Transgene flow from GE rice to weedy rice can result in diverse fitness effects, depending on the type of transgenes and the selective pressure to which the GE crop-weed hybrid descendants are exposed. In addition, other factors such as the genetic background of weedy rice populations that have obtained the transgenes may also influence the fitness effects of a particular transgene . Many studies have indicated that insect-resistance transgenes confer a fitness benefit for crop-weed rice hybrid progeny under high insect pressure . However, the same transgenes do not confer such a benefit to the hybrid progeny under low insect pressure. The fitness studies based on multiple generation descendants of GE crop-weed hybrids provide similar results . Therefore, we conclude that fitness change and evolutionary potential for transgene flow from GE insect-resistant rice to weedy rice populations are quite limited because of the low ambient insect pressure expected in extensively planted transgenic commercial rice production fields . In contrast, the movement of herbicide-resistance transgene to weedy rice populations appears to considerably change the fitness of the crop-weed hybrid progeny, both with and without the application of glyphosate herbicide sprays, and possibly the evolutionary potential of the hybrid progeny by altering their rate of biosynthesis and photosynthesis . This indicates that the movement of this specific herbicide-resistance transgene to weedy rice populations may result in increased weed problems.Our understanding of the fitness effects and expected evolutionary dynamics brought by transgenes, including those conferring herbicide resistance, drought and cold tolerance, hydroponic trays and stacked traits with diverse functions are still limited. It is clearly shown from the results of studies already done on the cultivated rice and weedy rice system that simple expectations from the transgene’s intended phenotype are not sufficient to predict what will occur under experimental conditions.

Are these results general? We sought to compare the results discussed above with a sample of results from similar field experiments involving different species and/ or different transgenes. We review a collection of such studies that are included in Table 2. Our sample involves twelve studies representing six crop donor species and eight recipient weedy/wild species. Four different transgenic phenotype classes are represented. The only generality that emerges is variability. Introgressed transgenes may or may not confer a fitness advantage under selective pressure associated with the intended transgenic phenotype. Without that selective pressure, the presence of the transgene may correlate with increased fitness, decreased fitness, or no significant fitness change. Taken collectively, the cultivated rice – weedy rice system case study reviewed above and the additional studies featured in Table 2 make it clear that the fitness changes associated with transgenic presence in unmanaged populations cannot be predicted a priori. While increased fitness in itself may not be sufficient to predict an environmental hazard, it does provide support for the conclusion that the transgene will persist and spread . Obviously, with regard to introgression-based transgene risk assessment, the current regulatory policy of case-by-case analyses informed by field-based research is sound and superior to predicting the fitness correlates of introgressed transgenes without such data.Glyphosate kills plants by inhibiting a particular enzyme, 5-enolpyruvyl shikimate-3-phosphate synthase. This enzyme is one of several in the shikimic acid pathway, which is how plants produce the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Amino acids are building blocks for the plant, so a plant not able to manufacture all amino acids is unable to grow and develop normally.

Plants also use these three specific amino acids to synthesize more complex structural compounds and a host of plant defense molecules , which together can make up 60 percent of a plant’s dry weight. Consequently, inhibition of this pathway causes serious consequences for a plant, and it helps to explain why glyphosate is such an effective herbicide. It is also an herbicide with a very low mammalian toxicity, as mammals do not have the EPSP synthase enzyme.Glyphosate is normally formulated as a salt, which is a compound that can split into positively and negatively charged portions when mixed with water. Glyphosate salts include potassium, diammonium, isopropylamine, trimethylsulfonium, and sesquisodium. Formulations differ in how much glyphosate ends up in the final product, due to the chemistry of the salt and the different adjuvants used by the various manufacturers. The amount of the glyphosate salt in the formulation is listed on the herbicide label as the active ingredient . In the case of glyphosate, however, only the glyphosate portion of the salt is actually herbicidal; the other portion of the salt is nonherbicidal. Why would a manufacturer formulate glyphosate as a salt? Glyphosate salts are better able to enter into plant tissues than is the free glyphosate acid, so these formulations provide better weed control. Since different salts have different molecular weights, it would be difficult to determine how much actual glyphosate is contained in different formulated products if we just look at the a.i. content, usually listed as pounds of a.i. per gallon or grams of a.i. per liter . When comparing different formulations of glyphosate, it is better to look at the acid equivalent , which is the amount of glyphosate in the negatively charged or acid portion of the salt, the part of the a.i. that binds with EPSP synthase. Therefore, using the a.e. is also the best way to select the appropriate application rate for various formulations, since the a.e.represents the amount of glyphosate needed to control certain weed species .

Surfactants are the most commonly used adjuvants; they modify the surface tension of water and, when in mixture with an herbicide, cause applied droplets to spread out on leavesand improve herbicide uptake. Most agricultural surfactants are nonionic, although crop oils are also widely used; other surfactants are organosilicon based. Most glyphosate formulations contain an adequate concentration of surfactant for general use, so additional surfactant is usually not necessary. Exceptions occur when applying glyphosate to weeds with dense hairs or thick cuticles on their leaves or when using a formulation that does not contain added surfactant, such as aquatic formulations of glyphosate. Read the label to determine whether adding a surfactant to a particular glyphosate formulation, or for a particular weed species, is necessary. Water-conditioning agents are another major type of adjuvant. Because glyphosate can exist as a negatively charged molecule after the herbicide is mixed with water, it can react with positively charged ions or molecules in the water. Water containing a high concentration of cations is commonly called hard water. Some common cations in hard water include sodium , potassium , calcium , magnesium , and iron . Cations with more than one positive charge bind strongly to glyphosate and reduce its ability to be absorbed into plant leaves. Water conditioners, such as ammonium sulfate or other proprietary adjuvants, help to soften hard water. When AMS is added to water, the compound splits into two ammonium ions and one sulfate ion . This ionized AMS helps improve glyphosate performance in two ways. First, if glyphosate binds to ammonium, the resultant molecule is much more easily absorbed through the leaf cuticle, through the cell wall, or across the plasma membrane of certain weed species than when glyphosate is bound to other cations, resulting in more herbicide penetrating the weed. Second, sulfate preferentially binds to calcium, magnesium, and iron cations in the water, thus removing them from the solution and leaving more glyphosate free to move into the weed. Studies show that translocation of glyphosate is increased when AMS is added, pipp mobile systems due to improved phloem mobility, probably because more glyphosate in plant cells increases phloem loading and translocation of the herbicide in the weed. The general recommendation is to add 1 to 2 percent of AMS by weight to glyphosate mixtures, which is equivalent to 8.5 to 17 pounds dry AMS or 2.5 to 5 gallons of liquid AMS per 100 gallons of spray solution. Buffering agents are another type of adjuvant. The pH of water is a measure of the hydrogen ion and hydroxide ion concentration. As the number of H+ increases relative to OH− , water becomes more acidic and pH decreases. As noted above, when glyphosate is unbound, it has a net negative charge and is absorbed more slowly across cuticles and cellular membranes than when it is bound to certain cations as a salt. At a lower pH, more glyphosate exists as a salt than as a free acid, so plant uptake of the sprayed solution is improved. Consequently, slightly acidic water is most suitable for mixing with glyphosate. When water pH exceeds 7, consider adding buffers or acidifiers to lower the pH.Since glyphosate binds tightly to soil particles, its application to dusty plants results in inactivation of much of the herbicide before uptake can occur.

Glyphosate activity is usually poorer on weeds growing in wheel tracks, probably due to dust or mud on the surface of the plant foliage. Also, weeds that have been run over by sprayers or other vehicles may not be healthy enough to translocate absorbed glyphosate to their growing points, resulting in poor control. For optimal weed control with glyphosate, weeds should be relatively dust free at the time of application. Applications are therefore best made prior to the onset of dusty conditions in the summer. If weeds are already dusty, irrigation may be an option to wash dust off the foliage, followed by glyphosate application after the foliage has dried.Weed control with glyphosate has sometimes been observed to be better when applied at low volumes than at high volumes. This may occur if the low volumes are achieved by using nozzles with small orifices, resulting in the production of smaller droplets and increased foliar coverage. Perhaps, too, lower volumes of hard water contain fewer cations to bind with glyphosate in the mixture. Also, smaller droplets are more likely to drift, reducing coverage of weed foliage and increasing the chance of crop injury, particularly when glyphosate is applied when the crop is bearing leaves and is actively growing.When other pesticides or additives such as fertilizers are mixed with glyphosate solutions, an opportunity exists for the chemicals to bind with otherwise-inactive glyphosate. Sometimes the mode of action of certain herbicides may also slow or prevent translocation of glyphosate. Metribuzin , carfentrazone , and sulfentrazone are herbicides that antagonize glyphosate activity on certain weed species, while certain anti-drift agents have also antagonized glyphosate. The best way to avoid antagonism is to mix glyphosate formulations only with other products listed on the glyphosate label. Applying herbicides in separate applications rather than in a tank mixture may also reduce antagonism between herbicides. Since tank mixtures may offer improved control of other weed species, however, antagonism observed in certain weed species may be an acceptable trade-off.Glyphosate absorption through treated foliage is affected by environmental conditions shortly before, during, and after glyphosate application. Glyphosate must translocate from foliage to the site in plant cells where shoots or roots are being actively produced. Therefore, weeds under stress due to cold, heat, or improper amounts of soil moisture or weeds displaying symptoms from plant disease or previous herbicide application are usually not actively growing and may not respond as quickly or as completely to glyphosate application. Excess leaf moisture from dew or rainfall too close to the time of application can also reduce glyphosate performance. Conversely, glyphosate activity is usually improved with higher relative humidity. Leaf cuticles are usually more hydrated under humid conditions, resulting in better herbicide uptake, provided that leaf surfaces are dry during and after the application.The stage of growth and the life cycle of targeted weed species are important to consider if maximal control with glyphosate is to be achieved. Annual weeds are best controlled when they are small, when less glyphosate is necessary for a lethal dose. If killed prior to flowering, seed production will also be prevented. Glyphosate is strictly a foliar herbicide and does not exhibit residual soil activity. Weeds that have not emerged at the time of application are not controlled, so multiple applications are usually necessary to fully control both early- and late-emerging seedlings. Tank mixtures or sequential applications with soil-residual herbicides may improve weed control while reducing the number of herbicide applications necessary to fully control weeds. Perennial weed species frequently become more problematic the longer a perennial crop is kept in production. Directed sprays or spot applications of glyphosate are usually necessary to gain adequate control while preventing crop injury.

The odds ratio analysis showed that the likelihood of occurrence of feral sorghum along a gravel or dirt road is 1.5 or 2.4 times greater than that of a paved road, respectively . Dirt and gravel roads are prevalent in rural areas surrounding farmlands and high likelihood for the presence of feral sorghum populations along these road types suggests that movement of farm equipment and production activities greatly contribute to sorghum seed dispersal into roadside habitats. Further, wash boarding, corrugation and any potholes on the surface of unpaved roads can increase vehicle bouncing and thus increase the chances of seed spill from seed transport trucks and farm equipment. Contrary to our findings, the higher frequency of feral oilseed rape along the paved compared to the gravel and dirt roads in France was attributed to the higher traffic intensity with commodity transport on paved roads. Although the Chi-square test suggested significant differences between the road types , the odds ratio analysis failed to detect such differences . This may suggest that all road types are equally likely to accommodate feral sorghum, following a seed immigration event. Although previous studies have found a strong relationship between the presence of feral crops and road type, this was not the case for feral sorghum in South Texas. One notable exception in the present survey was that feral sorghum populations were common along U.S. Highway 77 between Kingsville and Raymondville, TX where sorghum and crop production in generalis very sparse. The high intensity of grain transport via truck movement is likely contributing to sorghum seed dispersal along this highway. Feral sorghum was more likely to be present at the road shoulders as shown by the odds ratio values , but they were also present in the field shoulders and at field edges . However, cannabis drying system as the road and field shoulders are only separated by few meters in many cases, feral sorghum found along the road verges could also be sourced by sorghum production activities in the adjacent fields.

This postulation is supported by a lack of significant difference between the odds of feral sorghum presence at field shoulders relative to that of the road shoulders, as shown by the odds ratio estimates . However, a logical question could be why feral sorghum is less abundant at field shoulders or field edges if the adjacent fields could contribute to propagule immigration. Field edges are typically disturbed , disrupting the establishment and persistence of feral sorghum in these sites. Nevertheless, the microhabitats at the roadside could provide more moisture for the establishment of plants compared to field edges. The nearby land use had large effects on the presence of feral sorghum . Results showed that the odds for the occurrence of feral sorghum in sites adjacent to sorghum cultivation was larger than that of all other land uses; the likelihood of finding feral sorghum in a location closer to a sorghum field was almost twice as high as a location contiguous to corn, hay, pasture, shrubland, urban or fallow lands . Sorghum is one of the major crops grown in South Texas and results suggest that sorghum cultivation and seed transport activities in the region contribute to seed immigration and establishment of feral sorghum on roadside habitats. Further, the sorghum seed dispersed following harvest might germinate instantly due to the lack of seed dormancy and the warm environmental conditions in South Texas may allow feral sorghum to produce viable seed prior to killing frost and establish self-perpetuating populations. There is also a possibility for spring establishment of feral sorghum from the seeds entered into the soil post-harvest should they be able to survive during the fall and winter. Data on seed survival rate of sorghum coupled with early spring monitoring are needed to address this question.The population size of the feral sorghum at each site was scored based on visual estimations.

