Month: April 2025

Comparing the pollen-provision microbial communities of closely related specialist and generalist bees will help address how diet breadth affects microbe exposure and establishment. To help understand how diet breadth affects bee nest microbial communities, we characterized the pollen provision microbial communities of a pair of closely related bees collected across four sites: Osmia lignaria, a polylectic bee that prefers to forage on orchard trees in the family Roseaceae; and O. ribifloris, a specialist on blueberries and its relatives with Berberis serving as an alternate host. We address the hypothesis that, due to the importance of floral transmission for microbes found in the pollen provisions of megachilid bees, diet breadth affects pollen provision microbial composition. We predicted that the specialist bee would harbor fewer species of microbes and show less variation across sites compared to the generalist bee.We collected pollen provisions from the nest cells of two bee species native to the area of Logan, Utah, USA: Osmia lignaria propinqua and Osmia ribifloris biedermannii in Spring 2015. We chose four locations near Logan that varied in floral composition and interaction with other bee species: Site 1 was a cultivated floral garden located in a suburban area with a combination of native and introduced floral species, plastic planters wholesale including Rubus idaeus L. and Calluna vulgaris .

Site 2 was a bee-tight screenhouse that contained older, in-ground plantings of R. idaeus and Berberis fremontii , along with potted C. vulgaris. Sites 3 and 4 were natural areas of Logan Canyon that contained limestone cliffs and a mixed forest, and were approximately 1836 m apart, which is likely outside of the flight range of Osmia species. We note that the locations differed in the composition of their pollinator communities: Site 1 had a diverse pollinator community present, including numerous honey bee and bumble bee colonies. Site 2 was a research screenhouse that had no honey bees nor bumble bees present for at least five preceding years. Sites 3 and 4 had diverse pollinator communities without honey bees present, and we did not collect any O. ribifloris from Site 4. See supplemental Table S1 for the number of each bee species collected at each site. There were also differences in the sources of sampled Osmia nests between locations: We sampled natural Osmia populations from Sites 1, 3, and 4, while we sampled nests made by commercially acquired bees for Site 2. In this location, O. lignaria were sourced from Watts Solitary Bees and O. ribifloris were purchased from NativeBees.com . Within each location, we sampled the pollen provisions from cavities within wooden nesting blocks that were lined with paper straws and placed in each location for one week. As both species of bee will nest in these cavities at the same time, we were able to concurrently sample newly made nests. We used 1–6 nesting straws per bee species, then removed the straws from the nest blocks, numbered them sequentially, and X-rayed the straws to confirm that eggs were present and had not yet hatched.

Once the straws were collected, we carefully slit open the straws, removed the pollen provisions from each initially formed cells , then sterilely removed the eggs from nest cells before DNA extraction. We carefully excluded cell wall partitions from the collection of the pollen provision. Each cell was treated as a separate sample.We extracted DNA from the pollen provisions based on a modified protocol from Engel et al. 2013, Rothman et al., and Pennington et al. 2018. We used a Qiagen DNeasy Blood and Tissue kit for the DNA extractions, with slight modifications. We sterilely transferred entire pollen provisions to a 96-well tissue lysis plate , then added 100 µL glass beads, one 3.2 mm steel-chrome bead, and 180 µL buffer ATL to eachwell. We bead-beat the mixture at 30 Hz for three minutes, turned the plates over, then bead-beat for three more minutes. We incubated the mixture at 50 ◦C overnight, then followed the rest of the manufacturer’s protocol to finish the DNA extraction. We used the extracted DNA to prepare 16S rRNA gene libraries as in McFrederick and Rehan 2016, Rothman et al. 2019, and Pennington et al. 2017. Briefly, we used a dual-indexing approach to build an amplicon construct consisting of the universal primers 799F-mod3 and 1115R, a unique 8-mer barcode, and the Illumina adapter sequence as in Hanshew et al. 2013. We built the libraries in two rounds of PCR amplification: First, we used 4 µL of template DNA, 0.5 µL of 10 µM forward and reverse barcoded primers, 10 µL water, and 10 µL of 2× Pfusion DNA polymerase with an annealing temperature of 52 ◦C for 30 cycles. Next, we cleaned the PCR product with a MoBio UltraClean PCR cleanup kit . We then used the cleaned amplicons as template for another PCR reaction, with the following conditions: 1 µL template DNA, 0.5 µL of 10 µM primers PCR2F and PCR2R, 13 µL water, and 10 µL 2× Pfusion DNA polymerase with an annealing temperature of 58 ◦C for 15 cycles.

We cleaned and normalized the libraries with a SequalPrep Normalization kit , pooled 5 µL of each library, then cleaned and concentrated the libraries with a MoBio UltraClean PCR cleanup kit . Lastly, we used an Illumina MiSeq to sequence the libraries at 2 × 300 cycles in the UC Riverside Genomics Core Facility.Other studies have found that bee species identity, foraging patterns, and geography can affect the microbes found in pollen provisions. McFrederick and Rehan showed that the pollen provision microbiome of Ceratina calcarata co-varies with pollen usage across habitats, especially when considering fungi. Voulgari-Kokota et al. showed that bee species and foraging patterns drive the bacterial communities found in pollen provisions. Our findings add to the consensus of these other studies that floral transmission and pollen usage influence the composition of the pollen provision microbiome. Our study also extends these previous studies by adding an understanding of how diet breadth does and does not affect the pollen provision microbial community in these two species of Osmia. Whether the different bacterial variants found in the host species and sites across studies is due solely from different transmission networks, filtering of microbes via differential floral and pollen provision chemistry, or a combination of both requires further study. As in other bee species’ pollen provisions, Osmia spp. pollen provisions contain a wide diversity of microbes. In agreement with these previous studies, the bacteria identified here can also be found in the environment, in both flowers and soil. For example, four of the 18 ASVs that were found at all sites in our study belonged to the genus Acinetobacter. This genus is ubiquitous in the environment and has been found in association with plants and animals, in floral nectar, in sewage, and in water and soil. The small fragment of the 16S rRNA gene that we use for Illumina sequencing rarely allows species-level resolution of these bacteria, so we are unable to determine whether these taxa were sourced from the mud that the bees used to partition their brood cells or from the nectar they mixed into their pollen provisions. Acinetobacter, however, has been reported in association with pollen provisions of several different bee species, and it is therefore not surprising that it occurs at all study sites. One conspicuous group of bacteria that are found in many pollen provision microbiomes but were uncommon here are bacteria from the Apilactobacillus micheneri clade. These bacteria have been found in the pollen provisions of other megachilid species in North America and Europe, but it is becoming clear that they are unevenly distributed across species. For example, in Germany, Apilactobacillus spp. are abundant in the pollen provisions of Megachile spp., plastic plant pot at variable abundances in O. caerulescens provisions, at low abundance in Heriades truncorum provisions, and absent in O. bicornis and O. leaiana provisions. In Texas , we detected these same lactobacilli at high relative abundances in pollen provisions of Osmia chalybea, Osmia subfasciata and Megachile policaris. Here, we report that lactobacilli are present only at very low relative abundances in the pollen provisions of O. lignaria and O. ribifloris, and that the A. micheneri clade lactobacilli are absent. As these bacteria have been isolated from flowers in both Texas and California, we hypothesize that either foraging preferences or pollen provision chemistry drives the presence or absence of these lactobacilli, and understanding the apparently cosmopolitan phenomenon of uneven distribution of lactobacilli across wild and solitary bee species should be a priority for pollen provision microbial community studies.

Many of the bacteria that we identified in O. lignaria pollen provisions have also been found in association with O. lignaria adults. Cohen et al. found that adults that had been foraging in the environment had a different or more variable microbiome compared to bees that emerged under sterile conditions in the lab, again supporting the importance of environmental transmission for the wild and solitary bee microbiome. Many of the bacteria found in adult O. lignaria adults are found in the environment and in the pollen provisions that we studied here. For example, Massilia is a root-colonizing soil bacterium that has been found in O. bicornis nests. This bacterium may be found in adult O. lignaria and their pollen provisions due to the adult’s habit of collecting mud to build partitions between brood cells in their nests. Pantoea is a plant-associated microbe that is abundant in the environment but has also been reported in association with other solitary bees and honey bees. Surprisingly, Cohen et al. found lactobacilli associated with adult O. lignaria, and additionally found that the abundance of flowers at a site positively correlated with the relative abundance of lactobacilli associated with adult O. lignaria. Future studies examining the adult and pollen provision microbiome of O. lignaria will help unravel how the environment shapes the microbiome of these separate but connected niches. The sole bacterial family that we found to be differentially abundant by host species was Micrococcacea. Micrococcacea is a diverse family that occurs in the environment, includes a commensal but opportunistic human pathogen, and has been classified as a pathogen honey bees. As Micrococcacea represented around 4% of the reads in seemingly healthy O. ribofloris nests, it is unlikely that this bacterium is pathogenic to Osmia. The differential abundance between the two Osmia species may mean that it is somehow important for bee health, but may also be due to the differential abundance between sampling site too. Along this thought, we found that many bacterial families were differentially abundant between geographical location. This provides further support to the hypothesis of environmental and floral transmission for solitary bee microbes as has been shown in previous studies.Insects pollinate many plant species, including several major crops. Bees are the single most important insect pollinator group and can be a limiting factor for the success of plant reproduction. Consequently, there is strong inter- and intra-specific competition among plants for the attention of pollinators. With respect to insect-pollinated crops, pollinator visitation is required to obtain maximal seed and fruit production. Consequently, pollination facilitates higher yields even when a crop plant is self-compatible. Bumblebees are important pollinators of tomato and other Solanum species that utilize an unusual pollination system called ‘buzz-pollination’. Buzz-pollinated flowers provide excess pollen as a reward to foraging bumblebees that feed it to their developing larvae. Although domesticated tomato is to a large extent ‘self-fertilizing’, buzz-pollination by bumblebees or by manual application of mechanical vibration ‘wands’ is required for maximal seed production, which in turn promotes increased fruit yield . Cucumber mosaic virus , one of the major viral pathogens of tomato, is a positive sense RNA virus that encodes five proteins including the 2b protein, which is a viral suppressor of RNA silencing. Bees do not transmit CMV but the virus is vectored by several aphid species. Virus infection causes dramatic changes in plant host metabolism . CMV-induced metabolic changes include qualitative and quantitative alterations in the emission of volatile compounds and in certain host species this makes infected hosts more attractive to aphid vectors.It is not known if the virus-induced alterations in host volatile emission that influence aphid behavior can also affect plant-pollinator interactions. Most bee-plant interaction studies have focussed on the effects of visual cues. Therefore, the influences of floral and non-floral volatiles on bee-mediated pollination are less well understood.

