Patent Application
20180230554
The present invention relates generally to weed control, and in certain embodiments to methods and systems for controlling watermilfoil plants in lakes or other water bodies using chemical herbicides.
As further background, various methods exist for the control of aquatic weeds such as watermilfoil. The selection of an appropriate control method depends upon many factors such as environmental impact, cost effectiveness, efficacy, and the like. The various control methods available include physical controls such as mechanical harvesting, hand pulling or cutting, or the use of bottom barriers or water level draw-down. These methods can be both time consuming and labor intensive, and can create significant environmental disturbance, especially when considered on a large scale. Other mechanical controls such as rotovation have similar drawbacks.
Biological controls such as the use of organisms that feed on aquatic weeds can be desirable in some aquatic systems in that they reduce the use of equipment and have the potential for long term control. In temperate aquatic systems, the efficacy of such biological controls can also vary widely, and is dependent upon factors such as feeding preferences, metabolism, temperature, and stocking rate.
For these and other related reasons, the use of aquatic herbicides has become a common method for controlling invasive aquatic weeds. The use of herbicidal control nonetheless also presents risks and difficulties including the potential impact on the local environment, the potential for excessive decrease in the dissolved oxygen (DO) content of the waters due to rapid plant decay, and the potential for tolerance development, especially where an effective elimination of an invasive organism is not achieved during a treatment.
In light of this background, there is a need for improved methods for the control of aquatic weeds, including watermilfoil, with chemical herbicides. Such methods would desirably facilitate successful control of the target weed or weeds. The present invention addresses these needs.
In one embodiment, the invention provides a method for controlling watermilfoil in a lake. The method includes obtaining samples of target watermilfoil plants living in a lake, and obtaining genetic information from the samples. A relationship of the target watermilfoil plants to other watermilfoil plants, for example a phylogenetic or other biotype-grouping relationship, is inferred from the genetic information, wherein at least some of the other watermilfoil plants have continued tolerance to at least one chemical herbicidal agent. The method further includes treating the body of water with a selected chemical herbicidal agent in accordance with a treatment regimen based at least in part upon said inferring step.
In another embodiment, the invention provides a computer-based system useful for inferring a genetic relationship, for example a phylogenetic or other biotype-group relationship, of a target watermilfoil plant to other watermilfoil plants. The system includes a processing unit and one or more memory storage units coupled to the processing unit. The one or more memory storage units store (i) a routine for estimating a genetic relationship, for example for estimating a phylogenetic tree, (ii) first data representing genetic information from the target watermilfoil plant, and (iii) second data representing genetic information from multiple genetically (e.g. phylogenetically) differing watermilfoil plants and associating confirmed chemical herbicidal tolerance with at least some of said multiple differing watermilfoil plants. The routine, first data and second data are processable by the processing unit to estimate a genetic relationship, for example in the form of a phylogenetic tree, including the target watermilfoil plant and the multiple genetically differing watermilfoil plants.
In another embodiment, the invention provides a method for controlling target watermilfoil plants in a body of water. The method includes treating the body of water with a selected chemical herbicidal agent in accordance with a treatment regimen, wherein the treatment regimen has been based at least in part upon inferring from genetic information from the target watermilfoil plant a genetic relationship of the target watermilfoil plants to other watermilfoil plants. At least some of the other watermilfoil plants have confirmed tolerance to at least one chemical herbicidal agent. The genetic relationship, in certain inventive embodiments, can be a phylogenetic or other biotype-grouping relationship. In some embodiments, the selected chemical herbicidal agent is the at least one chemical herbicidal agent. The other watermilfoil plants can include Eurasian watermilfoil, North American watermilfoil, and hybrid watermilfoil plants, and/or the confirmed tolerance can be associated with at least some of the hybrid watermilfoil plants.
In another inventive embodiment, provided is a method useful for assisting in the management of a target watermilfoil plant population in a body of water. The method includes obtaining genetic information from one or more samples from the target watermilfoil plant population. The method also includes inferring from the genetic information a genetic relationship of the target watermilfoil plant to other watermilfoil plants, where at least some of the other watermilfoil plants are hybrid watermilfoil plants (typically Northern watermilfoil or Myriophyllum sibiricum×Eurasian watermilfoil or M. spicatum) having confirmed tolerance to at least one chemical herbicidal agent. The genetic relationship, in certain inventive embodiments, can be a phylogenetic or other biotype-grouping relationship. The method can also include generating a visible display of the genetic relationship including indicia representing the target watermilfoil plant and the other watermilfoil plants.
