Weed Control Through Cutting, and Herbicide Applications in Double Crop Soybeans
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ALTERNATIVE METHODS OF WEED CONTROL THROUGH CUTTING, AND HERBICIDE APPLICATIONS IN DOUBLE CROP SOYBEANS
Literature Review (Outline)
Objective:
- Develop recommendations for weed control in double-crop soybean systems with new dicamba-tolerant soybean technology
- Establish a baseline data for the integration of management practices (cutting and herbicide application), which may allow for an herbicide label change for growers.
Questions Theorized
- How does different harvest heights affect herbicide efficacy?
- Does plant regrowth affect the movement and efficacy of systemic herbicides?
- Do tank mixes provide synergistic or antagonistic effects when applied to distressed weeds?
- How do systemic herbicides affect not only the above biomass but also the below-ground biomass?
- How do weeds respond to physical stresses through plant resource allocation?
Studies (Answering Questions Theorized)
Non-crop height/timing study
This non-crop study allows for the two variables height, and timing to be better analysied in greater detail by allowing for varied heights and timings without damaging crops. Within this study, we would compare three different heights in comparison to herbicide efficacy to three different plant species Conyza canadensis, Ambrosia artemisiifolia, and Solidago Canadensis. The non-crop study also allows us to compare two different application timings this is significant because it will enable us to study and examine the effects of immediate herbicide effectiveness and delayed applications to allow for possible greater plant resource allocation. A substudy was also conducted within which involved tagging three C. canadensis within the respective three heights treatment zones this was done to study the three plants individually in respects to the herbicide efficacy on them this not only allows us to see the effect of the herbicides but also allowed us to go back later and harvest said plants for biomass analysis for their dry weight compositions further allowing us to better understand how the plants used their resource allocations in induced physical stress.
In Crop Tank-mix study
For our in-crop study two respective locations were established to allow for weed species spectrums commonly observed in southern Illinois. One of sites used to conduct our research was located at the Southern Illinois Research Center in Belleville Illinois under the guidance of Ronald Krausz, a renowned researcher in the field of weed science and one of the original authors for the protocol that started this study. The Belleville Research center was chosen for is diverse weed populations and differing biotypes of glyphosate resistance, along with its impeccably managed facilities on site. The second location for our study was established in Desoto Illinois due to its close proximity to southern Illinois university but also it was one of the first places in southern Illinois to have documented glyphosate resistance making it a catalyst for research. The in-crop aspect of our study allowed for a more realistic outlook of how a typical grower would utilize our purposed method of weed control in a double-crop system following wheat harvest. Our method also allowed us to implement an assortment of tank mixes typically used in burndown applications already used in full-season soybean cropping operations. Further studying our method becomes extremely important in giving us insight into how these tank mixes will interact with a mechanically damaged plant under physical stress while also seeing the implications of crop injury that may be associated with these types of applications on the desired soybean crop. To look at herbicide efficacy visual weed control ratings, were taken at 7, 14, 21, 35, and 42 days after applications on driver weeds such as Conyza canadensis, Ambrosia trifida, Ambrosia artemisiifolia, and Amaranthus rudis. Subsequently, visual ratings were also taken to see the effects of the grass weed species Panicum dichotomiflorum leading up to 35 days after application at this time a blanket application of SelectMax was applied to allow for more efficient ratings of broadleaf driver weeds. To justify our visual ratings and increase quantitative data weed counts were taken at both sites respectively.
Green House Study Plant Resource Allocation
Greenhouse study was established at the southern Illinois Research center greenhouses. Our objective for this study was to answer questions regarding plant resource allocation specifically what the interaction will be between shoot to root ratio in correlation to cutting heights and auxin herbicide applications of dicamba. We will attempt to answer the question of how does cutting affect shoot to root ratios we will establish three cutting heights of 6”, 12”, and no cut and to analysis the interaction alongside auxin herbicide applications of dicamba we will apply full rates of post applied Xtendimax with vapor grip technology at 0.5 lb. ae.