Oregon weather data were used to illustrate the impact of the different levels of parasitism on D. suzukii populations.In the U.S.A. and Italy, the current suite of parasitoids attack only late larval and early pupal life stages of D. suzukii, and field rates of parasitism are estimated to be around 2 % . In the biological control model runs, we made the simplifying assumptions that parasitism remains constant and that parasitism is attributable only to limited biological control agents based on field observations . For Oregon, we also assumed abundance of alternate hosts in surrounding vegetation from March 1, and that oviposition is possible as soon as the reproductive thresholds are met . Collections from South Korea show parasitism rates as high as 17 % . For these reasons, 2 and 15 % parasitism on the late larvalearly pupal life stages of D. suzukii were incorporated into the model on a season-long basis and compared to populations exhibiting no parasitism. These model runs would illustrate current and potential future population impact because of classical biological control using parasitoids.This research suggests that DD are very useful for estimating physiological time, and their use extends beyond phenology models. In addition, we describe the impacts of IPM strategies on early-season D. suzukii populations presumed to consist mainly of adults. The data presented are an alternative to the model developed by Wiman et al. to estimate populations of D. suzukii within environmentally known thermal thresholds. Previously, 10 plastic plant pots insects were forced to progress through the age-structured matrix with mandatory daily advancement without taking into consideration physiological age.

An advantage of the current model structure is that it allows flies to proceed through the physiological age-structured matrix contingent on accumulation of physiological time as approximated by DD. This refinement to the model allows survival and reproduction of concurrent cohorts comprising a population to be more precisely estimated. The two functions that describe mortality and fecundity within thermal thresholds in physiological time provided good fit using standard population survival and fecundity fitting techniques. We realize that a partial or complete shift of phenotypes inresponse to temperature extremes is not taken into account. However, future modeling efforts will be aimed at capturing phenotypic plasticity and its implications for survival and reproduction of populations experiencing extreme environmental conditions. We provided parameters of D. suzukii survival outside of thermal development thresholds, simulating a sudden transition to extreme cold. The fitted exponential function describes the survival rates of un-acclimated D. suzukii populations under prolonged environmental conditions. When exposed to suboptimal temperatures, un-acclimated D. suzukii populations clearly display high mortality rates. Theoretically, the horizontal asymptote of the fitted exponential function will not result in the extinction of winter or summer populations under these extreme conditions, and in the field, this phenomenon is observed as populations build during periods of favorable temperatures. Investigation of this observation, however, falls outside the scope of the current study where we focused on the growing season.

We therefore realize that the ability to estimate population response to a wide range of changing environmental conditions will further improve population estimation of adaptive invasive species such as D. suzukii. One step was made to more clearly understand how improving environmental conditions impact winter-surviving D. suzukii reproductive potential. Dissections of latedormant female D. suzukii from Oregon and Washington, U.S.A., and Trentino, Italy, displayed increasing levels of reproductive maturity as DD accumulated. Our laboratory data show that egg laying is initiated at 210 DD, whereas field increases in reproductive potential range from 50 to 800 DD. There may be various explanations for these differences, including year, microclimate, genetic variability, and trapping techniques. Although the timing of reproductive maturity with DD in these studies differed, the data nevertheless show a clear relationship between reproductive potential and physiological time. These data illustrate the importance of more suitable environmental conditions as a factor contributing to increased reproductive potential of D. suzukii. We realize that temperature is not the only factor contributing toward such increased reproductive potential, as the roles of humidity, alternate food sources, and host media during the late dormant period of D. suzukii may also need consideration . Our data suggest that in some regions, female sexual maturity may occur very early in the growing season before hosts become widely available. The lack of suitable fruit hosts in certain areas early in the growing season likely makes D. suzukii more dependent on alternate nutrient sources. These sources include pollen and nectar, which may be utilized by D. suzukii to increase reproductive potential and survival levels.

Such resource availability may be a factor resulting in variability of our data. For these reasons, it is important in the future to describe the contribution of these different factors to D. suzukii population dynamics. Clear parameters should be developed to more accurately model early-season D. suzukii population increase. We used a model to illustrate the importance of key periods when pest population structures can be exploited to the advantage of IPM . To the best of our current understanding of the structure of D. suzukii populations, adult females comprise the majority of overwintering individuals . Thus, early spring is a key period when only sexually maturing adults are present. Elimination of these initial adults before the population becomes established and spread among different ages would be ideal. In Parlier, California, U.S.A., a second key period exists during the summer when suboptimal hot temperatures prevail and D. suzukii populations decline. Insecticide A had a longer-lasting residual against adult stages and a shorter residual against immature stages as compared with Insecticide B, which explains the strongest effect on the early-season population. Insecticide B model runs simulated compounds designed to cause mortality at all life stages of D. suzukii . These compounds are of increasing importance as the population age structure becomes more diverse. In the early season, as in our simulation, adults dominate. During latter portions of the season, larger portions of D. suzukii populations are expected to be in immature life stages. During such latter portions of the growing season, there should be increased focus on pesticides that target all life stages of D. suzukii. The use of adulticides to prevent fruit rejections due to infestation before harvest when all ages of flies are present, however, will remain a key component of IPM programs. Additional considerations include the development of D. suzukii insecticide protocols to minimize the development of insecticide resistance. Genetic control using RNAi biopesticide technology with seven-day residual periods against all life stages resulted in minimal population reduction compared with untreated populations. Again, targeting of larvae makes more of a difference on structurally diverse populations. The utility of this technology to target immature life stages of D. suzukii may make it more effective at curatively managing pest populations compared to the adulticides during periods when immature life stages dominate the population structure. An additional advantage of such genetic pest management tools is that they can be designed to be species-specific and target D. suzukii only. Disadvantages currently include regulatory obstacles, wary public perception, and potential incompatibility with organic production practices. Both levels of biological control inputted into model runs resulted in D. suzukii suppression. Higher levels of parasitism will probably result in concomitantly lower levels of pest pressure during the harvest period. Currently, only low levels of biological control are found in most production regions . Model runs indicate that levels of parasitism close to 15 % will result in significant reductions of pest populations during the early portion of the season due to the loss of a portion of the initial crop-infesting population. Biological control is only effective, however, plastic pots large if it can suppress pest populations as the crop ripens. Model outputs estimate lower levels of suppression during the earlier portion of the season at the 2 % level of biological control. Clearly parasitism, particularly at the higher rate, helped to destabilize the D. suzukii population and is an effect that may be enhanced by additional compatible control measures.

Overall, these data suggest that biological control, as it currently stands, will not be effective as a standalone management tactic but will result in additive contributions to IPM programs targeting D. suzukii. Increased benefit will undoubtedly be gained from additional classic biological control introductions. We believe that the refined model presented here can be used as a comparative tool for practitioners and scientists, and such models will allow for the integration and optimization of multiple IPM technologies. This approach also illustrates that IPM practitioners should take advantage of environmental conditions that create vulnerability of the pest to management activities. During more suitable summer conditions, alternative factors such as pesticide use and biological control are considered as key management techniques. We realize that manipulations of D. suzukii populations are not the only factors that can be used in IPM strategies. Behavioral techniques including push–pull strategies, cultural methods, and insect barriers could also contribute to sustainable management of D. suzukii. In practice, IPM strategies often focus on single technologies including biological control, host plant resistance, chemical pesticides, or biopesticides. Typically, very little attention is given to the interaction or compatibility of the different technologies used and their timing . Future studies using stage-structured models such as the one presented here should be conducted to investigate these interactions. We realize that population modeling is only one approach for understanding how to manage this damaging pest. However, it can provide powerful insights into the relative performance of different tactics and combinations, and we expect that population modeling will allow rapid assessment of different integrated control programs and their expected performance under different environmental conditions.Cardiometabolic diseases encompass a cluster of cardiovascular, metabolic, prothrombotic, and inflammatory abnormalities that are recognized as disease states by the American Society of Endocrinology, the National Cholesterol Education Program, and the WHO . Food intake plays a key role in reducing the risk of cardiometabolic diseases, with data suggesting that >30% of all deaths could be prevented through dietary changes, particularly by increased consumption of plant based foods . Plant foods are rich sources of fiber and essential micronutrients, such as vitamins and minerals. They are also sources of a large group of bio-active compounds, which might not be essential throughout life or cause clinically manifested deficiencies, but when consumed with the diet, these phytochemicals may promote health and wellbeing in adulthood and the elderly population by reducing the risk of age-related chronic diseases . The major categories of dietary phytochemicals include polyphenols, such as flavonoids or phenolic acids, carotenoids, or plant sterols.Growing evidence from mechanistic studies, clinical trials, and prospective cohort studies suggest that these bio-active compounds may help in promoting health when consumed as part of the habitual diet. Polyphenols are secondary metabolites of plants and are found in fruits, vegetables, and their products . Carotenoids, including b-carotene, lycopene, lutein, and zeaxanthin, are phytochemicals found in many fruits and vegetables and account for the brilliant colors of these foods . Phytosterols are cholesterol-like molecules found in all plant foods, with the highest concentrations occurring in vegetable oils, but they can also be found in nuts, breads, or whole vegetables . Furthermore, caffeine ranks as one of the most commonly consumed dietary micronutrients; it is found in coffee beans, cacao beans, kola nuts, guarana berries, and tea leaves including yerba mate . Accumulating evidence from cohort studies suggests that an increased intake of polyphenols, which are the most abundant category of phytochemicals present in our foods, may reduce the risk of cardiovascular diseases . This evidence is supported by animal and clinical studies that have reported beneficial effects of the intake of polyphenol-rich foods or purified compounds on intermediate risk factors for CVD, including LDL cholesterol, blood pressure, and endothelial function . The most convincing clinical evidence for the cardioprotective benefits of the consumption of dietary polyphenols relates to their observed beneficial effect on endothelial function . For example, the consumption of suitable doses of plant sterols has repeatedly been shown in randomized controlled trials to reduce LDL cholesterol concentrations and thus reduce risk of subsequent CVD . Another phytochemical-rich source is coffee, one of the most widely consumed beverages worldwide. Coffee consumption may reduce the risk of type 2 diabetes mellitus and hypertension, as well as other conditions associated with cardiovascular risk , and epidemiological studies suggest that regular coffee drinkers have reduced mortality, predominantly as a result of their reduced risk of developing CVD .

