TOC1 and CO regulate circadian rhythm and day-length responses, while RGA represses floral growth in the absence of gibberellic acid. WUSCHEL genes have a well-characterized function in meristem organization and have been shown to interact with AGAMOUS in floral meristem development. AGL11 is important in ovule and seed development, and has been shown to interact with cytokinin to control fruit size. These floral development genes may have specific roles as “entry points” for the SDR into the floral development pathway to regulate the development of a particular sex . Cronk and Müller proposed that ARR17 may act as a feminizing master regulator in Populus through the suppression of PISTILLATA or APETALLA3 MADS-box genes. Importantly, there was no association of either PI or AP3 expression with genomic variation in the SDR, despite the fact that PI shows very high levels of expression in males , further suggesting that the mechanism of sex determination in Salix may be different from Populus. eQTL analysis revealed several loci with an exceptionally high number of trans-eQTL . Intriguingly, the hotspot with the greatest number of trans-eQTL is located in the region on chr07 homologous to the S. nigra SDR, which could implicate its role as an ancestral SDR in Salix, or explain a fitness advantage of a chr07 SDR in S. nigra when linked with sex-dimorphism genes in this region. Approximately 250 kb from this locus on chr07 is Sapur.007G068100, a homolog of AGL32,fodder growing system a MADS-box gene involved in ovule development. The expression of this gene is associated in trans with the chromosome-15 SDR region, further supporting its role as a top-level regulatory gene under direct regulation by the SDR.
Compounds produced from the phenylpropanoid and terpenoid pathways are well-characterized in Salicaceae, and there is evidence that floral volatile, terpenoid, and phenolic glycoside profiles differ substantially between males and females, which affects both pollinator attraction and herbivory, traits that are likely to be evolutionary drivers of diecy and affect cultivar yield. In support of this, we identified five terpenoid pathway genes and 15 phenylpropanoid pathway genes with eQTL in the SDR. These include both genes involved in the core phenylpropanoid pathway, and biosynthesis of specific compounds, including naringenin, flavenol glucosides, and sesquiter penoids . This provides evidence supporting a direct link between the SDR and synthesis of these compounds, by an as-yet unknown mechanism.Of the 45 identified miRNA loci, 18 were male differentially expressed and seven were female differentially expressed. Among the putative targets of these miRNAs were dicer-like genes, squamosa promoter-like genes, andauxin-response factors, and transcription factors providing evidence that miRNAs are likely to be a component of floral sex-dimorphism regulation. Notably, five miRNAs were identified that had no match with any small RNAs in the PmiREN database and could represent genus- or species-specific micro-RNAs. Furthermore, 13 miRNA loci with matches in the pmiREN database were not matched to a known P. trichocarpa miRNA, which is the closest species for which extensive small RNA data are available . This suggests that S. purpurea may utilize different sets of small RNAs in floral development relative to Poplar, which likely has implications on sex dimorphism and determination. Expression results showed that four miR156 and two miR172 homologs have greater expression in male floral tissue. In Arabidopsis, miR156 and miR172 interact to form a gradient that regulates vegetative-to-floral meristem transition through the targeting of squamosa promoter-like genes.
While all copies of both miR172 and miR156 show malebiased expression in catkins, the overall expression of miR156 is greater than miR172 in males. The majority of SPL genes that are targeted are female-up regulated in S. purpurea, including an SPL4 homolog on chr07 that also shows increased methylation in males in its promoter region. These data support that the miR156/172 pathway is upregulated in male catkins and may be responsible for sex dimorphisms . This pathway may play a role in male floral tissue development or differentiation. Importantly, one copy of miR156 is located in the SDR region unique to chr15Z. Alignment of the chr15Z miR156 precursor sequence to chr15W reveals that a single indel is responsible for this chr15Z-specific mapping , which may prevent transcription or processing of this small RNA from chr15W. This could indicate a dosage dependent response in males, which have four copies of this mature miR156 homolog, compared with only three in females. Female-upregulated miRNAs included miR403, which targets AGO2, and miR162, which targets a dicer-like gene. All of these are involved in small RNA signaling and DNA methylation, which is consistent with the enrichment of transcription regulation terms in the female-upregulated genes. This may point toward a role of genome-wide DNA methylation or RNA silencing in regulating sex dimorphism, possibly mediated by AGO4 or DRB1.Pest- and pathogen-free planting stock is essential for successful establishment and future productivity of new orchards and vineyards. Clean stock is also a requirement for intrastate, interstate and international commerce of tree, vine and garden rose planting stock. To ensure the quality of commercially produced nursery stock in the state, the California Department of Food and Agriculture enforces laws and regulations related to the production of certified nursery stock as outlined in the Nursery Inspection Procedures Manual .
