Focus on Weed Control: Natural Compounds as Next-Generation Herbicides

Herbicides with new modes of action (MOAs) are badly needed due to the rapidly evolving resistance to commercial herbicides, but a new MOA has not been introduced in over 20 years. The greatest pest management challenge for organic agriculture is the lack of effective natural product herbicides. The structural diversity and evolved biological activity of natural phytotoxins offer opportunities for the development of both directly used natural compounds and synthetic herbicides with new target sites based on the structures of natural phytotoxins. Natural phytotoxins are also a source for the discovery of new herbicide target sites that can serve as the focus of traditional herbicide discovery efforts. There are many examples of strong natural phytotoxins with MOAs other than those used by commercial herbicides, which indicates that there are molecular targets of herbicides that can be added to the current repertoire of commercial herbicide MOAs.The evolutionary forces driving the survival of species include chemical interactions between organisms, which function in positive interactions such as mutualistic and symbiotic relationships and negative interactions such as competitive and parasitic relationships. These processes have led to the emergence of novel secondary metabolic pathways (often through gene duplication), producing a vast array of structurally diverse and biologically active molecules (Moore and Purugganan, 2005; Ober, 2005; Flagel and Wendel, 2009; Jiang et al., 2013). This evolutionary process is similar to a high-throughput screen. However, unlike conventional in vitro screens, which test many compounds on a single biochemical target over a very short period of time, this natural high-throughput process selects molecules based on their whole-organism activities, involving numerous chemical interactions between countless organisms and target sites over millions of years. To date, approximately 200,000 secondary metabolites have been identified (Tulp and Bohlin, 2005), with many more expected to be discovered. Few of these compounds have been examined for phytotoxicity, and the modes or mechanisms of action (MOAs) of even fewer known phytotoxins have been elucidated.The negative chemical interactions between organisms are often characterized using anthropomorphic language, such as chemical warfare, referring to the production of phytotoxins used by plant pathogens to invade their host plants (Maor and Shirasu, 2005), and the novel weapons hypothesis, which is associated with the chemical-based advantage of some invasive plant species over native plant populations (Callaway and Aschehoug, 2000; Callaway and Ridenour, 2004; Callaway and Maron, 2006; Cappuccino and Arnason, 2006; Callaway et al., 2008). While simplistic, this terminology illustrates how these toxin-based interactions exploit biochemical weaknesses between an organism and its host or enemy/competitor to enhance its own survival (Verhoeven et al., 2009). In fact, these interactions can even be multitrophic, such as when exotic plants enhance their invasiveness by promoting the growth of certain native soil pathogens noxious to native plants (Mangla et al., 2008; Barto et al., 2011).As humans evolved from a nomadic hunter-gatherer subsistence existence to an agricultural lifestyle, they learned to utilize certain biologically active secondary metabolites to manage agricultural pests. Indeed, the concept that nature is an excellent source of natural pesticides is captured in the following ancient Lithica poem (circa 400 B.C.): “All the pests that out of earth arise, the earth itself the antidote supplies” (Ibn et al., 1781). Less than a century later, Greek and Roman treatises described practices to control agricultural pests that include the use of essential oils. Similar documents are found in Chinese literature, such as a survey describing plant species used to control plant pests (Yang and Tang, 1988). The mid-20th century ushered in the use of synthetic pesticides, which have revolutionized agriculture. Like pharmaceuticals (Harvey, 1999, 2008; Newman and Cragg, 2012), many pesticides are based on natural compounds. However, natural products have not played a major role in herbicide discovery (Copping and Duke, 2007; Hüter, 2011).     

Focus on Weed Control: Herbicides as Weed Control Agents: State of the Art: I. Weed Control Research and Safener Technology: The Path to Modern Agriculture

The purpose of modern industrial herbicides is to control weeds. The species of weeds that plague crops today are a consequence of the historical past, being related to the history of the evolution of crops and farming practices. Chemical weed control began over a century ago with inorganic compounds and transitioned to the age of organic herbicides. Targeted herbicide research has created a steady stream of successful products. However, safeners have proven to be more difficult to find. Once found, the mode of action of the safener must be determined, partly to help in the discovery of further compounds within the same class. However, mounting regulatory and economic pressure has changed the industry completely, making it harder to find a successful herbicide. Herbicide resistance has also become a major problem, increasing the difficulty of controlling weeds. As a result, the development of new molecules has become a rare event today.Modern industrial herbicide research begins with the analysis and definition of research objectives. A major part of this lies in the definition of economically important weeds in major arable crops (Kraehmer, 2012). Weed associations change slowly over time. It is important, therefore, to foresee such changes. Today’s weed associations result from events in the distant past. They are associated with the history of crops and the evolution of farm management. In Europe and the Americas, some large-acre crops such as winter oilseed rape and spring oilseed rape (canola), both derived from Brassica spp., and soybean (Glycine max) have attained their current importance only within the last 100 years. Other Old World crops, such as cereals, have expanded over a very long time span and were already rather widespread in Neolithic times (Zohary et al., 2012). The dominance of crop species in agricultural habitats only left room for weed species that could adapt to cultivation technologies. Changes in crop management and the global weed infestation have happened in waves. A major early factor in Europe was presumably the grain trade in the Roman period (Erdkamp, 2005). The Romans spread their preferred crops and, unintentionally, associated weed seeds throughout Europe, Asia, and Africa. A second wave of global vegetation change started in the 16th century after the discovery of the Americas. Crops and weeds were distributed globally by agronomists and botanists. Alien species started to spread on all continents. A third phase can be seen in the 19th century with the industrialization of agriculture and the breeding of competitive crop varieties. The analysis of weed spectra in arable fields grew from this historical background. Weeds are plants interfering with the interests of people (Kraehmer and Baur, 2013), which is why they have been controlled by farmers for millennia.Chemical weed control began just about a century ago with a few inorganic compounds, such as sulfuric acid, copper salts, and sodium chlorate (Cremlyn, 1991). The herbicidal activity of 2,4-dichlorophenoxyacetic acid was detected in the 1940s (Troyer, 2001). Büchel et al. (1977) and Cremlyn (1991), Worthington and Hance (1991). Targeted herbicide research began in the 1950s. In the early days, herbicide candidates progressed from screens purely on the basis of their having biology that would satisfy farmers’ requirements. Mode of action (MoA) studies did not play a major role in the chemical industry prior to the 1970s. Analytical tools were developed and the rapid elucidation of plant pathways and in vitro-based screen assays were used from the 1980s onward. However, in the 1990s and beyond, ever-increasing regulatory and economic pressures have changed the situation of the industry completely, and to satisfy the new requirements, selection criteria beyond biological activity have needed to be applied. Herbicide resistance in weeds has developed into a more serious problem that now constrains the application of certain types of herbicides in some markets. Finally, the introduction of crops resistant to cheap herbicides and of glyphosate-resistant soybean, in particular, took value out of the market and resulted in an enormous economic pressure on the herbicide-producing industry. As a result of this changing and more difficult landscape, the development of new molecules is now a rare event.