Viruses belonging to the recently established genus Coguvirus also infect plants and, although this genus has not been included yet in the family Phenuiviridae, many structural and molecular features and phylogenetic relationships with other phenuiviruses support its classification in this family. With respect to nsRNA viruses, there is only one preliminary report of tomato spotted wilt virus in this host ; however, later studies failed to transmit TSWV to grapevine. During the screening of two selections of Garan dmak and Muscat rose grapevines via HTS, partial sequences with homology to apple rubbery wood viruses 1 and 2 were identified. ARWV1 and ARWV2 are two novel nsRNA viruses with tri-segmented genomes recently discovered in apple trees displaying rubbery wood symptoms in Canada. Based on sequence identity and phylogenetic analysis, ARWV1 and ARWV2 were proposed to be representative members of a new genus, tentatively named Rubodvirus, within the family Phenuiviridae. Here, we report the natural occurrence and discovery of two novel nsRNA viruses, blueberry container size named grapevine Muscat rose virus and grapevine Garan dmak virus , in grapevine plants. Even more, this is the first evidence for the natural occurrence of phenui-like viruses in grapevine.

Later, reverse transcription PCR -based assays for the specific detection of both viruses were designed to investigate the prevalence in different grapevine populations. Phylogenetics and transmission of GMRV and GGDV were also investigated.In 2015, a new selection of grapevine, cultivar Garan dmak, was received as dormant cuttings from Armenia under a USDA Animal and Plant Health Inspection Service controlled import permit for inclusion in the Foundation Plant Services collection. Cuttings were propagated under mist and later transferred to single pots; all this within an insect-proof greenhouse enclosure. Six months after bud break, leaf tissue from the Garan dmak plant was collected for HTS as part of the plant certification program at FPS. Additionally, a Muscat rose grapevine at the USDA National Clonal Germplasm Repository but originated from Argentina was sampled and analyzed by HTS during a separate study about the genetic diversity of grapevine leafroll-associated virus 3. At the time of sampling, both grapevine cultivars did not display any known symptom associated with virus infection. Total nucleic acid extracts were prepared using a MagMax Plant RNA Isolation kit as per manufacturer’s protocol, but adjusting the amount of plant material based on the following molecular analysis. For RT-PCR, 0.2 g of tissue was homogenized in 2 mL of guanidine isothiocyanate lysis buffer PVP-40 using a Homex grinder. In the case of HTS, 0.7 g of tissue was homogenized in 7 mL of guanidine isothiocyanate lysis buffer. Subsequently, the quality of TNA extracts was verified using an 18S ribosomal RNA assay.

HTS analysis was performed as described by Al Rwahnih et al.. Briefly, aliquots of TNA from source samples were subjected to rRNA depletion and complementary DNA library construction. Later, cDNA libraries were sequenced using the Illumina NextSeq 500 platform using asingle-end 75-bp regime. Illumina reads were demultiplexed and adapter trimmed prior to analysis using Illumina bcl2fastq v2.20.0.422. Trimmed reads were subsequently de novo assembled into contigs using SPAdes v3.13. Generated contigs were compared against the viral database of the National Center for Biotechnology Information using tBLASTx. Novel virus contigs were initially identified by their conserved protein domains. Open reading frames were annotated using HMMER v3.1 to look for Pfam protein domains associated with viruses infecting land plants. In the case of Phenuiviridae-like contigs, the large RNAs were annotated with the bunyavirus RNA-dependent RNA polymerase domain. The medium RNAs were annotated with the viral movement protein domain. The small RNAs were annotated with the Tenuivirus/Phlebovirus nucleocapsid protein domain. The association between the small and the medium RNAs was investigated by BLASTn sequence similarity of their 50 ends. These contig sequences were subsequently confirmed by BLASTx hits to the NCBI nucleotide database which produced top hits to different accessions of ARWV2 and ARWV1. To complete the 50 end of each RNA segment present in GMRV and GGDV, the SMARTer RACE 5 0 /3 0 Kit was employed following the instructions provided by the manufacturer. In the case of the 30 ends, the methodology described by Navarro et al. was used, which involved the addition of a poly tail to the RNA template.In the last few years, as a consequence of the increasing application of HTS, many novel nsRNA viruses have been identified, most of which are from invertebrates. Thus, the classification of these viruses has been recently reassessed, with plant-infecting viruses now classified in the order Bunyavirales, Serpentovirales and Mononegavirales .

Recently, the tentative genus Rubodvirus has been officially proposed to classify two novel nsRNA viruses from apple trees, with a suggested species demarcation criteria of <95% aa identity for the RdRp. In the present study, HTS allowed the identification of two novel plant-infecting viruses with segmented nsRNA genome, GMRV, and GGDV, which are also the first nsRNA viruses identified and transmitted in grapevine. Although these viruses infect the same host species, the aa sequence identity between the putative proteins encoded by their genomic RNAs is always below 75% , indicating that GMRV and GGDV are two different viruses. Their genomes encode proteins showing the highest sequence identity with ARWV1 and ARWV2. GGDV and GMRV also share other traits with these viruses, including the number of genomic components, limited to three nsRNAs, identical terminal nucleotides in the genomic RNAs, and the lack of any ORF coding for glycoproteins. Moreover, close phylogenetic relationships between these four viruses are supported by the ML phylogenetic trees reported here, in which, independently of the considered protein , they always clustered in the same clade, which is significantly separated from all the other nsRNA viruses included in the analyses. Altogether these data support the classification of GMRV and GGDV as two novel species in the tentative genus Rubodvirus. When the other bunyavirales are considered, the ML phylograms inferred from the RdRps or the NPs show a close phylogenetic relationship between rubodviruses and coguviruses, which together with the arthropod-infecting LLV, form a superclade nested at a basal node, in closer proximity to arthropod-infecting viruses than other plant-infecting viruses. These data are consistent with the hypothesis, previously advanced for the coguviruses, that all the members of this superclade evolved from a common ancestor virus infecting arthropods. In this evolutionary scenario, the acquisition of the MP gene appears to be the key step in the adaptation of the ancestor virus to plants, an event that likely happened through the typical modular genome evolution process proposed for most eukaryotic viruses. However, the clustering of rubodviruses and coguviruses in two distant clades, observed in the ML tree inferred with MPs , supports the independent acquisition of the MP gene by the ancestor of the viruses included in these two taxa. These data are consistent with the hypothesis that the adaptation of invertebrate-infecting nsRNA ancestor viruses to plants happened several times through independent events during the evolutionary history of nsRNA viruses infecting plants. It is worthy of note that rubodviruses, laulaviruses, and coguviruses, although phylogenetically related, have divergent genome structures and gene expression strategies. In fact, the members of the first two genera have a genome composed of three monocistronic nsRNAs encoding different proteins;RdRp, NP, and putative MP in the case of rubodviruses, and RdRp, NP and a protein of unknown function in the case of laulaviruses. Instead, coguviruses have a bipartite genome consisting of one nsRNA encoding the RdRp , and one ambisense RNA , in which the ORFs encoding the NP and the MP are separated by a long intergenic region .

It has been shown that such an IR is AU-rich and self-complementary, thus assuming in both polarity strands a compact conformation containing a long hairpin predicted to serve as a transcription termination signal during the expression of viral genes. Taking this into consideration, growing raspberries in container the question arises of how viruses with such different genomic organizations may have evolved from the same ancestor virus. In this respect, it can be speculated that a recombination event between the viral and the vc strand of two genomic RNAs with long, AU rich, and almost identical 50 UTRs could generate ambisense RNAs containing an IR similar to those of coguviruses. Since nsRNA viruses with genomic RNAs showing structural features compatible with this possibility were not known previously, such a possibility appeared unlikely. However, the very long, AU-rich, and highly conserved 50 UTRs reported here for RNA 2 and RNA 3 of GMRV and GGDV, and also observed in the corresponding RNAs of ARWV1 and ARWV2, are the first clear evidence that nsRNA viruses with the structural features compatible with this evolutionary scenario may exist. Based on these considerations, the possibility that the bipartite genomes of coguviruses originated from a tripartite ancestor with genomic RNAs containing 50 UTRs similar to those observed in the rubodviruses appears feasible. No glycoprotein is encoded by rubodviruses , a feature previously reported also for coguviruses that, according to electron microscopy observations, are flexuous, non-enveloped viruses. In contrast, glycoproteins are expressed by most nsRNA plant viruses transmitted by arthropods . The lack of glycoprotein in the genome of rubodviruses and coguviruses opens the question on the existence of vectors, if any, involved in their transmission. In this respect, it is worthy of note that ophioviruses and varicosaviruses, which are plant-restricted or transmitted by fungi, also do not code for any glycoprotein. Vegetative propagation has been proposed as the prevalent transmission mechanism for ARWV1 and ARWV2. Whether, this is also the case for GMRV and GGDV needs further investigation. Most plant viruses code for viral suppressor proteins counteracting the plant antiviral defense mechanisms based on RNA silencing. Further studies are needed to ascertain whether one or more of the three proteins encoded by GGDV and GMRV and other rubodviruses may interfere with RNA silencing, thus showing multifunctional role, as already reported for VSRs of other viruses. GMRV and GGDV were identified in two different grapevines cultivars, Muscat rose and Garan dmak, that were tested by HTS; moreover, both viruses were found in association with other viruses and viroids. Interestingly, no obvious symptoms were observed in the two grapevines. Although infectivity of both viruses was ascertained by graft-transmission, only a Cabernet franc grapevine infected by GGDV developed symptoms post-grafting; further HTS analysis on this indicator plant reveals the presence of GRSPaV, GYSVd-1, and HSVd. Therefore, it was not possible to ascertain whether GGDV is associated with symptoms and additional studies on its pathogenicity are needed, likewise GMRV. An initial survey using several accessions of grapevine located in three different collections in California resulted in the identification of two and one plants infected by GGDV and GMRV, respectively; like the original sources of the viruses , these grapevines were symptomless. A more extensive survey, including other grapevine-growing regions in California and the USA, is necessary to determine the real distribution of these novel viruses. In that sense, the detection method developed in this study could be useful for virus testing and certification programs. Finally, during the review process of this manuscript, two novel mycoviruses related to coguviruses and rubodviruses were reported, which extends the host range of phenui-like viruses.Molecular networking1 , introduced in 2012, was one of the first data organization approaches to visualize the relationships between tandem mass spectrometry fragmentation spectra. In molecular networking, relationships between similar MS/MS spectra are visualized as edges. As MS/MS spectral similarity implies chemical structural similarity , chemical structural information can thus be represented as a network and chemical relationships can be visualized. This approach forms the basis for the web-based mass spectrometry infrastructure, Global Natural Products Social Molecular Networking which sees ~200,000 new accessions per month. Molecular networking has successfully been used for a range of applications in drug discovery, natural products research, environmental monitoring, medicine, and agriculture. To tap into the chemistry of complex samples through metabolomics, a subset of MS/MS spectra can be annotated by spectral library matching or by using in silico approaches. While molecular networking facilitates the visualization of closely related molecules in molecular families, the inference of chemical relationships at a dataset-wide level and in the context of diverse sample metadata requires complementary representation strategies. To address this need, we developed an approach that uses fragmentation trees and machine learning to calculate all pairwise chemical relationships.