Herbicides
- Xtendimax (Dicamba) TIR1 receptor, Growth Inhibitors
- Roundup PowerMax (glyphosate) ESPS inhibitors
- Authority First (cloransulam-methyl) ALS Inhibitors (sulfentrazone) PPO inhibitors
- Sencor DF (metribuzin) Photosystem II Inhibitors
- Rowel FX (flumioxazin) PPO Inhibitors (chlorimuron ethyl) ALS Inhibitors
- Sharpen (saflufenacil) PPO Inhibitors
Topics
- Double Crop Soybeans
- Wheat
- Plant resource allocations
- Glyphosate resistance
- Roundup Extend (Bean variety)
- No-Till
- ALS
- PPO
- Glutamine synthase inhibitors
- Photosystem II inhibitors
Introduction
With ever-growing populations of resistant biotypes in driver weeds such as Conyza Canadensis in part to the over application of glyphosate leading to the selection of said resistance biotypes causing detrimental effects on yield. C. canadensis is considered one of the ten most critical herbicide-resistant weeds, evolving resistance to triazine, amide, bipyridilium, imidazolinone, and sulfonylurea herbicides in more than ten countries worldwide (Heap 2004). To combat this increasingly troublesome resistance issue within our industry new technologies have been developed in the forms of new soybean herbicide-resistant varieties along with new formulations of herbicides, but this is only a band-aid that will only solve the problem for a short period of time. Therefore, our goal for this study is to hopefully provide producers an alternative tool for improved weed management. That may lead to increased control through the mechanical cutting of weeds through combine harvest.
Chapter 1
Review of Literature
Double-Crop
Double cropping is the agricultural practice of harvesting two crops from the same field in a given year (Borchers et al., 2014). Soybeans [Glycine max (Merr) L.] double cropped following wheat (Triticum aestivum L.) is a common practice in Illinois especially in southern part of the state (Association, 2017; McHarry and Kapuśta, 1979; Stucky, 1976). U.S. double-crop wheat/soybean production has accounted for around 6% of the total soybeans harvested in the nation of the past five years, ranging from a low of 3% to a high of 9% dependent on wheat acreage planted for the year (Ross Jeremy). Demographically double crop soybeans in the last five years have seen increased trends in the Mid-southern states such as Arkansas, Louisiana, Mississippi, and Tennessee compared to northern states like Illinois, Indiana, Kansas, Kentucky and Missouri (Ross Jeremy). A reason for the popularity of double cropping in southern states may be due to the longer growing season experienced in those areas compared to that of shorter northern growing seasons (Ross Jeremy). Traditionally double crop soybeans are planted in no-till wheat during mid to late June (Camper et al., 1972; McHarry and Kapuśta, 1979; Stucky, 1976; Triplett, 1978). No-till practices typically associated with double-crop soybeans can cause concerns when it comes to weed control. For the past several decades Preemergence and POST emergent herbicide programs have been the means for weed control management in double-crop production (Krausz and Young, 2001). Increasing research into the effects (PRE) followed by POST applications on double-crop soybeans has caused a shift in the way producers now manage weed control in this system (Krausz and Young, 2001). Study’s have shown that POST applied programs provided increased control compared to PRE-herbicides applied alone (Sims and Guethle, 1992). Alongside increased weed control POST applied herbicides are not as subjected to environmental conditions compared to that of PRE-applied herbicides that are highly dependent of rainfall (Krausz and Young, 2001). Whereas POST applied herbicides are increasingly dependent on the stage and height of both the desired crop and weed species (Krausz and Young, 2001). Increasing development of herbicide tolerant crops may facilitate in the increase of POST-applied herbicide applications. Weed control may not be the only confounding factor in a producer’s decision to implement double cropping practices. Double-crop soybeans can be subject to many abiotic, economic, and machinal accessibility issues that may sway producers in the choice to adopt this agronomic practice. Examples of these factors can include variable weather conditions, commodity prices, input costs, and the availability of equipment to plant double-crop soybeans (Ross Jeremy). Research has shown that producers don’t always employ as aggressive management strategies seen in full-season soybean production usually leading to declines in soybean yield (Association, 2017). In 2014 the Illinois Soybean Association and the Department of agriculture conducted a survey sent to 181 producers, and found that 57% invested the majority of their resources on wheat instead of the following soybean crop with only 12% allocating investment to the secondary crop (Davidson Daniel, 2015). Research has shown that double-crop soybeans have the opportunity to produce equal yields to that of full-season soybean production dependent on sound management strategies and favorable environmental conditions (Ross Jeremy). Double-cropping can provide many beneficial factors for producers such as providing good soil conservation, weed suppression in the form of cover crop characteristics from wheat, and increased market opportunities with the production of two crops annually (Association, 2017). Conversely this practice can be subject to many constraints in the forms of increased occurrence of populations of insect pest and disease pressure that are typically associated with later planting complications (Cooperative and Service; Mike, 2012; State and Service, 2016). Much of the research and production recommendations that we associate with double-cropping today come from research conducted during the 1900’s and 2000’s and have not been updated in the last ten years. While much of the past research is still valid and pertinent to double-crop production it leaves much to be updated and explored through future research (Ross Jeremy).
