PROJECT RESULTS

Multiple Herbicide Resistant Wild Oat: Occurrence and Evolutionary Mechanism

Applicant: 

Dr. Rene Van Acker

Department of Plant Science

University of Manitoba

Winnipeg, Manitoba  R3T 2N2  Canada

Table of Contents:

• Background and Objectives
• Procedure and Project Activities
• Results and Discussion
• Conclusions and Implications

ARDI Project:

#98-169

Project Status:

Completed February, 2004

Background and Objectives:

Over two years, 1994 and 1995, three fields with multiple herbicide resistant wild oat infestations were identified in the northwest agricultural region of Manitoba (near the town of Swan River).  These wild oats were/are resistant to the herbicides, Assert, Mataven, and Puma.  These multiple herbicide resistant wild oats were identified after producer reports of failure by Assert to provide satisfactory control in the field.  Although these wild oat infestations had previously been exposed to other herbicides (primarily Group 1 herbicides such as Puma), Assert had never before been applied.  In growth room experiments resistant plants were approximately 7 times more resistant to Assert and Mataven than susceptible plants.  The resistant wild oats exhibited a lower level of resistance to Puma of approximately 2.0-fold (however, a 2.0-fold level of resistance still resulted in a wild oat control problem in the field).  Subsequently, many additional fields have been identified (in all three Prairie provinces) with multiple herbicide resistant wild oat.

Because the multiple herbicide resistant wild oat populations (infestations) had not been previously exposed to Assert, it was of interest to investigate how this multiple resistance trait could become predominant in the wild oat population in the absence of direct selection pressure.  Investigating the genetics of inheritance of the multiple herbicide resistance trait in wild oat should provide information on this question.  Furthermore, the results of the genetics study should also provide some insight into the mechanism(s) of resistance and suggest agronomic practices to minimize the development of multiple herbicide resistant wild oat populations.

Procedure and Project Activities:

Multiple herbicide resistant wild oat seed was collected from the original three fields (described above).  These three multiple resistant wild oat populations will henceforth be referred to as Population 01, 02, and 03, respectively.  Wild oat plants from each one of these multiple resistant populations were hand-crossed with known herbicide susceptible (normal) wild oat plants to produce what is termed F₁ seed.  Also, the resistant populations were intercrossed.  Some seed on each parent plant was allowed to self-pollinate and this selfed seed was then screened for homozygosity (pure-breeding) of the multiple herbicide resistance trait.  ‘Screening’ in the context of this study refers to growing wild oat seedlings to an appropriate growth stage, applying a herbicide, and assessing the number of seedlings that were either killed by the herbicide or remained alive and continued to grow.  The ratio of dead to alive seedlings (after herbicide application) is termed a segregation ratio in genetics studies and is used to deduce the genetics of inheritance of the multiple herbicide resistance trait.  The three herbicides used in this study, Assert, Mataven, and Puma, were always applied in separate trials and not tank mixed, since prior observations indicated that tank mixing these herbicides does not result in normal herbicidal activity.  The screening of self-pollinated seed from parental plants confirmed that an individual wild oat plant does indeed possess (and exhibit) the multiple herbicide resistance trait, since self-pollinated seed from an individual parental plant was divided into three portions with one portion screened with Assert, one portion screened with Mataven, and one portion screened with Puma.

Wild oat F₁ seed from the initial crosses was planted and resulting seedlings grown to maturity in the greenhouse.  Seed on these F₁ plants was allowed to self-pollinate which produced what is termed F₂ seed.  Similarly, F₂ seed was planted and the resulting seedlings grown to maturity (self-pollinated) to produce F₃ seed.  Seedlings from both the F₂ and F₃ generations were screened with the three herbicides (Assert, Mataven, and Puma) to obtain segregation ratios.  In this study, over 30,000 individual wild oat seedlings were screened with the herbicides.  Because of limitations in personnel time and growth room space, crosses involving Population 02 were not fully screened and results involving Population 02 will not be discussed below.

