha'1, diflufenican (Brodal@) at concentrations ol O, 6.25, 12.5,25 and 50 g ai ha-1, clomazone (Command@) at concentrations of 0,
13g.2,278.4,556,8, 113.6 g ai ha'1and benzofenep (Taipan@) at concentrations of 0, 150,300, 600 g ai ha-1 mixed with 0.2% (v/v) BS 1000. After 24 hours glufosinate (Basta@) at concentrations of 0,
150, 3OO, 600 and 1200 g ai ha-1.was applied to these plants. ln each case a complete factorial experiment was set up with 4 replicates. Plants were harvested 30 DAT. The survival (%) and dry weights were determined.
4.2.2. Surfactant M ixes
Raphanus raphanistrum seedlings were grown outside the Waite campus as described in Chapter
3. When the plants were at 2-4 leaÍ stage they were treated with glufosinate formulated with different surfactants at rates of 0, 150,300,600 and 1200 g ai ha-1. Plants were harvested and survival and dry weights recorded 30 DAT. The formulations used were proprietoryexperimental formulations with varying mixtures of surfactants. The exact compositions of the formulations were not revealed.
4.2.3. Effect of Ammon¡um Sulfate
Baphanus raphanistrum seedlings were grown outside the Waile Campus during the winter months of 2001. The plants were treated with 10mM ammonium sulfate when they reached 2-4 leaf stage with a laboratory boom sprayer as described earlier. Glufosinate (Basta@) was then applied 24h
later. Plants were harvested and survival and dry weights were recorded 30 DAT.
68 Chopler 4: fmproving Glufosinote Efficacy ogoinsl R. raphanisfrun
4.2.4. Effect of EDTA
Raphanus raphanistrum seedlings were grown outdoors at the Waite Campus as described above.
When at the 2-4 leaf stage plants were treated with'1OmM EDTA mixed with glufosinate, 1OmM
EDTA applied 24 hours prior to glufosinate treatment and the control plants received glufosinate only. The same experiment was also conducted in a growth chamber under cool conditions where the day temperature was 1OoC, the night temperature was 5'C and the relative humidity was maintained between 60-70%. Plants received a 12h day with a light intensity of 5'15 pmoles m-t s''.
When the plants were at the 2 to 4 leaf stage they were sprayed with 10mM EDTA and glufosinate with the same treatments as described above. Plants were assessed after 30 days.
4.2.5. Absorption and Translocation of 14G-glufosinate in EDTA
Treated Plants
Haphanus raphanistrum plants were grown in the same growlh room as above. Al2-4leaf stage, plants were divided into two replicates. One was sprayed with 10mM EDTA 24 h prior to the glufosinate treatment (3OO g ai ha-l). The other had no EDTA but was sprayed with glufosinate.
The labeled glufosinate was applied as described in Chapter 3. Plants were harvested and were combusted as described in Chapter 3.
4.2.6. Effect of Kinetin
R. raphanisfrum plants were grown in a growth chamber under cool conditions where the day temperature was 1OoC, the night temperature was SoC and the relative humidity was maintained between 60-70%. Plants received a 12h day with a light intensity of 515 pmoles m-' s-t. When the plants were at the 2 to 4 leaf stage they were sprayed with kinetin (20 ppm), 24 hours prior to glufosinate as described above. Plants were assessed for survival after 30 days.
69 Chopler 4: fmproving Glufosinote Efficocy ogoinst P. raphanisfrum
14G-glufosinate 4.2.7. Absorption and Translocation of in Kinetin
Treated Plants
R. raphanisfrum plants were grown in the same growth room as above. At 2-4 leaf stage plants was sprayed with Kinetin (20 ppm) 24i'tprior to the glufosinate treatment (300 g ai ha-1). The labelled glufosinate was applied as described in Chapter 3. Plants were harvested and were combusted as described in Chapter 3.
4.2.8. Statistical Analysis
The survival and dry weight data of R. raphanistrum treated with amtrole, diflufenican, clomazone and benzofenap in conjunction with glufosinate were analyzed using 2 way analysis of variance with no blocking using GENSTAT. Synergistic effects would occur where the two herbicides in combination provide greater than additive effect. Antagonistic interactions occur where the two herbicides in combination give less than additive effects. The survival and dry weights of the dose response experiments conducted with R. raphanistrum with glufosinate containing different surfactants, ammonium sulphate and EDTA were analyzed using Probit analysis with GENSTAT.
4.3. Results
4.3.1. Herbicide lnteract¡ons: Carotenoid Biosynthetic lnhibitors
4.3.1 .1 . Amitrole plus Glufos¡nate
As shown in Figure 4.4, lhe dose response experiments conducted outside the Waite Campus showed in the absence of glufosinate, amitrole alone caused 20"/",70"/.,100% and 100% mortality at rates of 96, 1g2, 384 and 768 g ai ha-1 respectively. However, with increased concentration of
70 Chopter 4: fmproving Glufosinote Efficocy ogoinst R. raphanistrum
glufosinate the efficacy of the herbicidal activity was reduced as reflected by the increased plant survival.
The survival data on amitrole and glufosinate showed an antagonistic effect against R. raphanistrum at all concentrations except at the highest concentrations of glufosinate and amitrole (Figure 4.14).
However, the statistics (2-way analysis of variance on survival) showed there is no significant
interaction between the two herbicides (P< 0.056). The main effects showed that amitrole alone
had a highly significant effect (P< 0.001). No significant effect (P< 0.268) was observed when
glufosinate was used in the absence of amitrole. Figure 4.1 shows that as the amttrole
concentration was increased from 96 to 384 g ai ha-1, mortality increased from 2O/" to 100%. ln
contrast, when glufosinate concentration was increase from 150-1200 g ai ha-1 with amitrole
concentration of 192 g ai ha-l the survival ol R. raphanistrum increased from 10 to 50%. With
amitrole concentration 384 g ai ha'1 survival of R. raphanistrum remained low. With the highest
concentration of amitrole regardless of the glufosinate concentrations, 100% mortality was
achieved. When glufosinate was applied alone, the results showed H. raphanistrum was highly
tolerant to glufosinate (Figure 4.14). The survival remained at 100% up to 600 g ai ha-1 and the
highest rate of 1200 g ai ha-1 had 1O% mortality. The results of this experiment demonstrated that
the control o't R. raphanistrum was mainly due to amitrole and not due to an interactive effect of both
herbicides and that the two herbicides were antagonistic when used together against
R. raphanistrum.
Figure 4.48 shows the effect of amitrole and glufosinate on dry weights oÍ R. raphanistrum. There
was a signif icant reduction (P<0.001) of dry weights for glufosinate and amitrole treated plants when
compared to the controls. Similarly, in the absence of glufosinate, all amitrole treated plants
showed a significant reduction of dry weights. With 96 g ai ha-l of amitrole the dry weights of F.
raphanistrum decreased by 50% in the absence of glufosinate. With 192,984 and 768 g ai ha-l of
amitrole the dry weights reduced lo 21, 46 and 39% of control respectively. As the rate of
glufosinate was increased at this amitrole rate the dry weights also increased showing an
antagonistic effect.
71 Chapler 4: frnproving Glufosinote Efficocy ogoinsl P. raphonistrun
100 A
Amitrole BO (g ha-t) \o I o\ E0 õ60 .l o96
õ40 rl192
20 8384
r 768 0 0 150 300 600 1200 Glufosinate (g ai ha-1)
4 B
Amitrole 3 (g ha-t) ct)
U, E¡O 'õct) 2 tr96 = o t92 1 Et 384
t768 0 0 150 300 600 1200 Glufosinate (g ai ha'r)
Figure 4.1:Survival (A) and dryweights (B) of H. raphanistrum treated with varying dose rates of glufosinate and amitrole. Plants were grown and treated under winter conditions outside at the Waite Campus. Values are means t S.E. of 8 replicates.
72 Chopler 4: Irnproving Glufosinote Efficocy ogoinsl R. rophanisfrum
4.3.1 .2. Diflufenican plus Glufosinate
The interaction of diflufenican and glufosinate showed that the combination had a week significant
interaction (P= 0.044) on survival ol R. raphanistrum. However, when used alone both glufosinate
(P< O.OO1) and diflufenican (P< 0.001) showed highly significant effects on survival. Figure 4.24
shows that with glufosinate in the absence of diflufenican, 100% survival was achieved up to 600 g
ai ha-1. With the highest rate (1200 g ai ha-1) survival was reduced only lo 7O%. Addition of
diflufenican (O to 50 g ai ha-1) did not show a significant difference in survival until 600 g ai ha-1 of
glufosinate was used. At this rate of glufosinate, a synergistic effect on survival was observed in
plants treated with higher rates of diflufenican (25 and 50 g ai ha-1). Similarly the effect of
diflufenican was enhanced with plants treated with the highest rate of glufosinate (1200 g ai ha'1).
At this rate of glufosinate, mortality increased to 50,70 and 80% with 12.5,25 and 50 g ai ha-1 of
diflufenican.
The dry weights ol R. raphanistrum showed no significant effect (P= 0.271) of the interaction of the
two herbicides. The main effects showed a significant decrease of dry weights both with glufosinate
(P< 0.001) and amitrole (P< 0.001). Figure 4.2B shows a decrease in dry weights even with the
towest concentration of diflufenican. Dry weights were reduced to 63, 62, 30 and 50% of control
with 6.25,1 2.5, 25 and 50 g ai ha-1 diflufenican in the absence of glufosinate. With the highest
concentration of glufosinate (1200 g ai ha-1) and high concentrations of diflufenican, the dryweights
were significantly reduced. Therefore, it is possible a synergistic effect occurred with the interaction
of both herbicides at 1200 g ai ha-l of glufosinate and that the concentrations of diflufenican used in
the experiment was too low to cause a significant impact on survival ol R. raphanistrum. However,
the chance of damaging the crop would be high with increased concentrations of diflufenican.