Because of the potentially large and long-term impacts on the nursery crop as well as the subsequently planted orchards, vineyards and ornamental landscapes, control of plant-parasitic nematodes in nursery fields is a major focus of the nursery stock certification program. Producers of perennial crop nursery stock in California can meet nematode certification requirements by fumigating the field at the beginning of the nursery cycle using an approved treatment or by conducting a detailed inspection of soil and planting stock at the end of the production cycle. If growers elect to use inspection procedures instead of approved treatments and soil or plant samples are found to contain prohibited nematodes, further sampling is conducted to delineate the extent of the problem, and nursery stock from the affected area usually is destroyed. Preplant soil fumigation thus reduces the economic risk of a nonsalable nursery crop and is used in most tree and garden rose nurseries in California. Grapevine nursery stock also must meet phytosanitary requirements to be certified in California, but in contrast to tree and rose growers, many grape nursery producers elect to use the inspection procedures rather than fumigation. In practice, the risk of nematode occurrence in production of grapevine nursery stock without fumigants is reduced by spring planting, a relatively shorter nursery production cycle and market preference for smaller nursery stock. However, grape nursery operations with sandy soils or sites where grapes have been grown previously often use preplant fumigation practices comparable to tree and rose nurseries to reduce the economic and market risks of not meeting phytosanitary regulations. Most field-grown perennial nursery operations have used methyl bromide for preplant pest control because it effectively diffuses through the soil profile, penetrates roots and dependably provides effective pest control across a range of soil type and moisture conditions. Under the provisions of the U.S. Clean Air Act and the Montreal Protocol,chicken fodder system the import and manufacture of methyl bromide is being phased out because of its deleterious effects on stratospheric ozone. Perennial nursery producers have largely continued using methyl bromide under the critical use exemptions and quarantine/preshipment criteria . However, increasing production costs and international political pressure on CUE and QPS regulations have spurred efforts to identify economically viable alternatives to methyl bromide for the perennial nursery industry. Several factors limit the adoption of alternative fumigants in California nursery systems. First, there are very few fumigant or non-fumigant nematicides available . In the United States only a handful of fumigants are registered, including methyl bromide, 1,3-dichloropropene , chloropicrin, dimethyl disulfide , and methyl isothiocyanate generating compounds. Of these, DMDS is not currently registered in California and has had only limited testing in nurseries. Methyl iodide was registered in California in late 2010, but the federal registration was withdrawn by the manufacturer in early 2012.Of the fumigants registered in the state, only 1,3-D is an approved treatment in nurseries with medium- to coarsetextured soils . However, it is not approved for nurseries with fine-textured soils because the registered rates are not sufficient to provide acceptable pest control.
Most of the alternative fumigants are heavily regulated due to concerns about human safety and environmental quality related to emission of fumigants and associated volatile organic compounds . These concerns have led to a constantly changing regulatory environment, encompassing buffer zones, field preparation requirements, available compounds and rate limitations on a field and air basin level . Uncertainty within the nursery industry about current and pending fumigant regulations presents a continuing challenge to the adoption of methyl bromide alternatives in California. Although fumigation in the perennial crop nursery industry is driven by nematode certification, there are serious concerns that the level of secondary pest control provided by methyl bromide will not be matched by the alternatives. Weed control with many of the available alternatives is generally not as reliable as with methyl bromide . Although weeds can be addressed to a large extent with tillage, hand-weeding, and herbicides, there are likely to be environmental and economic impacts of greater reliance on these techniques. More importantly, many nursery producers are very concerned about the consequences of soil borne diseases that are currently controlled with methyl bromide or methyl bromide and chloropicrin combinations. Reliance on alternatives with narrower pest control spectrums may result in problems with new diseases or the resurrection of old ones. Research has been conducted in recent years to address issues limiting adoption of methyl bromide alternatives in California’s perennial crop nursery industry . As part of the USDA-ARS Pacific Area-wide Pest Management Program for Integrated Methyl Bromide Alternatives, two additional research and demonstration projects were implemented from 2007 to 2010. First, because current and pending regulations greatly affect how and when fumigants can be used, a research station field trial was conducted to simultaneously determine the effects of emission reduction techniques on pest control and fumigant emissions. Second, two trials were conducted in commercial nurseries to test and demonstrate pest control and nursery stock productivity with 1,3-D treatments in an effort to increase grower experience and comfort with available alternatives.A shank fumigation trial was conducted in 2007 at the UC Kearney Agricultural Center , near Parlier, to determine the effect of two fumigation shank types and five soil surface treatments on 1,3-D emissions and control of representative soil borne pests following removal of a plum orchard. Soil texture at the site was a Hanford fine, sandy loam with pH 7.2, 0.7% organic matter, and a composition of 70% sand, 24% silt and 6% clay. The experiment included 10 treatments with 1,3-D in a split plot design with surface treatments as the main plots and two application shank types as the subplots, as well as an unfumigated control and a methyl bromide plus chloropicrin standard for comparison . Individual plots were 12 feet by 100 feet, and each treatment was replicated three times.Fumigants were applied using commercial equipment on Oct. 2, 2007. Methyl bromide with chloropicrin was applied at 350 pounds per acre with a Noble plow rig set up to inject fumigants 10 inches deep through emitters spaced 12 inches apart while simultaneously installing 1-mil high-density polyethylene film. The 1,3-D treatments, at 332 pounds per acre, were applied using either a standard Telone rig with shanks spaced 20 inches apart and an injection depth of 18 inches or a Buessing shank rig with shanks spaced 24 inches apart and the fumigant injection split at 16- and 26-inch injection depths. The Buessing shank also had wings above each injection nozzle to scrape soil into the shank trace and minimize rapid upward movement of the fumigant . Following 1,3-D application, a disk and ring roller was used to level and compact the surface soil before surface seals were applied over the fumigated plots. Average soil temperature at 20 inches during fumigation was 70°F, and soil moisture was 8.2% to 10.5% weight per weight in the top 3 feet. Surface treatments included HDPE film; virtually impermeable film, VIF ; and a series of intermittent water applications . HDPE and VIF film was installed after the disk and rolling operation using a Noble plow rig.