Tongue length appears to be important for structuring foraging preferences but we are lacking experimental work that evaluates how traits such as tongue length influence pollinators’ response to competition. We systematically manipulated interspecific competition in bumble bee communities through targeted single species removals and examined patterns of species-specific plasticity in resource use in the remaining pollinators. Specifically we examined to what extent tongue length explains species-specific difference in the pollinators’ foraging behavior in response to release from competition. Our sites are self assembled communities with natural diversity varying in plant and bee community composition. Utilizing removals in these natural communities allowed us to examine if the identity of the most abundant bee species, influences bee foraging behavior. We focused on the foraging response of the remaining bees in the community with respect to floral fidelity, or, within plant species movements within a single foraging bout. Floral fidelity is critical for many plants species’ reproductive success because transfer of conspecific pollen must occur in order for fertilization to take place.Study Sites: We worked in 28 subalpine meadow sites in the landscape surrounding the Rocky Mountain Biological Laboratory , plastic potting pots in the Gunnison National Forest, western Colorado, United States.

Eachsite consisted of a 20 × 20-m plot, all with the same dominant plant species to minimize, as much as possible, plant-community-driven differences in foraging behavior. A minimum distance of 1 km separated any two sites. We collected data over three summer growing seasons , in 2010, 2011 and 2013. Manipulations. We assessed each plot in a control state, waited one day, and then assessed in a manipulated state. We kept the interval between control and manipulated states short because of the rapid turnover in flowering phenology in our high-altitude system, allowing us to keep the plant community constant in our control–manipulation comparisons . Manipulations reduce interspecific competition through the temporary, nondestructive removal of the most abundant bumblebee species in each plot. We determined the most abundant bee via inventory of Bombus species richness and abundance on the control day using nondestructive aerial netting, with two field team members netting for a 20-min period, not including handling time . To avoid double-counting, we kept each bee in an individual glass vial, identified to species, and kept in a cool, dark cooler until the inventory time period was over, at which point bees were released. On the manipulation day we removed the most abundant bee species . The removals were accomplished through targeted hand-netting, and we minimized disturbance of other bees and vegetation by carefully placing the insect net over entire inflorescence and allowing bee individuals to fly up into the net . Captured bees were transferred to vials and placed in a cooler during the manipulation and released unharmed afterward.

We used as much time as necessary to remove essentially all individuals of the target species from the sample plot and immediately adjacent area . We left a period of at least 30 minutes between manipulative bee removals and subsequent sampling to minimize the impact of the disturbance on the foraging activities of other bees. We recorded both the abundance of removed individuals, as well as the number of un-captured “escapees” that were observed during bee sampling. Each site was only used once in a manipulated and controlled state per year . Foraging observations: We directly followed the foraging sequences of Bombus individuals in both the control and manipulated states. We recorded the identity of each plant species visited in a foraging sequence. We discontinued an observation when the bee was lost from sight, when it ventured more than 5 m outside of the plot, when it had been observed for 10 full minutes, or when we had tallied 100 individual plants visited. We discarded observations of bees that visited fewer than five plants. The number of individuals observed per/state/site varied due to bee abundance .Tongue Length measurements: The data on proboscis lengths of workers was taken from published measurements of bumblebees collected on the Front Range of the Colorado Rocky Mountains and overlap with the sites and species that we used for this study . Measurements indicate the sum of the individual lengths of the prementum and glossa. The mean tongue length of each bumble bee species was assigned to each individual bee and used in the trait analysis .

Floral fidelity was measured as the binomial counts of individual bee foraging movements that were conspecific vs. heterospecific . We used GLMMs with binomial errors using the logit link in the lme4 package for R to model the floral fidelity response variable . Data from individual bees foraging within a site cannot be considered independent and therefore we used site as a random effect . Relative to a binomial distribution, our data were overdispersed, which we corrected by including an individual-level random effect . We used the R statistical programming language for all models.Our results demonstrate that bees vary in their floral fidelity and that tongue length explains a large part of this variation. Bees with shorter tongues move between plant species more often than bees with longer tongues. We did not find significant variation in the response of bee species to a reduction in interspecific competition, but rather saw a guild-wide reduction in floral fidelity in response to the removal of the dominant bee species . Finally, our results suggest that tongue length of the most abundant bee species, a site-level attribute, explains much of the site-to-site variation in pollinator foraging behavior. In particular, we found that as the tongue length of the most abundant bee increases, the site level foraging fidelity decreases. We found that bumble bee species vary in the degree to which they move between different plant species within a single foraging bout, and tongue length explains much of the variation. Some suggest that long tongued bees should exhibit broader resource usage patterns because their traits permit them access to a wider range of flower types . In contrast, short-tongued bees should act as specialists, with a more restricted range of resource use options, rarely able to access the nectar at the base of the flowers with long corollas . Our results suggest the opposite pattern, that shorter tongue bees are more labile with their foraging patterns and on average move between plant species within a single foraging bout more often than longer tongue bees. We suggest the following interpretation, because long tongues enable bees to access flowers with better rewards and maintain a monopoly on those rewards, they may have less incentive than short-tongued species to move between plant species while foraging. While longer tongue bumble bees are capable of foraging on flowers with short corollas it would provide less energetic gain, potentially making behavioral plasticity less profitable . In contrast, the shorter-tongued bees in our system tend to have smaller bodies and are more likely to depend on resources within a more restricted foraging range . These limitations could favor a labile foraging habit, with shorter tongue bees constantly assessing the resource availability and competitive context in their community. As such, shorter-tongued bees more readily switch between plant species. While we saw little variation in species-specific foraging responses to our manipulations, we found an overall reduction in floral fidelity across sites after the removal of the most abundant bee species. Our results build on the findings of Brosiand Briggs , reaffirming a guild-wide reduction in floral fidelity in response to a reduction in interspecific competition. This study adds an additional two years, 8 sites and 165 bee individuals to our previous study, confirming that the guild-wide results found in Brosi and Briggs are robust. Variation in bumble bee floral fidelity is largely explained by the tongue length of the most abundant bee species in each site. This means that pollinator foraging behavior is context dependent and is determined by the most abundant bee species. In general, raspberry container growing short tongued bees exhibit lower floral fidelity than long tongued bees, and when they are in a site that has a long tongued bee removal, their reduction in floral fidelity is magnified. Bumble bees are large bodied insects that require many floral resources to keep their hive growing throughout the growing season.

As such, we might expect strong competition between these species, and tongue length, arguably the trait most relevant for resource acquisition, could dictate how resources are partitioned within a community, ultimately drive the assembly of bumble bees within communities . Pyke proposed that bumble-bee species with similar tongue lengths could not exist in his altitudinal alpine transects presumably because the bees compete for floral resources. But later studies did not support this pattern , and, as in our study, found that bumble bee species with similar tongue lengths co-ocurred within a community. This has left researchers to wonder if the coexistence of many bee species with substantial overlap in their life history requirements is possible because bumble bee species compete for something other than flower resources allowing so many similar species to co-occur . Our study suggests that in our system, bumble bees do in fact compete for floral resources and that longer tongue bees seem to elicit competition that is experienced across the range of trait values seen in our sites . The willingness of short tongue bees to exhibit behavioral plasticity may allow for such a large number of seemingly similar bee species to coexist in a community. Future work should examine the extent to which this plasticity is adaptive and assess the fitness costs that may result from the willingness to switch floral resources in response to a reduction in interspecific competition. Most pollinators are generalist foragers that can switch between plant species within a single foraging bout . When pollinators move between plant species, they can transfer heterospecific pollen to plant stigmas which in turn can reduce plant reproduction . We found an effect in which competition from a long tongued bee changes the foraging behavior of the rest of the bees in a way that could be detrimental to plant reproduction. From a plant’s perspective, not only do short tongue bees exhibit behavior that likely results in the transfer of heterospecific pollen, but when short tongue bees are in communities in which a longer tongue bee is most abundant, they exhibit even greater floral infidelity, making the likelihood of heterospecific pollen deposition even greater .Pollinator species are on the decline globally . Bumble bees in particular are experiencing population range contractions due to climate change as well reductions in abundance due to disease , agricultural intensification and pesticide use . In a changing world where we are likely to experience an emergence of new interactions via range shifts, introduced species and climate change, exploring how the competitive landscape shapes foraging plasticity will help us generalize to other plant pollinator systems and begin to better predict the functional implications of competitive interactions.Pollinator losses are increasing across the globe which could have potentially strong negative effects on the plants that rely on them for pollination . Network-based simulations suggest that plant communities will be very robust to pollinator extinctions . This robustness is likely driven by two features of network structure. First, pollination networks are dominated by generalist interactions; most plants and pollinators each interact with several species from the other group over the course of their lives . Second, pollination networks have a nested structure in which specialist plants and pollinators tend to interact with a subset of the species in the other group that generalists interact with . Nestedness leads to an asymmetric interaction structure where specialists from one group tend to act with generalists, not specialists, from the other, which could reduce linked extinctions if specialists are vulnerable to stochastic extinctions . One feature of binary-graph plant-pollinator simulation models that may overestimate network robustness is that all interactions in a binary-graph network are positive. A typical binary-graph simulation modeling approach is that if at least one link remains between a plant and a pollinator, the plant will continue to persist . While the assumption of “all interactions positive” is a reasonable starting point in a mutualistic network model, empirical evidence suggests at least two ways in which pollinators can have negative consequences for the reproduction of plants they interact with . First, the benefit that pollinators have on plant reproduction is sensitive to how “faithful” pollinators are to particular plant species in a single foraging bout.