The results from the analysis of variance showed no significant relationship between all the measured factors and the population size of the feral sorghum at sampled sites expect for the vegetation cover . The largest feral sorghum population sizes were associated with the highest vegetation cover , a finding that is unexpected given that vegetation with higher canopy cover should be more resistant to invasion than those with low canopy cover. One possible explanation is that the roadsides are regularly treated with herbicides by the Department of Transportation and in some cases by county weed control specialists for controlling tall vegetation such as johnsongrass. It is likely that herbicides might have been recently applied at sites with low vegetation cover, thus reducing the chance of observing feral sorghum individuals.The co-occurrence of feral sorghum and johnsongrass was rare and both species were found together only in 48 of the 2,077 survey sites visited . Fig 4 shows co-occurrence of feral sorghum and johnsongrass in a roadside site near Corpus Christi, TX. Results from the logistic model showed a negative relationship between the occurrence of johnsongrass and feral sorghum . The likelihood of detecting feral sorghum at locations without johnsongrass was 4.3 times greater than the locations where johnsongrass was present. Three possible scenarios might explain this finding: the presence of johnsongrass in the site may have a negative influence on the germination and establishment of feral sorghum ; however, the data collected in this study was not sufficient to establish any causal relationship or there is no anecdotal evidence to support such a scenario in production fields, the dispersal of sorghum seed on roadsides as a function of intensive sorghum cultivation and seed transport occurs primarily in the much Southern parts of Texas from Victoria towards Brownsville, an environmental gradient increasingly less suited for johnsongrass as well as in the habitat suitability map for johnsongrass; thus, the cooccurrence of both species was perhaps naturally limited, and/or roadside herbicide applications that target johnsongrass may eliminate any feral sorghum plants present within these sites, while johnsongrass could regrow from rhizomes. It is very likely that the second and third scenarios have substantial influence on the co-occurrence of these two species. A followup observation conducted in summer 2017 has revealed supporting evidence for the third scenario in that several of the feral sorghum-johnsongrass complex sites we identified in the 2014 survey were severely impacted by roadside herbicide applications that typically target johnsongrass. In these sites, several johnsongrass plants survived, but almost all feral sorghum plants were eliminated. Given that feral sorghum and johnsongrass can hybridize, the co-occurrence of these two species may facilitate the persistence of feral sorghum through gene flow and introgression of adaptive traits from johnsongrass. In addition, cultivation of diverse sorghum lines, including sudangrass and sorghum-sudangrass hybrids, in the vicinity may enrich the diversity within the feral sorghum populations and thereby increase the adaptive ability of feral sorghum. Such an outcome has been reported for feral populations of oilseed rape and alfalfa. Since this is the first record of the presence of feral sorghum innature, no information is available on the diversity, population genetic structure and longterm persistence of feral sorghum populations.Although most of the feral sorghum populations were observed along the roadsides, they may have the potential for spread to their contagious natural and unmanaged areas. To investigate the potential for broader distribution of feral sorghum in South Texas, we calibrated a model using nearby land use type and regional habitat suitability for johnsongrass as reliable predictors.

These two variables were chosen because they were statistically significant and the georeferenced data for the entire region was available. The combination of these two variables effectively predicted the distribution of feral sorghum, with an increasing trend in abundance from the Upper Gulf Coast towards the Rio Grande Valley, which corresponded to an increasing intensity of sorghum cultivation and seed transport activities in the landscape. Further, in the more Southern areas of Texas, sorghum seeds germinating after the harvest season will have a high chance to produce mature seed prior to killing frost, if any. The projected map for feral sorghum distribution is shown in Fig 5, which corroborates with the overall trend observed in the survey. Conversely, growing tray the distribution of johnsongrass showed an opposite trend, with more abundance in the Upper Gulf Coast region than in the Rio Grande Valley, attributable to its habitat suitability as evident in the habitat suitability map .The current survey showed that roadsides and field margins are the initial niches for feral sorghum to establish outside of cultivated fields. We found that the occurrence of feral sorghum in South Texas is highly associated with sorghum cultivation in the nearby area, providing propagules for the establishment of feral populations in field edges and roadsides during planting and grain transport operations. We did not find any relationship between the frequency of feral sorghum and road characteristics . Although johnsongrass can be found commonly along the roadsides in South Texas, the co-occurrence of feral sorghum and johnsongrass was infrequent. Yet, there are significant opportunities for outcrossing to occur between the two species outside of cultivated fields. More research is necessary to understand the frequency of outcrossing between the two species and fitness of the progenies. Experiments are on the way to characterize, using phenotypic and molecular markers, the progeny of seed harvested from feral sorghum plants during this survey in sites where both species co-existed. Further, field surveys and monitoring are being carried out to confirm and characterize potential hybrid progenies in nature in these feral sorghum-johnsongrass complex sites.When transgenic plants were initially developed, most plant evolutionary biologists and geneticists considered spontaneous hybridization between species to be rare and of little importance in terms of evolution. This view extended to both crops and their wild or weedy relatives, but has now radically changed. More than twenty years of gene-flow research has shown that interspecific hybridization is very common in some groups of vascular plants and may be of considerable evolutionary significance. Hybridization may occasionally result in the extinction of a population, may trigger the evolution of plant invasiveness, or initiate speciation. A substantial body of evidence has now accumulated, demonstrating the high potential for interspecific hybridization between agricultural crops and their wild or weedy relatives. Transgenic crops are no exception, and empirical studies have provided evidence of transgene dispersal from GM crops to their weedy relatives. Many factors have been shown to influence the rate of hybrid formation between crops and their wild or weedy relatives. Population effects such as the local densities of the parental types and their relative frequencies, have been demonstrated in several cases. Mating system differences at the individual level due to, for example, selfing rates and apomixis, have also been found to affect hybridization rates. Moreover, several studies have shown that overlap in the flowering periods of crop and weed plants affect opportunities for hybridization. The aim of this study is to gain insight into the impact of hybridization with transgenic crops on the evolution of the weedy relatives by verifying that hybridization opportunities for weedy plants depend on their phenotypic traits , measuring the relative fitness of hybridizing weeds, and searching for associations between the transgenic trait and the phenotypic traits increasing hybridization opportunities in the offspring of weedy plants. We studied hybridization opportunities, phenotypic traits and offspring phenotype of weedy individuals in experimental plant populations cultivated under glasshouse conditions. Experimental populations were composed of weeds and transgenic plants in a 1:1 ratio. Transgenic plants were crop plants of the Brassica genus , F1 hybrids between B. rapa and B. napus, or first-generation backcrosses. Crop plants were all homozygous for the Btcry1Ac transgene from Bacillus thuringiensis, F1 hybrids were all hemizygous and first-generation backcrosses and consisted of an equal mixture of hemizygotes and null homozygotes. Hybridization opportunities for each weedy individual was calculated as the expected proportion of pollen received from transgenic plants based on the observed flowering schedules. This experimental system was ideal for addressing the question of interest in this study, for three reasons. First, despite barriers to interspecific mating such as apomixis or preferential exclusion of hybrid zygotes, numerous studies have shown that B. napus and B. rapa readily hybridize under controlled conditions, but also in the field.

The introduction of TYLCV into CR in 2012 was the second invasion event: a highly invasive OW monopartite begomovirus that can cause devastating losses to tomato production worldwide . Indeed, TYLCV has added a new challenge for tomato production in CR, as growers have reported increased losses due to begomoviruses disease since 2012 . However, what was less clear was how TYLCV interacts with the already existing bipartite begomoviruses ToYMoV and ToLCSiV. In Florida, the introduction of TYLCV led to a reduced incidence of the indigenous bipartite begomovirus ToMoV . Therefore, this provides an opportunity to investigate the invasion biology of these very different begomoviruses with the long-term objective of making predictions and management suggestions. In the infectivity experiments, interactions were revealed based on symptom severity and viral DNA accumulation. A general synergistic interaction was observed in which mixed infections resulted in more severe symptoms, with plants infected by all three viruses showing the most severe symptoms. A similar situations has been observed for the interaction of three indigenous begomoviruses in Brazil, as measured based on symptom severity . Furthermore, the interaction among begomoviruses detected in the present study is a type of neutral synergism, which has also been previously described, including for begomoviruses . For example, N. benthamiana and tomato plants co-inoculated with TYLCV and tomato yellow leaf curl Sardinia virus developed more severe symptoms than plants inoculated with either virus alone, curing cannabis and accumulation of each virus in co-infected plants was similar to that in single infections . Interestingly, we did detect an initial antagonistic or negative interference effect on ToYMoV and ToLCSiV accumulation at 7 dpi in all mixed infections, indicating it was not virus specific.

These results are consistent with a previous study showing a transient negative effect on viral accumulation early in mixed infections of tomato rugose mosaic virus and tomato yellow spot virus in N. benthamiana and tomato . The overall mechanism of antagonism in mixed infections remains to be elucidated, but may involve competition for host factors or stimulation of a more efficient defense response of the host . For example, potato spindle tuber viroid can interfere with TYLCSV accumulation in tomato by activation of the host DNA methylation pathways . The fact that TYLCV accumulation was not affectedduring mixed infections with NW bipartite begomoviruses in the present study could be due to the combine activities of multiple viral suppressor of gene silencing, e.g., C2, C4 and V2 . Taken together, our results with these three begomoviruses revealed the existence of temporary antagonism followed by a more sustained neutral synergism. We further showed that in mixed infections with TYLCV, TYLCD became dominant at 14 dpi and beyond, even in the presence of the two co-infecting NW tomato bipartite begomoviruses. Indeed, this dominant TYLCD phenotype has been observed in the tomato fields in Costa Rica since the introduction of TYLCV . Therefore, in terms of the invasion biology of these viruses, it appears they can effectively co-exist in tomato plants, which leads to more severe disease and the persistence of all three viruses in tomato production in CR. Another observation in mixed infections with TYLCV was that, although TYLCD symptoms eventually became dominant, symptoms induced by co-infecting bipartite begomoviruses appeared earlier , with TYLCD symptoms appearing at ~10 dpi. This observation can be explained in terms of the tissue tropism of these viruses. The more rapid appearance of mosaic/mottle symptoms in leaves may reflect the capacity to infect cells outside of the phloem, and more rapidly colonize and accumulate in plants.

The sap transmission of ToYMoV is evidence this virus is not phloem limited. In summary, we used the tomato begomovirus situation in CR to examine the invasion biology of three viruses, two of which were introduced. We first confirmed ToYMoV caused ToYMoD in CR and is a locally evolved NW bipartite begomovirus. We then used infectious clones to investigate interactions in mixed infections. We found that these viruses exhibited a neutral synergism, in which the viruses co-exist and induced more severe symptoms. In mixed infections with TYLCV, TYLCD became predominant. These results indicate that all three viruses are likely persisting in CR and causing more severe symptoms and losses, particularly in the presence of TYLCV. Thus, an effective management of these complexes will require an integrate approach, including the identification of varieties with resistance to all three viruses.The genus Begomovirus is comprised of a large and diverse group of plant viruses that possess a circular, single-stranded DNA genome encapsidated into twin quasi-icosahedral virions . These viruses infect dicotyledonous plants and cause numerous economically important diseases of fiber, fruit, ornamental and vegetable crops, mostly in tropical and subtropical regions of the world . Begomoviruses are transmitted, plant-to-plant, by whiteflies of the Bemisia tabaci cryptic species complex . The genome of begomoviruses is composed of either a single genomic DNA of ~ 2.8 kb or two ~2.6 kb DNA components , designated as DNA-A and DNA-B . The genomic DNA of monopartite begomoviruses is homologous to the DNA-A component of bipartite begomoviruses, and both are organized with overlapping virion -sense and complementary -sense genes transcribed in a bidirectional manner from an intergenic region , which contains the cis-acting elements involved in replication and gene expression .

In bipartite begomoviruses, an ~200 nucleotide noncoding sequence is shared between cognate DNA-A and DNA-B components, and this common region maintains the specificity of replication for these components. Otherwise, the sequences of the DNA-A and DNA-B components are different, and bothcomponents are needed for induction of typical disease symptoms . In terms of begomovirus evolution, continental drift is believed to have separated ancestral monopartite and bipartite begomoviruses, resulting in the predominance of monopartite begomoviruses in the Old World and bipartite ones in the New World . The subsequent independent diversification and evolution of OW and NW begomoviruses involved different combinations of mutation, recombination and acquisition and modification of foreign DNAs . For OW monopartite begomoviruses, acquisition of satellite DNAs has played a major role in evolution, whereas acquisition and modification of the DNA-B component was essential for bipartite begomoviruses, and allowed for pseudorecombination to act as an additional mechanism of evolution . Furthermore, the emergence of new begomoviruses has been facilitated by the global spread of the highly polyphagous B. tabaci species MEAM1, which can introduce mixtures of viral components/genomic DNAs into a diversity of plant species . Finally, human activities have led to the long distance intercontinental movement of numerous begomoviruses, blurring the geographic separation of OW and NW begomoviruses . The remarkable diversification of begomoviruses has been reflected in the appearance of diseases of crop and non-cultivated plants in tropical and subtropical regions worldwide. In these agroecosystems, it is common to observe non-cultivated plants showing striking golden/yellow mosaic symptoms, which are commonly associated with begomovirus infection. Inthe Caribbean Basin and other parts of Latin America, non-cultivated plants with these symptoms have been reported from species in the families Asteraceae, Capparaceae, Convolvulaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nyctaginaceae and Solanaceae . Importantly, characterization of begomoviruses associated with these diseases has revealed substantial genetic divergence from viruses that cause economically important crop diseases, although there are some exceptions such as the golden/yellow mosaic symptoms of Malachra alceifolia associated with tobacco leaf curl Cuba virus infection in Jamaica , and mosaic and crumpling symptoms of Nicandra physaloides infected with tomato severe rugose virus in Brazil . This suggests that begomoviruses infecting crops and weeds have co-evolved independently with their hosts, with the practical implication that most of these symptomatic weeds are not major sources of inoculum for crop-infecting begomoviruses. However, these begomovirus-infected weeds can serve as a mixing vessels for evolution of viruses with the potential to infect crops . The family Malvaceae, commonly referred to as mallows, is comprised of >4225 species of annual and perennial plants .