Wheat
No-till
No-till farming has been a well-adopted practice by producers in recent years in agriculture (Kapusta and Krausz, 1993). One of the reasons for the increased adoption of this practice was due to the release of glyphosate resistant crops to the United States in 1996 (Vangessel et al., 2001). Allowing producers to apply glyphosate after weeds had emerged in producers crops allowing for increased reliance of chemical control compared to that of conventional tillage (Martin, 1992). Many producers found chemical control more desirable in comparison to conventional tillage due to decreased input cost of fuel and labor (Brown et al., 1989; Griffith et al., 1986; Hairston et al., 1984; Kapusta and Krausz, 1993). Also, conventional tillage is extremely dependent on abiotic conditions of the environment such as weather and soil conditions making conventional tillage much risker in regards to proper weed control (Liebman et al., 2008; Mulder and Doll, 1993; Posner et al., 2008). Another reason for the increased adoption of no-till tillage practices may be due to the added benefits it imposes on overall soil health, such as increased nutrient retention, reduction of soil erosion from wind and water displacement, and increased soil organic matter through the degradation of the previous year’s cash crop (Blevins et al., 1984). No-till practices can have drastic implications on weed species population shifts in the environment (Staniforth and Wiese, 1985). Widely accepted shifts have been seen in regards to weed seed size with the transition from predominately large seeded species dominated fields of Xanthium strumarium , and Abutilon theophrati to small seed broadleaf weeds such as Amaranthus retroflexus, Panicum dichotomiflorum, and Chenopodium album respectively (Buhler and Oplinger, 1990). Weed seed physiological traits are not the only observed shifts seen in no-till systems, species life cycle changes have also been observed with the increase of perennial plant species populations seen in no-till systems (Kapusta and Krausz, 1993).
Conyza Canadensis (Marestail)
Conyza Canadensis (Marestail) is a weed species native to North American that has been an increasingly troublesome weed in over 40 cropping systems (Weaver, 2001). One of the main issues associated with this weed species is the increasing adoption of soil conservation practices such as no-till farming (Kapusta and Krausz, 1993). Marestail is considered a small-seeded broadleaf meaning that traditionally it has been controlled through conventional tillage that places the small seeds deep into the soil horizon, making germination increasingly difficult for this species (Holm, 1997). Furthermore C. canadensis also has many competitive attributes that allow it to compete with many agronomic crops. One of those characteristics is its diversification at the time of marestail seed emergence. Marestail has been classified as being both a summer or winter annual meaning it can germinate throughout the growing season of many agronomic crops (Weaver, 2001). Another competitive characteristic of marestail is its ability to travel vast distances through wind dispersion. The vast wind dispersion is due to marestail light seed mass allowing it to be carried by wind disturbance. Some studies have even documented some accounts that C. canadensis can be displaced into the earth’s atmosphere, although it has also been documented that 90% of the seeds land within 100 m of the parent plant (Dauer et al., 2007). Researchers have even assumed that C. canadensis may have similar wind dispersion characteristics to that of Taraxacum officinale L. (dandelion) because of their similar ratio of seed surface to seed biomass. (Andersen, 1993), found that C. canadensis had a lower settling velocity than that of T. officinale, therefore leading to the speculation that C. canadensis may have an increased wind dispersal ability compared to that of T. officinale. Alongside its adaptable traits, transgenic crops have played an important role in the history of this problematic weed. Following the introduction of glyphosate-resistant soybeans, resistance to glyphosate in C. canadensis shortly followed in just four years in the state of Delaware (Vangessel, 2001). Furthermore glyphosate-resistant biotypes, (trainer et 2005 ) also documentedsuspected ALS inhibitor resistance in 82 % of fields he tested. Multiple herbicide resistance to both glyphosate and ALS inhibitors has also been documented by Heap and Kruger. This leaves many producers few options for effective POST weed control due to both herbicides being in the top five most commonly used POST applied herbicides used in soybean production usda–nass 2008).