73 Chopter 4: fmproving Glufosinote Efficocy ogoinst P. raphanisfrun
100 Dif lufenican (g ha'')
BO E0 -oo\ õ60 Er 6.25 .ì ã+o N 12.5
ø25 20 r50 0 0 150 300 600 1 200 Gtufosinate (g ai ha-l)
5 B Dif lufenican 4 (g ha-t)
ct) EO aD 3 .9 tr 6.25 o = 2 o N 12.5
1 ø25
r50 0 0 150 300 600 1200 Glufosinate (g ai ha-1)
Figure 4.2: Su¡vival (A) and dry weights (B) of F. raphanistrum treated with varying dose rates of glufosinate and diflufenican. Plants were grown and treated under winter conditions outside at the Waite Campus. Values are means t S.E. of 12 replicates.
74 Chopter 4: frnproving Glufosinole Efficocy ogoinst R. raphanislrun
4.3.1 .3. Clomazone plus Glufosinate
Experiments conducted with clomazone and glufosinate showed an antagonistic effect against
R. raphanisfrum with most concentrations (Figure 4.3). Statistics on 2 way analysis of variance
showed a significant difference with the interaction of the two herbicides (P< 0.001) on survival. As
observed earlier with other interactions, R. raphanistruln was very poorly controlled wilh glufosinate
alone showing no significant difference on survival (P< 0.109). When glufosinate was applied in the
absence of clomazone, no mortality occurred up to 600 g ai ha-1. With the highest rate of 1200 g ai
ha'1, survival ol R. raphanistrum was reducedloT0/". However, as the concentration of clomazone
increased, survival significantly decreased (P< 0.001). ln the absence of glufosinate, the survival
was reduced to 88, gg,21 and 33% with 139, 278,564 and 1104 g ai ha-1 of clomazone. The rates
o1278,564 and 1104 g ai ha-1 of clomazone in the presence of 150 to 600 g ai hal of glufosinate
showed low survival rates. With 12OO g ai ha-l, survival was reduced to 79, 75,21 and 8.3% with
these clomazone concentrations.
Clomazone reduced dry weight of R. raphanistrum at all concentrations (Figure 4.38). ln the
absence of glufosinate the dry weights were reduced to 61 , 58, 55 and 54"/" of control with 139, 278,
564 and 1 104 g ai ha'1 of clomazone. All interactions with clomazone showed a reduction of dry
weights as glufosinate concentrations increased. Plants that received the highest concentration of
clomazone and glufosinate showed the lowest dry weights of 14o/" of the conlrol
75 Chapler 4: Improving Glufosinole Efficocy ogoinst P. raphonistrun
A 100 Clomazone (g ha-t) 80 E¡O -oo\ õ60 .l tr 139
J cn 40 N278
ø562 20 a1104 0 0 150 300 600 1200 Glufosinate (g ai ha-l)
5 B Clomazone (g ha-t) 4
ct) EO t, 3 .9 o'139 o = 2 N27B o ø562 1
11104 0 0 150 300 600 1200 Glufosinate (g ai ha-1)
Figure 4.3: Survival (A) and dry weights (B) of Æ. raphanistrum treated with varying dose rates of glufosinate and clomazone in combination. Plants were gown and treated under winter conditions outside at the Waite Campus. Values are means I S.E. of 8 replicates.
76 Chapter 4: fmproving Glufosinote Efficocy ogoinst R. raphonísfrun
4.3.1 .4. Benzofenap plus Glufosinate
Benzofenap had no significant effect on survival (P< 0.219) when used in conjunction with glufosinate in R. raphanistrum. As in previous experiments, glufosinate caused no mortality except at 1200 g ai ha-l (Figure 4.4A). However, the main effect on survival with glufosinate shows a significant decrease (P= 0.07) whereas benzofenap showed no significant (P= 0.57) effect on survival. The combination of benzofenap and glufosinate resulted in almost no mortality in any combination. With dry weights of R. raphanistrum the interaction of the two herbicides was not significant (P= 0.375). However, both glufosinate (P< 0.001)and benzofenap (P= 0.003) showed a significant effect on dry weights. Glufosinate reduced dry weights ol R. raphanistrum al all concentrations. The combination of benzofenap and glufosinate on dry weights were additive on F. raphanistru m (Figure 4.48).
77 Chopter 4: fmproving Glufosinole Efficocy ogoinst R. rophanislrun
100 Benzofenap (g ha-t) â80 E¡O G 't 0'150 tr300 u, 40 zt 600 20 r 1200 0 0 150 300 600 1200 Glufosinate (g ai ha'1)
3 B
Benzofenap (g ha-')
cD 2 Ø EO .c
.9(¡) B 150
= tr300 o 1
E¡ 600
r 1200 0 0 150 300 600 1200 Gtufosinate (g ai ha'l)
Figure 4.4: Survival (A) and dry weights (B) of R. raphanistrum treated with varying dose rates of glufosinate and benzofenap in combination. Plants were gown and treated under winter conditions outside at the Waite Campus. Values are means t S.E. of 12 replicates.
78 Chopler 4: fmproving Glufosinote Efficocy ogainsf R. raphonisfrun
4.3.2. Formulations of Glufosinate w¡th Different Surfactants
Two experimental formulations of glufosinate DS 6183 and DS 8165 (with different surfactants,
kindly provided by Hunsman Corporation Australia PTY LTD) were compared with the commercial
glufosinate formulation (Basta) to determine whether the experimental formulations would
demonstrate better herbicidal activity under winter conditions. As shown in Figure 4.5A, the results
indicated that DS 6183 and Basta had similar activity but DS 8165 efficacy was inferior compared to
the other two formulations. All three formulations showed 100% survival up to 300 g ai ha-1. With
the highest rate of glufosinate (1200 g ai ha-1) 8O% survival was observed with DS 8165 whilst DS
61 83 and Basta had 60% survival. ln spite of this ineff icient control based on the plant survival, the
dry weights of plants showed a continuous decline as the concentration of glufosinate was
increased for all of the three formulations used (Figure 4.58). A reduction of more than half in dry
weights was achieved at the highest (1200 g ai ha-l) glufosinate concentration, DS 8165 was
slightly less effective than DS 6183 and Basta in reducing R. raphanistrum dry weights.
4.3,3. Effect of Ammon¡um Sulfate
Dose response experiments conducted with glufosinate and ammonium sulfate outside at the Waite
Campus (during winter 2001) demonstrated that addition of ammonium sulfate did not improve
control of R. raphanistrum by glufosinate (Figure 4.10 A). Dry weights of treated plants showed a
continuous decrease with both treatments as the glufosinate concentrations were increased.
However, the addition of ammonium sulfate had additive effect (Figure 4.108).
79 Chapter 4: frnproving Glufosinote Efficacy ogoinst P. raphanislrun
100
80
òq r60.l ã40
20
0
0 300 600 900 1 200 Glufosinate (g ai ha-1)
5 B
4 ct) th c 3 'õctr 2 = o 1
0 0 300 600 900 1200 1500 Glufosinate (g ai ha'1)
Figure 4.5 Survival (A) and dry weights (B) of R. raphanistrum lrealed with different glufosinate formulations with different surfactants. Basta (^), DS 6183 (r), and DS 8165 (r). Values are means t S.E. of 6 replicates.
BO Chopter 4: lmproving Glufosinole Effícocy ogoinst P. raphonisfrum
A 100
80
òq õ60 .¿ õqo
20
0 0 300 600 900 1200 1 500 Glufosinate (g ai ha-l)
6 B
5
ct) 4 U' E .9 o 3 = o 2
1
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 4.6. Survival (A) and dry weights (B) of F. raphanistrum treated with varying dose rates of glufosinate with ammonium sulfate (o) and without ammonium sulfate (o). Values are means t S.E. of 8 replicates.
81 Chopter 4: fmproving Glufosinote Efficocy ogoinst R. raphanistrun
4.3.4. Effect of EDTA
Application of 10mM EDTA,24 hours prior to glufosinate treatment increased glufosinate activity against R. raphanistrum, grown outside at the Waite Campus during winter 2001 (Figure 4.64). At the field rate of glufosinate (600 g ai ha'1) EDTA pre-treatment increased plant mortality from 0%
(glufosinate only) to greater than 50% (glufosinate after pretreatment of EDTA). The LDso calculated by Probit analysis was reduced from 1438 I 268 to 669.8 I 61 .8 when EDTA was applied
24h prior to glufosinate. However, if EDTA was mixed with the glufosinate formulation and applied
as a single spray treatment, only a marginal increase in plant mortality was observed and the LDsq
value remained high (1263!497).
Dry weights of plants which received pretreatment of EDTA and were then treated with glufosinate
were much lower than those for glufosinate alone (Figure 4.78\. Glufosinate and EDTA as a
mixture also reduced dry weights but to a lesser extent.
Dose response experiments were also performed in a growth room where the plants were provided
with 12h, 10'C days and 12h, 5'C nights. Under these conditions the plants that were treated with
EDTA 24h prior to the application of glufosinate had a significant enhancement of efficacy when
compared to plants treated with glufosinate alone (Figure 4.7A). The LD5q values of the two
treatments showed that the plants that were treated with EDTA prior to glufosinate was only 423 t
87.3 compared with 1769 t 67.4for plants treated with glufosinate alone. The difference between
the EDTA treated and untreated plants was apparent throughout the experiment. The effect of
EDTA was particularly evident at low glufosinate concentrations. Plants treated with glufosinate at
300 g ai ha-1 had 100% survival, however, EDTA pre treatment increased mortality to B0%.
Glufosinate reduced dry weights of plants by a similar amount in the absence or presence of EDTA
(Figure 4.78). Both treatments showed a similar reduction in dry weights as the concentration of
glufosinate was increased.
82 Chopter 4: fmproving Glufosinote Efficocy ogoinst P. raphaníslrun
A 100
80 s r60.t
J AI\ U' ¿+U
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
þ B
E' 4 o
.9q)
= 2 o
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 4.7 Survival (A) and dry weights (B) of R. raphanistrum grown under winter conditions outside at the Waite Campus treated with EDTA and glufosinate as a mixture (r), EDTA 24h prior to glufosinate (tr) or glufosinate without EDTA (À). Values are means + S.E. of I replicates.