Members of this family are distributed worldwide, and occur in temperate, tropical and subtropical regions . Some species are important crops, such as cotton and okra ; others are grown as ornamentals or for medicinal purposes; and others are considered invasive weeds, e.g., Abutilon spp., Sida spp. and Malachra spp. . Moreover, weed dryer these malvaceous weeds are commonly infected by begomoviruses and develop striking golden/yellow mosaic symptoms . As part of a long-term study to characterize begomoviruses causing golden/yellow mosaic symptoms in weeds and assesses the potential of these viruses to cause diseases of crop plants in the Dominican Republic , we describe here the molecular and biological properties of two bipartite begomoviruses associated with these symptoms in Malachra sp. and Abutilon sp. plants on Hispaniola. Sequence and phylogenetic analyses together with infectivity studies with infectious clones were used to establish that the symptoms in Malachra sp. were caused by the crop-infecting bipartite begomovirus TbLCuCV, whereas those in Abutilon sp. were caused by a new species of weed-infecting begomovirus for which the name Abutilon golden yellow mosaic virus is proposed. Host range experiments showed that TbLCuCV also induced moderate to severe disease symptoms in Nicotiana benthamiana, tobacco and common bean plants plants. In contrast, AbGYMV induced mild or no symptoms in these plants, indicating a high degree of adaptation to Abutilon sp. from the DO and low potential to cause crop diseases. TbLCuCV and AbGYMV are closely related species in the Abutilon mosaic virus lineage of NW begomoviruses and we present evidence that recombination and pseudore combination play a role in the evolution of these viruses.To detect begomovirus DNA-A and DNA-B components, PCR tests were performed with the degenerate primer pairs PAL1v1978/PAR1c496 and PCRc1/PLB1v2040, respectively . PCR-amplified fragments were purified with the QIAquick gel extraction kit and directly sequenced with the PAL1v1978/PAR1c496 and PCRc1/PLB1v2040 primers. To estimate the number and genetic diversity of begomovirus DNAs present in the samples and to identify single-cutting restriction enzymes for obtaining full-length clones, restriction fragment length polymorphism analyses of circular DNAs generated by rolling circle amplification with Φ-29 DNA polymerase were performed . The RCA products were first digested with the fourbase-cutting enzyme MspI to generate RFLPs for estimating the number of begomovirus DNA components infecting the samples. Next, RCA products were digested with selected six-basecutting enzymes to identify sites in each DNA component for obtaining full-length clones. The linearized DNA components were ligated into pGEM11Z  or pSL1180 digested with the appropriate enzyme. Recombinant plasmids having the full-length DNA-A and DNA-B components were identified by restriction enzyme digestion and DNA sequence analyses. Based upon sequencing and RCA results, the begomovirus isolates from the M1 and M4 samples were selected for further studies. Thus, full-length DNA-A and DNA-B clones were obtained from sample M1 , sample M2 , sample M3 and sample M4 .The complete sequences of the cloned full-length DNA-A and DNA-B components of the bipartite begomoviruses from samples M1-M4 were determined and analyzed with Vector NTI advance software . Pairwise nt sequence alignments were performed with MUSCLE within the Species Demarcation Tool v.1.2, and with full-length DNA-A and DNA-B sequences of the ten begomoviruses with the highest identities revealed by the BLASTn search . The Vector NTI advance software was used to make more extensive comparisons, including individual open reading frames and non-translated regions from both components. The cis-acting elements involved in begomovirus replication were identified as described in Argüello-Astorga and Ruiz-Medrano .For the phylogenetic analyses, we used the complete nt sequences of the DNA-A and DNAB components of: the bipartite begomoviruses from the M1-M4 samples; TbLCuCV isolates from CU ; the ten most identical viruses revealed by the BLASTn search; and selected viruses representing the AbMV, Brazil, squash leaf curl virus , bean golden yellow mosaic virus and Boerhavia golden mosaic virus lineages of NW begomoviruses. Multiple sequence alignments for the DNA-A and DNA-B component sequences were generated with the MAFFT algorithm implemented in the Guidance2 Server . The alignment quality was analyzed, and unreliable regions were removed with the GUIDANCE algorithm . The resulting alignments were then exported as Nexus files. Phylogenetic trees were constructed with a Bayesian inference and Markov chain Monte Carlo simulation implemented in MrBayes V3.2 . The best-fit model of nt substitution for each data set was determined with the program MrModeltest V2.2 . The analyses were carried out by running 2,000,000 generations and sampling at every 100 generations, resulting in 20,000 trees. The first 10% of samples were discarded as a burn-in. Trees were visualized with Archaeopteryx tree viewer and exported in Newick format .

Weeds are the greatest biological constraint to rice yields, and farmer inputs towards weed management are expected to increase as herbicide resistance spreads worldwide . The potential yield lost to weed infestation is species dependent, and the practice of continuous rice monoculture in California has resulted in an abundance of highly competitive weeds that negatively impact rice yields . In California rice fields, weedy grasses are the largest predictors of overall yield loss. Late watergrass [Echinochloa phyllopogon . Koss] competition has caused rice yield losses as high as 59% . Studies in Arkansas have shown rice yield losses to be 79% from competition with barnyard grass [Echinochloa crus-galli Beauv.] and 36% from bearded sprangletop competition . Weedy rice densities of 30 to 40 plants m-2 can reduce rice yields by 60-90%, depending on the cultivar . In the United States Midsouth region, yield losses due to ducksalad infestations can reach 30% . Most California rice herbicides are limited in the spectrum of weeds controlled and the length of residual activity, requiring herbicide treatment plans to consist of multiple herbicides to enact weed control over a range of weeds . Continuous use of herbicides with the same mode of action aids in the development of herbicide resistance in a crop . Confirmed herbicide resistance from various populations of watergrass species and bearded sprangletop have been documented. California arrowhead and small flower umbrellas edge were the first confirmed instances of herbicide resistance in rice to bensulfuron-methyl, dry racking an ALS-inhibitor, in 1993 . Eight other rice weed species have since been identified with resistance to commonly used herbicides, some with resistance to more than one mode of action .

A direct result of herbicide resistance development to more than one mode of action is the necessity of using combinations of different modes of action to combat weeds in rice systems. Permanently-flooded rice agroecosystems are limited to few available herbicides in California, largely due to ecotoxicity and strict regulatory structure . As of 2019, there are 13 registered active ingredients for water-seeded rice in California and 9 modes of action registered for use . The rise in herbicide resistance has increased the cost and difficulty of weed management, necessitating demand for novel herbicide development to delay resistance expansion and assist the management of current herbicide-resistant weed biotypes . The following studies examined the crop response to chemicals not currently in use in California water-seeded rice. CHAPTER ONE describes field studies performed in 2019 and 2021 at the Rice Experiment Station in Biggs, CA. The efficacy of pyraclonil, a protox inhibitor, was explored alone and in combination with several currently available rice herbicides against common grass, sedge, and broadleaf weeds in California rice field. Combination treatments included pyraclonil at 0.3 kg ai ha-1 applied the day of seeding, in combination with or followed by recommended rates of propanil, clomazone, benzobicyclon plus halosulfuron, thiobencarb, bispyribac-sodium, penoxsulam, or florpyrauxifen-benzyl at their respective recommended application timings. Rice phytotoxicity and yield in response to pyraclonil and these registered herbicides was evaluated. Pyraclonil applied alone had mixed effects on weed control, but all pyraclonil herbicide combination treatments controlled watergrass species, bearded sprangletop, ricefield bulrush, smallflower umbrellas edge, ducksalad, and redstem consistently better than pyraclonil applied alone.

Pyraclonil applied alone caused minor visible rice injury that varied by year but did not reduce yields. This study determined that pyraclonil was effective as a base treatment herbicide and may prove to be a new useful tool for rice growers to incorporate into their weed management programs.CHAPTER TWO details greenhouse studies undertaken in 2021-2022 to evaluate the response of several rice genotypes to five different rates of foliar-applied metribuzin, a Photosystem II inhibitor herbicide not currently used in California rice systems. Short-grain rice cultivars as a group were found to be more susceptible to crop phytotoxicity than the long-grain or medium-grain rice lines. Crop injury from metribuzin was correlated with biomass reductions and plant height reductions . The results indicate that further research is needed to establish metribuzin’s candidacy for development as a POST emergence product in rice. This exploration of novel herbicides has characterized the activity of pyraclonil in California rice, both alone and in combination with other water-seeded rice herbicides. The efficacy of the herbicide, as well as the response of the target crop, has been identified and establishes pyraclonil as an herbicide with great potential for integration into existing rice weed management programs. The differential responses of various rice cultivars to increasing doses of foliar metribuzin has described heretofore unknown rice responses and identified areas of concentration upon which future researchers may focus. Introduction of novel herbicides and continued analysis of their activity in rice allows for development of alternate methods of sustainable weed control to contend with the rise of herbicide resistance amid the common weeds of California rice agriculture.Rice is the major calorie source for a large proportion of the world’s population and is one of the most commonly grown agricultural commodities in the world . California is the second largest rice-growing state in the USA, with approximately 200,000 ha of rice, most of which is concentrated in the Sacramento Valley. The majority of rice in California is produced using a continuously flooded, i.e., water-seeded system, where rice is pre-germinated and aerially seeded into fields with a 10-to 15 cm existing flood . The flooded conditions in which California rice is grown favor flood-adapted, competitive grass weeds such as watergrass species Beauv. spp.and bearded sprangletop [Leptochloa fusca Kunth ssp. fascicularis N. Snow] . The continuously flooded system also promotes sedges such as rice field bulrush [Schoenoplectus mucronatus Palla] and small flower umbrellas edge as well as aquatic broadleaf weeds such as ducksalad [Heteranthera limosa Willd.] and redstems . Weeds are the greatest biological constraint to rice yields, and farmer inputs towards weed management are expected to increase as herbicide resistance spreads worldwide . The potential yield lost to weed infestation is species dependent, and the practice of continuous rice monoculture in California has resulted in an abundance of highly competitive weeds that negatively impact rice yields . In California rice fields, weedy grasses are the largest predictors of overall yield loss . Late watergrass [Echinochloa phyllopogon . Koss] competition has caused rice yield losses as high as 59% . Studies in Arkansas have shown rice yield losses to be 79% from competition with barnyardgrass [Echinochloa crus-galli Beauv.] and 36% from bearded sprangletop competition . In the Midsouth region, yield losses due to ducksalad infestations can reach 30% . Most California rice herbicides are limited in the spectrum of weeds controlled and the length of residual activity, cannabis curing requiring herbicide treatment plans to consist of multiple herbicides to enact weed control over a range of weeds . Effective weed control in the state relies on combinations of herbicides to enact a complete spectrum of weed control Continuous use of herbicides with the same mode of action aids in the development of herbicide resistance in rice fields .