Plant Growth Regulators (Dicamba/Benzoic Acid)
Auxin herbicides consists of the extremely important phytohormone indole-3-acetic acid (IAA), which is an important natural hormone in plants that plays a role in almost every aspect of plant growth and development (Ross et al., 2001). Auxins have adverse effects on plants in relation to the concentration imposed on them (Taiz and Zeiger, 1998). Low concentrations of IAA have been observed to impart natural hormonal activities in plants such as cell elongation, and division, root formation, apical dominance, and vascular differentiation. Conversely at high concentrations auxins can cause plant growth to be interrupted leading to plant senescence (Grossmann, 2003). (Hansen and Grossmann, 2000) proposed that the interaction of induced ethylene and abscisic acid biosynthesis may be the cause for the herbicidal syndrome in sensitive dicotyledon species. Through the introduction into the worldwide market place after World War II and the discovery of the chemical structure of natural auxins in plants has led to the development of synthetic compounds that act in a similar manor to that of natural auxins in plants (Kelley and Riechers, 2007). Chemical classes so far developed from this discovery consist of phenoxycarboxylic acids, pyridine carboxylic acids, benzoic acids, aromatic carboxymethyl’s, and the quinolinecarboxylic acids (Grossmann, 2003). Dicamba was introduced in the early 1960’s by the company BASF and by the 1980’s it had already made its way into row crop production. Dicamba has been around for more than 40 years, where it has shown effectiveness to many broadleaf species (Vink et al., 2012). In recent years dicamba-tolerant soybeans have entered the U.S agriculture market in part of the need for an additional mode of action to control glyphosate-resistant biotypes in weed species. Although this new technology has shown to increase weed control it has not had an ideal reputation to its use due to its difficulty to handle, it has a history of being a very mobile herbicide through drift and temperature inversions. These unfortunate characteristics have caused the product to move from a location where crops are resistant to areas where crops may be susceptible causing agronomic and economic damage (Al-Khatib and Peterson, 1999). Scientist began to realize that this herbicide could be utilized for row crop systems especially in corn crops due to monocots ability to metabolize the active ingredient efficiently not harming the corn crop while controlling troublesome dicotyledons such as the driver weeds in pigweeds. The issue in this was that the formulation was still too unpredictable in response to its ability to move from site to site damaging neighboring crops. To alleviate this issue scientist in recent years have developed more stable formulations of the dicamba salt in products such as Xtendimax with vapor grip technology, Fexapan, and Engenia, being considerably less mobile formulations than that of its previous predecessors
Mechanical weed Management/Plant resource allocation
Mechanical weed management is one of the four major management practices when considering weed control alongside cultural, biological, and chemicals management (University of Illinois at Urbana-Champaign. Cooperative Extension, 1984). Mechanical weed control is not a new practice when it comes to weed management practices. Some activities associated with mechanical weed control could be considered as pulling weeds, tilling the soil before or after weeds emerge, and mowing/cutting (Service, 2009). The adoption of mechanical weed control in the last decade has gained increasing popularity in Europe, due to the adoption of organic farming (Dabbert, 2000). Organic farming requires very little to no use of chemical pesticides (Puech et al., 2014). Instead typically organic growers implement mechanical methods such as harrowing, rotary cultivation, or brushing (Rasmussen, 1995). Cutting emerged weeds can be beneficial to the suppression of weeds by removing top-growth while also decreasing soil disturbance usually caused by tillage. Weeds may remain an issue if multiple cuttings are not administered throughout the growing season due to plant regrowth (Andreasen et al., 2002). Adversely multiple cuttings may require multiple trips through the field in one growing season leading to soil disturbances that may promote the recruitment of addition cohorts through increased germination by stimulated scarification and increased seedling exposer to light (Andreasen et al., 2002). Additionally another negative effect that may be imposed through mechanical cuttings of weeds could be through a process called compensatory growth, where plants exhibit a positive response to injury. Compensatory growth has been described as being as small as the act of replacing damaged tissue of the injured plant all the way up to exceeding the net productivity of uninjured plants (Andreasen et al., 2002). (Andreasen et al., 2002) proposed that there may be a critical period of two months at the beginning of a row crops growing season. Where weed species have a competitive advantage to regrow and recover when the desired row crop is still in its adolescence stage of growth and development and has little to no competitive ability over the mature weed species. For many soybean