83 Chopler 4: fmproving Glufosinote Efficocy ogoinst P. rophonislrun
100 A
80 ro o\ 860 .z ã40
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
16 B
1 2 ctt U' -c 'õctt I = o 4
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-l)
Figure 4.8 Survival (A) and dry weights (B) of R. raphanisfrum treated with glufosinate under controlled growth conditions of 5/10"C. Glufosinate alone (n) or with EDTA 24h prior to glufosinate (r). Values are means t S.E. of 6 replicates.
84 Chopler 4: Improving Glufosinole Efficocy ogoinst P. raphanístrum
100
o Bo .gØ o f 'ã- Þg 60 :oJr9 -Oo0, cÌ 40 '{=oñ v CL
-o820
0 0 20 40 60 80 Time (HAT)
toO-glufosinate Figure 4.9 Absorption of into leaves ol R. raphanístrum following treatment with glufosinate alone (.) or with EDTA 24h prior to glufosinate (o). Values are means t S.E. of 5 replicates.
1aO-glufosinate ln the initial stages, absorption of was lower in plants pre treated with EDTA compared to plants treated with glufosinate alone. However, similar uptake of radiolabel was observed 24 HAT (Figure 4.8). Application of EDTA 24h prior to glufosinate altered the translocation of glufosinate in the first 24 HAT. Glufosinate translocation to the leaf tip of the treated plants continued to increase under both treatments with time. EDTA treated plants showed a reduction in translocation to the leaf tip in the first few hours after treatment (Figure 4.9A). The amount of labeled glufosinate retained in the treated part of the leaf ranged between 20-45% 1aC-glufosinate through out the experiment (Figure 4.98). When translocation of was investigated differences were noticed between plants that had received EDTA pre- treatment and those that 'l0h were treated with glufosinate alone. These differences were mainly restricted to the first of the experiment and included decreased translocation to the leaf tip (Figure 4.94), increased translocation to the leaf base (Figure 4.9C) and decreased amount of radioactivity retained at the
85 Chopler 4: frnproving Glufosinole Efficocy ogoinsl P. raphonísîrun treated zone (Figure 4.98). EDTA pre- treatment slightly decreased the amount of glufosinate translocated to the meristematic zone (Figure 4.9 D) but did not influence the amount translocated to untreated leaves (Figure 4.9E). From this experiment, it appears that EDTA pre- treatment influences the way that glufosinate is distributed with in the treated leaf.
86 Chopter 4: fmproving Glufosinote Efficacy ogoinst P. rophonistrun
80 A
60
40
20
0 B 60
40
20 a 8o c 8oo l¡(u пo-o o (ú c 'E zo : c,()0 :8 D
6
4
2
0 0.8 E
0.6
o.4
o.2
0 Tirne (HAT)
Figure 4.10: Time course of loC-glufosinate translocation in EDTA treated (r) and untreated (r)
R. raphanistrum plants exposed to cold conditions of 5/10'C. (A) Recovered from the tip of the treated leaf (B) recovered from the treated zone (C) recovered from the base of the treated leaf (D) recovered from the meristematic zone and (E) recovered from the untreated leaves. Values are means + S.E. of 5 replicates.
87 Chopter 4: frnproving Glufosinote Etficocy ogoinsf P. rophanislrun
4.3.5. Effect of Kinetin
Application of Kinetin (20 ppm), 24 hours prior to glufosinate treatment increased the glufosinate activity against R. raphanisfrum (Figure 4.104). At the field rate of glufosinate (600 g ai ha-1), plants treated with kinetin 24h prior to glufosinate had increased mortality from 10% (glufosinate alone)to greater than 45% (glufosinate plus kinetin). The LD5e values calculated with probit analysis were
1592+ 97 and 660 t 91 for glufosinate and for kinetin 24h prior to glufosinate. The dry weights of
plants from this experiment showed no differences between the glufosinate alone and glufosinate
plus kinetin treatments (Figure 4.108).
1aO-glufosinate Pre treating plants with kinetin reduced absorption of compared to those that were
sprayed with glufosinate alone (Figure 4.11). Translocation patterns of glufosinate were also
changed by pre-treatment with kinetin. There was little effect on translocation of glufosinate to the
leaf tip of the treated leaf compared to the glufosinate only treatment (Figure 4.12 A). Like wise the
amount of glufosinate retained at the point of application was similar between the two (Figure 4.12
B). Translocation of glufosinate to the base of the treated leaf was increased in plants treated with
kinetin compared to glufosinate treated plants for the first 10 HAT (Figure 4.12 C). Translocation to
the shoot meristematic region and to untreated leaves was greatly increased by 72 HAT in plants
that were treated with kinetin prior to glufosinate compared to plants treated with glufosinate alone
(Figures 4.12 D, 4.12 E). Kinetin appeared to increase early basipital translocation to the leaf base
promoting a subsequent greater translocation to the meristematic regions and to untreated leaves.
88 Chopter 4: frnproving Glufosinote Efficocy ogoinst R. raphanislrun
A 100
80 -o¡¡\ õ60 .l
Ø540 20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
7 B þ
E') 5 Ø 4 .9 o 3 = o 2
1
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 4.11 Survival (A) and dry weights (B) of B. raphanistrum trealed with kinetin 24h prior to glufosinate (r), or glufosinate alone (.). Values are means t S.E. of I replicates.
89 Chopter 4: fmproving Glufosinate Efficocy ogoinst R. raphanistrum
100
I(g '=, 80 eAtr' õHoo p;:o bO : -ioo '.=Oo\ g - ¡¡920
0 0 20 40 60 80 Time (HAT)
Figure 4.12. Absorption of radiolabeled glufosinate into leaves of R. raphanistrum treated with kinetin 24h prior to glufosinate (r), or with glufosinate alone (o). Values are means I S.E. of 10 replicates.
90 Chopter 4: fmproving Glufosinote Efficocy ogoinst R. raphanisfrun
A 60
40
20
0 B 60
40
20 go ¡¡ c äoo aD tt(g E¿o c) ca! 'õ 20 o :
:103o D I
6
4
2
0 5 E 4
3
2
1
0 o206080 Iime (HAT) 14C-glufosinate Figure 4.13: Time course of translocation in R. raphanistrurn plants pre treated with kinetin (r) or plants treated with glufosinate alone (o), exposed to cold conditions of 5/10'C. (A)
1aC-glufosinate recovered from the tip of the treated leaf, (B) recovered from the treated zone, (C) recovered from the base of the treated leaf, (D) recovered from the meristematic zone and (E) recovered from the untreated leaves. Values are means 1 S.E. of 10 replicates.
91 Chopler 4: frnproving Glufosinote EfÍicocy ogoinsl R. raphanistrun
4.4. Discussion
Strategic lmprovement of herbicidal efficacy may need to focus on methods to improve absorption
and subsequent translocation of the herbicide. ln the present study, several methods were tested to
enhance glufosinate efficacy against H. raphanistrum under southern Australian winter conditions.
These included the use of several co-herbicides, chelating agents, surfactants and a growth
regulator. Pre-treatment of plants with the above agents was compared with the use of glufosinate
alone to determine whether any additive or synergistic herbicidal activity could be achieved in
comparison to glufosinate use in isolation.
The results of interactive studies with carotenoid biosynthetic inhibitors in conjunction with
glufosinate showed a variable response against R. raphanistru¡n. Dose response data on amitrole
and glufosinate showed an antagonistic effect when used against R. raphanístrum. Similar results
were obtained from plants that were treated with clomazone and glufosinate. The interaction on
survival showed an antagonistic effect against H. raphanistrum. However, both herbicides when
applied alone reduced the dry weight significantly. When tested the interaction of diflufenican and
glufosinate showed a weak significant synergistic interaction (P= 0.044) on survival. However, the
dry weights of these experiments did not show a significant difference. The interaction of
glufosinate and benzofenap showed an additive effect on survival and dry weights.
Previous studies indicated that interactions between herbicides are antagonistic more frequently
than synergistic. However, when herbicides are synergistic, efficacy can be enhanced significantly.
According to Zhang et al. (1995) antagonistic interactions occurred no matter whether the
interacting herbicides were absorbed by the same or different mechanisms of uptake and
regardless of whether the target plants were annuals, perennials, crop or weeds" ln a similar study
(Wehtje and Walker 1997) investigated glyphosate and2,4-D for the control of several species of
lpomoea (morningglories) and showed that the response to both glyphosate and 2,4-D applied
alone was species and growth stage dependent and all combinations were either additive or
synergistic for many species. However, the response of small flower morningglory ranged from
92 Chopter 4: fmproving Glufosinote Efficocy ogoinst P. raphonislrun
toO-glyphosate antagonist¡c to synergistic. By contrast absorption of foliar applied and subsequent translocation of the radiolabel from the treated leaves to other parts were enhanced by the addition
of 2,4-D in all species except one. Our results established that inhibition of caroteinoid biosynthesis
did not increase the toxic effect of glufosinate except in the case of diflufenican. This contradicted
our hypothesis and suggested that glufosinate worked too fast and reduced absorption or
translocation of the caroteinoid biosynhesis inhibitors or caroteinoid biosynthesis inhibitors worked
too slowly to influence the translocation or the action of glufosinate. The only effective combination
proved to be glufosinate plus diflufenican. However, reasonably higher rates of diflufenican were
required to cause a significant increase in mortality in R. raphanistrum. Such higher rates of
diflufenican would be damaging to canola and therefore, not practicalto be used in field conditions.