In methods and systems described above and elsewhere herein, the genetic information can, for example, include amplified fragment length polymorphism (AFLP) data and/or simple sequence repeat (SSR) data and/or other molecular marker data. Also, the genetic information can be obtained by receiving a container containing the one or more samples, extracting DNA from the one or more samples, and obtaining the genetic information from the extracted DNA. Any or all of these data can be compared among a target watermilfoil plant and other differing watermilfoil plants. The other differing watermilfoil plants can include, but are not limited to, milfoil species/lineages such as Eurasian watermilfoil, Northern watermilfoil, and hybrid watermilfoils (for instance various Northern×Eurasian lineages or other lineages including crosses with variable watermilfoil (M. heterophyllum) or other watermilfoil species). The chemical herbicide tolerance can be associated with at least some of the hybrid (e.g. Northern×Eurasian) watermilfoil plants. The confirmed chemical herbicidal tolerance can be tolerance to one or more of auxin-mimic herbicides (including but not limited to 2,4-dichlorophenoxyacetic acid (2,4-D), triclopyr), bleaching mode of action herbicides (including but not limited to fluridone and/or another phytoene desaturase inhibitor, or other bleaching herbicide modes of action such as HPPD (hydroxyphenylpyruvate dioxygenase) inhibition), Photosystem I inhibitor herbicides (including but not limited to diquat dibromide), Protox inhibiting herbicides (including but not limited to carfentrezone or flumioxazin), ALS (acetolactate synthase) inhibitor herbicides (including but not limited to penoxsulam, bispyribac, imazamox, or imazapyr), and other potential aquatic herbicide modes of action including non-classified modes of action (e.g., endothall) or future registered aquatic herbicide modes of action. The methods or systems can involve or be useful in the selection of which chemical herbicide to use, and/or a concentration of a chemical herbicide to use, and/or a chemical herbicide contact duration to use, in the control of the target watermilfoil plants. This can for example be determined based on the relationship of the target watermilfoil plants to the known tolerant plants, e.g. whether the target watermilfoil is most closely related to plants known to be tolerant to the one or more chemical herbicides, e.g. in a phylogenetic cluster or other biotype group associated with such tolerance.
Additional embodiments as well as features and advantages of the invention will be apparent from the descriptions herein.
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments thereof and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.
As disclosed above, aspects of the present invention relate to methods and systems, and components thereof, to treat bodies of water to control watermilfoil or other similar aquatic weeds growing therein.
In one feature, it has been discovered that watermilfoil populations exhibiting reduced susceptibility (i.e. tolerance) to 2,4-d, fluridone and other chemical herbicides cluster together in phylogenetic trees or other similar comparisons (e.g. biotype groups) and are identified as genetically similar hybrid (Northern×Eurasian) watermilfoil plants. Thus, analysis of a target watermilfoil population in relation to such a phylogenetic tree or another genetic comparison can be used as a predictive tool for herbicide susceptibility of the target watermilfoil population.
Illustratively,
With reference to
For the purpose of promoting a further understanding of aspects of the present invention, as well as features and advantages thereof, the following specific Examples are provided. It will be understood that these Examples are illustrative, and not limiting, of embodiments of the invention.
Samples of 28 watermilfoil plant populations were collected from 19 lakes located in Wisconsin and Michigan, USA. All plants were washed thoroughly in distilled water in order to remove/reduce any potential contaminant DNA from symbiotic organisms such as periphyton, insects, snails, etc. Total genomic DNA was extracted from fresh submerged vegetative meristem tissue using DNeasy Plant Mini Kits (Qiagen), similar extraction kits, or a hexadecyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin of the Bontanical Society of America 19:11-15). AFLP reactions were prepared as described in Vos et al. (1995), Nucleic Acids Research, Volume 23, Issue 21, pp. 4407-4414, with some modifications.
For the restriction digestion, ˜100 ng of total genomic DNA was digested with EcoRI and MseI restriction enzymes using the following: 5 units EcoRI enzyme, 1 unit MseI enzyme, 4 μL 10×T4 DNA ligase buffer with ATP (New England Biolabs), 4 μL NaCl (0.5M), 2 μL BSA (1 mg/mL), and water to a final volume of 40 uL. Reactions were incubated at 37° C. for one hour.
For Adapter ligation, the EcoRI and MseI adaptors were ligated by adding the following to the digested genomic DNA: 1 unit T4 DNA ligase, 1 μL 10×T4 DNA ligase buffer with ATP, 1 μL NaCl (0.5M), 0.5 μL BSA (1 mg/mL), 1 μL annealed EcoRI adaptors (Applied Biosystems), 1 μL annealed MseI adaptors (Applied Biosystems), and water to a final volume of 504. Reactions were incubated at 37° C. for three hours.