It is now recognized that these mature hedgerow plants can be a source of locally grown elderberries and elderflowers to increase income and sustainability for the farm. However, to date there is no data on the concentration of the aroma or phenolic compounds in the flowers from this hardy heat-tolerant subspecies. The berries, flowers and bark of the elderberry plant have a long history of use by humans as both food and traditional medicine. Seeds have been found in archeological sites that date to the late stone age and their medicinal use is documented in the writings of The ophrastus , Pedanius Dioscorides and Gaius Plinius Secundus . Elderflowers are frequently used in medicinal and herbal teas, tonics, liqueurs, lemonades, and sparkling waters for their subtle and unique floral, fruity, and green aromas and medicinal properties. Infusions of elderflowers have been used in many cultures for the treatment of inflammation, colds, fever, and respiratory illness and for their diuretic and antidiabetic effects. Some studies have found evidence to support their use, such as antimicrobial activity of elderflower extract against Gram-positive bacteria and high vitro antioxidant activity. Much of the interest for using elderflower in health-promoting applications is based on the high content of biologically active phenolic compounds in the flowers. European and American elderflowers contain an array of phenolic compounds, such as phenolic acids , flavonols , flavonol glycosides [isorhamnetin-3-O-rutinoside , rutin ], flavan-3-ols [-catechin, -epicatechin], and flavanones. In European-grown elderflowers, nursery pots the dominant phenolic acid and flavonol glycoside include chlorogenic acid and rutin, although isoquercetin, isorhamnetin-3-rutinoside and kaempferol-3-rutinoside are also present. 

5-Hydroxypyrogallol hexoside and protocatechuic acid dihexoside were identified for the first time in elderberry, which could potentially serve as markers of this subspecies in products that use blue elderberry. There was considerable variation within and between hedgerows in both harvest years, but this appears to be a common attribute for the elderberry species. Blueelder berries have many ecological benefits for farms when planted in hedgerows, grow well in challenging environments, are not killed by wildfires and can therefore, serve as a sustainable source of an increasingly popular fruit.The elderberry is a deciduous, multi-stemmed shrub or small tree. It can grow several meters high and in diameter and produces hundreds of clusters of aromatic flowers in the spring, that mature into small berries in summer. The plant grows well in a variety of soils and climates, and is a native of Northern America, Europe, and parts of Asia. While there are many subspecies within Sambucus nigra, the primary subspecies widely grown and commercially cultivated include S. nigra ssp. nigra found across Europe, and the “American” subspecies S. nigra ssp. canadensis, which is native to the eastern regions of North America.56 The blue elderberry , is a drought-tolerant subspecies native to the western region of North America. The blue elderberry grows in riparian ecosystems from southern British Columbia, Canada to northwest Mexico.84 In California, there have been efforts for more than a decade to increase the levels of blue elderberry planted in hedgerows on farms because of its environmental benefits, such as improving the air, water, and soil quality, as well as providing food and shelter for pollinators. 

For example, in a study of European elderflowers grown in different locations and altitudes, the dominant class of phenolic compounds were the flavonols, namely rutin , whereas chlorogenic acid levels were lower . This study also found that the flowers contain four times more chlorogenic acid than the leaves or berries. The predominant phenolic compounds identified in elderflower syrup, a traditional herbal beverage, include chlorogenic acid and rutin . There has been only one study on the phenolic profile of the flowers of S. nigra ssp. canadensis which appears to be similar to the European subspecies, in that rutin and chlorogenic acid are the primary flavonol and phenolic acid identified, respectively. The aroma of the elderflower is derived from the volatile organic compounds in the flower and is an important characteristic to understand for consumer acceptance in applications.To date, only the VOCs of elderflowers from the European subspecies have been studied. The American subspecies S. nigra ssp. canadensis has not yet been investigated. As fresh flowers are highly perishable, many commercial products rely on dry, and in some cases, frozen flowers. Thus, it is important to understand how the organoleptic properties of elderflowers change in response to processing. The VOC profile of tea made with elderflowers of three European cultivars using dynamic headspace sampling revealed compounds important to the characteristic aroma to be linalool, hotrienol, and cis– and trans-rose oxide. Similarly, studies indicate that in fresh and dried flowers analyzed by headspace solid phase microextraction coupled with gas chromatography mass spectrometry , linalool oxides are the main aroma compounds. Linalool oxide has a floral, herbal, earthy, green odor. In hexane extracts of dry elderflowers analyzed via HS-SPME/GC-MS, cis-linalool oxide and 2-hexanone were the primary volatiles. The compound 2-hexanone has a fruity, fungal, meaty, and buttery odor. In syrups made from elderflowers, terpene alcohols and oxides were identified as the primary aroma compounds. Studies of the impact of drying on volatiles in the flowers demonstrate that nearly all types of drying change the volatile profile significantly. The aim of this study was to characterize the composition of phenolic compounds and VOCs in flowers of the blue elderberry , and to determine how these compounds change in response to drying and in the preparation of teas. Understanding how the aroma and phenolic compounds compare with current commercially available European and American subspecies will help to establish a role for blue elderflowers in commercial applications such as herbal teas and as a flavoring for beverages, as well as identify unique compositional qualities of this native and underutilized flower.LC/MS-grade acetonitrile and HPLC-grade hydrochloric acid were purchased from Fisher Scientific . HPLC-grade ethanol and acetonitrile were purchased from Sigma Aldrich . Purissimum grade phosphoric acid was purchased from Sigma Aldrich and filtered through 0.45 µm polypropylene filters under vacuum. Ascorbic acid was obtained from Acros Organics . Ultrafiltered water was obtained by a Milli-Q system . Analytical standards of rutin, quercetin, chlorogenic acid, and -catechin were purchased from Sigma Aldrich . A standard of n-butyl-d9 was purchased from CDN Isotopes . Kaempferol-3-O-rutinoside, isorhamnetin-3-O-glucoside, IR, and isoquercetin were purchased from ExtraSynthese .Elderflowers were harvested from hedgerows on a farm in Winters, CA in May and June 2021. The latitude and longitude coordinates of the hedgerow are 38.634884, -122.007502. Flowers were harvested between 8 and 10 am and were picked from all sides of the shrub. Picked flowers were placed in plastic bags, immediately put on ice, and transported to the laboratory at the University of California, Davis. Flowers were either dried at 25 °C for 24 h in a dehydrator or analyzed fresh. Once dry, stems were removed, and flowers were stored in oxygen-impermeable aluminum pouches. Triplicate samples of fresh flowers were analyzed for their moisture content by drying 1 g of fresh flowers at 95 °C until a consistent weight was achieved so that the same amount of dry matter could be used for fresh and dry flower analyses.

The optimal mixture of ethanol to water was determined by extracting flowers in 0, 25, 50, 75, large pots plastic and 100% ethanol. Solvents also contained 0.1% HCl and 0.1% ascorbic acid . For each extraction, 0.25 g dry flower material and 25 mL solvent were added to 50 mL Eppendorf tubes. The dry flowers with solvent were homogenized for 1 min at 7000 rpm with a 19 mm diameter probe head in the 50 mL tubes. Homogenized extracts were refrigerated overnight at 4 °C, then centrifuged at 4000 rpm for 7 min . The supernatant was filtered through 0.45 µm PTFE, then diluted 50% with 1.5% phosphoric acid before analysis. Three replicates were made for each extraction condition . Phenolics were extracted from fresh and dried flowers that were either whole or homogenized. Hence, four types of samples were made: fresh whole flowers , dry whole flowers , fresh homogenized flowers , and dry homogenized flowers . Flowers were mixed with the determined optimal extraction solvent and followed the same extraction process as described above, except whole flower samples were not homogenized and instead placed directly into the refrigerator to extract overnight.All sample extracts were analyzed via high performance liquid chromatography using an Agilent 1200 system with diode array detection and fluorescence detection . Separation of phenolic compounds was performed on an Agilent PLRP-S column at 35 °C, using a previously published method. 116 Mobile phase A was 1.5% phosphoric acid in water and mobile phase B was 80% acetonitrile, 20% mobile phase A . The flow was set at 1.00 mL min-1 . The gradient used was as follows: 0 min, 6% B, 73 min, 31% B, 78-86 min, 62% B, 90-105 min 6% B. Most phenolic compounds were detected using a at 280 nm , 320 nm , and 360 nm . Flavan-3-ols were detected using a fluorescence detector . Compounds were quantified using external standard curves employing surrogate standards for each group of phenolic compounds [-catechin for flavan-3- ols, chlorogenic acid for phenolic acids and simple phenols, quercetin for flavonol aglycones, and IR for flavonols]. Standards were prepared at concentrations of 200, 100, 50, 10, and 5 mg L -1 , except IR which included an additional concentration of 500 mg L -1 . Triplicate analyses of each concentration were performed .Compounds were separated using HPLC-DAD-FLD as described above and identified using authentic standards to check retention time and absorption spectra. Several peaks in the chromatograms did not match tR or spectra of authentic standards. Therefore, fractions of these peaks were collected. Fractions were dried and reconstituted in 1% formic acid in water. These samples were then subjected to high resolution mass spectrometry using an Agilent 6545 quadrupole time-of-flight mass spectrometer , using conditions previously established for elderberry phenolic compounds. Data were then analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 . To tentatively identify compounds, the mass to charge ratio of the precursor and fragment ions were compared to online libraries of compounds and using formula generation for the peaks in the spectra.Volatile compounds were analyzed by headspace solid phase microextraction gas chromatography mass spectrometry . The equilibration and extraction parameters were optimized using ground dry flowers, prepared using a spice grinder, pulsed 25 times . A 1 g sample of ground dry flowers was placed into a 20 mL glass vial and the vial was sealed by a crimp-top cap with a Teflon septa. Various incubation temperatures , equilibration times , and extraction times were evaluated to optimize for the highest total peak area and unique compounds identified from samples. The fiber used for all analyses was a divinylbenzene/carbon wide range/polydimethylsiloxane , 23 Ga, 1 cm length, with 80 µm phase thickness . After extraction, the fiberwas injected into the GC and volatile compounds were desorbed at 250 °C for 5 min. Compounds were then separated on a DB-Wax column . Helium was used as a carrier gas at 1 mL min-1 . A temperature program was used with the following steps: 35 °C for 1 min, 3 °C min- 1 to 65 °C, 6 °C min-1 to 180 °C, 30 °C min-1 to 240 °C, hold at 240 °C for 5 min. Total run time was 37.167 min. Compounds were detected with a single quad, triple axis mass spectrometer . The mass range for acquisition was 30 to 300 m/z. The MS transfer line temperature was 250 °C, the source temperature was 230 °C, and the quad temperature was 150 °C. The electron ionization was set to 70 eV. To have the same volume of headspace in fresh and dry flower samples, 0.5 g of fresh whole flowers or 1.5 g ground dry flowers were placed in the 20 mL clear glass vials. For tea samples, 4 mL tea was placed in 20 mL vials. To each sample, 10 µl of 1-butanol-d9 in methanol was added as an internal standard. Volatile compounds were identified using Agilent Mass Hunter Unknown Analysis , using the NIST17 library requiring an ≥ 80% match and that compounds were identified in at least three of the five to be considered a volatile compound in the samples.