Changing the formulation of glufosinate by altering the surfactant did not improve efficacy against R.
raphanistrum. Formulation DS 6183 behaved similarly to Basta, whereas formulation DS 8165
provided less control than Basta. Previous studies with surfactants indjcated that they play an
important role in formulating herbicides as emulsifiers, wetting and suppressing agents as modifiers
and activate the herbicide by physicochemical interactions between the surfactants, the herbicide
and the target species (Holloway and Stock 1990). Much of the previous work in this area has been
done with glyphosate. According to Liu and Zabkiewicz (1999) water soluble surfactants TX-100,
A'lO and MON 0818 being smaller droplet spreders enhanced glyphosate penetration significantly in
wheat (53 to 58% within 6 hours after treatment). However, TX-45 and L-77 being greater droplet
spreaders caused low absorption into wheat foliage (17 to 28y.,6 hours after treatment). Previous
research also showed that surface-active organosilicon surfactants such as the trisiloxane
polyethoxylate (Silwet L-77) allow direct penetration of agrochemicals into the leaves via open
stomata (Stock and Holloway 1993). These surfactants are capable of reducing the equilibrium
surface tension of the solution (Stock and Holloway 1993). Many authors confirm that the addition
of surfactants to the original Roundup formulation has often enhanced uptake and efficacy (Gaskin
and Zabkiewicz 1989, Sandbrink et al. 1993, Wales and Griffiths 1995)" Balneaves et al (1993)
showed that gorse was controlled more effectively when an organosilicon surfactant was added to
glyphosate spray mixture. Glyphosate absorption is normally slow and incomplete. Therefore,
surfactants can have a significant effect on glyphosate efficacy by increasing efficacy of the
93 herbicide. The situation ¡s somewhat different for glufosinate on F. raphanistrum. Glufosinate is fairlywell absorbed into leaves ol R. raphanistrum (Chapter3). Therefore, increasing absorption by changing surfactants is unlikely to influence efficacy significantly.
Our experiments peformed with glufosinate and ammonium sulfate against R. raphanistrum showed no differential response to plants treated with glufosinate alone. However, addition of
ammonium sulfate to the spray solution has been shown to improve the efficacy of glufosinate in
some weed species in previous studies (Maschhoff et al. 2000). Ammonium sulfate is assumed to
improve herbicide performance by either softening water or reducing the apoplastic pH. lt is not
clear which mechanism is involved in the improved absorption of glufosinate into leaves. However,
the mechanism seems to be species dependent, as ammonium sulfate did not improve glufosinate
efficacy in R. raphanistrum.
EDTA is a chelating agent that binds divalent cations. Such cations present outside of leaves may
bind glufosinate, inhibiting the rapid absorption of the herbicide. As EDTA had a significant effect on
glufosinate efficacy, the impact of EDTA on absorption and translocation of glufosinate was studied. laC-glufosinate Pre treatment of plants with EDTA inhibited the absorption of rather than promoting
it (Figure 4.8). The addition of EDTA reduced the initial absorption of glufosinate by 20% when
compared to the plants that were treated with glufosinate alone. However, by 24h all plants had
absorbed same amount of glufosinate. By 72h after treatment almost 100% of the applied
glufosinate was absorbed into the treated leaf, regardless of pre treatment. EDTA pre treatment
only slightly altered the translocation pattern of glufosinate in F. raphanistrum. lt is possible EDTA
reduced early absorption of glufosinate, but increased early basipital translocation to the leaf base.
However, this did not promote greater translocation to the meristematic regions or to other leaves.
Enhanced efficacy of glufosinate was observed in growth room and outdoor dose response
experiments following treatment with EDTA. However, EDTA did not increase absorption of
glufosinate. lt would seem that EDTA increased biochemical activity of glufosinate in some other
way. The reason why EDTA increased glufosinate efficacy were not further investigated and
remained unclear. Application of EDTA prior to glufosinate enhanced efficacy of glufosinate in
94 Chopler 4: fmproving Glufosinote Effrcocy ogainst R. raphanislrun
R. raphanistrum. However, the mixture of glufosinate and EDTA did not improve the herbicide efficacy (Figure 4.10 A). EDTA is a cation chelater and would be expected to reduce the ability of calcium and other cations to bind glufosinate. lt is possible by chelating calcium and other divalent and monovalent cations present on the cell wall surface, EDTA allows less chance for the negatively charged glufosinate molecules to be bound to cations on the cell walls, hence more uptake into cells. Translocation to the base of the treated leaf was greatly increased in plants. Despite this, increased translocation to the meristematic regions was not observed.
Application of kinetin 24h prior to glufosinate treatment also showed an enhancement of glufosinate efficacy against R. raphanistrumin dose response experiments. Further studies on absorption and 1aO-glufosinate translocation with showed that kinetin reduced the absorption of glufosinate but promoted increased translocation to the meristematic zone and to untreated leaves in F. raphanistrum. Kinetin is a cytokinin known to promote cell division, cell differentiation, chlorophyll senescence and apical dominance in plants (Mok 1994). Therefore, it is possible that kinetin stimulated apical development in F. raphanistrum even under cold conditions. This may have caused higher flow of photosynthates to the meristematic regions causing more indirect translocation of the herbicide to the meristematic regions. There may be an opportunity to improve glufosinate efficacy by the addition of a kinetin like molecule to the spray mixture. Whether this will be practical or economically viable remains to be determined.
95 Chopler 5: pH Effect on Glufosinote uploke in R. raphanistrumond C. austrolís
Ghapter Five: pH lnfluence on Glufosinate Absorption in Raphanus raphanistrum and Chara australis
5.1. Introduction
The work presented in the previous chapters showed that the poor control of ß. raphanistrum is mainly due to the lack of translocation of glufosinate to the shoot meristematic region. Several attempts were made to improve glufosinate absorption and subsequent translocation without much success. The current study was initiated to determrne the ability to enhance the cell uptake of glufosinate to facilitate more phloem translocation.
Herbicide Efficacy and Cell Uptake: For a phloem active herbicide to be effective, there needs to be efficient uptake into cells, loading into the phloem, retention and unloading at the appropriate sinks. lnformation on the mechanisms of herbicide absorption across plant membranes is scarce and particularly the understanding on glufosinate absorption across membranes is limited.
According to Sterling (1994), for a herbicide to be effective it needs to be transported to the site of action. Normally, the site of action is located within the intracellular organelles (e.9. GS2 in chloroplast). Once the herbicide reaches the plant cell, it needs to be transported through the cell wall, the plasma membrane and organelle membrane to reach the site of action. Passive or active
movement across these membranes is often controlled by several factors such as the
physicochemical characteristics of the herbicide molecule, the structure of plant cell membranes
and the electrochemical potential across plant cell membranes (Sterling 1994).
Most herbicides move across the plant membranes via non{acilitated (passive) diffusion, due to a
mechanism by which solute moves down an electrochemical gradient (Hess 1985, Nobel 1991).
Herbicides that are absorbed through non{acilitated diffusion are divided into two classes
depending on the physicochemical characteristics of the herbicide molecule. These are lipophilic-
96 Chopter 5: pH Effect on Glufosinote uptoke in P. raphanistrunond C. ausfralís neutral molecules and lipophilic molecules with a funct¡onal group sensitive to pH, which can dissociate into a less lipophilic ion. According to Sterling (1994), lipophilic neutral herbicide molecules diffuse passively into plant cells and their lipophilic nature allows them to move rapidly across the membranes down their concentration gradient to reach their equilibrium concentration between the external environment and the cell interior. Previous work carried out wilh the herbicides amitrole, monuron, norflurazon, oryzalin and triazines is in agreement with this hypothesis (Donaldson et al. 1973, Lichtner 1983; Mersie and Singh 1987; Nobel 1991;Price and
Balke 1982; Upadhyaya and Nooden 1980). lt was demonstrated through these studies, that passive uptake of herbicides into cells is a rapid process and it depends on the herbicide and the targeted species. These studies suggest that the plasma membrane is not a barrier to lipophilic, neutral herbicide absorption (Sterling 1994).
Several investigations have been carried out on the herbicide efflux out of cells or tissues containing herbicides. Atrazine diffused rapidly out of excised Zea mays (Price and Balke 1983),
Setaria faberi (Orwick et al. 1976) and Solanum tuberosum tuber discs (Peterson and Edgington
1976) and leaf slices (Price and Balke 1983). ln these studies, S0-90% of the initial atrazine present in tissues, effluxed within 30 min.
The Role of lon Trapping in Cellular Uptake of Herbicides: Lipophilic, ionic herbicides are either weak acids or weak bases that accumulate in cells by the mechanism of ion trapping.
Herbicide molecules are absorbed into cells down a concentration gradient via passive diffusion.
However, in contrast to lipophilic-neutral herbicides, weak acid herbicide absorption saturates over time with the herbicide reaching cellular concentrations greater than external concentrations
(Darmstadt et al. 1983, Devine et al. 1987, Gronwald et al. 1993). Undissociated acids diffuse
across the plasma membrane since cell membranes are more permeable to undissocialed neutral
molecules compared to the charged molecules. However, once in the alkaline compartments of the cell the acid dissociates and is unable to pass back through the membrane. The net result is
that the herbicide accumulates with in the cell (Sterling 1994). Previous studies showed that the
absorption of several weak acid herbicides by various plant species and tissues was linearly
related to external herbicide concentrations (Grimm et al, 1985, Stoller 1969, Devine et al. 1987,
97 Chopter 5: pH Effect on Glufosinote uptoke ín P. raphanistrunond C. australis
Devine et al. 1987, Jonson and Bonner 1956, Wedding and Erickson'1957, Van Ellis and Shaner
1988, Smith and Vanden Born 1992, and Ooka and Balke 1991).
The Role of pH and Herbicide Uptake: The studies based on weak acids have reported that pH dependent uptake supports ion trapping as higher concentrations of the undissociate exist at acidic rather than neutral pH and the absorption of weak acid herbicides by plant cells and tissues was greater at a pH close to the pKa of each herbicide. According to Liebl et al. (1992), the extracellular pH had a significant effect on bentazon (pKa = 3.45) uptake by cultured soybean cells.
At pH 6.6 uptake of bentazon was very poor at 0 to 1 nmol g-t fr wt throughout the experiment.
However as the extra cellular pH was reduced to pH 5.6 there was a 3 to 4 fold increase in uptake.
