WO2001086303A2

WO2001086303A2 - Rapid tests for herbicide resistance in grasses

Info

Publication number: WO2001086303A2
Application number: PCT/GB2001/002068
Authority: WO
Inventors: John Reade; Andrew Howard Cobb
Original assignee: Harper Adams University College
Priority date: 2000-05-11
Filing date: 2001-05-11
Publication date: 2001-11-15
Also published as: GB0011268D0; WO2001086303A3; AU2001256481A1

Abstract

A method for use in detecting the presence of a target polypeptide in a weed, wherein the target polypeptide is connected with resistance of the weed to herbicides. The method comprises delivering to a sample of the weed an antiserum which is directed against the target polypeptide. The antiserum identifies and binds with the target polypeptide and the bound antiserum is then visualised by an appropriate method. The target polypeptide is preferably a glutathione S-transferase and the antiserum is preferably a monoclonal antiserum raised against polypeptide.

Description

RAPID TESTS FOR HERBICIDE RESISTANCE IN GRASSES

The present invention relates to a test method for determining whether weeds, in particular black-grass and other grasses, have developed resistance to particular herbicides.

Cultivation of cereal crops is of considered importance in the UK. The maximum yield possible is desired by farmers. However, weeds compete for light, water, nutrients and space which can result in a lower yield. Problems are encountered from the growth of weeds when cultivating cereal crops. Weeds can be divided into broadleaf and grass weeds.

Grass weeds are treated with herbicides but problems arise in finding herbicides which can distinguish the grass weeds from the grass-type cereal crops. Therefore the range of herbicides available is not extensive. Use of the same field every year to grow the same crop and treatment with the same herbicide may lead to the weed population becoming resistant to that herbicide. Farmers are unwilling to use crop rotation as some areas of land are better suited to a particular crop.

The problem of herbicide resistance in grass weeds in the UK was first noted in black-grass in a field in Essex and has spread north over the last 20 years with the resistance now having reached the Scottish borders. The presence of a grass weed in a cereal crop field can reduce the yield of the cereal crop by 40-50%.

There are two types of herbicide treatment available for grass weeds such as black-grass. The first type of treatment is post-emergence of the crop.

The second type of treatment is pre-emergence treatment where the field is treated with herbicide before the cereal crop germinates and the crop grows in a weed-free area. However, pre-emergence treatment is not used routinely as it uses chemicals which are less environmentally friendly and are more expensive.

It would therefore be beneficial to produce a way of testing black-grass and other grass weeds in a crop field to determine whether they are resistant to herbicides or not in order that they can be adequately treated i.e. treated with more effective chemicals and pre-emer gents, but only where necessary.

Work has been carried out at Harper Adams University College on herbicide resistant black-grass plants to try to determine some of the mechanisms involved in herbicide resistance. Glutathione S-transf erases (GSTs) metabolise herbicides in some plants and the investigations carried out by Harper Adams University College were based on this group of enzymes.

Glutathione S-transf erases (GSTs, EC 2.5.1.18) are a group of enzymes which catalyse the nucleophilic substitution reaction of a wide variety of substrates with the tripeptide glutathione. The hydrophobic substrates are all electrophilic and are usually foreign to the organism. GSTs are found throughout the animal and plant kingdoms. In animals they have been extensively studied and are implicated in many roles, including toxin and drug metabolism. Although studied to a lesser extent in plants, GSTs have been implicated in herbicide resistance, crop tolerance to herbicides, various stress responses, and also in secondary metabolism. GSTs have been studied in maize (Zea mays L) , soybean (Glycine max (L) Merr) , and wheat (Triticum aestivum L) . In maize multiple GSTs with subunit MWs of 29, 27 and 26kDa have been identified. GSTs composed of these subunits exhibited activity towards the herbicides atrazine, alachlor, metolachlor and fluorodifen. Activity against these herbicides was also detected in a variety of associated weed species. In soybean, a GST containing 26 kDa subunits has been identified with activity towards metolachlor. Activity against the herbicides fomesafen, acifluorfen and chlorimuron-ethyl have also been reported in this species. Wheat is also reported to contain GST activity against the herbicides fenoxaprop and fluorodifen, and contains a safener-induced GST with 26 kDa subunits. The involvement of GSTs in the metabolism of many herbicides has generated interest in a potential role for these enzymes in herbicide resistance in weeds and crops. Herbicide resistance may arise due to an altered target site, increased metabolism or increased compartment- alisation of the herbicide away from its site of action. Thus, an inherited increase in the amount and/or activity of enzymes responsible for herbicide metabolism may lead to a weed or crop population demonstrating resistance to herbicides metabolised by the enzyme in question.