producers, multiple cuttings throughout the year are just not realistic. The administration of multiple cuttings could be detrimental to producers crops because of the potential damage that could incur with multiple trips through the field with machinery. One solution that I and many of my collages agree on is the combination of both mechanical and chemical weed management practices. Combination of these two management practices would include the suppression of top growth before planting followed by applications of chemical herbicides. Throughout the history of weed control, it has been widely believed that applying herbicides to “cut” or “damaged” weeds has less control efficacy. The previously mentioned notion may not be entirely accurate for all types of herbicide modes of action because by cutting plants this allows a direct pathway for herbicide translocation through the opened stem via the xylem or phloem. Direct applications to the xylem and phloem may lead to increased control of systemic herbicides that move bipedally within plant systems. Bipedal movement in plants is the movement downward in plants. Many believe that the reason that this method provides less efficacy then applications customarily made to leaf foliage because typically after plants have been damaged or cut the area where the damage was inflicted will soon after harden decreasing permeability in the plant system. Research on the interaction between annual weed species and plant regrowth from mechincal cutting is sparse and irrespectively not observed leaving many questions to be asked and analysied (Andreasen et al., 2002).
Photosystem Inhibitors
Photosystem inhibiting herbicides are known to inhibit or modify photosynthesis this process works by blocking photosynthetic electron transport of the reducing side of photosystem II (John, 1996). Many of the main chemical groups within this herbicide are urea’s, amides, triazines, triazinones, pyridazinones, carbamates, and nitrophenols. All these herbicides share a commonality in that they block photosystem II-dependent Hill reactions (Pfister and Charles, 2014). Herbicides associated with this group have been known to be effective at controlling many annual broadleaf weeds along with certain grass herbicides (Senseman et al., 2007).
PPO
Foliar-applied PPO inhibiting herbicides have developed resistance in recent years to many weed species such as waterhemp making type of application ineffective in many instances. “The causal mechanism of PPO-R in waterhemp originates from a codon deletion on the nuclear-encoded gene (PPX2L) coding for the PPO enzyme, which is dual-targeted to the mitochondria and the chloroplasts” (Patzoldt et al. 2006). Through research studies it has been seen that the PPO inhibiting chemistry can still work as a soil-applied herbicide conveying that this targeted site of action may still be inhibited when a plant is still young in the early emerging phases depending on the active ingredient applied (Harder et al. 2012; Shoup et al. 2003). Of the PPO chemical families Saflufenacil is a relatively new PPO preplant herbicide used in burndown situations with excellent control of broadleaf weeds. This new herbicide may provide growers with a valuable tool for managing multiple herbicide resistant populations of C. canadensis anonymous 2008.
Roundup Extend System
The introduction of biotechnology-derived, herbicide-resistant crops has contributed many advancements in the profitability of weed control, increased production, and increasingly environmentally sound practices (Behrens et al., 2007). With the commercialization of glyphosate-resistant soybeans in 1996, the united states use of glyphosate on those hectares has increased ten-fold when used as preplant, or postemergence herbicide applications were applied (Johnson et al., 2010). This monoculture of herbicide use has led to the development of several herbicide resistance herbicide resistance in driver weeds such as giant ragweed (Ambrosia trifida), common ragweed (Ambrosia artemisiifolia), waterhemp (Amaranthus tuberculotus), Palmer amaranth (Amaranthus palmeri), horseweed (Conyza canadensis) (Heap, 1997). To address this growing concern many are targeting the development of dicamba-resistant crop plants (Behrens et al., 2007).Dicamba is considered environmentally friendly by not having a persistent soil life, it is low costing, and nontoxic to both human and animal (Behrens et al., 2007). Monsanto Company has developed a genetically modified soybean (MON 87708) which contains a demethylase gene from stenotrophomunasmaltophiliathat encodes for the dicamba monooxygenase protein (Taylor et al., 2017). Before monsanto’s development of dicamba resistant crops, dicamba had been exclusively used in PRE applied and burndown herbicide application with successful results in controlling glyphosate resistant biotype weed species (Byker et al., 2013). A concern with use of dicamba PRE plant or in a burndown situation is that dicamba may not provide full season control due to its low activity as a soil residual herbicide (Eubank et al., 2008; Main et al., 2004) Development of dicamba resistant soybeans provides growers with the ability to apply dicamba POST as a means of control for glyphosate resistant weed species giant ragweed (Ambrosia trifida) (Vink et al., 2012) and C. canadensis (Byker et al., 2013).