This was further enhanced when the extra cellular pH was reduced to pH 4.6. At this pH the absorption increased 12 to 15 fold when compared to the uptake at pH 6.6. This showed that, the bentazon uptake into cells was increased remarkably when the pH was closer to the pKa. Similar results have been reported for other weak acid herbicides (Sterling 1994)'
lon trapping does not apply to glyphosate uptake (Sterling 1994). Glyphosate exists as both monovalent and divalent anions at physiological pH as it's pKa values are from 2.21o 2.6 (pKar),
5.5 to 5.9 (pKa2) and 10.1 to 10.9 (pKa.). Glyphosate absorption by Beta vulgaris (Gougler and
Geiger 1981) and Vicia faba (El lbaoui et al. 1986) leaf discs and by wheat leaf protoplasts
(McCloskey 1991) followed a linear relationship between glyphosate concentration and the uptake.
However, evidence for nonfacilitated diffusion and carrier mediated uptake of glyphosate have
been seen in some plant species (Burton and Balke 1987). Further studies showed that
glyphosate absorption occurred in wheat leaf discs, independent of the solution pH (pH 3.5 to 9.7)
and weak acid accumulation did not take place (El lbaoui et al. 1986, Gougler and Geiger 1981).
According to Sterling (1994), the plasma membrane presents a barrier to glyphosate absorption
and accumulation as a weak acid. Studies conducted with isolated plasma membrane vesicles
from Chenopodium album showed glyphosate absorption was very low compared to absorption of
the amino acid glutamate and other herbicides such as atrazine and bentazon (Sterling 1994).
Studies conducted on glyphosate uptake revealed that it can be competitively inhibited by either
98 Chopter 5: pHEffeci on Glufosinote uPtqke in P. rophantstrumand C. auslralis
phosphate and phosphonoform¡c acid. Therefore, it is assumed that glyphosate can be absorbed via a phosphate transporter of the plasma membrane (Denis and Delrot 1993).
lnvestigations on glufosinate absorption in Lemna gibba showed a biphasic uptake of the herbicide with respect to time and concentration (Ullrich et al. 1990). The uptake of the herbicide was strongly enhanced below pH 5 but remained unchanged between pH 5 and 7 (Ullrich et al. 1990)'
It was also reported that the addition of glufosinate after or in the presence of some amino acids
(e.g. glutamate, alanine and aspartate) caused a partial inhibition of glufosinate uptake. According to the above authors glufosinate induced a continuous K* release, instead of glutamate. Because of it's immediate membrane depolarizing effect, lhe anion glufosinate is assumed to be taken up by a proton colransport mechanism and at lower pH it was observed that additional undissociated acid is transported by diffusion.
The objective of the present study is to develop strategies to increase the cell uptake of glufosinate in order to facilitate more phloem translocation. lt was hypothesized that lowering the pH of the commercial formulation of glufosinate would promote the cellular mechanism of ion trapping which may increase the intracellular uptake of glufosinate. An increase in cellular uptake would enhance the phloem translocation and improve glufosinate efficacy. These hypotheses were tested in F.
raphanistrum and in the cells ol Chara australis.
5.2. Materials and Method
5,2.1. Dose Response Studies
Dose response pot experiments were performed using a single population ol H. raphanístrum
grown at the Waite Campus, with no previous history of herbicide use. The seeds were
germinated as described in Chapter 3. Germinated seedlings were transplanted into 15 cm square
pots filled with potting soil. Each pot contained four F. raphanistrum seedlings. The plants were
99 Chopler 5: pH Effect on Glufosinote uPtoke in R. rophonistrumond C. dustrdlis
grown outdoors during winters of 2OOO and 2001 as well as ¡n a growth chamber set at 12h, 10'C,
480 pmol m-2 s-1 day and a12h,5"C night where the relative humidity ranged from 60-70'/..
5.2.2. Dose Response Pot Experiments
At the 2 to 4 leaf stage, seedlings were sprayed (for both outdoors and growth chamber experiments) with solutions of glufosinate (Basta@) with altered pH. To reduce the pH of glufosinate, hydrochloric (pH 2.5) or phosphoric acids (pH 2.5 and 5) were used. To increase pH
(pH 7), potassium hydroxide was used. Glufosinate solutions of different pH were applied with a laboratory moving boom sprayer equipped with T-jet fan nozzles at a speed of 'l ms-1. The output of the sprayer was calibrated at 125 L ha'1 at a pressure of 250 Kpa. Dose rates ranged f rom 0-
12OO g ai ha-1. Plants were returned outside or to the growth chamber after spraying and assessed for survival 30 DAT. Plants were harvested and shoot dry weights were determined after drying the shoots at 60"C for 48h.
5.2.3. Absorption and Translocat¡on Studies: Effect of pH
Experiments to determine absorption and translocation of glufosinate were peformed using 2 to 4
leaf H. raphanistrum seedlings, which were germinated and grown as described in Chapter 3.
Absorption and translocation experiments were performed as described in Chapter 3. For these 14C experiments, one set of plants was sprayed with glufosinate (300g ai ha-1) and the solution applied was prepared in the normal glufosinate solution. The other set of plants was sprayed with 1aC glufosinate solution adjusted to pH 2.5 with hydrochloric acid and the solution applied was
prepared in the pH adjusted glufosinate solution.
5.2.4. Uptake into Leaf Slices
Seedlings ol R. raphanistrum were germinated as for the dose response experiments under cold
conditions of 5/10'C (Chapter 3, Material and Methods). Once the plants were in 2lo 4leat stage,
leaves were harvested and sliced into 1mm slices immersed in buffer. Leaf slices were placed into 1aO-glufosinate eppendorf tubes (1.5m1) containing solution (O.5mL) with concentrations ranging
100 Chopler 5: pH Effect on Glufosinote uptoke in P. rdphaníslrunond C. auslrolis
lrom 7-2OO¡tM. The tubes were incubated in a water bath at 25"C lor 30 min. The radio labeled solution was removed from the tube and the leaf slices were washed in 600p1 of 200pM glufosinate at 4'C for 15 min. Fresh weights were taken and the leaf slices were wrapped in silicon paper and the parcels were dried in an oven set at 60'C over night. The dried samples were combusted in a biological sample oxidizer as described in Chapter 2 and the amount of radioactivity per sample
14C was analyzed by liquid scintillation spectroscopy. The amount of radioactivity present in the incubation and the wash solutions was also determined by liquid scintillation spectroscopy.
5.2.5. Cellular Uptake of Glufosinate
Chara australis cells (a gift from Dr Robert Reid, Department of Plant Science, Adelaide University) were cultured in artificial pond water (APW) supplemented with micronutrients, 1 mM NaCl, 0.1 mM
KCI and 0.5 mM CaClzat pH 6 (buffered with 2 mM MES). The cells were grown under constant light at 25'C in large containers under laboratory conditions. APW was used in the uptake experiments. For the time dependent uptake experiments 35 large cells were transferred into 20 mL APW solution. A radio labeled solution of glufosinate (200 pM) was prepared with a specific 1aO-glufosinate activity of 125 Bq. The experiment was started by the addition of solution to the
APW (20mL) solution containing the cells. After 10,20,30,40,60 and 90 min intervals, S cells were taken out and were washed in APW solution for 1 min and pat dried. The length of each cell was measured and the ends of each cell were cleaved using a sharp blade. The cytoplasmic contents were emptied into a scintillation vial containing 10mL scintillation fluid. Similarly the laO-glufosinate remaining cellwall was placed in 10 mL scintillation fluid. The in cellsamples was measured using liquid scintillation spectroscopy. The experiment was repeated several times.
Several experiments on concentration dependent uptake were performed with C. australis. The cells were incubated in a 1aC-glufosinate solution with 10,30,50, 100, and 200pM glufosinate concentrations for 30 min. Similarly, several experiments were performed with different solutions where the external (buffer) pH was adjusted by using Tricine (pH 8.5), Hepes (pH 7.5), MOPS (pH
6.5) or MES (pH 5.5) buffers.
101 Chopter 5: pHEffecl on Glufosinote upïoke in P. raphanístrunand C. australis
5.3. Results
5.3.1. Dose Response Pot Experiments
Whole plant dose response experiments conducted outdoors at the Waite Campus during winter of
2O0O and 2001, treated with pH adjusted spray solutions with phosphoric acid showed poor control of H. raphanistrum with alltreatments (Figure 5.1A). The percentage survival remained near 100% with 300 g ai ha-1, irrespective of the solution. However, as the rates of glufosinate increased, there was better controlwith low pH solutions compared to higher pH solutions. The LD5s values of the survival data showed a decrease in efficacy as the pH was increased. The LD5e as estimated at 1061 , 1046, 1371 , and 1 593 for pH 2.5, 3.5, 5 and 7 respectively (Table 1 ). The dry weights did not show a marked difference between the treatments (Figure 5.18).
When the same experiment was conducted under controlled environmental conditions in the growth room at cool temperatures of 5/10'C, greater control ol H. raphanistrum was observed compared to application outdoors (Figure 5.2A). However, there were only small differences in efficacy for the solutions of different pH with a greater trend for greater activity of solutions of pH
2.5 or pH 7. The LDue values showed that both pH 2.5 and 7 had similar efficacy on H. raphanistrum. However the LD5q values for pH 3.5 and 5 were significantly higher than pH 2.5 and
7. Dry weights of the plants grown in the growth room were approximately 5 fold higher than those that were grown outdoors during winter (Figure 5.28). Dry weights decreased as the concentration of glufosinate increased in alltreatments and there were no differences between treatments.
102 Chopter 5: pH Effect on Glufosinale uptoke in R. raphonistrumond C. ousfralis
A 100
80 o\-o õ60
ã40
20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
4 B
3 ctt
at, .9 o 2 = o 1
0 0 300 600 900 1200 1500 Glufosinate (g ai ha'1)
Figure 5.1: Survival (A) and dry weights (B) of F. raphanistrum treated with glufosinate solutions adjusted with phosphoric acid to pH 2.5 (r), pH 3.5 (o), pH 5 (o) or pH 7(o) outdoors during winter at the Waite Campus. Values are means t S.E. of 15 replicates.