Table I.

History of chemical weed control innovationsPost, Postemergence application; Pre, preemergence application, based on data from Cremlyn (1991), Worthington and Hance (1991), Büchel et al. (1977), Herbicide Resistance Action Committee (www.hracglobal.com), and others.

Focus on Weed Control: Indaziflam Herbicidal Action: A Potent Cellulose Biosynthesis Inhibitor

Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad-acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam-treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within <1 h of treatment and in a dose-dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). Arabidopsis mutants resistant to known CBIs isoxaben or quinoxyphen were not cross resistant to indaziflam, suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management.Cellulose is a composite polymer of β-1,4-linked glucan chains and is the main load-bearing structure of plant cell walls (Jarvis, 2013). Although cellulose is a relatively simple polysaccharide molecule, its synthesis is quite complex. The principle catalytic unit is a plasma membrane (PM)-localized protein complex referred to as the cellulose synthase complex (CSC; Davis, 2012). In plants, the CSC, visualized with freeze fracture microscopy, is a solitary, hexagonal rosette-shaped complex (Herth and Weber, 1984; Delmer, 1999) and at least three of the catalytic CELLULOSE SYNTHASE A (CESA) proteins are required in each CSC for the production of cellulose (Desprez et al., 2007; Persson et al., 2007). In addition to CESAs, several accessory proteins have been discovered to be necessary for the production and deposition of cellulose, such as KORRIGAN (Lane et al., 2001), COBRA (Roudier et al., 2005) and CELLULOSE SYNTHASE INTERACTING1 (CSI1; Gu et al., 2010), as well as several others that are yet to be identified. The loss of function in any of the aforementioned proteins causes complete or partial loss of anisotropic growth in cells undergoing expansion, resulting in radial swelling. Severe radial swelling in rapidly expanding tissue is also a common symptomology observed in seedlings treated with cellulose biosynthesis inhibitors (CBIs). Therefore, numerous potential herbicidal targets exist (mechanisms of action) for the broad group of known CBIs.Classification of an herbicide to the CBI designation was traditionally achieved by short-term [¹⁴C]radioisotope tracer studies focused on the incorporation of Glc into cellulose (Heim et al., 1990; Sabba and Vaughn, 1999). More recently, time-lapse confocal microscopy of reporter-tagged CESA proteins (Paredez et al., 2006) has been used to further classify CBIs. CBIs can be classified into at least three primary groups based on how treatment disrupts the normal tracking and localization of fluorescently labeled CESAs (for review, see Brabham and DeBolt, 2012). The disruption is, it can be assumed, the result of the inhibitory mechanism of the CBI. In the first group, isoxaben and numerous other compounds cause YELLOW FLUORESCENT PROTEIN YFP):CESAs to be depleted from the PM and concomitantly accumulate in cytosolic vesicles (called small CESA compartments or microtubule-associated cellulose synthase compartments; Paredez et al., 2006; Crowell et al., 2009; Gutierrez et al., 2009) The second group, consisting only of dichlobenil (DCB), causes YFP:CESAs to become immobilized and hyperaccumulated at distinct foci in the PM (Herth, 1987; DeBolt et al., 2007b). The third group influences CSC-microtubule (MT)-associated functions resulting in errant movement and localization of YFP:CESAs (DeBolt et al., 2007a; Yoneda et al., 2007). These different disruption processes suggest that each CBI group targets a different aspect of the complex cellulose biosynthetic process.A lack of evolved weed resistance in the field suggests that CBIs are potentially underutilized tools for weed control (Sabba and Vaughn, 1999; Heap, 2014). CBIs have also been useful research tools in decoding fundamental aspects of cellulose biosynthesis. An exogenous application of a CBI provides spatial and temporal inhibition of cellulose. Resistance screens to CBIs have uncovered key genes in cellulose biosynthesis (Scheible et al., 2001; Desprez et al., 2002). Furthermore, CBIs such as isoxaben have also been effective in linking accessory proteins with CESAs in the CSC (Robert et al., 2005; Gu et al., 2010). Therefore, it is important to extend our range of CBI compounds. Indaziflam (Fig. 1A), an herbicide introduced by Bayer Crop Science, was recently proposed to be a CBI and was reported to have a photosystem II inhibition value of 9.4 (Meyer et al., 2009; Dietrich and Laber, 2012). Indaziflam is labeled for use in turf, for perennial crops, and for nonagricultural situations for preemergent control of grasses and broadleaf weeds (Meyer et al., 2009; Brosnan et al., 2011). The aim herein was to investigate indaziflam as a CBI and to characterize its inhibitory effect on cellulose biosynthesis.Open in a separate windowFigure 1.Indaziflam is a fluoroalkytriazine-containing compound that inhibits elongation in seedlings of P. annua and Arabidopsis. A, Chemical structure of indaziflam. B to D, Images of 7-d-old seedlings treated with increasing concentrations of indaziflam. B shows light-grown P. annua seedlings (indaziflam concentrations from left to right are 0, 100, 250, 500, 1,000, 5,000, and 10,000 pm). C and D show light-grown and dark-grown Arabidopsis seedlings, respectively (indaziflam concentrations from left to right are 0, 100, 250, 500, 1,000, and 2,500 pm). Indaziflam treatment induced swollen cells. E, Representative images of the primary root of P. annua grown in plates for 4 d with and without 10 nm indaziflam. F, Transgenic Arabidopsis seedlings expressing GFP:PIP2 were examined by laser scanning confocal microscopy and images represent visualization of the primary root grown vertically for 7-d plates without and with 250 pm indaziflam. PIP2, Plasma membrane intrinsic protein2. Bar = 10 mm in B, 5 mm in C and D, 2 mm in E, and 50 μm in F.     