However, due to high costs of development and registration, few additional herbicides are currently available for California rice growers, particularly herbicides that target grass weeds . As of today, there are 13 registered active ingredients for water-seeded rice in California that belong to 9 modes of action . The rises in herbicide resistance have made weed management more difficult and more costly to California rice growers . Herbicide resistance has also been a major biological issue, with confirmed resistance from various populations of watergrass species and bearded sprangletop . California arrowhead and small flower umbrellas edge were the first confirmed cases of herbicide resistance in rice to bensulfuron-methyl, an ALS-inhibitor, in 1993 . Eight other rice weed species have since been identified with resistance to commonly used herbicides, some with resistance to more than one mode of action . A direct result of herbicide resistance development to more than one mode of action is the necessity of using combinations of different modes of action to combat weeds in rice systems. Pyraclonil is a broad-spectrum herbicide with protoporphyrinogen oxidase inhibitor mode of action that is new to California. Carfentrazone, which is a currently registered protox-inhibitor, is a viable herbicide for California water-seeded rice but lacks activity on grass weeds . Pyraclonil is presently in use in Japan and has shown efficacy against sulfonylurea-resistant broadleaf biotypes of Lindernia procumbens Borbas, grasses, and sedges . Currently, there is no record of protox inhibitor resistance in California rice weeds. Protox inhibition takes place inside the chloroplasts of plant cells. As the last enzyme in the common tetrapyrrole biosynthesis pathway prior to heme and chlorophyll synthesis, protoporphyrinogen IX oxidase catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX . Pyraclonil inhibits the conversion of protogen to proto by blocking protox activity. When protox is inhibited, excess protogen accumulates in the chloroplast until protogen leaks to cytoplasm . In cytoplasm, leaked protogen is oxidized into proto and is unable to reenter the chloroplast . When proto is exposed to light and molecular oxygen in the cytoplasm, it produces toxic oxygen species, which are responsible for lipid peroxidation and membrane disruption, resulting in overall plant death . A formulation of pyraclonil has been developed by Nichino America Inc. as a preemergent granular form that is suitable for aerial application in California water-seeded rice agroecosystems.Therefore, the objectives of this research were to determine the grass, sedge, and broadleaf control of pyraclonil alone and in partnership with other commonly used herbicides in water-seeded rice systems and determine the rice response to the granular formulation of pyraclonil.Field experiments were conducted during the 2019 and 2021 growing seasons at the Rice Experiment Station in Biggs, CA, USA . Soils at the study site are characterized as Esquon-Neerdobe silty clay with a pH of 5.1, and 2.8% organic matter. The study site weed seedbank has been previously described in Brim-DeForest et al. and contains watergrass species, bearded sprangletop, rice field bulrush, small flower umbrellas edge, ducksalad, and redstem.Seeds of medium-grain rice cultivar ‘M-206’ were soaked in water for 24 hours for pregermination and then drained and aerially seeded at a rate of 168 kg ha-1 into a 10 cm flooded field. Seeding dates were June 13, 2019, and June 1, 2021. Plots were 3 m by 6 m and surrounded by small levees to prevent herbicide cross contamination to other plots . Pyraclonil was applied as a granular formulation of 1.89% pyraclonil at a rate of 0.3 kg ai ha -1 at day of seeding . Pyraclonil was also applied in combination with propanil, clomazone, benzobicyclon plus halosulfuron, thiobencarb, bispyribac-sodium, penoxsulam, and florpyrauxifen-benzyl .Treatment applications were timed on rice emergence or development stages according to manufacturer labels. Granular herbicides were evenly broadcast by hand. Foliar applied herbicides were applied with a CO2-pressurized boom sprayer with a 2 m boom equipped with six 8003XR flat-fan nozzles calibrated to deliver 187 L ha-1 at 180 kPa. For the combination treatments including propanil, the spray mixture included 2.5% v/v crop oil concentrate . For the combination treatment including bispyribac-sodium, the spray mixture included a multifunction adjuvant of 0.37 ml ha-1 . Several of the contact herbicide treatments required the 10 cm permanent flood to be lowered in order to reveal the weeds. For the treatments containing propanil, bispyribac-sodium, and florpyrauxifen-benzyl, the plots were drained to reveal 70% of the weeds prior to that herbicide application and were reflooded to 10 cm 48 hours after application, according to the manufacturer labels.Visual ratings measuring weed control were conducted for watergrass species, bearded sprangletop, rice field bulrush, small flower umbrellas edge, ducksalad, and redstem at 14 and 42 DAT . Ratings consisted of a 0 to 100 scale, where 0 = no weed control, and 100 = no weeds present, or full control. Visual crop phytotoxicity ratings were conducted at 14 and 42 DAT on a 0 to 100 scale, where 0 = no injury and 100 = plant death, as compared to the non-treated control plots. Phytotoxicity ratings consisted of stunting and chlorosis ratings. Rice grain was harvested from each plot with a small-plot combine with a swath width of 2.3 m . Rice grain yield for both years was adjusted to 14% moisture.

Currently, six resistance loci have been mapped in tomato . The Ty-1 and Ty-3 genes are allelic and were introgressed from S. chilense . The Ty-2 gene originated from S. habrochaites and encodes a nucleotide-binding site-leucine-rich repeat containing protein . The Ty-4 gene is located on the long arm on chromosome 3, and was introgressed from S. chilense . The recessive Ty-5 gene encodes the messenger RNA surveillance factor Pelota, and is believed to have been introgressed from S. peruvianum . Finally, the Ty-6 gene is located on the long arm of chromosome 10, and was introgressed from S. chilense . The Ty-1 is a partially dominant resistant gene that was introgressed from the S. chilense line LA1969 . The Ty-1 locus is composed of three allelic variants, and Ty–1-like alleles are present in both resistant and susceptible tomato lines . Expression analyses of these alleles revealed that the Ty–1 gene expression was significantly higher compared to that of the ty- 1 allele, and that these differences correlate with the resistant phenotype . The Ty-1 gene encodes an RNA-dependent RNA polymerase of the γ-class, but with an atypical DFDGD amino acid motif in the catalytic domain located at the Cterminus . The γ-RdRp is found in plants and fungi, but no clear function has been established for these proteins . Investigation of the Ty-1 function has shown that the accumulation of TYLCV in inoculated tissues and the long-distance movement of the virus were impaired in Ty-1 lines,suggesting a role in suppression of cell-to-cell movement . However, vertical farm equipment it was later demonstrated that TYLCV-specific small interfering RNA levels were higher in resistant Ty-1 plants compared with those in susceptible ty-1 plants .

Furthermore, the presence of these siRNA was correlated with cytosine methylation of the viral genome . This suggested that the Ty-1 gene is involved in transcriptional gene silencing . So far, little is known about the functional properties of the Ty-1 protein, and the nature of the signal transduction pathway involved in the defense response. Here, we generated cDNA libraries from samples of inoculated stems and newly emerged leaves of the near isogenic lines LA3473-R and LA3474-S, respectively. We used an RNASeq approach to identify differentially expressed genes during the resistant and susceptible responses to TYLCV infection. Finally, we cloned and sequenced the Ty-1 gene and determine some properties of the Ty-1 protein to provided further insight into how some of the newly identified DEGs participate in the TYLCV-mediated defense response. These results will be discussed in terms of the mechanism of resistance and associated signal transduction pathways.NILs of tomato with the Ty-1 gene or ty-1 gene were used for these experiments . Seeds of these NILs were obtained from the Tomato Genetics Resource Center, UC Davis. Tomato plants of both lines were grown in a greenhouse, allowed to be self-pollinated and fruits were harvested. Seeds were extracted and treated with 2.7% sodium hypochlorite for 30 min and rinsed with MilliQ water. Treated seeds were planted into sunshine mix 1 potting mix in a controlled environment chamber .To confirm the resistant and susceptible phenotypes of plants produced from increased seeds, NILs LA3473-R and LA3474-S, respectively, seedlings at the three- to five-leaf-stage were agroinoculated with cell suspensions of a strain of Agrobacterium tumefaciens containing a binary plasmid with the multimeric infectious clone of TYLCV from California by needle puncture inoculation of the stem just beneath the shoot apex .

The positive control in these experiments were seedlings of the susceptible tomato plants cv. Glamour agroinoculated with TYLCV, whereas the negative control was seedlings agroinoculated with the empty vector . Inoculated plants were maintained in a controlled environment chamber, and symptom development was assessed visually and recorded at 7, 14 and 21 d post infection based on the severity scores described in Lapidot et al. . Absolute viral DNA accumulation was quantified at 7, 14 and 21 dpi by quantitative polymerase chain reaction tests according to the protocol described by Mason et al. . Total genomic DNA was extracted from newly emerged leaves according to the method of Dellaporta et al. . A virus-specific primer pair for qPCR detection was designed to direct the amplification of an ~150 base pair fragment from the capsid protein gene of TYLCV . The specificity of this primer pair was predicted based on BLAST search , and confirmed experimentally by conventional PCR with DNA extracts of tomato plants agroinoculated with TYLCV and non-inoculated plants. The PCR-amplified TYLCV fragment was cloned into pCR-Blunt II-TOPO to generate a standard for qPCR assays. Recombinant plasmids containing the cloned PCR-amplified TYLCV fragment were quantified with a NanoDrop1000 spectrophotometer , and plasmid copy number was adjusted to 107 copies/µl with the Avogadro’s constant . Standard curves for qPCR were prepared with tenfold serial dilutions ranging from 101 to 106 copies of plasmid DNA. These standard curves were used to estimate the viral Cn for each sample. The qPCR was conducted on a QuantStudio™ 6 Flex Real-Time PCR System with 100 ng of total genomic DNA in a 20-μl reaction mix with the SsoFast EvaGreen Supermix kit . To detect the presence of the Ty-1 or ty-1 genes in the NILs LA3473-R and LA3474-S, respectively, restriction fragment length polymorphism analyses of the amplified fragments with a marker to Ty-1/ty-1 alleles were performed.

Here, leaf samples were collected from non-inoculated leaves of LA3473-R and LA3474-S plants and total genomic DNA was extracted as previously described. PCR tests were performed with the C2_At5g61510- F/C2_At5g61510-R primer pair , which direct the amplification of an ~1.0 kb fragment. PCR-amplified fragments were purified with the QIAquick gel extraction kit and digested with the five-base-cutting enzyme HinfI and the reaction analyzed by agarose gel electrophoresis.For the RNA-sequencing experiments, a cell suspension of an A. tumefaciens strain containing the multimeric infectious clone of TYLCV-[US:CA:06] or the empty vector was used for agroinoculation of seedlings of the NILs LA3473-R and LA3474-S as previously described. A total of three plants per treatment were inoculated per experiment, and the experiment was repeated three times. Tissue samples of the resistant and susceptible lines were individually collected and pooled together in each treatment from inoculated stems and, from other inoculated plants, from newly emerged leaves for a total of eight-time points . Samples were frozen immediately in liquid nitrogen. High-throughput RNA-seq library preparation was performed according to the method described in Kumar et al. . Briefly, double-stranded complementary DNA was prepared with random hexamer priming, and the resulting cDNA was fragmented, end-repaired and A-tailed. DNA barcodes for multiplexing Illumina DNA-Seq libraries were added to the cDNA fragments during adapter ligation, and the adapter-ligated cDNA libraries were enriched with 13 cycles of PCR amplification followed by size selection of ~200-500 bp fragments. A total of 96 cDNA libraries we generated, which included the eight-time points each for the resistant and susceptible lines and for the three independent experiments. Finally, barcoded libraries for each independent experiment were pooled together and sequenced on two lanes on Illumina HiSeq 2000 platform at the UC Davis Genome Center.Raw sequence data were filtered/trimmed for low-quality reads and technical sequences with Trimmomatic . Trimmed RNA-Seq reads were aligned to the high quality, non-redundant database generated from the tomato genome by the International Tomato Annotation Group with Hisat2 . The latest ITAG database was obtained from Sol Genomics Network . Feature Count was used to count the number of reads mapped to each gene . DEGs were identified with Cuffdiff, a method that estimates the relative transcript abundance . Gene expression levels were normalized with fragments per kilobase of exon per million mapped reads values, and the false discovery rate was used to determine the differentially expressed p-value threshold. In addition, an independent DEGs analysis was performed with DEseq and EdgeR methods , commercial indoor growing systems and data were visualized with Galaxy . In the present study, genes were considered to be differentially expressed only when their absolute value of log2 fold change was >1.5 and p-value was <0.05. To identify the potential function of DEGs involved in the Ty-1 resistance response to TYLCV infection, the functional classes of DEGs were identified with gene ontology enrichment analyses with PANTHER within the Gene Ontology project .