Results and Discussion:

Screening of the self-pollinated seed from the parental wild oat plants provided the first indication that the multiple herbicide resistance trait (resistance to Assert, Mataven, and Puma) exhibited by an individual wild oat plant is not conferred by a single gene.  While all parental plants were homozygous (pure-breeding) for Assert resistance, a number of parental plants were heterozygous (not pure-breeding) for the Mataven and Puma resistance traits.  These heterozygous parental plants, and all crosses based on these heterozygous parental plants, were then eliminated from the study.

Results of screening F₂ seedlings (derived from resistant (R) x susceptible (S) crosses) indicated that for each of the three herbicides, Assert, Mataven, and Puma, individuals in the majority of F₂ families segregated in a 3R:1S ratio.  This segregation pattern is expected when one dominant nuclear gene controls the resistance trait (or three separate dominant nuclear genes – one for each herbicide).  Since reciprocal crosses for all three herbicides had similar segregation ratios, the gene(s) are part of the nuclear DNA and not cytoplasmic (i.e. the resistance trait is carried by pollen and is not inherited in a maternal fashion).  Screening results for R x S F₂ derived F₃ families (F_2:3) generally supported the F₂ screening results; however, there were some unclear results particularly for those crosses involving Population 03.

The purpose of the R x R intercrosses was to determine whether the resistance trait is carried at the same gene locus in the multiple resistant populations (i.e. whether a single, common genetic mutation per herbicide confers resistance across several wild oat populations).  If the resistance trait was carried at the same gene locus then all progeny of the R x R crosses would be resistant and survive herbicide application.  However, this was not the case.  For both Assert and Mataven, F₂ seedlings from R x R crosses segregated into resistant (alive) and susceptible (dead).  The fact that there was segregation of F₂ seedlings indicates that there are different genes for resistance in each population.  A segregation ratio of 15:1 generally fit the data, which indicates separate dominant nuclear genes in each population.  Because the multiple resistant populations were not homozygous for Puma resistance in the parental generation, the R x R crosses were not screened with Puma.

The R x S F_2:3 results provided further evidence (in addition to the parental screening results) for separate genes conferring resistance to each herbicide within a single population.  F₂ families did not always exhibit consistent results when screened with the herbicides (e.g. some families were resistant to Assert but either segregating or susceptible to Mataven – and vice versa).  These segregation results were used to calculate possible linkage between the genes.  When the Assert and Mataven screening results for Population 01 are considered together, the recombination frequency value indicates a relatively close linkage between the two genes.  As of the date of this report, data is still being analyzed and interpreted for Population 03.  While population genetics theory indicates that multiple genes conferring resistance in an individual plant should be a very rare phenomenon (i.e. an accumulation of several rare mutation events), the linkage results are in agreement with population genetics theory.  The three multiple resistant wild oat populations were exposed to or selected mainly by Group 1 herbicides such as Puma; when Assert was applied for the first time on these populations in farmers’ fields, it did not provide control.  If all three resistance traits are linked and normally segregate as a group of genes, this would explain how the repeated application of Group 1 herbicides over a number of years also selected for resistance to Assert and Mataven.  It does not, however, explain the accumulation of three rare mutation events in an initial individual multiple herbicide resistant ‘founder’ plant.

Conclusions and Implications:

1. More than one gene is involved in conferring multiple herbicide resistance in individual wild oat plants.

2. The gene(s) conferring resistance to a specific herbicide differ between multiple resistant wild oat populations (i.e. they do not occur at the same gene locus).

3. The resistance genes appear to be linked and normally segregate as a group which confers the multiple herbicide resistance trait even in the absence of selection by a specific herbicide.

Since other research/survey results have indicated that multiple herbicide resistant wild oat is relatively widespread throughout the Prairie provinces (i.e. not rare), and the results of this genetics study indicate that resistance to several different herbicide groups can be inherited as a linked ‘block’ of genes in wild oat, then the reliance by farmers on in-crop selective herbicides as the primary method of controlling wild oat is an unsustainable practice in the long term.  Rather, an integrated approach to controlling wild oat infestations incorporating non-herbicidal control measures whenever and wherever possible is required.

Acknowledgements:

This project was possible due to funding from the Governments of Manitoba and Canada through the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI).

These funds were matched by the following companies (in total): AgrEvo, BASF, Bayer, Cargill, DuPont, Monsanto, Novartis, Rhone-Poulenc, UGG, Westco, and Zeneca.

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