Focus on Weed Control: The Impact of Herbicide-Resistant Rice Technology on Phenotypic Diversity and Population Structure of United States Weedy Rice

The use of herbicide-resistant (HR) Clearfield rice (Oryza sativa) to control weedy rice has increased in the past 12 years to constitute about 60% of rice acreage in Arkansas, where most U.S. rice is grown. To assess the impact of HR cultivated rice on the herbicide resistance and population structure of weedy rice, weedy samples were collected from commercial fields with a history of Clearfield rice. Panicles from each weedy type were harvested and tested for resistance to imazethapyr. The majority of plants sampled had at least 20% resistant offspring. These resistant weeds were 97 to 199 cm tall and initiated flowering from 78 to 128 d, generally later than recorded for accessions collected prior to the widespread use of Clearfield rice (i.e. historical accessions). Whereas the majority (70%) of historical accessions had straw-colored hulls, only 30% of contemporary HR weedy rice had straw-colored hulls. Analysis of genotyping-by-sequencing data showed that HR weeds were not genetically structured according to hull color, whereas historical weedy rice was separated into straw-hull and black-hull populations. A significant portion of the local rice crop genome was introgressed into HR weedy rice, which was rare in historical weedy accessions. Admixture analyses showed that HR weeds tend to possess crop haplotypes in the portion of chromosome 2 containing the ACETOLACTATE SYNTHASE gene, which confers herbicide resistance to Clearfield rice. Thus, U.S. HR weedy rice is a distinct population relative to historical weedy rice and shows modifications in morphology and phenology that are relevant to weed management.Weedy rice (Oryza sativa), a conspecific weed of cultivated rice, is a global threat to rice production (Delouche et al., 2007). Classified as the same species as cultivated rice, it is highly competitive (Diarra et al., 1985; Pantone and Baker, 1991; Burgos et al., 2006), difficult to control without damaging cultivated rice, and can cause almost total crop failure (Diarra et al., 1985). The competition of cultivated rice with weedy rice can lead to yield losses from less than 5% to 100% (Kwon et al., 1991; Watanabe et al., 2000; Chen et al., 2004; Ottis et al., 2005; Shivrain et al., 2009b). Besides being difficult to control, weedy rice persists in rice fields because of key weedy traits, including variable emergence (Shivrain et al., 2009b), high degree of seed shattering (Eleftherohorinos, et al., 2002; Thurber et al., 2010), high diversity in seed dormancy (Do Lago, 1982; Noldin, 1995; Vidotto and Ferrero, 2000; Burgos et al., 2011; Tseng et al., 2013), and its seed longevity in soil (Goss and Brown, 1939). Weedy rice is a problem mainly in regions with large farm sizes where direct-seeded rice culture is practiced (Delouche et al., 2007). It is not a major problem in transplanted rice culture, where roguing weeds is possible and hand labor is available. The severity of the problem has increased in recent decades because of the significant shift to direct seeding from transplanting (Pandey and Velasco, 2002; Rao et al., 2007; Chauhan et al., 2013), which is driven by water scarcity (Kummu et al., 2010; Turral et al., 2011), increasing labor costs, and migration of labor to urban areas (Grimm et al., 2008).The herbicide-resistant (HR) Clearfield rice technology (Croughan, 2003) provides an option to control weedy rice in rice using imidazolinone herbicides, in particular, imazethapyr. Imidazolinones belong to group 2 herbicides, also known as ACETOLACTATE SYNTHASE (ALS) inhibitors. Examples of herbicides in this group are imazamox, imazapic, imazaquin, and imazethapyr. Developed through mutagenesis of the ALS locus (Croughan, 1998), Clearfield rice was first commercialized in 2002 in the southern U.S. rice belt (Tan et al., 2005). Low levels of natural hybridization are known to occur between the crop and weedy rice. Gene flow generally ranges from 0.003% to 0.25% (Noldin et al., 2002; Song et al., 2003; Messeguer et al., 2004; Gealy, 2005; Shivrain et al., 2007, 2008). After the adoption of Clearfield technology, resistant weedy outcrosses were soon detected in commercial fields (Fig. 1), generally after two cropping seasons of Clearfield rice, where escaped weedy rice was able to produce seed (Zhang et al., 2006; Burgos et al., 2007, 2008). Similar observations have been reported outside the United States, in other regions adopting the technology (Gressel and Valverde, 2009; Busconi et al., 2012).Open in a separate windowFigure 1.Suspected herbicide-resistant weedy rice in a rice field previously planted with Clearfield rice along the Mississippi River Delta in Arkansas. More than 10 morphotypes of weedy rice were observed in this field, with different maturity periods. In the foreground is a typical weedy rice with pale green leaves; the rice cultivar has dark green leaves. The inset shows a weedy morphotype that matured earlier than cultivated rice.Despite this complication, the adoption of Clearfield rice technology is increasing, albeit at a slower pace than that of glyphosate-resistant crops. After a decade of commercialization, 57% of the rice area in Arkansas was planted with Clearfield rice cultivars in 2013 (J. Hardke, personal communication). Clearfield technology has been very successful at controlling weedy rice, and polls among rice growers suggest that farmers have kept the problem of HR weeds in check by following the recommended stewardship practices (Burgos et al., 2008). The most notable of these are (1) implementation of herbicide programs that incorporate all possible modes of action available for rice production; (2) ensuring maximum efficacy of the herbicides used; (3) preventing seed production from escaped weedy rice, remnant weedy rice after crop harvest, or volunteer rice and weedy rice in the next crop cycle; (4) rotating Clearfield rice with other crops to break the weedy rice cycle; and (5) practicing zero tillage to avoid burying HR weedy rice seed (Burgos et al., 2008).Clearfield rice has gained a foothold in Asia, where rice cultivation originated (Londo and Schaal, 2007; Zong et al., 2007). Clearfield rice received government support for commercialization in Malaysia in 2010 (Azmi et al., 2012) because of the severity of the weedy rice problem there. Dramatic increases in rice yields (from 3.5 to 7 metric tons ha⁻¹) were reported in Malaysia where Clearfield rice was planted (Sudianto et al., 2013). However, the risk of gene flow and evolution of resistant weedy rice populations is high in the tropics, where up to three rice crops are planted each year, and freezing temperatures, which would reduce the density of volunteer plants, do not occur.In the United States, where Clearfield technology originated and has been used for the longest time, the interaction between HR cultivated rice and weedy rice is not yet fully understood. Two main populations of weedy rice are known to occur in the southern United States and can be found in the same cultivated rice fields. These populations are genetically differentiated, are largely distinct at the phenotypic level, and have separate evolutionary origins (Reagon et al., 2010). One group tends to have straw-colored hulls and is referred to as the SH population; a second group tends to have black-colored hulls and awns and is referred to as the BHA population (Reagon et al., 2010). Genomic evidence suggests that both groups descended from cultivated ancestors but not from the tropical japonica subgroup varieties that are grown commercially in the United States. Instead, the SH group evolved from indica, a subgroup of rice commonly grown in the lowland tropics, and the BHA group descended from aus, a related cultivated subgroup typically grown in Bangladesh and the West Bengal region (Reagon et al., 2010). Weed-weed and weed-crop hybrids are also known to occur, but prior to Clearfield commercialization, these hybrids had occurred at low frequency (Reagon et al., 2010; Gealy et al., 2012). With the advent and increased adoption of Clearfield cultivars, the impact on U.S. weedy rice population structure and the prevalence of the SH and BHA groups are unknown.Efforts to predict the possible consequences of HR or genetically modified rice on weedy rice have been a subject of discussion for many years. Both weedy rice and cultivated rice are primarily self-fertilizing, but, as mentioned above, low levels of gene flow are known to occur. Additional environmental and intrinsic genetic factors can act as prezygotic and postzygotic mating barriers between cultivated and weedy rice and influence the possibility and levels of gene flow between these groups (Craig et al., 2014; Thurber et al., 2014). However, once gene flow occurs between cultivated and weedy rice, and if the resulting hybrids are favored by selection, the resulting morphological, genetic, and physiological changes in weedy rice populations can alter the way that weedy rice evolves and competes. For example, herbicide-resistant weed outcrosses in an experimental field have been observed to be morphologically diverse (Shivrain et al., 2006), with some individuals carrying major weedy traits and well adapted to rice agriculture. Such weedy plants could be more problematic than their normal weedy counterparts. Thus, introgression of crop genes into weedy populations has the potential to change the population dynamic, genetic structure, and morphological profile of weedy plants. This, in turn, must alter our crop management practices. To increase our understanding of the impact of HR rice on the evolution of weedy rice, in this article we aim to (1) assess the frequency of herbicide resistance in weedy rice in southern U.S. rice fields with a history of Clearfield use; (2) characterize the weedy attributes of resistant populations; and (3) determine the genetic origins of herbicide-resistant weeds in U.S. fields.     