As set out above Alopecurus myosuroides Huds (black-grass) is a major problem weed in autumn-sown cereal crops in the UK and its presence can cause reductions in both crop quality and yield. This grass weed is chemically controlled using urea herbicides (e.g. chlorotoluron, CTU) and more recently with aryloxyphenoxypropionates (e.g. f enoxaprop-ethyl) . However, in the early 1980s populations of black-grass were reported to be resistant to CTU and this has become an increasingly observed problem, with reports of resistance in the UK, Germany, France, the Netherlands, Spain and Israel. Cross -resistance and multiple resistance have also been observed. Cross-resistant biotypes display resistance to two or more herbicides due to a single resistance mechanism, whereas multiple resistant biotypes possess two or more distinct resistance mechanisms. Laboratory investigations have suggested that increased metabolism may be, at least in part, the mechanism responsible for resistance in black-grass, involving both cytochrome P450 monooxygenases and GSTs. The applicant has shown that the black-grass biotype Peldon (resistant to CTU and fenoxaprop) has approximately twice the GST activity towards l-chloro-2,4-dinitrobenzene (CDNB) of susceptible biotypes. This raised activity is constitutive, not requiring herbicide treatment to be expressed. However, it is important to recognise that CDNB is only a model substrate. Indeed, not all GSTs metabolise CDNB and it is possible that isoforms that metabolise herbicides may not metabolise CDNB, and vice versa. A purification scheme for GSTs from black-grass has been developed in order that their role in herbicide metabolism in this important grass weed can be further evaluated. (See specific example section) .

Purification of GSTs by the authors (Reade and Cobb) from a susceptible biotype revealed one, 27.5 kDa, polypeptide possessing GST activity. This polypeptide was also present in the Peldon biotype along with a 30 kDa polypeptide. The smaller polypeptide appeared to be present at higher amounts in the resistant population. Antisera raised against the 27.5kDa polypeptide detected the 27.5kDa polypeptide in Peldon extracts indicating this to be the same protein. The antisera raised against the black-grass polypeptide did not detect the 30 kDa polypeptide in Peldon extracts. This indicated that the larger polypeptide is a distinct protein rather than a pre-processed version of the 27.5 kDa polypeptide. It also suggests that the 27.5 kDa polypeptide is not a breakdown product of the 30 kDa polypeptide. Increased GST activity in the Peldon biotype may be a result of increased amounts of the 27.5 kDa polypeptide or the combination of this with the presence of the 30 kDa polypeptide. It would seem likely from comparison with other weed and crop GST profiles that both increase in the amount of the 27.5 kDa polypeptide and the presence of the 30 kDa polypeptide, suggested to be a GST subunit, contribute to the higher GST activities in the resistant biotype Peldon.

In conclusion, a purification scheme for a GST from black-grass has been developed. A 27.5 kDa polypeptide with GST activity has been identified in herbicide-susceptible black-grass. Herbicide-resistant black-grass yielded this polypeptide in greater amounts and in addition, a 30 kDa polypeptide. Antisera study suggests that the 30 kDa polypeptide is not a precursor of the 27.5 kDa polypeptide. It is postulated that this polypeptide is a separate GST subunit that may play a role in herbicide resistance in the Peldon biotype.