103 Chapler 5: pH Effecl on Glufosinote uPtoke ¡h P. raphanistrunond C. ausfrdlis
A 100
80 \o o\ r60 .à õ40
20
0 0 300 600 900 1200 1500
Glufosinate (g ai ha-1)
25 B
20
ct)
U, 1 5 -c .9 o = 1 0 o 5
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-1)
Figure 5.2: Survival (A) and dry weights (B) of F. raphanistrum treated with glufosinate solutions adjusted with phosphoric acid to pH 2.5 (r), pH 3.5 (tr), pH 5 (o) or pH 7(o) under controlled growth conditions of 5/10'C. Values are means I S.E. of 6 replicates.
104 Chopter 5: pH Effect on Glufosinote uptoke in P. raphonistrumond C. ouslralis
Table 5.1: LDso values for glufosinate for R. raphanistrum. Herbicide solutions were adjusted with phosphoric acid to pH 2.5,3.5 5 or7. Plants were grown and treated outdoors in winter or under controlled growth conditions of 5/10'C.
Solution pH LD5e for glufosinate on R. raphanistrum mean + SE
Out doors Growth Room
pH 2.5 1061 I 70 788 ! 120
pH 3.5 1046 I 108 1179!203
pH5 1371 t 195 867 I 105
pH7 1593r70 758 + 92
The efficacy of glufosinate against B. raphanisfrum adjusted with hydrochloric acid or phosphoric acid to reach pH 2.5 were compared with plants treated with the commercialformulation (Basta@) in three dose response experiments conducted under conlrolled environmental conditions of
5/10"C. As shown in Figure 5.34 the plants treated with the commercialformulation of glufosinate had considerably less mortality than those treated with the herbicide at low pH. The highest concentration of glufosinate (1200 g ai ha-1) resulted in 47% mortality. ln contrast, this rate gave
100% mortality when pH was adjusted with HCI to 2.5. When phosphoric acid was used to adjust the pH of the herbicide solution to pH2.5 mortality was less reaching 65%. The LD5e value for glufosinate adjusted to pH 2.5 with HCI was 490 g ai ha-1 compared to 788 g ai ha-1 for phosphoric acid.
The dry weights of plants were decreased by glufosinate regardless of pH (Figure 5.38). However, dry weights of R. raphanistrum treated with glufosinate adjusted to pH 2.5 with HCI were lower when compared to the other treatments.
105 Chopler 5: pHEfÍecl on Glufosinole uPtoke in R. raphonistrumand C. oustrolís
100
80
ÐoorO õ .¿
U'=40 20
0 0 300 600 900 1200 1500 Glufosinate (g ai ha-l)
20 B
1 5 ctt tt,
ctt 'õ 1 0 B o 5
0 0 300 600 900 1 200 1 500 Glufosinate (g ai ha'1)
Figure 5.3: Survival (A) and dry weights (B) of R. raphanistrum trealed with the commercial formulation of glufosinate (r), or glufosinate adjusted to pH 2.5 with hydrochloric acid (u), or phosphoric acid (o). Values are means 1S.E. of 12 replicates.
106 Chopler 5: pH Effecf on Glufosinote uploke in R. rophanistrunond C. ousfralis
5.3.2. Absorption and Translocation: PIants Treated w¡th Low pH
Glufosinate
14C-glufosinate Experiments with conducted under controlled growth conditions of 5/10'C showed that the absorption of glufosinate remained similar in plants that were treated with the commercial formulation of glufosinate and those treated with glufosinate adjusted to pH 2.5 with hydrochloric 1aO-glufosinate acid. A rapid absorption of was evident under both conditions and by 72 HAÍ nearly allthe applied herbicide had been absorbed into the treated leaf (Figure 5.4).
1aC-glufosinate Translocation of within the plants was similar regardless of whether the plants had been treated with the normal formulation of glufosinate or glufosinate adjusted to pH 2.5 with HCL
(Figure 5.5). Most of the herbicide was found in either the tip or treated part of the treated leaf with very small amounts translocated to the meristem or to other leaves.
100 c) (ú 'i, 80 o fE ,? Ëuo :-ã ! üoo eeo- b20 ¡¡U'
0 020406080 Time (HAT)
1aO-glufosinate Figure 5.4 Absorption of in R. raphanistrum treated with the commercialformulation of glufosinate (r), or glufosinate adjusted to pH 2.5 with HCI (o). Values are means t S.E. of 15 replicates.
107 Chopïer 5: pH Effect on Glufosinote uPtoke in R. raphonístrunand C. auslralis
60 A
40
20
0 60 B
40
20
E o 0 t¡ 60 o c u, .cl G' S 40 o GI 20 .g .n o ¿ 0 6 1 0 () D I b 4 2 0 1 0 E I 6 4 2 0 o20406080 Time (HAT)
Figure 5.5 Time course of 14C-glufosinate translocated in plants treated with the commercial formulation of glufosinate (r) or glufosinate adjusted with HCI to pH 2.5 (o) in R. raphanistrum toO-glufosinate 1aC-glufosinate grown at 5/10"C. Translocation of to upper leaf (A), retained in the treated leaf (B) toO-glufosinate translocated to the base of the treated leaf (C), to the shoot meristem (D) or to other leaves (E). Values are means 1 S.E. of 10 replicates.
108 Chopter 5: pHEffect on Glufosinole uPtoke in R. raphanistrumand C. ousîralis
5.3.3. Uptake of Glufosinate into Leaf Slices
Experiments were conducted with leaf slices ol R. raphanistrum in an attempt to determine the
1aC- possible intra cellular uptake of glufosinate. These experiments showed the amount of glufosinate absorbed remained low throughout the time course of 30 min. Absorption was linear laC-glufosinate for the concentrations used, indicating that the uptake of is mainly via non- facilitated diffusion. Experiments conducted at different external pH demonstrated that pH 1aC-glufosinate influenced uptake into leaf discs. There was more absorption into leaf disks at low pH. ln our experiments there was a significant (P<0.001) increase in uptake occurring with the external pH 5 when compared to pH 7.5 (Figure 5.6).
12
10 =E) oU' I o E CL 6 c .9 CL 4 o Ø .ct 2 t
0 0 50 100 150 200 250 Glufosinate (p mo¡es)
Figure 5.6: Concentration dependent uptake of glufosinate into leaf slices of R. raphanistrum in Hepes buffer-pH 7.5 (r) and in acetate buffer-pH 5 (o). Values are means t S.E. of B replicates.
109 Chopter 5: pH Effect on Glufosinote uptoke in P. rdphanistrun ond C. auslralis
5.3.4. Uptake of Glufosinate into Cells
Subsequent experiments were conducted with cells of the giant alga Chara australis because cell walls and cell contents could be easily separated in this species. The experiments on time dependent uptake of glufosinate into C. australis suggested that the highest rate of uptake of glufosinate was after 10 min. As the time increased, the rate of absorption decreased (Figure
5.74). After 10 min, the uptake of the labeled glufosinate amounted to 50 nmoles m-'s-'. When this was further analyzed by separating the cytoplasmic contents from the cell wall, the rate of transfer across the plasmalemma was calculated at 10 nmoles m-'s-t. The remainder of the radioactivity was absorbed on to the cell walls (Figure 5.78). Experiments examining the concentration dependant uptake of glufosinate into cells of C. australis showed a slow rate of uptake at low concentrations. However, with higher concentrations of glufosinate the rate of uptake increased rapidly. Analysis of glufosinate retained in the cell wall and that translocated through the plasmalemma showed that at low concentrations the rate of glufosinate translocation to the cytoplasm was low. As the external concentration of glufosinate was increased, the rate of absorption to cell walls and uptake into cells increased (Figure 5.8). Changing the buffer pH in
uptake experiments with C. australis showed that there was an increase in the rate of uptake as pH
increased from 6.5 to 7.5 (Figure 5.94). However, at pH 5.5 and 8.5 the total rate of uptake was
lower than with pH 6.5 and 7.5. Much of the increased rate of uptake by C. australs cells at pH 6.5
and 7.5 was due to increased absorption by cell walls. However, there was also an increase in rate
of uptake across the plasmalemma (Figure 5.98).
110 Chopter 5: pH Effect on Glufosinote uPtake in P. raphonislrunond C. ouslrolís
70
60 o .E.nr ^so o tt, Eî-40cDtr öo : 930 _eE -20 f,å 10
0 10 30 60 120 180 240 300 Time (min)
taO-glufosinate Figure 5.74: Time dependent rate of uptake of into cells of Chara australis. Values are means + S.E. of 10 replicates.
50
.9 '6840 Or; =ol- +- 30 Jr6 b ?ro
E-9(ú ä10 f
0 10 30 60 120 180 240 300 Time (min)
toO-glufosinate Figure 5.7B: Time dependence of rate of uptake of into cell walls (n) and cytoplasmic contents (t) ol Chara australis. Values are means t S.E. of 10 replicates.
111 Chopter 5: pH Effecl on Glufosinote uptoke in P. raphdnistrumand C. australis
40
o G c 'õ 30
e"fN
o)cl¡- 20 oooo otr lto-9 (ú 10 o. Ð
0 0 50 100 150 200 250 Concentration (pM)
14C-glufosinate Figure 5.8: Concentration dependent uptake of into (o) cell walts and (r) cell contents of Chara australís. Values are means + S.E. of 10 replicates.
100 Ø o¡ E 80 oU' o E¿ 60 t- fL o- 40 () ìt o lÉ (ú 20 CL f 0 5.5 6.5 7.5 8.5 Buffer pH
Figure 5.94: Total rate of uptake of glufosinate into cells of Chara austral¡s at different pH. Values are means t S.E. of 10 replicates.
112 ChopTer 5: pH Effect on Glufosinole uPtoke ¡n R. raphanistrumond C. ousfralís
100
rto (\ 80 E U' -9 o E 60 l- F o. o. 40 Ëo o ll (u 20 o. f 0 5.5 6.5 7.5 8.5 Buffer pH
laO-glufosinate Figure 5.98: Rate of uptake of into cell walls (a) and cytoplasmic contents (r) of Chara australis at different pH. Values are means t S.E. of 10 replicates.