The content of tartaric acid in grapes can vary depending on cultivar, ripeness, and environmental conditions; for example, Kliewer et al. reported a tartaric acid content ranging from 3.7 to 13.2 g/L in different cultivars and from 3.4 to 9.2 g/L in early- vs. late-harvested cultivars. The doses used here covered these reported ranges. Each treatment had 20–22 replicates, and each replicate started with 10 D. suzukii eggs from the laboratory culture that were placed in drosophila vials over the diet. The number of developed adults was recorded. A sub-sample of 25 pupae from each treatment was measured for pupal length and width , and the volume of each pupa was estimated based on the formula 2.A separate analysis with 10 different cherry cultivars did not yield a significant effect of the Brix on the percentage of eggs that successfully developed to adults, although we could not rule out the possibility that Brix and other chemical properties may affect other fitness parameters of the developed flies. Many of the differences in chemical traits among different fruits could be attributed to geographic location and differences in environmental and cultivation conditions rather than inherent varietal properties, such as in cherries. In the current study, drainage gutter chemical differences were controlled to some extent, as cherry cultivars used were grown in the same plot with the same fertilization and irrigation regimes. The physical properties of different cultivars did not seem to affect the fly’s oviposition.

The percentage of eggs that developed to adults decreased with the increasing egg density per gram of fruit probably due to intra-specific competition, and this was further confirmed by manipulating the egg density and using the same ‘Bing’ cherry cultivar as the tested host. Females preferred larger fruit for oviposition, which is consistent with the density-dependent survival as the large fruit support higher numbers of fly larvae per fruit. It is well known that many fruit flies employ a variety of fruit characters to assess host quality and tend to be more attracted to larger fruits. Female D. suzukii appears to be able to assess host quality based on fruit size, and this behavior would likely increase foraging efficiency per unit time. Though we recovered very low numbers of D. suzukii from damaged citrus fruits, our laboratory study showed the fly can oviposit into and develop from freshly damaged or rotting navel oranges. Kaçar et al. showed that D. suzukii overwinter in citrus, surviving 3–4 months when fresh oranges were provided as adult food or ovipositional medium, and field-emerged adults from soil-buried pupae could produce and oviposit viable eggs on halved mandarin fruit. Thus, citrus fruit likely play an important role as reservoirs in sustaining the fly populations during San Joaquin Valley winter seasons, and in the spring, those populations may migrate into early season crops, such as cherries. We did not observe grape infestation in our field collections, and our laboratory trials showed a low survival rate of D. suzukii offspring on grapes when compared to other fruits . The oviposition susceptibility and offspring survival could vary among varieties or cultivars due to the variations in skin hardness and chemical properties. For example, Ioriatti et al. demonstrated that oviposition increased consistently as the skin hardness of the grape decreased.

Chemical properties, such as sugar content and acidity levels, may play a role in host susceptibility. In the current study, we found that although table grapes had a tougher skin than raisin or wine grape cultivars tested , females were able to lay eggs into all three types of grapes, often through the fruit surface or near the petiole . The sugar levels of all tested grapes were either equal to or considerably higher than other fruits tested. We also found that tartaric acid concentration negatively affected the fly’s developmental performance. Still, about 20% eggs successfully developed to adults in the diet mixed with the highest tartaric acid, whereas only 4.5% eggs developed from the wine grape cultivar tested. It is thus possible that other unknown chemical traits might also affect larval performance. Overall, our results are consistent with other reported studies that grapes are not good reproductive hosts for D. suzukii .California’s San Joaquin Valley is one of the world’s most important fruit production regions, with a diverse agricultural landscape that can consist of a mosaic of cultivated and unmanaged host fruit crops. Such diverse landscapes result in the inevitable presence of D. suzukii populations that represent a difficult challenge for the management of this polyphagous pest. We showed that only the early seasonal fruits, such as cherries, seem to be at greatest risk to D. suzukii Many of other later seasonal fruits are not as vulnerable to this pest, because either their intact skin reduces oviposition, they ripen during a period of low D. suzukii abundance, or their flesh has chemical attributes that retard survival. However, some of these alternative hosts—such as citrus and damaged, unharvested stone fruit—may act as shelters for overwintering populations and provide sources for early populations moving into the more susceptible crops.

Consequently, area-wide management strategies may need to consider fruit sanitation to lessen overwintering populations, suppressing fall and winter populations by releasing natural enemies, and reducing pest pressure in susceptible crops through ‘border-sprays’ and/or ‘mass trapping’ to kill adults before they move into the vulnerable crop. Alternative and sustainable area-wide management strategies such as biological control are highly desirable to naturally regulate the fly population, especially in uncultivated habitats. An understanding of the temporal and spatial dynamics of the fly populations would be of aid in the optimal timing of the future release of biological control agents to reduce the source populations in the agricultural landscape.The woody plant pomegranate produces colorful flowers and fruits with ornamental and culinary values. Different pomegranate tissues have historically been used for alleviating symptoms or treating various diseases due to the accumulation of a wide diversity of bioactive metabolites . In recent years, pomegranate fruits and juice have been pursued by consumers for their favorable nutritional quality, contributed by the abundant phenolic compounds, e.g., hydrolyzable tannins and flavonoids, in these tissues and products. Genetic variations underlying different metabolite profiles reportedly exist in pomegranate and have been utilized for breeding cultivars with desirable traits. Complementary to the classic breeding approach, new molecular techniques, such as genome editing, can enable targeted modification of key metabolic genes for improved nutritional and commercial quality of pomegranate fruits and products. Among the various genome-editing technologies, Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR-associated protein 9 has gained increasing popularity for its efficiency and ease of use. In this method, a single guide RNA directs the Cas9 nuclease to the target gene sequence upstream of a protospacer adjacent motif. Cas9 creates a break in the double-strand DNA, which is then ligated by homology-directed repair or non-homologousend joining. In general, five genotypes can be obtained from the CRISPR/Cas9-mediated genome editing in a diploid species, including wild type , homozygous mutant , heterozygous mutant/monoallelic , biallelic , and chimera . Initially used for disruption of gene function, there has been rapid advancement in the CRISPR/Cas9 technology for more precise and versatile genome editing. Although CRISPR/Cas9 has been successfully adopted in many plant species , its application has not been reported in pomegranate. In consideration of the time and effort required for transformation and regeneration of pomegranate plants, plastic gutter we chose a hairy root system for testing the feasibility and efficacy of CRISPR/Cas9-mediated genome editing in pomegranate. This is because hairy roots can be induced from different pomegranate explants, accumulate HTs and other phenolic compounds, are transformable, and produce sufficient amounts of tissues for molecular and metabolite analyses within 3 months of transformation. Commerce via global trade and transport provides a mechanism for introduction of invasive species to new territories, extending pest habitats outside of their native regions . Invasive species threaten biodiversity, habitat, nutritious food, clean water, resilient environments, sustainable economies, and human health . Agricultural production systems are continuously challenged by invasive species that attack high-value crops, thereby significantly hampering the ability of food industries to maintain profitability . The geographic range of agricultural crops provides the potential for invasive species to colonize regions on a global scale . Factors that aid expansion include short life cycle, fast growth rate, high plasticity, and resiliency to a wide range of environmental conditions .

Such factors are drivers of rapid evolutionary change, population increase, and global colonization . Practitioners and stakeholders should aim to implement new strategies to manage such new invasive species in agricultural production . Drosophila suzukii Matsumura is an invasive species native to Southeast Asia. Passive transportation is the main reason of the dispersal of this species . It was first detected in North America and Europe in 2008 , and later in South America in 2013 , and Northern Africa in 2017 . The long-serrated ovipositor of D. suzukii enables it to oviposit inside fresh fruit, which creates a challenging management problem . When D. suzukii became established in the U.S. during 2008, the total annual revenue losses for the West Coast berry and cherry industries were estimated at over $500 million . Currently the situation is not changed in term of economic impact . This particular insect is challenging to manage due to its high dispersal potential, ability to survive and adapt to harsh environmental conditions, and ability to attack a wide host range. For these reasons, D. suzukii is a key pest of these fruit industries worldwide. In the last decade, conventional insecticide uses on affected crops significantly increased to manage D. suzukii fruit damage. Typically used insecticides include spinosyns, pyrethroids, and organophosphates . Intensive use of insecticides poses a tremendous risk to non-target organisms such as pollinators, natural enemies, and humans . In addition, frequent insecticide applications likely resulted in resistance development . These factors require development of an IPM program that includes alternatives to conventional insecticides for managing D. suzukii. Non-insecticidal control methods including cladding, irrigation, netting, mulching, pruning, monitoring and mass trapping have been implemented against D. suzukii . While each method provides some relief to D. suzukii pressure, they provide limited reductions in crop damage . Behavioral control of D. suzukii on susceptible fruit indicated promise for industry adoption. The food-grade gum possesses tactile and odorant cues resulting in reduced egg infestation. The food grade gum makes use of physical properties to mimic fruit, resulting in D. suzukii laying their eggs in a soft gel-like substrate, instead of the fruit itself. The food grade gum is a mixture offood-grade ingredients which is highly attractive to D. suzukii and competes with the ripening fruit throughout the season . To the best of our knowledge, the food-grade gum modifies various D. suzukii behaviors, ultimately resulting in a significant decrease in fruit damage. The product diverts D. suzukii away from ripening fruit, which results in significant retention of the pest, keeping it away from fruit. Third, the food-grade gum acts as an egg sink. Since the D. suzukii eggs laid in this medium cannot develop, this translates in a substantial reduction of the pest population growth . The aim of this work was to determine the potential of the foodgrade gum to reduce D. suzukii damage in large-scale commercial open-field and screenhouse fruit production units on blueberry, cherry, raspberry, blackberry, and wine grape. The hypothesis was that food-grade gum would reduce D. suzukii damage in small fruit, tree fruit and grapes under semi-field and small-scale field conditions. These studies were conducted during 2019 and 2020 in California and Oregon in the western United States.In all field trials, GUM dispensers were placed at least 27 meters away from untreated control plots to minimize volatile plume interaction between treatments. In the current study, cotton pads were used to apply ~1.8 g of GUM on each dispenser at the rate of 124 dispensers per hectare under commercial production conditions . Cotton pads were placed directly on the ground close to irrigation drippers to provide adequate daily moisture. Earlier work illustrated that dispensers have a field longevity of 21 days and for this reason, dispensers were therefore deployed 1 to 4 times depending on the duration of crop ripening and susceptibility. In three trials , egg laying data were collected in buffer plots that were located between UTC and GUM plots to determine the active range of released volatiles beyond treated areas.