A test to determine the extent of herbicide resistance in black-grass populations would aid in its control. Present tests for resistance utilise seeds collected from black-grass prior to crop harvest. Hence, seed is returned to the soil and resistance control measures can only be carried out in the following season. Alternative tests involve glasshouse spraying of black-grass plants that have survived herbicide treatment in the field, and can be costly and time consuming. Thus, existing testing can only be accomplished with seed or transplanted plants over a minimum of 6 weeks. The development of a quick resistance test that can be carried out prior to post-emergence herbicide application, so allowing alternative measures to be adopted, where necessary, during the same season as detection would be beneficial. It is envisaged that this test should give results only hours after collection of plants.

Accordingly the present invention provides a method for use in detecting the presence of a target polypeptide in a weed which is connected with the resistance of the weed to herbicides comprising the steps of :- a) delivering to a sample of the weed an antiserum which is directed against the target polypeptide to identify and bind to the polypeptide; and

b) visualising the bound antiserum

The antiserum used is preferably a monoclonal antiserum raised against the 30 kDa polypeptide.

Preferably the weed is a grass weed, most preferably black-grass. Alternatively the grass weed could be rye grass or wild oats.

Preferably the target polypeptide is a glutathione S-transferase (GST) , most preferably a 30 kDa GST subunit of GST.

Preferably the N-terminal sequence of the 30kDa GST polypeptide is as follows :-

ala-pro-val-lys-val-phe-gly-pro-ala-met-ser-thr-asn-val-ala-arg-val-ile- leu.

Amino acid key ala = alanine pro = proline val = valine lys = lysine phe = phenylalanine giy = glycine met = methionine ser = serine thr = threonine asn = asparagine arg = arginine ile = isoleucine leu = leucine gin = glutamine

The visualisation step preferably further comprises the addition of a visualisation means. The visualisation means may include the use of enzymes such as peroxide or alkaline phosphatase, alternatively fluorescent compounds. Particulate materials or radio labelled compounds may be used. The avidin bio ten complex method may be used for visualisation.

An amplification means may be used in addition to the visualisation means.

The visualisation is preferably carried out using ELISA technology. In ELISA:

a) The target polypeptide is diluted to lμg/50μl and lμg samples are bound to wells in ELISA plates overnight at 4°C;

b) The plates are blocked with milk protein for 3 hrs at 37°C.

c) The Proteins are challenged with monoclonal antisera raised against the target polypeptide overnight at 4°C; d) Following a wash step the protein-antibody complex is challenged with second antibody conjugated with a detection means (for example, goat ani-mouse alkaline phosphatase) for 2 hrs at 21°C to amplify and detect the results; and

e) Following a second wash step visualisation is carried out by colour development of alkaline phosphatase.

The present invention further provides a test kit for use in detecting the presence of a target polypeptide in a weed which is connected with the resistance of the weed to herbicides, the kits contain a supply of an antiserum which is directed against the target polypeptide.

The kit may further include a supply of a detection means. A supply of an amplification means may also be included in the kit. Also included may be a supply of a visualisation means.

The kit may further comprise a supply of a reference or control material.

The kit may include instructions.

Further provided is a test kit as set out above which is suitable for use on automated test apparatus.

The present invention also relates to the use of an antiserum which is directed against a target polypeptide connected with resisitivity of weeds to herbicides in the detection of the polypeptide in accordance with the method set out above or using the test kit as set out above. The present invention will now be further described with reference to the example and the figures in which :-

Figure 1 Behaviour of proteins extracted from biotypes of black- grass on gel filtration column;

Figure 2 Behaviour of proteins extracted from biotypes of black- grass on agarose affinity column;

Figure 3 SDS-PAGE analysis of protein extracted from biotypes of black-grass;

Figure 4 GST activity of black-grass from four different field trial plots; and

Figure 5 ELISA response for black-grass samples from four different field trial plots.

1) Investigation of GST activity in herbicide resistant plants.

A) MATERIALS AND METHODS

Al) Plant materials and growth conditions

Black-grass seeds from plants susceptible to CTU were obtained from Herbiseed Ltd, Berkshire, UK, and seeds from plants resistant to CTU and fenoxaprop were collected by hand at Peldon, Essex, UK (July 1996) . The Herbiseed biotype is a black-grass population grown without any herbicide application for at least seven generations (Herbiseed Ltd, Berkshire, UK, pers comm) . Seeds were sown in J Arthur Bower's peat-based multi-purpose compost and watered from below. All plants were grown under glasshouse conditions, 20°C day, 15°C night ( ± 5°C), with supplementary lighting from high-pressure 250-W sodium lamps for 16h each day. Plants were harvested at the three-leaf stage and either processed immediately or frozen in liquid nitrogen and stored at -70 °C until needed.