113 Chopter 5: pH Effect on Glufosinote uPloke ¡n P. raphanistrumand C. australís
5.4. Discussion
The present study tested the hypothesis that glufosinate absorption might be important at lower pH as a result of increased acid trapping, and investigated the role that solution pH may play in glufosinate uptake by cells. A better understanding of these processes may lead to improved efficacy of the herbicide. Therefore, the possibility was tested that reducing the pH of the spray solution might provide an additional driving force for glufosinate molecules to transport through the cell walls and the plasmalemma of B. raphanistrum cells. Whole plants were sprayed with glufosinate adjusted with different pH, with HCI or phosphoric acids and with potassium hydroxide.
The results of these studies indicated that solution pH 2.5 had enhanced glufosinate efficacy and that when R. raphanísfrum was treated with glufosinate solutions adjusted with HCI (pH 2.5) efficacy was enhanced. There were differences between experiments conducted in growth rooms compared to those outdoors. Changing pH appeared to have a lesser impact on herbicide efficacy for plants grown outdoors. lt is unclear why this was so.
Studies on the concentration dependent uptake into leaf slices showed an increase in uptake of lac-glufosinate as the external glufosinate concentration was increased with a linear uptake suggesting that glufosinate, an analogue of glutamate, is taken up via non facilitated diffusion at
higher concentrations in F. raphanistrum. Experiments conducted with glufosinate by Ullrich et al.
(1990) showed similar results to this finding. They showed that the uptake of glufosinale in Lemna
gibba proceeded biphasically with respect to time and concentration and it was strongly enhanced
below pH 5 but remained unchanged between pH 5 and 7. These authors suggested that the
reason for this could be associated with dissociation of the carboxyl group of glufosinate playing an
important role in transport by allowing diffusion of the uncharged acid at low pH.
Because of the immediate membrane depolarizing effect, the anion glufosinate is assumed to be
taken up by a proton co-transport mechanism and at lower pH it was observed that additional
undissociated glufosinate is transported by diffusion (Ullrich et al. 1990). Other studies in this area
show similar results. According to Novacky et al. (1978) and Etherton and Rubinstein (1978) the
characteristics of glufosinate uptake and primary depolarization resemble those of glutamate or
114 Chopter 5: pH Effect on Glufosinote uPtoke in R. raphonístrumond C. ouslrolis
alanine uptake. Ullrich et al. (1990) stated that in their experiments the uptake rate of glufosinate was approximately 5"/" of that of glutamate. According to Denis and Delrot (1993) glyphosate, a herbicide structurally similar to glufosinate, is taken up into the cells of Viciafabavia a phosphate transporter in the plasma membrane. Concentration dependent studies showed glyphosate uptake exhibited a saturable phase at low glyphosate concentrations (0.5 to 3 pM) superimposed by a linear uptake at higher concentrations (up to 100 pM) (Denis and Delrot 1993). Kinetics indicated that the saturable component of glyphosate transpoft was completely inhibited by either phosphate or phosphonoformic acid (Denis and Delrot 1993).
The experiments with C. australis showed that uptake of glufosinate into cells was complete within 10 min and only about a quarter of the absorbed herbicide was taken up through the plasmamembrane. Concentration dependent uptake experiments showed that at low toQ-glufosinate 14C- concentrations, in the cytoplasm remained very low. This suggests that not all glufosinate taken up through the cell wall is translocated across the plasmalemma. The rate of labeled glufosinate translocated across the plasmalemma remained minute until external concentration was 100¡rM. With 2O0pM of glufosinate, the rate of uptake increased to 6.05 nmoles m-t s-t. At this concentration the rate of absorption by the cell wall was more than f ive fold the rate of transport into the cytoplasm. This indicates that translocation through the plasmalemma remains poor for glufosinate. Changing the pH of the glufosinate solution resulted in a change in the rate of uptake through the cell wall. At pH 6.5 and 7.5 the rale of uptake more than doubled compared to that at pH 5.5. However, when the rate of glufosinate transport across the
plasmalemma was examined, this remained low. Therefore, changing the pH of the solution
appeared to mainly increase the rate of uptake into the cellwalls.
From leaf slice experiments, glufosinate uptake into F. raphanistrum increased at lower pH,
however, only a marginal effect on glufosinate efficacy was observed by reducing the pH of the
spray solution. Taken together with the results from experiments on C. australrs, these results
suggest that lower solutions pH greatly increase partitioning of glufosinate into cell walls, but only a
marginal increase in transport of glufosinate across the plasmalemma occurs. ln addition,
115 glufosinate transport across the plasmalemma is generally poor and this is probably reflected in the poor translocation of glufosinate in phloem tissues.
116 Chapler 6: Generol discussion
Chapter six: General Discuss¡on
The proceeding five chapters described investigations on reasons for poor efficacy of glufosinate against R. raphanistrum and L. rigidum under southern Australian winter conditions. Absorption and translocation of glufosinate in these weeds were studied in some detail under different environmental conditions. Attempts were then made to improve glufosinate efficacy by changing surfactants or adding chelating agents, ammonium sulfate or other herbicides. Studies were also under taken to understand the uptake of glufosinate through cell membranes.
Problems associated with glufosinate in the control of different weed species were discussed in
Chapter 1. Although glufosinate is described as a non-selective foliar active herbicide with many advantages for the user, recent studies have demonstrated that some weed species are poorly controlled by this herbicide. Glufosinate is soon to be registered in Australia for weed control in genetically modified canola cultivars known by the name lnVigor@, but is known to provide less than adequate control of two major weeds of cropping, R. raphanistrum and L. rigídum (Michael
Clark pers. comm.). Similar findings have been reported in relevance to several other weed
species. Hass and Muller (1987) after studying the effect of glufosinate on several weed species
pointed out that Calopogium mucunoides (L) was the least sensitive to glufosinate. According to
Pline et al. (2000), Asclepias syriaca, was highly tolerant to glufosinate compared lo Setaria faberi.
Burgos et al. (2000) showed that glufosinate did not provide sufficient control ol Sida spinosa,
Echinoctoa crus-gattiand Ameranthus palmeri. Such differential sensitivity to glufosinate was also
reported in several other species (Ridley and McNally 1985; Mersey et al. 1990)'
As described in Chapter 1, the mechanisms for the variation of efficacy of glufosinate have not
been fully elucidated. However, it is known that the differential sensitivity of glufosinate was not
based on the differences in molecular target sites in weeds (Ridley and McNally 1985) or due to
differential metabolic degradation (Hass and Muller 1987, Mersey et al. 1990; Komossa and
117 Chapter 6: Generol discussion
Sandermann (1992). Reasons for variable efficacy have been explained as a result of rapid phytotoxicity occurring in the plants after spray appl¡cation (Beriault et al. 1999) and such intra- specific differences in activity may relate to differences in uptake and translocation (Mersey et al. lggo). Previous studies also showed that glufosinate efficacy is influenced by environmenlal conditions and the herbicide application rates (Carlson and Burnside 1984).
The possible influence of environmental conditions on glufosinate efficacy was discussed in
Chapter 1 and also in Chapter 3. ln the southern regions of Australia, most of the cereal crops are sown during autumn and grown in the winter to spring months. Hence, most of the post emergence weed control occurs during winter. The weather pattern in this region of Australia is very similar to the Mediterranean region where dry summers are followed by wet winter months.
Weeds such as R. raphanistrum, L. rigidum and A. sterilis having originated in the Mediterranean regions, are well suited to the climatic conditions of the southern regions of Australia.
Differential efficacy of glufosinate was evident in the experiments presented in Chapter 2 where the
herbicide efficacy was compared between L. rigidum and A. sterilis. The results showed that L'
rigidum was poorly controlled when compared to A. sterilis. Dose response experiments
conducted under controlled growth conditions as well as outdoors under winter conditions showed
that GRso of A. steritis was significantly lower than that ol L. rigidum. Studies on metabolism of
glufosinate using thin layer chromatography to separate glufosinate from degraded products
showed that there was no significant metabolic degradation of glufosinate in either species.
There was no significant difference in the retention of glufosinate between these species and no laO-glufosinate difference was observed in absorption of into leaves. However, subsequent studies
on translocation showed differential patterns of glufosinate translocation in the apoplastic and
symplastic streams of the two species. ln L. rigidum relatively more glufosinate translocated
towards the leaf tip of the treated leaf when compared lo A. sterilis. However, significantly more
glufosinate translocated to the meristematic regions ol A. sterilis when compared to L. rigidum.
This study indicated that efficacy of glufosinate in the two model species was determined by the
amount of herbicide translocating in the symplastic stream towards the apical meristem and the
118 Chopler 6: Generol discussion
roots. A similar finding has been reported in relevance to paraquat against the resistant biotype of
Hordeum leporinum, where the herbicide resistance was reduced under warm conditions (Purba et al. 1995). This study showed that the enhanced herbicide efficacy was due to enhanced basipital translocation. The apical meristem of grasses is located at the base of the plant. For foliar active herbicides to control grasses, sufficient herbicide need to translocate to the apical meristem so that newly developing leaves contain a lethal dose of the herbicide. Therefore, it is conceivable that the efficacy of a herbicide in grasses could be related to the ability of the herbicide to translocate to the meristem
Efficacy of glufosinate under differential environmental conditions on ¡?. raphanistrum was analyzed in Chapter 3. These results confirmed the originalfinding of Michael Clarke (1999)that glufosinate gives poor control ol R. raphanistrum under southern Australian winter conditions. According to
Michael Clarke (1999), glufosinate has a better control of B. raphanistrum in Europe. However, when the H. raphanistrum population from England was grown under southern Australian winter conditions, this population was poorly controlled by glufosinate. The results of this experiment suggest that the poor sensitivity to glufosinate may not be related to the genetic variation of
R. raphanisfrum populations but determined by the environment.
Experiments conducted with P. raphanistrum under different temperature regimes showed that efficacy of glufosinate was enhanced by increasing temperature. ln contrast, light intensity had no impact in enhancing the efficacy of the herbicide. Absorption and translocation experiments performed under cold (5/10'C) and hot (20125C) controlled growth conditions showed that glufosinate was absorbed rapidly into leaves ol R. raphanistrum regardless of the temperature. A significantly higher proportion of the labelled glufosinate translocated to the tip of the treated leaf
under cold conditions compared to warmer temperatures. ln contrast, basipetal translocation was four times higher in plants that were exposed to warm conditions than those exposed to cool
conditions. Under cold conditions, translocation to the meristematic regions was less than 1% of
the absorbed herbicide by72HAT. ln contrast, underwarm temperatures, labeled glufosinate in the meristem accumulated to 45% by 72 HAf . Similarly, the label glufosinate that was
translocated to other leaves was lower than 1% under cold conditions.