In order to investigate the transcriptional changes in tomato stems and leaves during TYLCV infection, we performed RNA-Seq experiments with inoculated stems and systemically infected leaves in the NILs LA3473-R and LA3474-S. Overall, ~1140 million read pairs were obtained for 96 libraries, with an average of ~12 million read pairs per library . The reads from these libraries were trimmed and then aligned to the tomato genome in the ITAG database. Time course comparisons of DEGs in the NILs revealed that the largest transcriptional changes in LA3473-R occurred at early stages of infection in inoculated stem tissues. A total of 797 and 660 genes were differentially expressed at 12 and 24 hpi, respectively, and dropped to 42 DEGs at 48 hpi . In the systemically infected leaves, there were substantially fewer DEGs, with 0, 44 and 10 genes differentially expresses at 7, 10 and 14 dpi, respectively . Most of the transcriptional changes observed in the LA3474-S plants occurred from 12 to 48 hpi in inoculated stem tissues and at 7 dpi systemically infected leaves. A total of 264 , 540 and 355 genes were differentially expressed at 12, 24 and 48 hpi, respectively . In systemic infected leaves, 356 genes were differentially expressed at 7 dpi, whereas 99 and 2 genes were differentially expressed at 10 and 14 dpi, respectively . Interestingly, a similar number of DEGs were induced in both resistant and susceptible responses, with 1553 and 1616 DEG in the resistant and susceptible lines, respectively. However, the percentage of upregulated genes in the susceptible LA3474-S line was higher, whereas more genes were downregulated in the resistant LA3473-R line . In order to gain insight into the mechanism or signal transduction pathway involved in resistance to TYLCV infection, we analyzed the transcriptome of resistant vs susceptible tomato plants at the eight time points during the TYLCV infection response following agroinoculation. DEGs in the resistant vs susceptible response to TYLCV infection were identified with three different statistical algorithms, and the analyses revealed 10751 genes differentially expressed by Cuffdiff, 5051 by Deseq, and 1462 by EdgeR, respectively . Moreover, a total of 679 genes and 58 lncRNAs were identified as differentially expressed in LA3473-R during TYLCV infection by all three methods . A large number of genes were differentially expressed at 24 hpi , but the largest transcriptional changes occurred at 7 dpi in systemically infected leaves, a time when TYLCV is accumulating in emerging leaves . In contrast, a total of 50 and 17 genes were differentially expressed at 10 and 14 dpi, respectively . To further study the TYLCV-mediated defense and susceptible responses in tomato during early and late infections, DEGs in protein families involved in stress responses in plants were selected for further analyses . Of the DEG identified by all three methods, 12 genes were associated with protein families involved in tolerance to abiotic stress response and plant-pathogen interactions, such as WRKY transcription factors, nucleotide binding site-leucine rich repeats proteins, receptor-like protein kinases , leucine-rich repeat receptor kinases and chloroplast proteins. In comparisons between resistant and susceptible responses to TYLCV infection, the mannose-1- phosphate guanyltransferase was upregulated 3.1-fold at 48 hpi, the heavy metal transport/detoxification protein upregulated 5.4-fold at 10 dpi and the WRKY transcription 46 upregulated 5.8-fold at 14 dpi . Comparisons between susceptible and the control revealed that Ycf68 chromosomal protein was upregulated 4.1-fold at 14 dpi; whereas the WRKY transcription factors 45 , 46 and 55 , two NBS-LRR genes , two LRR-RKs genes and one RLK gene were downregulated 1.6 to 6.7-foldat 14 dpi . Additionally, two lncRNAs were downregulated 2.5 to 4.0- folds at 14 dpi in the susceptible response to TYLCV infection . For a better understanding of the transcriptional responses to TYLCV infection in LA3473- R plants and to reveal putative functions of DEGs, GO enrichment analyses were performed with PANTHER and the tomato reference genome sequence. In these analyses, >80% of the DEGs, respectively, were annotated. Within the biological process class, the majority of DEGs belong to the category of cellular processes, metabolic processes and biological regulation .

Barb goatgrass has a higher rating due to its more limited distribution and, therefore, greater opportunity for containment than medusahead. Both of these species commonly occur with other exotic annual grasses . As annual grasses, barb goatgrass and medusahead produce seed at the end of the growing season for subsequent germination in the fall. Inhibiting this seed production is crucial to suppressing or controlling both weeds. Unfortunately, barb goatgrass and medusahead are prolific seed producers, making management to reduce their abundance possible but eradication difficult and unlikely. Barb goatgrass compounds this problem because in addition to producing seed for the following season, it also produces a dormant set of seed that will not germinate until the second growing season after seed production, making 2 years of control necessary to reduce infestations. For these reasons, grazing has not yet proven to be a fully successful strategy for depleting stands of barb goatgrass. Mowing prior to seed maturity may be a better mechanism to control barb goatgrass. Fortunately, medusahead populations can be drastically reduced with a single event that suppresses seed production, lending it to both grazing and mowing treatment methods. Both of these methods require appropriate timing so that the mechanism matches the biological susceptibility of the plant for successful management. The timing of growth events, including major biological changes in plant growth and development through the season, is referred to as phenology. As applied to managing invasive annual grasses, the important events to consider include germination, vertical grow rack vegetative growth phases, and a series of reproductive growth phases that end in the production of viable seed.

As barb goatgrass and medusahead develop and change visually, other changes are less apparent, though important to consider, related to the nutritional quality for livestock grazing, ability of the individual plant to recover from defoliation , and the ability of the seed to continue to develop and later germinate after it is detached from the plant. While these changes are roughly predictable, variation from year to year, across regions, and even within pastures occurs due tovariation in weather, climate, landscape, presence of grazing, soil differences, and genetics. This means that predicting the timing for control can be somewhat imprecise, making the use of phenology observations imperative to optimize control treatments. University of California research conducted over multiple years describes how observations of plant growth stage can help to optimally time grazing and mowing treatments.The stages in table 1 provide a framework to describe how both of these species progress and change in their physical characteristics over the course of the growing season. The 12 stages are broken into vegetative stages V1 to V3, reproductive stages R4 to R9, a mature stage M10, a summer dry stage D11, and dead residual in the subsequent growing season at stage L12. Defining these stages helps to optimize the timing of grazing and mowing control treatments by defining the windows of susceptibility.During the first two vegetative stages the plants are very small and inconspicuous. They are often unnoticed when viewed on a landscape level and provide very little forage for grazing. When plants are grazed during these stages they will readily recover with new flowering stems; thus, little to no control is achieved. In order to impact plants heavily enough to prevent further reproductive stages from progressing, targeted grazing of the infested areas should begin during the late vegetative stage 3 or boot stage . Plants are affected by grazing from stage V3 until the reproductive stage 4 , when awns fully emerge, and neither grass species is palatable to grazing animals thereafter .

Crude protein of the vegetative grasses drops significantly from approximately 10 to 11 percent at the V3 stage to 7 to 8 percent at the R4 stage in the R8 stage , the seed is able to continue to develop and become viable if spikes detach from the plant. This can be visually approximated as the point that plant leaves and stem are turning brown, but the seeds and seed head are still green. The plant is fully mature once shades of red, brown, and green are apparent on the seed head . The window for treatment is much longer for mowing than grazing since palatability is not an issue.To better understand the timing of phenology of these two grasses in California, UC researchers measured the proportion of barb goatgrass and medusahead at various times through the growing season. Included were 18 locations in 11 counties from Shasta to Monterey at elevations from 80 to 990 feet above sea level during the growing season from 2006 to 2010. Whereas medusahead was present at all sites, barb goatgrass was present in samples taken from Tehama County to Yolo County. The limited range of sample locations for barb goatgrass reflected the more restricted range of this species compared with medusahead .Medusahead was sampled over a larger geographic range than barb goatgrass, and we therefore expected and observed more variability in the timing of phenology of medusahead relative to barb goatgrass. Medusahead became susceptible to grazing treatments from early or mid-April to early or mid-May, depending on the site and year. The transition from grazing susceptibility to only mowing susceptibility reliably occurred in early May and was usually complete in mid-May. Medusahead tends to enter the stage of maturity that is too late for effective mowing treatment between the last week of May and the first week of June. Some cooler locations in the Central Coastal valleys can be about a week later. In addition, there seem to be some locations with warmer winter temperatures and less moisture overall where this transition to maturity occurs much earlier, such as early May. Select sites in Glenn, San Joaquin, and Shasta Counties were consistently 2 to 4 weeks earlier than other sites. This may be due to a number of factors, including locally warmer spring temperatures. Barb goatgrass became susceptible to grazing treatments from mid-March to mid-April; however, grazing is not recommended at this time due to the existence of a dormant seed bank and less complete control compared with mowing, making this approach challenging. In 2007, barb goatgrass began developing notably later; 2007 was the only year when barb goatgrass collections made in late March and early April were not yet susceptible to grazing. During other years, the first collections made in early April were already susceptible to livestock grazing. The transition from susceptibility of grazing to only mowing reliably occurred in early May and was complete by mid-May. Generally, at all sites, barb goatgrass entered the stage of maturity that is too late for effective mowing treatment around June 1, with a variance of about 5 days. One year seemed to have more variability, with several sites maturing earlier and others sites later than other years. At the pasture scale, some individuals and patches of medusahead will mature more quickly than others due to variation in soils, slope, and aspect. In more-uniform pastures, most individual and patches of plants may be well synchronized, while in morevariable pastures there may be a greater range of stages at any given time in the spring. This can lead to patchy areas, with some grasses that are too early for treatment success and others that are too late for treatment success. Variable effects within pastures are amplified when they are not grazed. In these cases, managers will need to keep in mind that grazing should be considered a long-term approach that will not be fully successful every year .While the start of susceptibility varies substantially, commercial vertical hydroponic systems ranging from late March to early May, the duration of susceptibility for both barb goatgrass and medusahead tends to vary across its range and from year to year, but in somewhat predictable patterns.

The period of susceptibility of medusahead to targeted grazing is 2 to 3 weeks, while susceptibility to mowing of both species is about 5 weeks. The length of these periods varies by year, location, precipitation, and soils . Cooler spring seasons tend to lengthen the period of susceptibility relative to drier, warmer sites and years. Livestock stocked at moderate or light stocking rates will avoid medusahead and goatgrass particularly as plants transition fromV3 to R4. To overcome this aversion, heavy stocking rates that far exceed rates considered normal for annual rangelands are required to encourage enough consumption to impact seed production. For grazing to be successful, plants must be impacted enough to prevent the onset of seed set. This is possible because these two species tend to mature so late in the season that soil moisture is not present in adequate amounts for plant recovery , although this is not the case every year. The rate is determined by the forage biomass present, but it often exceeds 1 to 2 animal units per acre during the critical V3 and early R4 stages. This high rate is necessary because of the short window and amount of forage that needs to be removed. The objective is to consume forage biomass to, or even slightly below, 500 pounds per acre . This makes treatment site-specific within most ranches because of the difficulty of fencing, watering, and providing enough cattle to impact an area, as well as the increased management time. The short period of susceptibility for successfully implementing a grazing treatment can be overcome with mowing treatments. When implemented correctly, mowing is more likely to be a successful treatment than grazing because the timing will almost, but not always, prevent subsequent seed production. However, mowing faces other challenges, including the inability to cover steep terrain, rocks that damage the mower, and fire potential, which are very common scenarios on California rangelands. It is important to ensure that these treatments are applied as effectively as possible to get the most benefit out of them. By understanding the phenology of barb goatgrass and medusahead, these treatments can be better planned so that the timing and intensity of treatment corresponds to when they will most effectively reduce the abundance of these noxious annual grasses in the future.Grazing and mowing are two of many successfully tested weed control methods that can be used in combination with other methods for medusahead and barb goatgrass control. Using multiple methods is highly suggested for barb goatgrass areas. Ultimately, treatment decisions are site and management specific. Specifics on the effectiveness and implementation of the various other treatment options, such as burning and herbicide application, are available from sources such as Davy et al. 2008, DiTomaso and Kyser 2013, and Kyser et al. 2014. In addition, articles by Aigner and Woerly and James et al. have also assessed the effectiveness of different treatment tools on barb goatgrass and medusahead, respectively.Crops that grow vigorously can often outcompete weeds. Weeds grow best where competition is sparse; for instance, between rows or in gaps in a crop stand. Crops that are well adapted to their planted areas are often better competitors since they will tend to occupy a site rapidly. If you increase the density of the crop by decreasing the in-row spacing or by reducing the space between rows you will improve the crop’s competitiveness. A close-planted crop will close the canopy more rapidly, reducing the weeds’ ability to compete. Some crops compete effectively with weeds if given an early competitive advantage, while others never establish a competitive canopy. The use of transplants give the crop an advantage over the weeds because transplants enter the field larger and more developed that the weeds. With help from subsequent cultivation or hand weeding operations, a transplanted crop can develop a full canopy and crowd out weeds.Practices that reduce the production of weed seed also reduce weed pressure and can help keep weeding costs down over time. In an ideal situation, no weed would be allowed to go to seed. Any that do go to seed can aggravate weed problems for many years to come. As an example, common purslane seed has been shown to remain viable for over 20 years in the soil, and black mustard seed survives for over 40 years. The longevity of weed seed, together with the large numbers of seed produced by individual plants , can lead to the long-term build-up of enormous seed banks in the soil. If you make it a policy to remove weeds prior to seed production, you can reduce weed pressure in subsequent seasons.

CE Farm Advisors serve as a valuable link between land grant universities and growers, remaining relevant by developing programs that address stakeholder problems, issues, and concerns . However, land-grant universities continue to face funding uncertainties, which has affected CE programming and staffing. CE currently operates with approximately half the staff positions it did in 1990 . With less funding from its traditional sources, such as the USDA, state government, and county governments, less Farm Advisors are hired, and therefore each advisor must cover more territory and a broader range of crops than in the past. In addition, self generated funds have increased by 63% over the past five years, placing a greater burden on UC staff to find their own funding to carry out research and outreach . More recently, the COVID-19 crisis has forced the reevaluation of spending priorities in the state budget. Making the case for CE moving forward will require a robust coalition of agricultural interests and the demonstration of results and impact . CE recognizes the importance of using evaluation data to demonstrate program value and set priorities for future programming based on input from a range of stakeholders . Needs assessment generally refers to methods, efforts, and activities involved in or used for identifying needs, providing a method to learn what has already been done and what gaps in understanding remain . In many cases, needs assessments are surveys used to identify stakeholders’ challenges and/or concerns, and help CE understand how they can respond with programs and services . Needs assessments are important because what one person identifies as a need might be irrelevant to another person and needs are subject to change over time . Therefore, vertical farming systems for sale research indicates that targeted strategies for developing CE programs are more likely to be effective than approaches broadly directed toward the general population .