A2) Glutathione S-transferase activity assayed toward CDNB

GST activity was extracted and assayed. Approximately 0.5g of frozen tissue was ground to a powder in a pestle and mortar and thawed in potassium phosphate buffer (0.1M, pH 7.0; 5ml) containing sodium ascorbate (lOmM) and diethylenetriaminepentaacetic acid (DTP A; 5mM) . Polyvinylpolypyrrolidone (PVPP) was added at 40glitre ^x prior to the addition of the extract to inhibit polyphenoloxidase activity. The extract was homogenised for 20s using an Ultra-turrax homogeniser and centrifuged (15000g, 15 min at 4°C). The supernatant was desalted on a 10ml Sephadex G-25 column previously equilibrated with potassium phosphate buffer (0.1 M, pH 7.0; 25 ml) containing DTPA (0.25 mM). The sample (2.5 ml) was loaded onto the column and eluted with 3.5 ml of the equilibration buffer. Samples were either used for immediate study or stored at -70 °C.

GST activity was determined using CDNB as an artificial substrate. Black-grass extract (50 μl unless otherwise stated) was added to potassium phosphate buffer (0.1M, pH 6.5; 950 ml) containing reduced glutathione (GSH) and CDNB both at a concentration of 1 mM in a final assay volume of 1 ml. Conjugate formation was monitored spectrophotometrically at 340 nm for 4 min at 20° C. Non-enzymic conjugate formation was determined by replacing the plant extract with an equal volume of assay buffer. Activity was calculated as nmol CDNB min ¹ mg^"1 total protein using the molar extinction coefficient of conjugate formed (9.6mM ¹ cm ¹) . Protein content of extracts was determined by the method of Bradford. (Anal.Biochem 72:248-254 (1976)).

A3) Kinetic studies Michaelis constants for CDNB and GSH were determined from Lineweaver-Burk plots. The assays were carried out as stated above with either the crude tissue extract or purified GST fractions. The concentration of each substrate was varied independently over a range of 0.25mM to 4 mM. Each was carried out four times from three extracts.

A4) Purification of GSTs

Frozen tissue (32 g) was ground to a powder in a pestle and mortar and thawed in 196ml buffer A [Tris HCl (0.1 M, pH 7.5) containing Na₂EDTA (1 mM), 2-mercaptoethanol (14mM)] and PVPP (SOglitre ¹) . All subsequent steps were carried out at 4°C. The extract was homogenised for 20 s using an Ultra-turrax homogeniser, filtered through four layers of muslin and centrifuged (15000g, lOmin). The supernatant was adjusted to 80% saturated ammonium sulfate and stirred for 3h. Precipitated protein was collected by centrifugation (15000g, 10 min), resuspended in 24 ml of buffer B [Tris HCl (20 mM, pH 7.8) containing 2mercaptoethanol (14mM)] and dialysed against this buffer overnight. After clarification of the dialysed extract by centrifugation (as above) proteins were loaded onto a 4-ml Q-Sepharose column (Econo-Pac High Q, Bio-Rad Laboratories Ltd, Hemel Hempstead, Herts, UK), previously equilibrated with buffer B, at a flow rate of 1 ml min ¹. The column was washed with 20 bed volumes of the buffer B, then eluted with 10 bed volumes each of the buffer B containing 0.2, 0.4, 0.6, 0.8 and l.0 M NaCl. All GST activity was found to be eluted in the 0.2M NaCl fractions. Fractions containing GST activity were pooled and buffer exchanged on a 45 ml Sephadex G-75 gel filtration column, previously equilibrated with buffer C [Tris HCl (20mM, pH 7.0) containing NaCl (0.4 M), dithiothreitol (1 mM) , DTPA (0.2mM)] , at 1ml min ¹. Fractions containing GST activity were pooled and loaded onto a 45-ml GSH agarose column (Sigma-Aldridge Company Ltd, Poole, Dorset, UK), previously equilibrated with buffer C, at a flow rate of 0.5 ml min ¹ followed by 50 ml buffer C at 0.5 ml min ¹ followed by 50 ml buffer D [potassium phosphate buffer (20 mM; pH 7.2) containing dithiothreitol (ImM) , DTPA (0.2mM)] at 1 ml min ¹. Proteins were eluted by reverse elution using buffer E [potassium phosphate buffer (20mM, pH 6.0) containing glutathione (10 mM) , DTPA (0.2mM)] at 1ml min ¹. Fractions containing GST activity were pooled and retained for later study. Protein concentrations in column elutants were monitored spectrophotometrically at 280nm. GST activity was assayed using CDNB as described above.