Preselective PCR amplification employed primers EcoRI-A and MseI-C and therefore amplified only those digested segments that contained a 3′ A or C on the EcoRI and MseI ends of the restriction-ligation fragments, respectively. The preselective amplification reactions consisted of: 1 μL preselective primers (Applied Biosystems), 154 AFLP Core Mix (Applied Biosystems), and 44 restriction-ligation product (after diluting five-fold in water). Preselective thermal cycling conditions consisted of one cycle at 72° C. for 2 minutes, followed by 20 cycles of: 94° C. for 2 min; 56° C. for 30 s, and 72° C. for 2 min; with a final extension at 60° C. for 30 minutes. Preselective products were run out on an agarose gel (˜4%) to ensure that amplification had occurred.
Selective PCR amplification was employed to add a fluorescent label to the EcoRI fragments and further limit the number of digested products amplified by employing primers that added an additional two nucleotides to the 3′ end of the preselective primers.
The current work used only one pair of primers (EcoRI-ACA and MseI-CAT), but additional primers could also be used. Selective amplifications consisted of: 1 μL of each selective primer, 15 μL AFLP Core Mix, and 3 μL preselective product (after 20-fold dilution in water). Reaction conditions for the selective amplification consisted of one cycle at 94° C. for 20 seconds, 66° C. for 30 seconds, and 72° C. for 2 minutes; the annealing temperature was then lowered 1° C. each cycle during the next 10 cycles (i.e., 56° C. in the tenth cycle). Twenty additional cycles were performed using an annealing temperature of 56° C., followed by a final extension at 60° C. for 30 minutes. Selective amplification products were run on an ABI 3130xl automated DNA sequencer at AWRI using the internal size standard MapMarker1000 ROX (BioVentures, Inc.).
AFLP genotype data were scored with GeneMapper v4.0 (Applied Biosystems).
The analysis was limited to fragments between 80 and 500 bp in length. The binset was constructed using a peak height threshold (PHT) of 200 relative fluorescence units (rfu) and a bin width of 0.75 bp. Each sample was then automatically scored for this binset using a PHT of 30 rfu. All allele calls were also visually checked and edited.
Structure v2.3.2 (Pritchard et al., Inference of population structure using multilocus genotype data, Genetics, 155:945-959 (2000); Falush et al., Inference of population structure using multilocus genotype data: dominant markers and null alleles, Molecular Ecology Notes (2007)) was used to identify genetically distinct groups (biotypes) and individual membership to these groups. Briefly, Structure uses an iterative Bayesian Markov Chain Monte Carlo (MCMC) method to simultaneously evaluate the number of distinct genetic groups within a dataset (K) and assign a proportion of each individual's genome to each of the K distinct groups. The number of distinct genetic groups in a dataset is evaluated by comparing likelihood scores for runs at different values of K whereas the proportion of an individual's genome attributed to each value of K is determined by the posterior probability of membership to each group.
Structure can employ a variety of different models for population structure, including allowing for admixture (hybridization) among different groups, correlated allele frequencies among groups, and estimating the admixture proportion (a) separately for each group. Models employing all possible combinations of the above parameters were evaluated. A typical run of the MCMC was >75,000 generations, preceded by a burn-in period of 25,000 generations. Likely values of K for the dataset were identified by (i) graphing −ln likelihood of data vs. K, (ii) determining which values of K consistently contained individuals with high assignment probabilities, and (iii) evaluating clusters identified with AFLP in the context of patterns identified with inter- and intra-specific genetic variation in ITS.
The results of comparative genetic analyses, which can be generated with the assistance of well known commercial software (e.g. for generating phylogenetic tree-based outputs), are displayed in
As can be seen, it has been discovered that confirmed or highly suspect herbicide tolerant hybrid (Northern×Eurasian) watermilfoil plants occur in a highly phylogenetically related cluster. Correlation of herbicide tolerance to relative position on the phylogenetic tree enables the use of genetic molecular analyses of target watermilfoil populations to predict susceptibility to a given herbicide(s), and thus the design of a treatment regimen taking into account potential tolerances in the target watermilfoil populations. A new target watermilfoil population can be sampled, and genetic data such as those described above can be obtained and used to infer a position of the new target population on the phylogenetic tree of
All publications cited herein are hereby incorporated by reference in their entirety as if each had been individually incorporated by reference and fully set forth.
The use of the terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/447,347 filed Feb. 28, 2011, which is hereby incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61447347 | Feb 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13407064 | Feb 2012 | US |
| Child | 15786027 | US |