Focus on Weed Control: Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.Glyphosate [N-(phosphonomethyl) Gly] is the most widely used agricultural pesticide globally (Duke and Powles, 2008). Originally, being a nonselective herbicide, its use was limited to vegetation management in noncrop areas; however, introduction of glyphosate-resistant (GR) crops in the late 1990s, coupled with their exceptional adoption, led to accelerated use totaling approximately 128 million ha worldwide in 2012 (James, 2012). GR crop technology has made a significant contribution to global agriculture and the environment, as it not only increased farm income by $32.2 billion (Brookes and Barfoot, 2013), but also moderated the negative environmental impacts of mechanical weed management practices (Gardner and Nelson, 2008; Bonny, 2011). Glyphosate offers a simple, effective, and economic weed management option in GR crops. In addition, it provides immense value in no-till crop production systems by enabling soil and moisture conservation. However, due to intensive glyphosate selection pressure, several weed populations globally have evolved resistance through a variety of mechanisms. Globally, herbicide resistance, in particular the recent proliferation of glyphosate resistance in weed species, is a major crop protection threat; nearly two dozen GR weed species have been reported in the last 15 years (Heap, 2014).Glyphosate, an aminophosphonic analog of the natural amino acid Gly, nonselectively inhibits 5-Enolpyruvylshikimate-3-Phosphate synthase (EPSPS) in plants, preventing the biosynthesis of the aromatic amino acids Phe, Tyr, and Trp (Steinrücken and Amrhein, 1980), resulting in the death of glyphosate-sensitive individuals. In plants, EPSPS is one of the key enzymes in the shikimate pathway (Herrmann and Weaver, 1999), and glyphosate inhibits EPSPS by binding to EPSPS-shikimate-3-P binary complex forming an EPSPS-shikimate-3-P-glyphosate complex (Alibhai and Stallings, 2001). Bradshaw et al. (1997) hypothesized against the likelihood of weeds evolving resistance to glyphosate, primarily because of its complex biochemical interactions in the shikimate pathway and also due to the absence of known glyphosate metabolism in plants. Nonetheless, several cases of glyphosate resistance, as a result of difference in glyphosate translocation (Preston and Wakelin, 2008) or mutations in the EPSPS, were confirmed (Baerson et al., 2002). More importantly, duplication/amplification of the EPSPS appears to be the basis for glyphosate resistance in several weeds (Sammons and Gaines, 2014). Here, we use duplication to refer to the formation of first repetition of a chromosomal segment and amplification to refer to increase in number of the repetitions (more than two repetitions of a chromosomal segment) under positive selection. The first case of EPSPS amplification as a basis for glyphosate resistance was reported in an Amaranthus palmeri population from GA (Gaines et al., 2010). In this A. palmeri population, there is a massive increase (>100-fold relative to glyphosate-susceptible [GS] plants) in EPSPS copies, and these copies are dispersed throughout the genome (Gaines et al., 2010).Field-evolved GR Kochia scoparia populations were first reported in western Kansas in 2007 (Heap, 2014). We previously determined that evolution of GR populations of K. scoparia in the U.S. Great Plains is also due to amplification of the EPSPS (A. Wiersma and P. Westra, unpublished data). Unlike in GR A. palmeri, we found relative EPSPS:acetolactate synthase (ALS) copies ranging from three to nine in GR K. scoparia populations. While it quickly became widespread in the region, its presence was reported in another five Great Plains states by 2013 (Heap, 2014). GR K. scoparia populations we tested were 3- to 11-times resistant (population level) to glyphosate compared with a GS population (Godar, 2014), and EPSPS expression positively correlated with genomic EPSPS copy number (A. Wiersma and P. Westra, unpublished data). Here, we reveal the genomic organization of the amplified EPSPS copies in two GR K. scoparia populations, an alternative mechanism of gene amplification to that reported in GR A. palmeri.     

Clarifying the Nomenclature in Microbial Weed Control

Correct terminology is essential to promote the concepts of biological control. In the current literature there are many terms used interchangeably and often inappropriately. The purpose of this paper is to examine the overlapping and sometimes contradictory terms used in biological weed control and to suggest a standardized usage to clarify existing terms. This is achieved through a hierarchical classification of existing terms (bioherbicide, mycoherbicide) and the introduction of new terms such as biopestistat (an inundatively applied, living organism which reduces the competitive ability of the target weed to below a desired threshold) and microbially derived phytotoxin (secondary metabolite used in weed management practices as an analogue of a chemical herbicide).     

Focus on Weed Control: In Vivo 31P-Nuclear Magnetic Resonance Studies of Glyphosate Uptake,Vacuolar Sequestration,and Tonoplast Pump Activity in Glyphosate-Resistant Horseweed

Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. ³¹P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate [AMPA] and N-methyl glyphosate [NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. ³¹P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells.Glyphosate is arguably the world’s most important herbicide (Duke and Powles, 2008). Environmental factors affecting its uptake and translocation in higher plants have been widely studied (Kells and Rieck, 1979; Coupland, 1983; Devine et al., 1983; Masiunas and Weller, 1988; Zhou et al., 2007). Notably, the role of light is important for effective uptake and translocation, suggesting that metabolic energy plays a role in this process (Simarmata et al., 2003; Shaner et al., 2005). Death of the whole plant requires effective glyphosate translocation from source to sink tissue, a process requiring ATP to maintain Suc gradients, which drive phloem movement (Bromilow et al., 1990; Dill et al., 2010).Weed species have developed glyphosate-resistant (GR) biotypes during the past decade (Heap, 2014). This has spurred interest in factors that may contribute to resistant attribute(s) as well as methods that can be used to screen plants for herbicide toxicity (Shaner, 2010). Resistance mechanisms have been reported for horseweed (Conyza canadensis; Feng et al., 2004; Zelaya et al., 2004; Ge et al., 2010, 2011), Palmer amaranth (Amaranthus palmeri; Gaines et al., 2010), and ryegrass (Lolium rigidum and Lolium multiflorum; Powles et al., 1998; Perez et al., 2004; Ge et al., 2012).Since glyphosate is foliar applied, glyphosate toxicity involves a multistep delivery process. Glyphosate must traverse the nonliving structures of the leaf cuticle and the cell walls of the epidermis, apoplast, and mesophyll prior to accessing the phloem for transport to sink tissues (Bromilow et al., 1990; Bromilow and Chamberlain, 2000). Indeed, restriction of glyphosate delivery to the plant cell cytoplasm (and chloroplast) by any means is, in itself, a resistance mechanism (Shaner, 2009; Ge et al., 2013). Elucidation of key factors governing delivery to the intracellular milieu of plant source leaves is critical for developing a complete understanding of the mechanism(s) of resistance to glyphosate.Glyphosate’s phosphono group offers the opportunity to employ in vivo ³¹P-NMR spectroscopy to track glyphosate movement and metabolism, additionally including monitoring of cellular pH, and gradients therein, and ATP levels, both indicators of tissue viability (Roberts, 1984). This laboratory has extended the ³¹P-NMR approach initially used by Gout et al. (1992) with suspension-cultured sycamore (Acer pseudoplatanus) cells. The initial findings, that source and sink leaf tissue from GR horseweed rapidly and avidly sequestered glyphosate within the vacuole compartment and that leaf tissue from glyphosate-sensitive (GS) horseweed did not, led to the hypothesis that vacuole sequestration was a key, perhaps the dominant, component of the resistance mechanism (Ge et al., 2010). It was then shown that GR horseweed acclimated and maintained at cold temperature (approximately 10°C–12°C) did not rapidly and avidly sequester glyphosate within the vacuole. Importantly, under such conditions, GR horseweed succumbed to the toxic effects of glyphosate. In short, by preventing glyphosate sequestration, GR horseweed became glyphosate sensitive, a laboratory finding confirmed in the field (Ge et al., 2011).The proposition that, by limiting the herbicide available for translocation, glyphosate vacuole sequestration could serve as an important if not dominant resistance mechanism was further strengthened by experiments that showed vacuolar glyphosate sequestration correlated with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe (Ge et al., 2012). However, ³¹P-NMR studies of other weeds revealed that in some species, for example, Palmer amaranth, waterhemp (Amaranthus tuberculatus), and johnsongrass (Sorghum halepense), resistance correlated strongly with a lack of glyphosate uptake into the plant cell, a frontline resistance mechanism (Ge et al., 2013).Throughout these previous ³¹P-NMR studies, the finding that plants could regulate the compartmental access of glyphosate led us to speculate that the apoplast, tonoplast, and perhaps chloroplast possessed glyphosate-active transporters whose up-regulation or down-regulation and/or expression would confer resistance (Ge et al., 2010, 2011, 2012, 2013). This hypothesis motivated additional in vivo ³¹P-NMR experiments to further describe the determinants of glyphosate delivery in horseweed leaf tissue. Specifically, experiments with GR horseweed were designed with the goal of probing the transporter hypothesis.Findings from these experiments are reported herein and are consistent with the existence of a tonoplast transporter that is responsible for glyphosate resistance via vacuole sequestration. As described here, vacuole sequestration requires ATP, is active for multiple substrates, and shows substrate competition. Furthermore, glyphosate entry into the cell can be markedly inhibited by vanadate pretreatment. These characteristics are similar to those of ATP-binding cassette transporters in plants (Hetherington et al., 1998; Rea, 2007; Verrier et al., 2008; Prosecka et al., 2009; Conte and Lloyd, 2011) and mammalian cells (van de Ven et al., 2009; Ernst et al., 2010).     