A5) Analysis of purity

For analysis of purity of GSTs extracts were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), using a Bio-Rad Mini-Protean II apparatus, at 200 V. All resolving gels were 10% acrylamide. Proteins were visualised using Coomassie blue protein stain.

B) RESULTS

Bl) Purification of GSTs from black-grass biotypes susceptible and resistant to herbicides

A procedure was developed to purify GSTs from both black-grass biotypes. After tissue disruption and ammonium sulfate precipitation, proteins were resolubilised by dialysis. Initial separation was carried out by anion exchange on a 4-ml High-Q column (BioRad Laboratories Ltd, Hemel Hempstead, Herts, UK). For both biotypes (Herbiseed and Peldon) one peak of GST activity was eluted from this column with 0.2M NaCl. No further GST activity could be eluted by increasing the NaCl concentration. The second column purification step utilised a 45-ml Sephadex G-75 gel filtration column. This allowed a buffer exchange to be carried out prior to affinity chromatography. Both biotypes gave one peak of GST activity with this column. These peaks were associated with the first of two peaks of protein eluted from this column (Fig 1) .

Final purification was achieved using a 45-ml GSH agarose affinity column. The majority of protein did not bind to this column and was eluted during loading and the subsequent wash stages. Some GST activity was eluted at this stage, either because the column was overloaded or because binding conditions did not favour a particular GST isoenzyme. Bound proteins were eluted by combining 10 mM GSH with a change in column conditions to pH 6.0. The column was reverse-eluted in order to tighten the eluted peaks. Elution resulted in one peak of GST activity from both biotypes (Fig 2). Purification data for each biotype is given in Table 1, which demonstrates that greater GST activity resulted from the Peldon biotype.

GST-containing fractions were pooled and analysed for purity by SDS-PAGE (see Fig 3). Herbiseed (susceptible) samples revealed one, 27.5 kDa, polypeptide. Peldon extracts revealed two polypeptides, one 27.5 kDa and one 30 kDa. Similar amounts of starting material were used for extraction and the 27.5 kDa polypeptide appeared to be present at greater amounts in the Peldon extracts. These polypeptide profiles compare well with those previously detected in crude extracts of black-grass using anti-wheat GST antisera. Purification of these polypeptides now allows GST activity to be ascribed to the 27.5 kDa polypeptide and suggests that the higher GST activity in Peldon may be due, at least in part, to the additional presence of the 30 kDa polypeptide.

Table 1. Purification tables for GST activities from (A) Herbiseed and (B) Peldon black- rass biotypes. Data are from purification from equal amounts of black-grass tissue.