119 Chopler 6: Generol discussion
Results presented in Chapter 3 showed that glufosinate efficacy against R. raphanistrum can be altered by changing environmental conditions. Similar findings have been reported by other workers on the efficacy of glufosinate in other weed species. Steckel et al (1997) reported that differential glufosinate sensitivity in Setaria faberi, Echinochloa crus-galli, Abutilon theophrasti, and
Chenopodium album was caused by differential translocation of the herbicide. Xie et al (1996) also showed that basipital translocation of imazamethabenz-methyl was increased in Avena fatua by high temperatures whilst low temperature (5/10"C) decreased total translocation and translocation efficiency of this herbicide. Harker and Dekker (1988) showed evidence that increase in temperature enhanced basipital translocation of several herbicides (glyphosate, sethoxydim, cloproxydim, fluazifop-butyl) in Agropyron repens. These studies suggest that translocation of herbicides in the plant is vital for efficacy of the herbicidal activity. The results presented in Chapter
3 show glufosinate translocation to the meristamatic region is criticalfor better herbicide efficacy.
These two studies have produced a consistent pattern with regard to glufosinate control of weeds. ln both cases poor control was correlated with increased accumulation of glufosinate in the leaf tips. This suggests increased movement of glufosinate in xylem tissues and hence reduced
uptake into cells. Better control was correlated with increased translocation of glufosinate to the
meristematic zone. Such translocation from the point of application must have occurred via the
phloem. This suggests that better uptake into cells and hence movement in the phloem is
essentialfor good control by glufosinate.
Chapter 4 considered a range of approaches to improve glufosinate efficacy against
R. raphanistrum. Herbicides such as amitrole, diflufenican, clomazone and benzofenap were used
in combination of glufosinate in several dose response studies. These herbicides were chosen as
they were carotenoid biosynthesis inhibitors and might be expected to enhance the activity of
glufosinate. The results showed that the carotenoid biosynthetic inhibitors amitrole and clomazone
were antagonistic with glufosinate in the control of Æ. raphanistrum. Benzofenap and glufosinate
were additive in effect where as diflufenican and glufosinate were slightly synergistic.
120 Chopler 6: General discussion
Dose response experiments performed with alternative formulations contain¡ng different surfactants (DS 6183 and DS 8165) did not show an enhanced efficacy in R. raphanistrum.
However, application of 'lOmM EDTA 24 hours prior to glufosinate application increased the mortality of H. raphanistrum by more than two fold at the field rate of glufosinate (600 g ai ha'1) though a mixture of EDTA and glufosinate did not improve the efficacy. EDTA is a chelaling agent that binds and removes cations such as calcium from the solution. Calcium ions would bind to negatively charged herbicide molecules such as glufosinate at the cell wall surface and may prevent or reduce absorption. As a consequence, more intracellular absorption may be possible.
However, experiments examining glufosinate absorption and translocation failed to determine how
EDTA improved glufosinate efficacy. Further studies will be useful to focus on the effects of EDTA on the cell wall and plasmalemma to determine the influence of EDTA on the movement of glufosinate in the symplastic stream and in the phloem.
The addition of ammonium sulfate to the spray solution has been shown to improve the efficacy of a number of herbicides including glufosinate in previous research (Mashhoff et al. 2000). However, our experiments performed against R. raphanisfrum showed ammonium sulfate was unable to improve the efficacy of glufosinate. The reason for the failure to improve the efficacy may be related to the weed species. Mashhoff et al. (2000) reported that ammonium sulfate at 20 g L'1 increased the efficacy of glufosinate on Echinochloa crus-galli, Setaria faberi and Abutilon
theophrastibut not on Ameranthus rudls or Chenopodium album. ln a similar study Pline et al
(2000) failed to improve glufosinate and glyphosate control ol Asclepias syriaca, Solanum carolinense or Setaria faberiwilh ammonium sulfate.
Kinetin, a cytokinin herbicide that helps control plant growth process including stimulating cell division, proved to be an effective addition to glufosinate in controlling Æ . raphanistrum (Chapter
4). ln this case kinetin slightly decreased glufosinate absorption into leaves, but greatly increased
glufosinate translocation to the meristem and to other leaves. Therefore, the improvement of
glufosinate efficacy by kinetin is correlated with increased translocation in phloem. Kinetin may
have caused the meristem to be a greater sink for carbohydrates and hence increased
121 Chopler 6: General discussion
translocation of other compounds. lt does not appear that cytokinins have been investigated as herbicide adjuvants but they show some potential.
Chapter 5 investigated the mechanisms of glufosinate uptake into leaf slices of R. raphanistrum and into individual cells of C. australis. Previous studies conducted with glufosinale in Lemna gibba showed that absorption of glufosinate was strongly enhanced below pH 5 (Ullrich et al. 1990).
They also reported that the addition of glufosinate after or in the presence of glutamate, alanine and other amino acids caused a partial inhibition of glufosinate uptake. Studies conducted with a range of herbicides such as bentazon, bromoxynil (Grimm et al. 1985) and 2,4-D (Jonson and
Bonner 1956, Wedding and Erickson 1957) showed that the enhanced uptake could occur with reduced pH and this pH dependent uptake supports ion trapping. The present study by comparing glufosinate solutions adjusted to different pH values (2.5 to 7.5) showed similar results to previous studies, that lower pH improved glufosinate efficacy and that the efficacy could be further enhanced by reducing the pH with hydrochloric acid and phosphoric acid. Studies on concentration loO-glufosinate dependent uptake into leaf slices showed an increase in uptake of as the external glufosinate concentration was increased. The uptake was proportionately increased with increasing concentrations of glufosinate. This suggests glufosinate uptake depends on non- facilitated diffusion in R. raphanistrum. Experiments conducted with glufosinate by Ullrich et al.
(1990) showed similar results. According to these authors, glufosinate may be taken up by a proton co{ransport mechanism, using the same carrier as neutral and acidic amino acids and at low pH, additional undissociated acid was transported by diffusion. Studies with other herbicides also showed such enhanced uptake due to low pH. Despeghel and Delorot (1983) after studying the uptake of U-14C threonine and 'oC aminoisobutyrate by leaf discs ol Vicia faba showed that
uptake of these compounds was strongly pH dependent with an optimum at pH 4 and exhibited
biphasic saturation kinetics.
Experiments with C. australis showed that uptake of glufosinate into cells was completed within 10
min and approximately a quarter of the absorbed herbicide was taken up through the plasma
membrane. The remainder of the herbicide was located in the C. australis cell wall. Concentration 1aO-glufosinate dependent uptake experiments showed that at low concentrations, in the cytoplasm
122 Chopler 6: Generol discussion
taO-glufosinate remained very low. This suggests that not all absorbed by the cell wall was translocated across the plasmalemma. At pH 6.5 and 7.5, the uptake of glufosinate was more than doubled in C. australrs compared to pH 5.5, However, importantly, it was observed that the amount of the labelled glufosinate translocated across the plasma lemma remained low irrespective of the pH of the herbicide solution. lt is known that intracellular uptake of herbicides is criticalfor phloem loading. It can be suggested that poor translocation of glufosinate in to the meristamatic area may have resulted due to poor intracellular uptake of the herbicide. lnformation derived from the C. australis study may help understanding the reason for greatertranslocation of glufosinale into leaf tips of R. raphanistrum. The C. australis study, showed that most of the absorbed glufosinate remained bound to the cell wall which would promote greater movement of the herbicide in the apoplastic stream and possibly accumulation in the leaf tip.
ln summary, this project addressed the important question of why the major weed species such as
R. raphanistrum and L. rigidum are poorly controlled by glufosinate under soulhern Australian winter conditions. This represents an important industrial problem in relevance to introduction of the genetically modified canola cultivar lnVigor@ to southern Australian cropping regions. The genetical modification of lnVigor@ was targeted at creating a glufosinate resistant canola variety, in order to gain the specific advantage of controlling the major Brassicaceae weeds such as F. raphanistrum, which belong to the same family as canola. Results of the present study showed that the poor efficacy of the herbicide may not be related to genetic differences between
R. raphanístrum populations, effect of altered target site, increased metabolism or due to the uptake of the herbicide. The results showed that the poor efficacy under cold temperatures was primarily due to the poor translocation of the herbicide in to the meristematic regions of the weed.
ln species such as R. raphanistrum and L.. rigidum, only a small percentage of labeled glufosinate translocated to the meristematic regions. Therefore, not enough herbicide was available to provide a lethal dose in newly developing leaves. ln contrast, in A. sterilis, where greater translocation of glufosinate to the meristematic regions was achieved correlated to the successful control of the weed by glufosinate.
123 Chopter 6: Generol discussion
ln Australia, canola is cultivated during winter months. The absorption and translocation studies showed that it is likely that the cold winter conditions of southern Australia aggravate the poor translocation even further. The results showed that although glufosinate is effective at causing bleaching and necrosis in existing leaves, it is unable to prevent survival and new leaf growth of the plant. To achieve better control of R. raphanistrum with glufosinate it is necessary to apply glufosinate under conditions that promote translocation of glufosinate to the meristem. At present that means applying the herbicide under warmer conditions. This may mean applying the herbicide earlier or later in the season when conditions are warmer or in the more northerly regions of the wheat belt. Another option would be to explore the inclusion of compounds in the formulation that might increase phloem translocation. This latter idea seems an area of fruitfulfuture research.
124 References
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