Focus on Weed Control: Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance.Although extensive monoterpenol (especially linalool) oxidative metabolism has been described in many plant species, leading to fragrant and bioactive compounds as diverse as alcohols, aldehydes, acids, and epoxides (Williams et al., 1982; Matich et al., 2003, 2011; Luan et al., 2005, 2006; Ginglinger et al., 2013), pyranoid or furanoid linalool derivatives (Pichersky et al., 1994; Raguso and Pichersky, 1999), and geraniol-derived iridoids and secoiridoids (Dinda et al., 2007a, 2007b, 2011; Tundis et al., 2008), limited information is available on the enzymes generating these oxygenated compounds. Involvement of a cytochrome P450 (P450) enzyme extracted from Vinca rosea (now renamed Catharanthus roseus) in the hydroxylation of geraniol and nerol was suggested as early as 1976 (Madyastha et al., 1976). The first plant P450 gene to be isolated, CYP71A1 from avocado (Persea americana) fruit, was later shown to encode an enzyme with geraniol/nerol epoxidase activity (Hallahan et al., 1992, 1994). To our knowledge, a connection with compounds formed in the fruit has not yet been established. The geraniol 8-hydroxylase (often named geraniol 10-hydroxylase) CYP76B6, involved in the biosynthesis of secoiridoids and monoterpene indole alkaloid anticancer drugs in C. roseus, was found to belong to the CYP76 family in 2001 (Collu et al., 2001). The catalytic function of this enzyme was recently revised, and was shown to include a second oxidation activity, the conversion of 8-hydroxygeraniol into 8-oxogeraniol (Höfer et al., 2013). The same work also revealed a geraniol 8- and 9-hydroxylase activity of CYP76C4 from Arabidopsis thaliana. More recently, another CYP76 enzyme (CYP76A226) from C. roseus was found to metabolize oxidized geraniol derivatives and to have an iridoid oxidase activity, catalyzing the triple oxygenation of cis-trans-nepetalactol into 7-deoxyloganetic acid for the biosynthesis of secoiridoids and terpene indole alkaloids (Miettinen et al., 2014; Salim et al., 2014). Not all CYP76 enzymes seem to be devoted to the metabolism of monoterpenols. In most cases, however, CYP76s seem to be involved in terpenoid metabolism. CYP76Ms from monocots were found to metabolize diterpenoids for the synthesis of antifungal phytocassanes (Swaminathan et al., 2009; Wang et al., 2012; Wu et al., 2013), CYP76AH1 from Salvia miltiorhizza and its ortholog CYP76AH4 from rosemary (Rosmarinus officinalis) were shown to hydroxylate the norditerpene abietatriene in the pathway to labdane-related compounds (Zi and Peters, 2013), whereas CYP76Fs from sandalwood (Santalum album) were found to hydroxylate the sesquiterpenes santalene and bergamotene (Diaz-Chavez et al., 2013). CYP76B1 from Helianthus tuberosus was, however, found to metabolize herbicides belonging to the class of phenylurea (Robineau et al., 1998; Didierjean et al., 2002), but its physiological function was not reported. Other P450s from soybean (Glycine max; CYP71A10; Siminszky et al., 1999) or tobacco (Nicotiana tabacum; CYP71A11 and CYP81B1; Yamada et al., 2000) were also reported to metabolize phenylurea, but their physiological function was not investigated.A. thaliana ecotype Columbia-0 (Col-0) emits no geraniol and only tiny amounts of linalool, and extensive volatile profiling of different tissues detected only minor amounts of lilac aldehydes (oxygenated linalool derivatives; Rohloff and Bones, 2005). However, ectopic expression of a linalool/nerolidol synthase of strawberry (Fragaria × anannasa cv Elsanta) revealed a potentially efficient oxidative linalool metabolism in A. thaliana rosette leaves (Aharoni et al., 2003). Only recent work started to explore linalool metabolism in A. thaliana, which was found mainly localized in the flowers (Ginglinger et al., 2013). This work demonstrated the existence of two linalool synthases producing different enantiomers, and the concomitant involvement of two P450 enzymes, CYP76C3 and CYP71B31, with predominance of CYP76C3, in linalool oxidation. It also suggested the presence of partially redundant enzymes that may contribute to floral linalool metabolism.A family of eight CYP76 genes is detected in the A. thaliana genome. We report here an evolutionary and functional analysis of this family. We show that members of the CYP76C subfamily, when successfully expressed in yeast (Saccharomyces cerevisiae), all metabolize monoterpenols with different substrate specificities. Although CYP76Cs seem specific to Brassicaceae, they share common functions with CYP76s from other plants, such as CYP76B1 from H. tuberosus and CYP76B6 from C. roseus. These functions include not only monoterpenol oxidation, but also metabolism and detoxification of herbicides belonging to the class of phenylurea. Because of this property, CYP76Cs can be used simultaneously for monoterpenol oxidation and as selectable markers for plant transformation.     