A recently conducted needs assessment survey of 150 dairy producers in California demonstrated the importance of regionally targeted strategies, with the top 5 CE priority topics identified by respondents differing based on region . The northern San Joaquin Valley and greater Southern California regions were found to have greater similarities in priorities than the Northern California region, perhaps explained by differences in average herd size, type of production system, and climate . Because California agriculture is diverse and each cropping system will respond to change differently, adaptation research and effective stakeholder engagement should be regionally focused . In the rapidly changing context of California agriculture, identifying the relative importance of different topics is critical for prioritizing extension activities and making the best use of limited resources, while incorporating feedback from clientele will help to increase the effectiveness and impact of extension programs. Many forces beyond the farm level shape what is or is not possible on the farm, and there is a pressing need to understand how these forces intersect . New legislation, including the Sustainable Groundwater Management Act , which is the state’s first law regulating groundwater use in its history; new reporting requirements for the Irrigated Lands Regulatory Program; and new or impending agrochemical bans will shape the future of farming in California. Currently, it is unclear which issues are most pressing regarding grower management decisions and information needs. Equally important, the level of satisfaction with current extension activities is not well understood. Therefore, documenting the concerns and needs of growers, consultants, and allied industry will highlight the most important topics for research and extension to focus on, and guide policymakers and administrators on where resources and funding should be allocated.

Increases in California’s agricultural productivity have long been sustained by expanding water supplies, increasing use of fossil fuel energy, and new technology – all of which are now under pressure because of scarcity, cost, and public opposition . Now, more than ever, UCCE would benefit from a statewide understanding of common goals, challenges, and preferences for research and extension across different regions and crops to determine how innovative collaborations and partnerships might be established to meet clientele needs. While individual CE Advisors have conducted needs assessments for their clientele, to our knowledge there have been no prior efforts to comprehensively gather statewide information. Therefore, the primary objective of this study was to set research and extension priorities for agronomic crop production in California based on feedback and input from growers, their consultants, and allied industry professionals. The specific objective was to conduct a survey to i) identify top concerns and management challenges, ii) understand the motivations for growing agronomic crops and priorities considered in management decisions, and iii) prioritize information needs that can be addressed through research and extension efforts in the future.The needs assessment designed for this project was an online survey developed by members of the UCCE Agronomy Program Team and administered using Qualtrics survey software . The first step in developing questions was to collect and summarize previous needs assessments shared by individual members of the Agronomy Program Team for their specific crop or region. Based on overarching themes from past needs assessments and bearing in mind the objectives of this collaborative effort, questions were drafted and reviewed by a team of CE advisors and UC Davis faculty working in agronomic crop production.

Prior to launching the survey, it was piloted by 10 growers and other industry professionals. In depth phone conversations with pilot participants allowed for robust feedback that was incorporated into a final version of the survey. The final survey included a total of 21 questions, covering the areas of management challenges, concerns for the agronomic crop industry, motivation, importance of extension topics and level of satisfaction with UCCE. We also asked respondents who they communicate with about crop production practices and how they prefer to receive information. The survey was reviewed by the Institutional Review Board and approved as “exempt”. The needs assessment survey was a cross-sectional census survey attempting to collect as many responses as possible from anyone currently involved in agronomic crop production in California. We tried to ensure that we were getting accurate representation of California agronomic crops clientele by including a screening question. The survey link took respondents to a page asking if they grow, consult on, or work in allied industry of agronomic crops in California. If they responded yes, they were taken to the survey, and if they responded no, they were not able to continue. The first question on the survey following the screening question asked respondents to identify their primary vocation between “grower”, “consultant”, “allied industry”, or “other”. Depending on their response, we were able to direct management related questions specifically to growers, while still gaining insight from consultants and allied industry on broader topics. To identify concerns and challenges faced by those working in agronomic crop production, respondents were asked to rank their level of concern from a list of 15 topics that influence crop production in California. Next, respondents who identified as growers or consultants were asked to select their highest priority management challenges from a list of 8 common management challenges identified by our internal team of CE Advisors and CE Specialists. To understand the motivations for growing agronomic crops and priorities considered in management decisions, vertical farming equipment we asked respondents who identified as growers to rank how often certain factors affect their management decisions for field crop production . We also asked growers to select their primary reasons for growing field crops from a list of 9 commonly cited reasons, as determined by our internal team. To prioritize information needs that can be addressed through research and extension, we used Importance-Performance Analysis . This method is a quantitative approach for measuring how people feel about certain issues . The analysis generates a picture of how important specific topics are to clientele in comparison with how satisfying they are – or in this case, how satisfied clientele are with UCCE’s delivery of information on these topics . Typically, the visual output of this method is an IPA matrix created by plotting importance and satisfaction on a two-dimensional graph having four quadrants . The boundaries of the quadrants are based on the means of the two measures and each quadrant is interpreted as having implications for prioritization of information. The idea is that focus should be placed on topics found in the “high priority” quadrant, while resources can be allocated away from the “lower priority” quadrants . Focus should remain on topics that fall into the high importance and high satisfaction quadrant; however clientele is seemingly satisfied with UCCE’s work in disseminating information on these topics. Importance and satisfaction were each measured through a Likert-type scale, where participants were given a list of 19 topics commonly addressed by CE and asked to select if these topics were of “high priority”, “medium priority”, “low priority” to them. They also had the option to select “no opinion”, which received a score of zero. With the same list of topics, respondents were asked to select “high satisfaction”, “medium satisfaction”, “low satisfaction”, or “no opinion” based on how satisfied they were with UCCE’s delivery of information on these topics. High priority and satisfaction were given a score of 3, medium priority and satisfaction were given a score of 2, and low priority and satisfaction were given a score of 1. Scores for priority and satisfaction were averaged and plotted to create an IPA matrix. 2.2 Survey dissemination. The target audience of our online survey was all California agronomic crop growers, their consultants, and allied industry. Because no comprehensive list of such individuals exists, contact lists from individual agronomic program team members were compiled and duplicates were removed. In July 2020, stakeholders were sent an email invitation to take the online survey. The survey was open from July 23, 2020 until September 1, 2020 with three reminders sent to those on the centralized contact list, as suggested by the Dillman method to maximize response rate . The first 100 participants to complete the survey were also offered an incentive of a $10 gift certificate. As stated on the survey, all information was kept anonymous, and respondents were informed that the survey would be used to better guide UCCE research and extension efforts by highlighting the most important issues facing agronomic crop production in California and helping set priorities for future programming. While the centralized contact list contained statewide representation, the team decided that an aim of this needs assessment was also to reach people who UCCE might not already be serving. Therefore, to avoid excluding any potential respondents, the team developed a list of influential groups or organizations external to UCCE that could distribute the survey. This list included commodity boards, crop associations, Farm Bureaus, County Agricultural Commissioners, Water Quality Coalitions, and input distributors. These partner stakeholders were contacted and asked if they would be willing to share the survey with their clientele. If they agreed to share the survey, an anonymous link to the survey was sent to them for dissemination. The survey software was able to track which responses came from the original centralized contact list and which responses came from the anonymous link. However, with the anonymous link, the response rate could not be measured. Since our goal was to gather responses from a wide range of participants, we accepted this limitation in our methodology.Concerns varied by crop and region . For instance, the top categories for anyone identifying as a rice grower and rice consultant were “very concerned” about included regulations on chemical use , input costs , and regulations on water quality . The top categories that those growing or consulting on alfalfa were “very concerned” about included regulations on water use , water costs , and water quality . Respondents growing or consulting on wheat were “very concerned” about the commodity price of their crop , consumer demand , and availability of quality labor . Finally, corn growers and consultants were “very concerned” about regulations on water quality , availability of quality labor , and regulations on chemical use . All regions ranked “regulations on water use ” and “water costs” as the top concerns relative to other concerns. Based on mean responses, the greatest concern for regulations on water use was seen in the Southern San Joaquin Valley , the Intermountain region , and the Northern San Joaquin Valley , while greatest concern for water cost was observed in the same three regions: SSJV , Intermountain , and NSJV .

Therefore, substantial effort should be made to clean and disinfect all equipment used in a field with broomrape infestation. Equipment sanitation should begin with removal of plant and soil debris manually, as debris not only can contain seeds but can also reduce the effectiveness of disinfectants. Once most of the debris has been removed, chemical disinfection agents can be used on the equipment to kill any remaining seed and pathogens. According to Hershenhorn et al. , several quaternary ammonium products are available for disinfestation of farm equipment, such as didecyl dimethyl ammonium chloride, alkyl dimethyl benzyl ammonium chloride, dioctyl dimethyl ammonium chloride, octyl decyl dimethyl ammonium chloride, and ammonium bromide. Commercial products may contain one or a combination of these chemistries. For example, New Development Process is an example of a commercially available product, containing multiple quaternary ammonia, that can be used for disinfection of farm equipment, clothing and shoes against broomrape seeds. Soil fumigation using methyl bromide is one of the most effective tools to kill broomrape seeds, but due to its environmental toxicity the chemical has been banned and is no longer generally available. Even if methyl bromide were allowed under quarantine restrictions, the cost of treatment would be prohibitive. Other soil fumigants, such as chloropicrin, dazomet, metamsodium, metam-potassium and 1,3-dichloroproprene, may also provide different control levels of broomrape seeds . However, at this time, industrial drying rack few of these fumigants have been evaluated experimentally under California conditions.Herbicidal control of broomrape can be undertaken using pre-plant and post-plant herbicide applications and/or chemigation .

This is an area of on-going research in California and builds on programs developed in other regions. In processing tomato in Israel, for example, herbicides have been used to effectively and economically manage broomrapes in highly infested fields where eradication is no longer feasible . Growers found that pre-plant herbicide applications followed by complimentary post-transplant applications of acetolactate synthaseinhibiting herbicides such as sulfosulfuron provided control of Egyptian broomrape at both preand post-attached stages in tomato . The use of rimsulfuron as a pre-plant incorporated herbicide with a complimentary post-emergence application also provided good suppression of broomrape without causing significant damage to tomato plants . Some herbicide application protocols are based on the level of severity of broomrape infestation in tomato. For example, researchers in Israel have developed a thermal time-based decision support system named PICKIT that takes into account infestation levels and growing degree days since planting to guide the timing and rate of multiple herbicide applications for control of Egyptian broomrape; the system has been applied on a broad commercial scale . For severe infestations , growers apply sulfosulfuron three times post-planting at 200, 400 and 600 GDD, followed by overhead irrigation complemented by two foliar-applied doses of imazapic at a later growth stage. The DSS suggests that a medium level of broomrape infestation requires a single pre-plant incorporation of sulfosulfuron before planting tomato, followed by drip chemigation of imazapic at 400, 500, 600, 700 and 800 GDD, with two additional foliar imazapic applications at a later growth stage.

A similar DSS system is being tested on branched broomrape infestations in processing tomatoes in Chile and California with promising initial results .In California, only the rimsulfuron component of the PICKIT system is currently registered for use in processing tomato. Crop safety and registration support research is ongoing in California in an effort to register additional herbicides and application techniques in the event that branched and/or Egyptian broomrape problems expand in scale . A preliminary result from this research suggests that no visual injury and yield loss are associated with the use of the PICKIT system in local tomato fields .Cultural practice, such as rotating tomato plants with false hosts or non-host crops, could help with seedbank depletion, provided branched broomrape seed is not re-introduced to the field from outside. A trap crop is a species with root exudates that induce broomrape seed germination but the crop does not allow attachment or support broomrape seedling growth and survival. Potential trap crops for branched broomrape that can be used in a rotation are alfalfa , cowpea , green pea and flax . Tomato and other host crops should be excluded from the rotation for several years to encourage further depletion of seedbank with no chance of seed production. Since broomrape seed is very sensitive to flooding, incorporation of flooded rice into the crop rotation may also accelerate the depletion of soil seedbank . Soil fertility management can contribute substantially to the management of branched broomrape. Direct contact with fertilizer, such as urea and ammonium, may be toxic to broomrape, inhibiting seed germination and seedling growth . The negative effect of ammonium on broomrape is due to the plant’s limited ability to detoxify the ammonium compound using glutamine synthetase . Application of adequate fertilizer will not only ensure unhindered growth of the tomato plant; it will also minimize the release of the plant’s strigolactone, a root exudate that stimulates broomrape germination .