USDA Sustainable Agriculture Research and Education director Jill Auburn reports that it is a struggle to maintain emphasis on the social “third leg of the sustainability stool” and to talk about social issues in a way that resonates with rather than alienates farmers . Still, the Center’s position is that we need to be able to work with both radical and mainstream groups to effect change. Currently, different groups have varying abilities to claim and provide resources in university programs, which has implications for who are considered legitimate audiences of these programs and which kinds of research agendas are likely to be funded. This creates a cycle of declining emphasis in certain areas. Since universities increasingly base decisions on the value of a person or program’s work on their ability to attract funding, this results in increased power and legitimacy for those able to do so. One consequence is that researchers are drawn to court those with money, who by definition are not marginalized people. University research agendas are shaped to a large extent by extramural funding. Traditionally, public funds are used to cover the base costs of the public research system, with private funding often covering discretionary research costs. For example, large round plant pots biotechnology companies now exert significant influence on the type of research that takes place through agreements with major campuses such as UC Berkeley.

Private or profit-driven funders are unlikely to fund research on the environmental or social justice issues that comprise the Center’s research agenda. Yet, precisely because groups such as farmworkers, the hungry, and capital-limited farmers are as members of a public university, with our salaries paid all or in part by the citizens of California, what is our role in the University of California system? What issues should we be addressing in our research? What audiences should we be serving in our teaching and outreach efforts? These are questions we continually ask ourselves as we plan our research and education programs. In the cover article, associate director Patricia Allen addresses these issues as we discuss the way that the Center’s work has taken on the challenges inherent in trying to meet the needs of those who have often been overlooked by traditional agricultural programs. This article is based on a paper that Allen and I prepared for a seminar I presented at Yale University’s colloquium series. In a related topic, social issues researcher Phil Howard addresses the idea of community-based research , in which community members identify problems for study. This type of research provides a way for academics to address pressing social and environmental issues, and to make their work more relevant to the broader community. Howard discusses some of the ways that the Center has worked with local growers and community groups to address their questions and concerns. Our work in developing resources for those teaching sustainable agriculture topics continues to grow.

This spring, curriculum specialist Albie Miles completed an online resource that links educators to a comprehensive course outline, catalogue description, and annotated resource lists for post-secondary instructors . This project grew out of Miles’s work developing our two popular teaching manuals, Teaching Organic Farming & Gardening: Resources for Instructors, and Teaching Direct Marketing & Small Farm Viability: Resources for Instructors. Another outgrowth of the curriculum development work is an upcoming conference focusing on sustainable agriculture education at two- and four-year colleges and universities . Convened by the Center and by UC Davis’s College of Agriculture and Environmental Sciences and Student Farm, the conference will address the status of sustainable agriculture education, course and program content, teaching methods, and much more. Our efforts to reach the gardening audience with the message of sustainable techniques also continue to evolve, thanks this time to a grant from the Stanley Smith Horticultural Trust. A portion of this grant funded a booklet on organic rose care for home gardeners, written by garden manager Orin Martin and excerpted here .Community-based research is “research that is conducted by, with, or for communities” . It stands in contrast to most research, which primarily addresses the needs of private businesses or focuses on esoteric scholarly subjects. Community-based research, on the other hand, is intended to benefit non-profit organizations or local governments that will use the results for practical and positive change .

One example of a community-based research process is the Dutch science shop system. In the Netherlands, every public university has a center that accepts research questions from community groups, and involves faculty or students in answering these questions . The first science shops were established in the late 1970s by Dutch faculty and student volunteers. They were active in social movements, such as opposition to nuclear power, and wanted to make the production of knowledge more democratic. These early science shops gained popular political support, which led to their expansion, as well as a small amount of government funding. There are currently 50 science shops in the Netherlands and they answer approximately 2,000 questions a year . The types of projects have ranged from requests from environmental groups to estimate the risks from pollutants to the exploration of social concerns such as understanding the causes of teenage disaffection. For a question to be addressed it must come from a group that can demonstrate it lacks the resources to conduct the research, and that it will make good use of the results. While early research was conducted primarily by faculty members, much of the work is now conducted by students who receive academic credit for their efforts. Because academic requirements typically require research and writing papers anyway, addressing community research questions has not increased the workload for students or professors. Unlike many student papers that end up in the recycle bin or on the shelf to gather dust, students’ research papers are used by the community organizations and government agencies that have commissioned them. This benefits these organizations, which often lack the resources of large corporations and governments, and students gain valuable real-world experiences .The Center for Agroecology and Sustainable Food Systems has been involved in a number of community based research efforts. For example, Center researchers have worked with growers to conduct field trials of organic production techniques and pest control strategies, and graduate students have been awarded grants by the Center to conduct surveys with the close participation of the farmers involved. In 2004, researchers at the Center worked with other UC researchers and nongovernmental organizations to establish the Activist Researcher Consortium . The purpose of ARC is to build an ongoing forum for discussion, research, and education among California academics and community organizations with an interest in working together toward a socially just food system. Research questions that were prioritized at the initial ARC workshop included – What are the barriers, or perceived barriers, to socially just businesses? Are businesses that try to incorporate social justice into their practices more likely to fail? How do socially just businesses that are economically successful internalize their ethical commitments? How does access to food differ by ethnicity and socioeconomic status? How does farmworker access to food compare with that of other groups of people? Efforts are currently underway for activists and researchers to begin addressing these important questions collaboratively. Center researchers are also working with community groups on the Central Coast of California to identify areas where large populations have limited access to food resources that are healthy, plant pots round culturally appropriate, affordable and sustainable. The results will help organizations target programs to increase food security in the region and may also identify promising markets for small-scale growers who have limited marketing opportunities. Center staff members are also participating in a studentled coalition at UCSC that is building support for a more ecologically sustainable and socially just campus food system. Finally, representatives of the Center worked with other organizations in Santa Cruz County to organize a community forum on the food system in February 2005.

This day-long meeting had an agenda that was determined entirely by the participants, and they raised a number of questions that they continue to investigate. Some of these inquiries included – What food policies from other areas would work well in our county? How can we help local farms while also getting healthy food to low-income people? How can we increase support for school gardens? The Center for Agroecology and Sustainable Food Systems plans to remain involved in community-based research as funding allows . However, as mentioned, there are very limited funds available for this type of research and for research on sustainable food systems in the United States. Currently, the community-based research conducted by the Center is funded primarily through a grant from the U.S. Department of Agriculture to examine Central Coast food and agricultural systems. Expanding this work will require additional support. With tax increases unlikely, this would mean a reallocation of existing public research funds and an increase in support for community-based research on the part of private donors and foundations.The Center’s social issues research was well represented at the annual joint meetings of the Agriculture, Food, and Human Values Society and the Association for the Study of Food and Society in Portland, Oregon this spring. Social issues specialist Patricia Allen, and researchers Phil Howard and Jan Perez presented results from a variety of Center projects. Allen and Perez discussed their study of consumers’ and food-system activists’ concepts of social justice in the food system—how these groups define social justice, their vision for it, how they think about it, and what aspects of social justice are important to them. This information will help organizations that are developing criteria for defining social justice within the food system, particularly those working to develop social justice-based criteria as part of certification labels. Howard and Allen presented results from a mail survey administered to consumers in the Central Coast region. In part, the survey measured whether consumers felt they had more power in a locally-based food system. Their findings showed that consumers who obtain foods at least once a week from direct, local sources scored higher on an index measuring “empowerment” as it relates to the food system. Those respondents who frequently purchased organic food did not score higher on the empowerment index. The index included such factors as the consumers’ perceived knowledge about their food, availability of information about their food, availability of food they wanted, satisfaction with choices available, and ability to change the food system. Allen, along with Carolyn Sachs of Penn State University, gave a paper that explores the conflict and ambivalence women experience in providing food for their families, their households, and themselves. They argue that food processors, supermarkets, and restaurants are profiting from the time squeeze that many women experience as they come to rely on convenience foods and pre-cooked meals. At the same time, the food industry and the fashion industry send conflicting messages to women, who are encouraged to indulge in junk food but expected to have “perfect” bodies. The conference also included a session focusing on Allen’s latest book, Together at the Table: Sustainability and Sustenance in the American Agrifood System, published last winter by Penn State University Press. The book distills more than ten years of Allen’s research on the discourses, agendas, and strategies of U.S. alternative food movements and institutions. Panelists highlighted the theoretical, substantive, and practical contributions of Allen’s book. “This workshop showed that both academics and activists find this research to be accessible and important to the future of alternative agrifood movements and institutions. It is extremely gratiflying to know that Together at the Table is considered both an important scholarly contribution and relevant for those working to create change in the American agrifood system,” says Allen.In the agricultural fields above Elkhorn Slough in northern Monterey County, a checkerboard of strawberry, vegetable, and cover cropped plots is yielding clues as to how best to grow strawberries organically while maintaining the health of the agroecosystem. UC Santa Cruz researcher Joji Muramoto oversees the complex study, which involves rotating strawberries with vegetable crops and cover crops in various intervals , and comparing strawberry yield and soil health with plots where strawberries are grown in consecutive years .

The utilization of entomopathogens against thrips is not a new concept; entomopathogenic fungi, such as, Metarhizium anisopliae Sorokin , Neozygites parvispora Remaudière & Keller , Verticillium lecanii Viegas , and Paecilomyces fumosoroseus Brown & Smith have also been used in laboratory and greenhouse trials with much success, whereas field trials have shown limited successes. However, various strains of B. bassiana have been shown to effectively control western flower thrips on greenhouse ornamentals and peppers , and several reports indicated that F. occidentalis, Thrips palmi Karny and T. tabaci Lindeman were successfully controlled under field or laboratory conditions .In conclusion, both citrus and avocado thrips can be infected by B. bassiana but high doses may be required, especially for avocado thrips. These high doses are difficult to obtain outside the laboratory and application of such doses would be costly. We believe B. bassiana is not a sufficiently effective alternative to traditional insecticides to warrant further study with avocado thrips, particularly because the commercially available strain GHA gave poor control on avocado thrips, but it may have potential against citrus thrips in an integrated pest management program. Further studies are warranted to determine if GHA could be used in field control of citrus thrips.Citrus thrips, Scirtothrips citri , plant plastic pots has been recognized as a major pest of California citrus since the 1890s and is also known to scar mango fruits .