Glyphosate Resistance
Herbicide resistance has been a significant challenge for producers worldwide. Glyphosate has been especially prevalent, due to the extremely popular adoption of glyphosate-resistant cropping systems introduced by Monsanto in 1974. This has been especially problematic in row crops such as cotton, corn (Zea mays L.), and soybean [Glycine max(L.) (Bagavathiannan et al., 2013). Glyphosate works by killing plants by blocking 5-enolpyruvylshikimate-3-phosphate synthases (EPSPS; EC 2.5.1.18), which is an important enzyme involved with the synthesis of aromatic amino acids in plants (Jiang et al., 2013). The first documented case of glyphosate resistance was recorded in 1996 to Lolium rigidum (ridged ryegrass) in Victoria Australia (Heap, 1997). At this time in glyphosates infancy, many disregarded the belief that weed species could genuinely develop resistance to what seemed to be an invisible herbicide. It would not be until 2000 that many began to truly understand the vulnerability of this herbicide to the development of resistance. This realization was due to the first documented resistance of Conyza canadensis (marestail) in Delaware (Heap, 1997) once the phenomenon was credited research began to try and better understand the cause of the developed resistance. Scientist realized that when herbicides are used in excesses that they can apply extreme selection pressures for herbicide resistant biotypes, and in essence selecting for dominated populations of resistant biotypes (Neve, 2008). Factors associated to the development of resistance have been genetic, biological, and operational factors, and that no one factor dominates the selection alone but rather they are influenced by interactions between each other (Neve, 2008).
Justification
Increased adoption of double-cropping systems in U.S agriculture due to the need for increased demand for world food supplies, brings the importance for increased weed control in these systems to provide increased yields. Currently there has been little investigation into practices that may increase yield production within this system. Given recent interest in to double crop soybean production, this study aims to compare wed control in combination with the effects of cutting on said weeds, at three different locations. One location will be with no-tillage and be in a non-crop situation to analysis detailed effect of weed height, and application timing. In the next to locations they will also be subject to no-tillage practices but be planted to with soybeans to observe the interaction between treatments and yield.
Non-crop study Hypothesis:
Hypothesis I: I would expect that increased cutting heights of weeds would have a positive correlation regarding percent weed control independent of herbicide treatment applied. Hypothesis II: I would anticipate that earlier application timing would have a greater interaction regarding weed control compared to that of a later application. Hypothesis III: I would expect that systemic herbicides used with multiple MOA’s would have increased herbicide efficacy than products used alone or treatments of the contact herbicide variety.
Materials and Methods
In crop study
In crop field experiments were conducted in the 2017 and 2018 growing seasons across two locations in southern Illinois, located in Belleville (38°30’58.3” N 89°50’20.6” W), and De Soto (37°47’45.0” N 89°15’45.1” W) IL. Both fields received no tillage practices to replicate no-till cultural practices done by a typical grower. Additionally, both fields were planted with a drill planter
Non-crop Study
A non-crop experiment was established in 2017 and 2018 in Carbondale IL, at the Agronomy research center (37°41’56.6” N 89°14’34.3” W). The objective of the non-crop study was to look at the effects of three different cutting heights and two timings in combination with 10 different treatment’s. The study was laid out with 10 by 30-foot plots, to observe different cutting heights we separated the 30-foot plots into three 10-foot sub plots with a 6-inch cut height, 12-inch cut height, and in the back 10 feet being noncut. To observe our timing factor in this study we applied the first 10 pesticides at 3 days after cutting (DAC) and then applied the following 10 treatments 5 DAC. Cutting heights for the 6 inches and 12-inch sub plots were made with a sickle bar mower that was setup on corresponding blocks for each respective height. Weed control rating were administered at 7, 14, 21, and 35 days after treatment for each timing. At the end of the study above and below ground dry biomass weights were recorded for three marestail plants per sub plot, to keep track of each plant at the beginning of the study plants of 12 inches were tagged with plastic rings and marked with different colors of spray paint.
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