For example, it has been demonstrated that phosphate fertilization negatively impacts branched broomrape seed germination in tomato fields because of reductions in strigolactone exudation . Soil solarization has been shown to be an effective alternative to fumigation in reducing broomrape seed viability in areas with sufficiently hot climate. Solarization can significantly increase top soil temperatures up to 6 inches [15 cm] in depth when moist soil is covered with transparent polyethylene sheets for a period of one to two months. Dahlquist et al. reported 100% seed mortality of several weed species with solarization that raised soil temperature above 45°C for at least 96 cumulative hours. Mauro et al. found that soil solarization for two consecutive summers provided 99% mortality of viable seeds of branched broomrape in the seedbank without any negative impact on tomato yield. A recent field study conducted at UC Davis confirmed that soil solarization plus organic amends of either tomato pomace or plowed-down tomato plants can be used to substantially reduce the weed seedbank in general in tomato fields , although broomrape was not present at this site. One challenge in using this approach is the need to take tomato fields out of production for several months during the summer growing season in California. Additionally, it is not currently known if the elevated temperatures from solarization would penetrate deeply enough into the soil to provide adequate control of broomrape seed throughout the tomato root zone in an open-field production system. Other thermal methods of soil disinfestation, such as soil steaming, are another alternative to chemical fumigation. Soil steaming has been shown to be effective in controlling seeds of several weeds and other soil pest in California strawberry production . High soil temperatures of 158°F for 30 minutes can be regularly achieved in the field to a depth of 0 to 10 inches . This treatment seems to be sufficient to kill seeds of many weeds . Although the effect of this technique on broomrape seed mortality has not been studied, the small seed size of broomrape plants and their lack of protective tissues suggest that broomrape could be vulnerable to steam heating. However, like solarization, it is not known whether the depth of control from soil steaming would be sufficient as part of an eradication strategy for a quarantine pest like branched broomrape.Physical weed removal, such as hand weeding, particularly for a small infestation, can be part of an integrated approach to broomrape control. California is a state where hand removal of broomrape may be an option given the limited infestation level and widespread use of farm labor. The efficacy of hand weeding is highly dependent on thorough scouting and detection, drying rack for cannabis which can be very difficult given the plant’s small stature and the short period between its emergence and seed set . Deep inversion plowing would bury broomrape seeds to a depth below the soil layer where attachment to tomato root can occur . However, the dormancy and durability of broomrape seed in the soil seedbank would increase the risk of later broomrape re-occurrences. Physical removal and deep burial could be part of a management strategy if broomrape became too widespread for quarantine and eradication efforts to be feasible; however, because broomrape is an A-listed pest , physical removal and deep burial are not likely to provide a sufficient level of control alone.Biological control involves the use of biological agents or processes to damage seed, kill weedy plant or interfere with parasite-host relationships.

An insect herbivore, Phytomyza orobanchia, is known to be specific for broomrapes and feeds on broomrape ovules and seeds, thereby reducing broomrape seed production . Pathogens such as Fusarium sp. can be incorporated into the soil to control broomrape through an induced cytoplasm metabolism and endosperm cell wall degradation that breaks seed dormancy, thereby depleting the broomrape seedbank . Pathogen-based herbicides have been reportedly used to control young seedlings of parasitic weeds , and these bioherbicides can provide complete control of all emerged broomrapes if formulated with multiple pathogens . However, to date, no research on the applicability of these approaches in California cropping systems and broomrape infestation levels has been conducted, and they are not currently available for use. Cultivation of resistant tomato varieties would also be an effective approach to prevent parasitic effects of broomrape. Resistance to branched broomrape might be achieved by incorporating traits that prevent haustorium attachment and penetration, or tubercle formation; this approach has been demonstrated in broomrape-resistant sunflower . A group of scientists at UC Davis are currently screening a wide range of tomato varieties to determine their resistance to branched broomrape; results from this study could help to determine if enough genetic variability exists in tomato to use conventional breeding approaches to breed for broomrape resistance. Although screening is effective in small plots and is promising in the longer term, at present there are no effective commercial biological measures for broomrape control in tomato.The re-emergence and spread of branched broomrape are of great concern in tomato and other susceptible crop production systems in California. At this point in time, the problem is still relatively small. Current efforts are focused on quarantine and eradication using a regulatory approach and soil fumigation. These approaches depend on the reporting of new infestations and generally result in total crop loss to the grower and extremely high treatment costs. Therefore, success will depend on significant funding from state or industry sources to offset grower costs in order to ensure grower participation and reporting. In the event that broomrape problems in California expand beyond what can realistically be managed using quarantine approaches, management and mitigation approaches will be needed just like with other widespread weeds. Other countries have successfully demonstrated that an integrated approach on a long-term basis, involving outreach to growers, field scouting and detection of new infestations, mapping of contaminated areas and fields, equipment sanitation, manipulation of cultural practices and carefully timed herbicide treatments, among other treatments, can effectively reduce yield losses caused by branched broomrape. Significant research efforts are being made by a group of university, industry and regulatory scientists to develop detection and management approaches for branched broomrape and to modify existing approaches from other regions for adaptation in California.Across California, annual rangelands cover approximately 16 million acres and are among the most species-rich ecosystems in the state, supporting thousands of plant and animal species . California’s modern-day rangelands are largely dominated by nonnative annuals, which some believe replaced previously diverse native forb and grass communities . These naturalized annuals now provide a majority of the state’s livestock forage base. Currently, several noxious weed species are driving another transformation of California’s rangelands and pose a continued and growing threat to rangeland ecosystem functions and services . The spread of invasive weeds changes plant community composition and can lead to shifts in soil moisture and nutrient availability as well as the suppression of both native plants and other desirable and more palatable nonnatives, thereby reducing herbaceous diversity, wildlife habitat, forage quality and agricultural productivity . Across California’s annual rangelands, noxious weeds have been estimated to reduce livestock carrying capacity by as much as 50% to 80% .Two of the most prominent invasive species of concern are medusahead and yellow starthistle .

Tobacco growing requires heavy applications of fertilizers, and pesticides like methyl dibromide and ethyl-bromide that harm workers and pollute drinking water. In Pakistan, “Up to 48 different chemicals are used between the processes of sowing the seed to its implantation at the sapling stage. Inadequately trained and lacking in proper gear the farmers continue to expose themselves to the dangers of chemical and pesticide exposure year after year.” Nicotine poisoning threatens adults and children who cultivate tobacco . In Mexico children aged 0-14 years who work in tobacco fields are exposed to potentially harmful and toxic amounts of pesticides . Children and adults are harmed by polluted drinking water from pesticide run-off. Most tobacco families in Mexico are financially unable to afford protective clothing and bottled drinking water. Information on injuries, accidents, and fatalities of child laborers in tobacco farming needs to be collected, analyzed and disseminated. Children who work in tobacco fields experience backaches, broken bones, snake bites and other risks. Research is needed on risksfacing child workers and the influence of risks on their educational and psychological development. Researchers can apply Helmut Geist’s multi-method approach of statistical analysis, meta-analytical study and narratives to conduct investigations of child laborers in tobacco growing developing countries. Researchers need to devise measurements to determine soil degradation and downstream effects of pesticides and use the measurements to understand tobacco-related destruction of soil nutrients and pollution of water tables. Research findings could be used to devise workshops and study circles on health and pesticide education, rolling benches for growing building on worker education infrastructure already created by agricultural trade unions. Studies are needed on tobacco industry corporate social responsibility schemes focused on child labor and deforestation.

The studies need to analyze how actual tobacco industry practices contradict corporate schemes and their messages. Research is also needed to understand farmer and consumer perceptions of “ethically produced” cigarettes and how tobacco companies through these cigarettes undermine health policy, pass on misinformation, and build public faith in tobacco. Research is needed on how health policymakers and advocates view and participate in tobacco industry responsibility schemes. Research is needed on the direct links between tobacco industry practices and child labor, deforestation, and other realities of tobacco farming that clash with farmer welfare. Do tobacco companies knowingly purchase tobacco produced with child labor? What evidence is needed to verify that tobacco companies knowingly purchase tobacco produced with child labor? To what extent do companies’ policies and practices allow them to buy leaf produced with child labor? Policymakers and advocates need to examine opportunities for excluding imports of tobacco produced with child labor.Health policymakers and tobacco control researchers need to find a balance between building corporate accountability and recognizing tobacco companies’ efforts to cultivate tobacco and sell cigarettes. How should public health and tobacco control policymakers attempt to make tobacco companies accountable to child labor and other socially disruptive behavior without pressuring companies to move into more vulnerable societies where labor costs are lower and environmental standards are less restrictive or non-existent? What are experiences of tobacco farmers who contract directly with leaf companies and cigarette manufacturers? Is there transparency in contract agreements between farmers and tobacco companies? What remedies exist for tobacco farmers who have been entrapped through debts for marked up inputs from tobacco companies? What is the impact of contract farming on social development and environmental health in tobacco farming communities? Policymakers and researchers need to pressure tobacco companies to publicize details of tobacco farming contracts, average and enforced prices for inputs, and loans granted and collected to ensure fairness in contract arrangements. Cultural attitudes that support child labor need to be examined. What cultural attitudes, practices, and beliefs of tobacco farmers justify or sustain child labor? What cultural changes need to happen to mainstream, standardize, and normalize tobacco growing free from child labor and environmental destruction?

Research is needed on experiences of tobacco farmers and tobacco farm workers, recognizing that these economic groups have contradictory and overlapping interests. How many casual or day laborers work in the global tobacco growing sector? To what extent do farm workers use child labor and harm environments?How can public health policymakers and tobacco control advocates overcome ambivalence toward trade unions of tobacco farmers and farm workers that promote fair and decent work? Do health policymakers, advocates, and researchers develop partnerships focused on food security and sustainable agriculture with tobacco farm worker trade unions that lend support to tobacco industry social responsibility child labor projects? To what extent do health policymakers call upon trade unions that accept tobacco industry money and promote living wages to justify their policy of accepting tobacco money?The best practices for addressing tobacco-related child labor, deforestation and poverty involve equity and inclusivity. Equity in social protections such as quality education, health care, and housing and inclusivity of tobacco farmers in policy making processes and research activities in tobacco farming are major goals of best practices. The aims of best practices are to ensure prosperity and welfare of tobacco farmers, reduce the influence of tobacco companies on child labor and environmental projects, and in cases where tobacco companies financially support projects, obtain commitment from companies to support a program of outside, independent monitoring of compliance with global standards such as the International Labor Organization Convention No. 182 on the Worst Forms of Child Labor, 1999. Best practices to reduce tobacco-related child labor, deforestation and poverty are most effective when balanced with specific country experiences and policy priorities. Child labor in Malawi and child labor in India are different, requiring analyses of local contexts, stakeholder interests, and country needs. Deforestation in tobacco growing sectors in Tanzania and Brazil is not the same. The best practices below need to be examined in specific country contexts and implemented to ensure compatibility between best practices and policy environments.The International Labor Organization, International Program on the Elimination of Child Labor with projects in 88 countries, including many tobacco growing countries, is an example of best practices to address child labor in tobacco growing. The Dominican Republic provides a representative case of ILO-IPEC tobacco related research. In 2004, research was conducted to generate data on the extent and nature of youth and their families working in tobacco plantations in the Dominican Republic. One hundred children performing tobacco-related jobs were interviewed and fifty focus groups discussions were conducted on 35 farms. The main finding of the study is that child laborers perform poorly in school and have low attendance rates in schools because of their involvement in tobacco cultivation. The researchers recommended that non-tobacco agricultural development needs to be created and mechanisms to monitor and inspect child labor on tobacco plantations are required. The study provides a best practice approach to research that could provide basic information on the child labor problem in order to assess the extent and impact of child labor in tobacco growing countries. ILO-IPEC works in partnership with and receives financial support from global tobacco companies through the Elimination of Child Labor in Tobacco Growing Foundation , a tobacco industry funded group, raising the issue that tobacco control policymakers and researchers need to weigh the advantages and disadvantage of involvement with social, development, cannabis dry racks and environmental groups that collaborate with tobacco companies. Beginning in 2002, ECLT financially supported ILO-IPEC projects to reduce tobacco-related child labor in countries such as the Dominican Republic, Indonesia, and Tanzania. ILO-IPEC/ECLT studies appear to document child labor problems in a reasonable manner.

The major weakness of ILO-IPEC/ECLT studies is the absence of information and comment on tobacco companies’tobacco growing practices that harm farmers, children and environments, and companies’ strategies to use corporate social responsibility schemes to build faith in the tobacco and deflect criticism of tobacco companies’ practices. ECLT on its website states that the International Labor Organization plays an advisory role to ECLT. On ILO-IPEC website, ECLT is listed as a donor to ILO-IPEC in 2002-3 and 2006-7. ECLT through ILO involvement obtains legitimacy for ECLT and tobacco companies social responsibility schemes focused on child labor to sidestep labor exploitation in Malawi and other countries where ECLT operates child labor projects. The WHO is not a participant to ILO-IPEC. Industry funded child labor projects create a unique problem for health policymakers and tobacco control researchers that support WHO’s Framework Convention on Tobacco Control. Involvement of health policymakers and researchers in ILO-IPEC/ECLT projects could enhance legitimacy of tobacco industry efforts to promote goodwill and build public faith in tobacco through child labor projects. Refusal of health policymakers and researchers to participate in ILO-IPEC/ECLT child labor schemes creates a gap between the goals of policymakers and researchers to promote farmer prosperity and resources to reduce inequalities and improve living standards on tobacco farms.The hazard rating matrix developed to assess work performed by children in vegetable farming in the Philippines provides a simple tool tobacco control policymakers and researchers could use to assess work performed by children in tobacco cultivation . The hazard rating matrix is a specialized checklist and classification scheme comprised of work environment, materials and equipment used, and contact with social and water. The hazard rating matrix of the degree of safety of working conditions and the intensity of work could allow policymakers and researchers to identify hazardous work of children in tobacco growing that should be banned.78Promoting the creation and dissemination of documentary films about tobacco in Argentina as well as films about tobacco related child labor, deforestation, pesticide pollution and nicotine poisoning in Malawi, Tanzania, Mexico, Brazil, and Bangladesh. The Instituto de Ciencia y Tecnologia Regional in Jujuy, Argentina, coordinates projects to develop leadership among the youth regarding tobacco control through research, identify risk factors such as poverty that factor in the uptake of tobacco use in displaced aboriginal youth, and to raise community awareness and support for improved livelihoods of tobacco farmers in Argentina. In 2004, the Instituto de Ciencia y Tecnologia Regional produced the documentary film “Tabaco, Voces Desde El Surco” on tobacco farmers and workers in Jujuy to educate Argentineans and the international community about the social and environmental costs of tobacco farming. The video is available for viewing on the Internet, providing visual imagery of human experiences of tobacco farming to researchers, policymakers, and individuals with Internet access throughout the world. In the video, a tobacco farmer standing with a hoe in a tobacco field says, “One starts learning from very young when you are eight or nine years old and gets together with friends. We play to put the tobacco leaves on the cane [drying sticks], and in this way you are brought up doing this work. Then, when you are twelve you do the work of an adult.” The video imagery of farming, child labor, and environmental destruction from tobacco farming augments text-based reports and statistical analyses of tobacco work to more fully assess the extent and characteristics of tobacco-related child labor and biodiversity loss. In Malawi, the Guernsey Adolescent Smokefree Project established in 2006 the project “Ana a topa” to support children who work in the tobacco farming sector. Guernsey is a British Crown dependency in the English Channel near Normandy, France. “Ana a topa” involves a partnership between the Guernsey Adolescent Smokefree Project and the Tobacco Tenant and Allied Workers Union of Malawi, the main tobacco farm worker organization in the country. “Ana a topa” is in its beginning stages of a crop diversification scheme that directly supports children in Malawi and a research project with local advocates to assess the frequency of child labor abuses in Malawi.The project is a unique tobacco farmer union-public health group alliance to raise awareness of child labor, reduce the factors that force parents to send their children to tobacco fields instead of schools, and strengthen the tobacco farm worker union’s child labor committees in tobacco farms to confront the child labor problem. The project is cross-national and involves a media campaign in Guernsey to educate youth on the working practices imposed by the tobacco industry on Malawi and the demands placed on children to work in tobacco fields. In Uganda in 2004, the Environmental Action Network developed a project to create a database of information on deforestation and other issues affecting tobacco farmers. The project filled a local knowledge gap on environmental problems relating to tobacco by systematically collecting and organizing data specific to Uganda, allowing researchers and advocates to reduce dependency on data from other countries.