Total Specific

Volume activity activity Purification Yield

(ml) (μmol (μmol % min ^■') min-' mg-' total protein)

(A) Crude extract 40 2.968 0.040 1.0 100

Post-Q Sepharose column 12 1.993 0.096 2.4 67

Post-G-75 Sephadex column 26 1.508 0.056 1.4 51

Post-GSH-agarose column 18 0.364 3.181 79.5 12

(B) Crude extract 37.5 4.790 0.071 1.0 100

Post-Q Sepharose column 12 3.396 0.187 2.6 71

Post-G-75 Sephadex column 32 3.107 0.155 2.2 65

Post-GSH-agarose column 26 2.329 5.973 84.1 49

B2) Enzyme kinetics Michaelis constant data for GSTs from susceptible and resistant black-grass plants are given in Table 2. Comparison of V_max data for crude and purified GST samples suggests a purification of approximately 65 times. This is backed up by analysis of samples taken throughout the extraction processes (Table 1) . This purification value appears low when it is considered that a single polypeptide is present in the final GST pools from susceptible plants. The purified GSTs were found to be very unstable in the post-affinity column samples. All activity was lost from Herbiseed samples within 24 h of purification. This loss of activity may explain the low purification value. It is possible that the higher GSM K_m values in purified samples, indicating a lower affinity of enzyme for substrate, may also be due to instability of the purified GSTs in vitro. It also must be considered that, as GSTs are dimeric, the kinetic data may be the result of more than one GST isoform. Hence, care must be taken when interpreting the results. However, if increased GST activity was due to increased abundance of a single isoform, it would be expected that the K_m values for crude extracts of susceptible and resistant black-grass would be similar. As this is not the case for K_m(CDNB) (Table 2) it implies that the increased GST activity in extracts from the resistant biotype may be due to the presence of a different GST isoform.

Table 2 Michaelis constants for crude and purified GSTs from Herbiseed and Peldon black-grass biotypes.

Crude GST extract Purified GST fraction

GSH CDNB GSH CDNB

K_ra GSH K_m CDNB K_m GSH K_n CDNB

(mM) V_raa-" (mM) V_raax ^» (mM) V_ma (mM) V-V

Herbiseed 0.64 64 3.67 149 1.07 5270 1.56 6990

(suseptible)

Peldon 0.66 164 2.57 308 1.15 4460 2.32 10345

(resistant)

B3) N-Terminal Sequence

Polypeptides are composed of a chain of amino acids linked together by peptide bonds. Each amino acid has a carboxyl and an amino group and it is between these groups that a peptide bond is formed. Polypeptide chains hence have one amino acid with a free carboxyl group and one amino acid with a free amino group found at either end of the polypeptide chain. The end of the polypeptide with the free carboxyl group is termed the C terminal and the end with the free amino group is termed the N terminal. N-terminal protein sequencing involves determination of the N terminal amino acid using the Edman degradation technique. In doing this the N terminal amino acid is removed to reveal a new N terminal amino acid. The process is then repeated to identify the new N terminal amino acid. Continual cycling of this process can often determine up to 20 amino acids in the order they occur at the N terminus of a polypeptide chain.

The N-terminal sequence has now been obtained for the 30kDa polypeptide purified from the black- grass biotype Peldon. This is the unique sequence of a polypeptide constitutively produced in the herbicide-resistant biotype Peldon. The first 19 amino acids are:

ala-pro-val-lys-val-phe-gly pro-ala-met-ser-thr-asn-val-ala-arg-val-ile-leu.

Direct sequencing of this black-grass GST polypeptide has not been carried out previously.

B4) Tryptic Digest

After a tryptic digest and HPLC purification of the resultant polypeptide fragments an internal polypeptide sequence from the 30kDa GDST was obtained:

asn-pro-phe-gly-gln-ile-pro-ala

2) Investigation of possible test for plant resistance to herbicides.

A) MATERIALS AND METHODS

Al) Plant material

Black-grass plants were collected from three UK field sites (courtesy of

Novartis Crop Protection UK Ltd.) . Sites 1 and 3 had received various treatments on separate plots (Table 1), and were sampled once. Site 2 received no herbicide treatment this season (1998-1999) , but had been treated with a variety of herbicides on separate plots for the previous 6 years (Table 3). This site was sampled four times between February and May 1999.

Table 3. Herbicide treatments at the three field sites. Sites 1 and 3 received treatments on 4th November and 16th December 1998 respectively, when black-grass was at GS 11-13. Site 2 received no treatment during the season of the study, but received the treatments described below for 6 years preceding the study.