Comparative Efficacy of Weed Control Practices for Parthenium Weed and Sunflower Crop under Varying Tillage Systems

Parthenium poses serious threat to modern crop production system and necessitate evaluating control practices for its effective management. Efficacy of different weed control practices for controlling parthenium was explored in conventional and deep tillage systems in the field conditions. Hand hoeing (20 and 35 days after emergence), S-Metolachlor (pre-emergence herbicide), sorghum straw mulch @ 5 tons ha^-1 and combination of hand hoeing and sorghum straw mulch (hand hoeing at 20 and straw mulch at 35 days after emergence) were used as weed control practice. Weedy check where no weed control measure was applied was also included in this experiment for comparison. Results concluded that the all weed management treatments significantly reduced parthenium density, its fresh and dry biomass during both the years of study as compared to weedy check. Maximum sunflower achene yield was recorded in hand hoeing (20 and 35 days after emergence) in combination with deep tillage. So, mold bold plough used for the purpose of deep tillage should be encouraged for better control of parthenium and higher achene yield of sunflower crop (3293.3 kg ha^-1 in 2017 and 3221.3 kg ha^-1 in 2018). Moreover, is also inferred that total dose of herbicide might be reduced by using hoeing and mulching in an integrated way.     

除草剂"大田净"防除烟田杂草的效果

通过除草剂"大田净"的不同时期和不同浓度施用,观测其对烟田杂草的防除效果.试验结果表明,揭膜培土时(移栽后25d)喷施杂草防除效果较好;喷施不同浓度间,400倍液、550倍液比800倍液防治效果好.建议在揭膜培土时喷施550倍"大田净"进行除草.     