The water flowed by way of gravity into a 125 mL beaker, leaving debris behind. Filter papers were changed periodically as needed. Then, 50 mL were allocated from the filtered sample and placed in storage at -20° C until analysis. A standard for pendimethalin, ACS-grade hexane and MS-grade acetonitrile were obtained from Fisher Scientific. Liquid-liquid extraction methods were modified from USEPA . High pressure liquid chromatography tandem mass spectrometry was employed to analyze for residue in water samples. Fifteen mL samples were extracted three times with 3 mL of hexane and placed on a rotary platform shaker for 5 minutes, then set aside for 15 minutes. Hexane extracts were pooled and 3 mL were then dried under a nitrogen gas stream. Then, volumes of 500 µL acetonitrile were added to the dried sample and vortexed. Volumes of 500 µL 0.4% formic acid was then added and vortexed for a final concentration factor of 15. A Shimadzu LCMS-8040 triple quadrupole mass spectrometer was used equipped with electrospray ionization on positive mode. The desolvation line temperature and heat block temperature were 250° and 400° C, respectively. Nebulizing gas and drying gas were set at a flow of 3 L min-1 and 15 L min-1 , respectively. The mobile phase flow rate was 0.4 mL min-1 and an injection volume of 10 µL. The C18 column was Phenomenex Kinetex polar, 100 by 3.0 mm and 2.6 µm particle size. The multiple reaction monitoring ion transitions for the quantifier ionwere 282.0 > 212.1 m/z in a dwell time of 10 ms and for the qualifier ions were 282.2 > 43.1 m/z and 282.2 > 194 m/z in a dwell time of 5 ms. The limit of detection was 0.006 µg L-1 and the limit of quantification was 0.008 µg L-1. Multiple calibration curves were implemented for the low concentration range and for the high concentration range using Shimadzu LabSolutions and MacCoss Skyline software for small molecules. Method recovery was performed by spiking five non-treated collected water samples with 0.20 µg L-1 of pendimethalin before extraction . A low concentration of pendimethalin below 0.05 µg L-1 was present in the collected non-treated samples, therefore, cannabis dryingn racks the peak areas of the control samples without standard spiking were subtracted from the spiked samples.

The recovery in water samples was on average 79%.A significant increase in sweet potato [Ipomoea batatas Lam.] production area in the southeastern United States has occurred in the past decade, increasing from 33,548 ha in 2007 to 51,800 ha in 2017 . Sweetpotato has proven to be a valuable crop with a national farm gate value of $705.7 million in 2016, up from $298.4 million in 2006 . North Carolina is the largest sweetpotato-producing state, accounting for 54% of U.S. production . North Carolina, California, Mississippi, and Louisiana account for 94% of sweetpotato production in the United States . Unfortunately, due to its prostrate growth habit and relatively slow growth, sweetpotato does not compete well with problematic weeds, resulting in reduced yields . Palmer amaranth and large crabgrass [Digitaria sanguinalis Scop.] are among the top five most common weeds in North Carolina sweetpotato, with A. palmeri being identified as the most troublesome weed . Amaranthus palmeri has been reported to be taller, to have a faster growth rate and greater leaf area, and to produce more overall biomass when compared with other Amaranthus species . Season-long A. palmeri interference is seen in vegetable crops, with reduced yield of 94% in bell pepper , 67% in tomato , 36% to 81% in sweetpotato , with the greater yield losses associated with higher A. palmeri densities. Limited herbicide options exist for use in sweetpotato . Growers rely on PRE herbicides, which do not always provide efficacious weed control and require rainfall for activation. POST herbicide options for A. palmeri control in sweetpotato are limited to between-row applications of carfentrazone or glyphosate . The lack of POST herbicides forces growers to use tillage for control of weeds until row closure, at which time growers have no additional control options for dicotyledonous weeds other than mowing weeds above the cropcanopy and hand weeding, which is a costly control measure . Digitaria sanguinalis is commonly found in fruit and vegetable crops but has not been highly ranked as a problematic weed due to efficacious POST herbicides such as clethodim, fluazifop, or sethoxydim . Although these graminicides can be effective, grasses escaping herbicide application or sprayed after substantial establishment may continue to compete with the crop and reduce yields.

Furthermore, herbicide resistance management for D. sanguinalis should be considered, as resistance to acetyl-CoA carboxylase herbicides, including those registered for use in sweetpotato has been reported . While its impact on sweetpotato has not been reported, season-long, D. sanguinalis reduced yield in bell pepper by 46% , snap bean by 47% to 50% , and watermelon [Citrullus lanatus Matsum. & Nakai] by 82% . A better understanding of the interactions of A. palmeri and D. sanguinalis with sweetpotato would allow for better decision making regarding their control. Thus, the objectives of this study were to determine the effect of five densities of A. palmeri and D. sanguinalis on sweetpotato biomass and storage root yield and quality, the intraspecific response of A. palmeri and D. sanguinalis across five densities with and without sweetpotato, and the effect of sweetpotato on growth of A. palmeri and D. sanguinalis.Field studies were conducted with ‘Covington’ sweetpotato at the Horticultural Crops Research Station near Clinton, NC on a Norfolk loamy sand with humic matter 0.31% and pH 5.9 in 2016 and an Orangeburg loamy sand with humic matter 0.47% and pH 5.9 in 2017. Nonrooted ‘Covington’ sweetpotato 20- to 30-cm-long cuttings were mechanically planted approximately 7.6-cm deep into ridged rows 1 m apart in the entire study at an in-row spacing of approximately 30 cm on June 9, 2016, and June 12, 2017. At 1 d after transplanting, sweetpotato plants were removed by hand in the no-sweetpotato treatments. On the same day, treatment rows assigned A. palmeri or D. sanguinalis were broadcast seeded on the soil surface and lightly raked to a depth of approximately 1.0 cm. After weed seeding, the entire study was irrigated with 1.3 cm of water using overhead irrigation to aid in weed seed establishment. No additional irrigation was applied, in either year, after the initial irrigation event. Treatments consisted of a single weed species at five weed densities grown with and without sweetpotato arranged in a randomized complete block design with three replications . Amaranthus palmeri and D. sanguinalis were hand thinned to treatment densities of 0 , 1, 2, 4, and 8 and 0 , 1, 2, 4, and 16 plants m−1 of row, respectively, when A. palmeri was approximately 8 cm tall, and D. sanguinalis had two expanded leaves. At the time of weed thinning, sweetpotato averaged one to two newly expanded leaves on each plant. Densities of A. palmeri and D. sanguinalis were based on those used in previous research . Plots consisted of two bedded rows, each 1-m wide by 5-m long, with the first row being a weed-free buffer row planted to sweetpotato and the second row a treatment row. Treatment rows were maintained at specific weed treatment densities, and border rows were maintained free of weeds season-long by weekly removal by hand. Season-long rainfall and growing degree day data are presented in Table 1. Two days before sweetpotato harvest, 5 sweetpotato plants and 5 plants of each weed species were randomly harvested at the soil level from each plot to determine aboveground biomass. Samples were placed in 2-ply paper yard waste bags measuring 40 by 30 by 89 cm and fresh biomass was recorded. Samples were then placed in a propane-heated, forced-air drier for 96 h at 80 C. Once dry, samples were removed and weighed immediately to determine dry biomass. To determine fresh and dry sweetpotato and weed biomass on a per plant basis, total sweetpotato or weed biomass within a treatment and replication was divided by the number of plants harvested. To determine dry biomass per meter of row, individual weed biomass was multiplied by sweetpotato plant and/or weed number in 1 m of row, respectively.

Sweetpotato storage roots were harvested at 113 d after transplanting in 2016 and at 107 DAT in 2017. In both years storage roots were harvested with a tractor-mounted two-row chain digger and hand sorted into jumbo , no. 1 , and canner grades and weighed. Total marketable yield was calculated as the sum of jumbo and no. 1 grades. Data for crop biomass, vertical growing weed individual weed biomass, weed biomass per meter of row, yield, and quality were subjected to ANOVA using PROC MIXED in SAS . Treatment, year, and treatment by year were considered fixed effects, while replication within year was treated as a random effect. Year was treated as a fixed effect to further evaluate components of the year by treatment interaction, such as year by weed density and year by crop presence or absence. If the treatment by year interaction was not significant, a contrast statement was used to test for a linear trend for dependent variables with increasing weed density, calculated separately for each weed species. All response variables, except canner yield, were square-root transformed to reduce both data skewness and variance heterogeneity before carrying out the mixed model ANOVA.Marketable yield decreased as the density of A. palmeri or D. sanguinalis increased. No treatment by year interaction for sweetpotato yield was observed ; therefore, data were combined over years. Marketable yield loss associated with A. palmeri density ranged from 50% with 1 A. palmeri plant m −1 of row to 79% with 8 plants m−1 of row, respectively, when compared with the weed-free check . Marketable yield reduction by D. sanguinalis was similar to marketable yield reduction caused by A. palmeri but at higher weed densities. Marketable yield was reduced by 35% and 76% with 1 and 16 D. sanguinalis plants m−1 of row, respectively . Loss of jumbo yield is a significant contributor to overall marketable yield loss at weed densities as low as 1 plant of either species m−1 . Jumbo grade had greater yield loss with 1 plant m−1 for A. palmeri and D. sanguinalis than the no. 1 gradefor both weed species at the same density . Results for estimated marketable yield loss per weed as weed density approaches zero for A. palmeri and D. sanguinalis were 119% and 61%, respectively. The higher estimated marketable yield loss as weed density approaches zero for A. palmeri relative to D. sanguinalis indicated higher competitive capacity of A. palmeri at low densities. These results for A. palmeri are consistent with another study in sweetpotato but higher than in soybean [Glycine max Merr.] , peanut , and corn . Estimated yield loss as weed density approaches zero in the present study indicates that A. palmeri and D. sanguinalis, even at low densities, can greatly reduce sweetpotato marketable yield. The initial yield loss as weed density approaches zero for D. sanguinalis was less than A. palmeri at lower densities. However, sweetpotato yield loss from interference byD. sanguinalis was higher than yield loss reported in snap bean . For parameter A, the asymptote of the regression model estimating the maximum yield loss due to weed density was 87% for A. palmeri and 83% for D. sanguinalis. Meyers et al. estimated a maximum marketable yield loss of 90% at A. palmeri densities of 6.5 plants m−1 of sweetpotato row. Findings from our study further support the findings of Meyers et al. , who also reported the highly competitive nature of A. palmeri with sweetpotato. To reduce interference of A. palmeri and D. sanguinalis, which are commonly reported in sweetpotato, growers should use a combination of efficacious PRE herbicides, as outlined by Meyers et al. , in combination with tillage, hand removal, and mowing . Although POST herbicides for A. palmeri are limited, POST herbicide options for selective grass control in sweetpotato are available and should be used when D. sanguinalis is less than 10 cm to minimize yield loss. If D. sanguinalis resistance is suspected, then alternative methods should be analyzed for control. Growers should not dismiss the impact of either weed, as a single A. palmeri or D. sanguinalis per meter of row reduced marketable yield by 50% and 35%, respectively . Reduction in marketable yield loss was due to a decrease in weight of no. 1 and jumbo sweetpotato grades.