Plot

Site

IPU (2500 g.a.i ha) Dicolfop (900 as Auger g.a.i/ha) as

(5 litres/ha) Illoxan-European

(2.4 litres/ha)

IPU (2500 g.a.i ha) Clodinafop (30 g Fenoxaprop-P (69g as Auger a.i./ha) as Topik a.i./ha) as Cheetah

(5 litres/ha) (0.125 litres/ha) S (1.25 litres/ha)

IPU (2500 g.a.i ha) Diclofop (900 as Auger g.a.i/ha) as Illoxan-

(5 litres/ha) European (2.4 litres/ha)

All above ground biomass was harvested from ten plants, where possible, from each plot. Tissue was frozen on dry ice for transportation to the laboratory.

A2) Protein extraction and GST assay

Proteins were extracted and GST activities determined using the method in 1) above. Each plant was extracted in 100 mM phosphate buffer (pH 7.0) , desalted and stored at -80 °C. Dilutions for ELISA detection were carried out using the extraction buffer. Protein determinations were carried out using an adaptation of the method of Bradford (1976) (reference given on page 10 above).

A3) ELISA detection of GST polypeptides in extracts

Antisera were raised against a 30 kDa GST subunit purified from the herbicide resistant biotype Peldon (detailed in 1) above) . These antisera were used to detect polypeptides in extracts from site 2 (Feb 1999) . Immunodetection was carried out on 96 well plastic plates. Extracts were diluted to a protein concentration of 1 μg/50 μl and 1 μg was loaded per well overnight at 4 °C. Wells were then blocked for 3 h using 3 % (w/v) milk powder (Marvel Original, Premier Beverages, Stafford, UK) at 37° C. Antiserum raised against the 30 kDa GST subunit purified from the black-grass biotype Peldon was incubated with the immobilised protein overnight at 4°C. Following a wash step, the wells were incubated with goat anti-mouse alkaline phosphatase conjugate (Dako Ltd. , Cambridgeshire, UK) for 2 h at room temperature with shaking. Following a second wash step, the antibody conjugate was detected using 1 mg/ml p-nitrophenyl phosphate. Colour development was stopped using 3 M NaOH. Absorbance was measured immediately at 405 nm. At least three replicates were carried out for each sample.

RESULTS

Plants treated as described in Table 3 were sampled in February 1999 and assayed for GST activity.

All three sites demonstrated higher GST activities in plants surviving herbicide treatment (Table 4) . At all sites, plants surviving IPU treatment possessed mean GST activities of between 2.1 and 3.4 times that of plants from untreated plots. In the two sites where diclofop was studied, plants surviving this treatment possessed mean GST activities of between 1.7 and 2.4 times those of untreated plots. Plants in plots that had been treated with fenoxaprop-P and clodinafop, but were not sprayed during the 1999 season, had average GST activities of 3.3 and 3.4 times those of untreated plots, respectively (Table 4).

Table 4. Mean GST activities in plants sampled in February 1999, expressed as μmol CDNB/min/g fresh biomass. n= 10 except * n = 7. Figures in brackets are ratios to untreated.

Site Untreated IPU Diclofop Clodinafop Fenoxaprop- P

1 0.44 0.90* 0.74 2 0.18 0.62 0.62 0.60 3 0.34 0.99 0.80*

Time Course of GST Activity

Plots at site 2 were resampled four times between March and May 1999. Data for mean GST activities are shown in Figure 4.

Detection of polypeptide using anti-GST antibodies Results are shown in Figure 5.

At all sites the range of GST activities from untreated plots showed some degree of crossover with the ranges for treated plots. However, the lower GST activities in the untreated plots were absent from treated plots. In sites 1 and 3 raised activities could be a result of herbicide treatment as opposed to innately higher GST activities in the population. However, site 2, which did not receive herbicide treatment during the season of study, also showed raised activities. Populations in site 2 are a result of repeated use of single types of herbicide year after year. These results suggest that populations surviving herbicide treatment consisted of plants with higher GST activities, even with respect to herbicides that are not metabolised by this enzyme family. Hence, the GST activity profile of a black- grass population may allow a prediction of how well that population will be controlled by herbicide. Further sampling of site 2 (in March and May) indicated that the initial observed difference in GST activities between treated and untreated plots (in February) was not present later in the season. GST activities in untreated plots were similar to those in treated plots by early March, and the activities in untreated plots remained similar to treated plots on subsequent samplings. This suggests that any prediction of herbicide resistance utilising GST activities would have to be carried out early in the season.