Focus on Weed Control: A Novel Rice Cytochrome P450 Gene,CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.The mechanism of herbicide tolerance can be classified roughly into two groups: target-site and non-target-site herbicide tolerance (Powles and Yu, 2010). Target-site herbicide tolerance is caused by the prevention of herbicide binding to the target enzyme, caused by point mutations occurring in the latter. It is relatively easy to elucidate the molecular mechanisms of target-site herbicide tolerance, because it is regulated mostly by a single gene encoding a target enzyme harboring point mutations. On the other hand, non-target-site herbicide tolerance is caused by reduction to a nonlethal dose of herbicide reaching the target enzyme, caused by mechanisms such as activation of herbicide detoxification, decrease of herbicide penetration, and herbicide compartmentation in plant cells (Yuan et al., 2007). Among these mechanisms, the oxidization of herbicides by endogenous cytochrome P450 monooxygenase is thought to be a major pathway in plants (Werck-Reichhart et al., 2000; Siminszky, 2006; Powles and Yu, 2010). From the point of view of weed control, non-target-site herbicide tolerance is a greater threat to crop production and in the evolution of herbicide-resistant weeds, because it is often involved in resistance to multiherbicides that inhibit different target proteins, including never-used and potential plant growth regulators (Yuan et al., 2007; Powles and Yu, 2010). Conversely, it is expected that multiherbicide-tolerant crops could be produced easily by the application of non-target-site herbicide tolerance. Moreover, information gained from study of the molecular mechanisms of non-target-site herbicide tolerance can be applied to the research and development of novel herbicides and plant growth regulators.Acetolactate synthase (ALS; also known as acetohydroxy acid synthase) plays a key role in the biosynthesis of branched-chain amino acids such as Val, Leu, and Ile in many organisms. ALS is the primary target site for at least four classes of herbicides: sulfonylurea, imidazolinone, pyrimidinyl carboxylates, and triazolopyrimidine herbicides (Shimizu et al., 2002, 2005). These herbicides can inhibit ALS activity, resulting in plant death caused by a deficiency of branched-chain amino acids. ALS-inhibiting herbicides control many weed species in addition to exhibiting high selectivity in major crops and low toxicity to mammals, which lack the branched-chain amino acid biosynthetic pathway. However, various mutations in ALS that confer ALS-inhibiting herbicide tolerance have been found in many weeds (Shimizu et al., 2005; Powles and Yu, 2010). Similar mutations in ALS have also been reported in crops (Shimizu et al., 2005). To date, crops that show tolerance to ALS-inhibiting herbicides have been produced by various approaches, such as conventional mutation breeding, conventional transformation, and pinpoint mutagenesis via gene targeting based on information obtained from analyses of ALS mutants (Shimizu et al., 2005; Endo and Toki, 2013). On the other hand, weeds that show tolerance to ALS-inhibiting herbicides by cytochrome P450-mediated detoxification have also been reported (Powles and Yu, 2010). However, compared with target-site herbicide tolerance, little is known of the molecular mechanism of herbicide metabolism mediated by cytochrome P450. In rice (Oryza sativa), an herbicide-sensitive mutant has been produced by γ-ray irradiation (Zhang et al., 2002). This mutant showed 60-fold higher sensitivity to bensulfuron-methyl (BSM), a sulfonylurea herbicide, compared with wild-type rice (Pan et al., 2006). Genetic mapping and complementation tests revealed that a cytochrome P450, CYP81A6, is involved in BSM tolerance (Pan et al., 2006). As far as we know, this is the only example of the isolation and characterization of a cytochrome P450 gene involved in nontarget herbicide tolerance in rice.Bispyribac sodium (BS), a pyrimidinyl carboxylate herbicide, is effective in controlling many annual and perennial weeds, with excellent selectivity on direct-seeded rice (Shimizu et al., 2002). Recently, it was reported that japonica rice varieties show higher sensitivity to BS compared with indica rice varieties at the early stages of plant growth (Ohno et al., 2008; Taniguchi et al., 2010). A mutated ALS gene confers BS tolerance in plants including rice (Shimizu et al., 2005; Endo and Toki, 2013). However, the deduced amino acid sequences were shown to be highly conserved among japonica and indica rice varieties, and ALS levels of sensitivity to BS were similar in japonica and indica rice varieties (Taniguchi et al., 2010). These results suggest the possibility that indica rice varieties might show higher tolerance to BS due to the acquisition of nontarget herbicide tolerance.In this study, we isolated and characterized a novel cytochrome P450 gene, CYP72A31, involved in BS tolerance in rice. We also demonstrated that overexpression of CYP72A31 confers tolerance to ALS-inhibiting herbicides, including BS and BSM, in Arabidopsis (Arabidopsis thaliana).     

Integration of Herbicides with Arthropod Biocontrol Agents for Weed Control

Classical biological control of weeds using arthropods is being attempted on a large scale in a number of countries, sometimes with spectacularly successful outcomes. However, in many cases biocontrol is not completely effective and use of herbicides on weeds continues to occur, either in the presence of biocontrol agents or as an alternative to them. The ways in which the two techniques may interact are discussed, including direct toxicity of herbicides to biocontrol agents, responses to death of host plants and responses to sublethal changes caused by herbicides with different modes of action. A literature review for selected weed taxa showed that the great majority of publications relate to either chemical or to biological control techniques separately, with integration of the two seldom addressed. Possible reasons for this situation are discussed and some suggestions for future priorities are made.     

第一届国际杂草防除大会在墨尔本市召开

由国际杂草学会(International Weed Science Society)组织并由澳大利亚维多利亚杂草学会(Weed Science Society of Victoria)主办的第一届国际杂草防除大会于1992年2月17日-21日在澳大利亚墨尔本市南郊的摩拿士大学(Monash University)召开。参加这次大会的有来自美国、英国、加拿大、丹麦、印度、意大利、法国、日本、荷兰、菲律宾、瑞士、泰国、芬兰、尼日利亚、巴西、澳大利亚、新西兰、中国、埃及等30多个国家的536名代表。其中来自澳大利亚和美国的代表占一半以上。欧洲有十几个国家的代表参     

Formulation of Bacteria for Biological Weed Control Using the Stabileze Method

Two plant pathogenic Pseudomonas spp. were formulated using the 'Stabileze' method which involves the incorporation of bacteria in a water-absorbent starch matrix with oil and sucrose, then granulating the matrix with hydrated silica. In one experiment, P. syringae pv. tabaci formulated with the standard Stabileze formula was evaluated for storage viability at -15, 2 and 22°C. Bacteria stored for 1 year at -15 and 2°C lost only 0.2 and 0.5 log 10 colony forming units (CFU) g -1 respectively compared to a loss of log 10 3.5 CFU at 22°C. In a second experiment, the same pathogen was evaluated using variations of the formula with and without oil, and with and without sucrose. P.s. pv. tabaci formulated with sucrose and oil in combination, and sucrose and oil alone survived better than the formulation without oil or sucrose. A third experiment tested the effect of four levels of oil and four levels of sucrose (4 ×4 factorial) on survival of P.s. pv. tagetis over a 28 month period. Sucrose alone enhanced survival more than oil alone, and the beneficial effect of the sucrose was reduced when it was combined with oil. These experiments suggest that the Stabileze protocol is effective for stabilizing bacteria, but there are differences in response to different formulation components between species of bacteria.