Detection of polypeptides using antisera raised against a purified GST subunit from black-grass indicated that raised GST activities may be a result of the presence of more GST polypeptide. ELISA detection of immunoreactive material from four plants from each plot at site 2 indicated that all but one plant from treated plots had more detectable polypeptide than three of the four plants from untreated plots.

From this study it is evident that either GST activity (against CDNB) or ELISA detection using antisera may be used to predict the response of a population to herbicide treatment. As activity studies take less than one day and ELISA study can be completed within three days, these tests would give results very quickly. In addition, as the tests would be carried out as soon as black-grass plants were at the two-leaf stage, results could be obtained before application of post-emergence herbicides. This would allow alternative control measures to be adopted where herbicide resistance was indicated.

Claims

1. A method for use in detecting the presence of a target polypeptide in a weed which polypeptide is connected with the resistance of the weed to herbicides comprising the steps of:-

a) delivering to a sample of the weed an antiserum which is directed against the target polypeptide to identify and bind to the polypeptide; and

b) visualising the bound antiserum

2. A method according to Claim 1 wherein the target polypeptide is glutathione S-transferase (GST).

3. A method according to Claim 2 wherein the target polypeptide is a 30 kDa GST subunit of GST.

4. A method according to claim 3 wherein the N-terminal sequence of the 30kDa GST subunit is ala-pro-val-lys-val-phe-gly-pro-ala-met-ser-thr- asn- val- ala- arg- val-ile-leu

5. A method according to any preceding claim wherein the antiserum used is a monoclonal antiserum raised against a 30 kDa polypeptide.

6. A method according to any preceding claim wherein the weed is a grass weed.

7. A method according to any preceding claim wherein the weed is black-grass.

8. A method according to any of claims 1-6 wherein the weed is rye grass or wild oats.

9. A method according to any preceding claim wherein the visualisation step comprises the addition of a visualisation means.

10. A method according to Claim 9 wherein the visualisation means includes the use of enzymes such as peroxidase or alkaline phosphatase.

11. A method according to Claim 9 wherein the visualisation means is a fluorescent compound, a particulate material or a radio labelled compound.

12. A method according to claim 9 wherein the avidin bioten complex method is used for visualisation.

13. A method according to Claim 9 wherein the visualisation step comprises :-

(a) diluting the target polypeptide to lμg/50μg samples and binding them to wells in ELISA plates overnight at 4°C;

(b) blocking the plates with milk protein for 3 hrs at 37°C;

(c) challenging the proteins with monoclonal antisera raised against the target polypeptide overnight at 4°C;

(d) washing the protein-antibody complex then challenging it with a second antibody conjugated with a detection means (for example, goat anti-mouse alkaline phosphatase) for 2 hrs at 21°C to amplify and detect the results; and

(e) carrying out a second wash step before visualisation is carried out by colour development of alkaline phosphatase.

14. A method according to any preceding claim wherein an amplification means is used in addition to the visualisation means.

15. A test kit for use in detecting the presence of a target polypeptide in a weed which polypeptide is connected with the resistance of the weed to herbicides, the kit containing a supply of an antiserum which is directed against the target polypeptide.

16. A kit according to Claim 15 wherein the kit further includes one or more of a supply of a detection means, a supply of an amplification means and a supply of a visualisation means.

17. A kit according to Claim 15 or Claim 16 wherein the kit further comprises a supply of a reference or control material.

18. A kit according to any of Claims 15-17 wherein the kit includes instructions.

19. A test kit according to any one of Claims 15-18 which is suitable for use on automated test apparatus.

20. The use of an antiserum which is directed against a target polypeptide connected with resisitivity of weeds to herbicides in the detection of the polypeptide in accordance with the method set out above or using the test kit as set out above.