Historically, highbush varieties of blueberries could only be grown in regions too cold for citrus production . However, breeding efforts to cross the northern highbush blueberries with several other Vaccinium species led to the development of heat-tolerant highbush blueberry varieties . This has enabled the establishment of a blueberry industry in the San Joaquin Valley, a region where both citrus and citrus thrips flourish . The known host range of citrus thrips has broadened and in recent years, they have become a significant pest of blueberries planted in the San Joaquin Valley of California . Citrus thrips feed on blueberry foliage during the middle and late portions of the season causing distorted, discolored, and stunted flush growth and poor development of fruiting wood required to obtain the subsequent crop. Repeated pesticide applications of the few effective and registered pesticides to reduce thrips populations pose a concern regarding pesticide resistance management, and this issue is relevant not only to the blueberry industry but also for the 108,665 ha of California citrus which has experienced repeated documented cases of pesticide resistance in citrus thrips populations . Currently, there are no integrated pest management plansavailable for control of citrus thrips in blueberry, probably due to the recent nature of this crop-pest association. With a limited number of pesticides available for thrips control and the frequency of insecticide resistance shown by thrips, populations should be monitored carefully, treatments limited to populations of economic concern, and applications timed optimally . Appropriate cultural practices and conservation of natural enemies should be practiced in concert with the use of pesticides only on an as-needed basis.

Understanding citrus thrips’ life history in the blueberry system to determine where and if susceptible stages could be exploited, is one of the first steps in the development of alternative methods to the use of traditional insecticides. In citrus, citrus thrips pupation occurs on the tree in cracks and in crevices, however, the majority of thrips drop as late second in stars from trees to pupate in the upper layer of leaf litter below trees and move upward onto the plant after adult eclosion. Propupae and pupae are rarely seen, move only if disturbed, and do not feed. Pupation in the upper layers of the soil surface may create the ideal interface for control using the entomopathogenic fungus Beauveria bassiana Vuillemin due to this vertical movement of the citrus thrips. However, blueberry plants have much different plant architecture than citrus trees and citrus thrips pupation behavior has yet to be studied on blueberries.In the U.S., pressure is increasing to move away from broad-spectrum insecticides and focus on alternative methods of control. Earlier work with B. bassiana determined that the commercially available strain, GHA , was the most effective of six strains tested in laboratory trials against citrus thrips . The goal of this study was to determine if this strain of B. bassiana could be utilized effectively against citrus thrips in California blueberry production. To achieve this objective, several factors of importance to fungal efficacy were evaluated before commencement of our field trial: 1) location of citrus thrips pupation in commercial blueberry plantings, 2) field sampling locations and methods, 3) fungal formulation and timing of application, and 4) density of product used and method of thrips infection.

We then conducted a field trial evaluating the potential utility of the GHA strain of Beauveria bassiana in commercial blueberries for citrus thrips management as a possible alternative to the use of traditional insecticides.Source of insects for greenhouse studies. Citrus thrips were collected in Riverside County, Riverside, CA from wild laurel sumac, Malsoma laurina , a suspected major host for this species before citrus was introduced into the state . Thrips were collected via aspiration the morning of the bioassay and held in 15-dram plastic aspiration vials with a copper mesh screened lid. A small sumac leaf, just large enough to fit in the vial, was included to allow the insects to settle on the leaf and feed. In experiments where late second instar thrips were needed, i.e. thrips that were close to pupation, selected thrips were large and had darkened in color. Their abdomens appeared plump and the overall color of the thrips was a deep yellow with almost no opalescence. Early to mid second instar thrips show limited abdomen distention and have an overall pearlescent hue. When adult females were used, selected females were of unknown age. Location of citrus thrips pupation in potted blueberries. Because of the complex arrangement and number of blueberry canes arising from the rhizome of commercial blueberry plants, we first evaluated movement of second instar citrus thrips on potted single cane blueberry plants in the laboratory. Known numbers of late second instar citrus thrips were released onto the leaves of potted blueberry plants in the lab. Paper sprayed with Tangle Trap sticky coating was placed a) at the base of the plants with a ring of sticky tape around the base of the stem and partially on the stem of the plant to capture any insects crawling down, and b) extending from the base of the plant horizontally outward above the pot surface to ensure complete coverage of the area covered by the plant canopy . This experiment was replicated on a single potted plant over time on 7 dates . Data were analyzed using Fisher’s exact test using SAS 9.2 . Field sampling of thrips pupation sites. At our planned field trial site that would later be used in the B. bassiana trial, pupation emergence cages were used to sample insects moving off foliage towards pupation sites and later emerging out of the soil after pupation. Cages were made from Schedule 40 white PVC pipe with a diameter of 10.2 cm with cages cut to a height of 5.1 cm. The cage was then topped with a double-sided sticky card cut to fit, black plastic pots which was fixed into place with two elastic bands. Four lines of four cages were pushed into the soil to a depth of approximately 1 cm immediately adjacent to each other at the base of a blueberry plant and oriented in a cardinal plane to determine which direction showed the most thrips activity. The four adjacent cages in a particular plane were used to assess thrips movement in the understory of the blueberry plant in each directional. The study was replicated on 5 plants on a single date and conducted just prior to the commencement of the field trial. Data were analyzed with a nested ANOVA using SAS 9.2. Fungal formulation and timing of application. In a greenhouse trial, Mycotrol O ® was applied directly to the soil surface as raw spores and compared to the same product colonized onto millet seed, also using soil application. Millet seed colonization used the Stanghellini and El-Hamalawi method as described below. The colonized millet seed, when allowed to imbibe water and incubate in the laboratory, can support 1.0 x 106 conidia/seed . Based on Stanghellini et al. with modification, we held the GHA colonized millet seed in containers such that the seed mat was at a depth of no greater than 2.54 cm.

The seeds were wet with the consistency of very thin slurry and were gently stirred three times per day for four days to ensure they imbibed water properly so that mycelial growth and sporulation would occur. Sporulation was confirmed by slide mounting random sections of mycelia and checking for condia formation under the microscope. Once spores were initially observed, the seed was held an additional three days so that sporulation could continue before use of the colonized seed in the field study. Mycotrol O® was applied in the maximum recommended field rate for high thrips levels of 2.84 L of material in 378.5 L of water. Thrips avoidance of colonized millet seed. The colonized millet seed was tested in the greenhouse to determine if late second instar citrus thrips would become infected if they crawled over or through the seed when it was placed at the base of a laurel sumac seedling. A single small laurel sumac seedling, about ~10 cm tall, was placed into each of ten, 9.5 x 9.5 x 18 cm styrene cages with 6 cm diam air holes on all four sides that were covered with ultra fine mesh screening . Small holes were made in the bottom of the container and covered with pebbles to allow for drainage, then soil was added to a depth of 7.62 cm and the top of the container was covered with a removable lid. The base of each plant was completely surrounded by either B. bassiana colonized millet seed or with uncolonized seed . A minimum of 20 late second instar thrips were released onto the leaves of each plant, and were left until enough time had passed for the thrips to molt to the propupal stage. The seedling was then cut at the soil line and examined for pupating thrips; the removable lid of the cage was sprayed with Tangle Trap sticky coating to collect any emerging adults after 5 days so infection could be measured. The study was replicated on 5 dates . Data were analyzed using 1-way ANOVA with time as a factor and means were separated using Tukey’s Least Significant Difference test using SAS 9.2. Density of colonized millet seed to use. To determine the optimum number of colonized millet seeds needed for close to 100% infection when thrips were seeking pupal refuges off the plant, varying amounts of colonized seed were evaluated in a greenhouse trial based on the size of the seed once it had imbibed water and sporulation had occurred. After water inhibition, nine seeds completely filled one square cm of soil surface. There was a 0.5 cm buffer area around all sides of the cage, which was kept clear of seed to provide a 9 x 9 cm grid of seed on the soil surface below the plant. All but two leaves were plucked from the seedling. Small holes were made in the bottom of the container, which was covered with pebbles to allow for drainage. The 9 x 9 cm2 grid was created from wire screen and differing amounts of sporulating seed or seed alone were placed on the light imprint made from the wire screen on the soil surface. Two replicate seedlings per treatment were set up per date in a complete block design . Plants were watered every third day. A minimum of 20 late second instar thrips were placed onto the leaves of the plant, and were left until enough degree-days had passed for the thrips to molt to the propupal stage, typically about 5 days. The seedling was then cut at the soil line and examined for pupating thrips; the removable lid was sprayed with Tangle Trap sticky coating to collect any emerging adults after another 5 days.

Volatile chemicals stimulate chemosensory receptors in tephritid flies when assessing a potential novel host and trying to expand . Therefore, chemosensory-related genes are involved in the initial process of host plant expansion for tephritids. Olfactory-related genes of tephritids are one type of chemosensory gene that includes several gene families of odorant-binding proteins , chemosensory proteins , odorant receptors , ionotropic receptors , and sensory neuron membrane proteins , which are primarily involved in the identification of volatile chemicals, including volatiles of host fruits. After receiving odor chemical signals, these olfactory-related genes are triggered to transduce cascades that send information to specific regions of the brain, which ultimately leads to specific behavioral responses . OBP genes play an important role in the first step of chemosensory identification of insects, including tephritids . OBP genes direct odorant-binding proteins to bind volatile odor molecules specifically by distinct expression to related olfactory receptors that are bound to olfactory receptor neurons in antennae . CSP genes are regarded as playing a similar role as OBP genes involved in the initial process of chemosensory signal transmission to corresponding receptors . OBP and CSP genes are major gene types that lead tephritid flies to respond to different chemosensory chemicals, including volatile chemicals of host plants . Except for these two categories of genes, large plastic growing pots some odor receptor genes also play important roles in host odor recognition of tephritids, such as genes related to odor receptors and ionotropic receptors .

Odorant receptors of insects are composed of at least two proteins: a conserved coreceptor as an ion channel and a specific OR subunit , which determines the ligand specificity and forms structurally ligand-gated ion channels . The OR genes mediate odorant receptors of insects transmitting the odorant molecules they receive into electric signals that are transmitted to a higher-order neural center . IR genes are related to ionotropic glutamate receptors , which are regarded as ion channels . They also play important roles in odor chemical perception . The sensory neuron membrane proteins gene encodes transmembrane domain-containing proteins that belong to a large gene family of CD36 receptors . SNMPs regulates the corresponding proteins to identify chemosensory signals, mainly pheromone chemicals . The GR family is another type of chemosensory protein that is a ligand-gated ion channel broadly expressed in gustatory receptor neurons in taste organs and is mainly involved in taste recognition of CO2 , sugar, and bitterness . When receiving taste signals, GR genes are involved in identifying taste and ingestion. Among tephritid flies, Bactrocera dorsalis and Ceratitis capitata are well-known polyphagous species that have expanded their host plants to more than 250 species . However, Bactrocera minax and Z. cucurbitae are oligophagous species that mainly attack citrus fruits and cucurbit plants, respectively. Bactrocera oleae , Procecidochares utilis , and Carpomya vesuviana are monophagous species infesting olive , crofton weed , and jujube , respectively, and all have limited host plant species . Compared to several major olfactory-related gene families, the two polyphagous species have more genes, with 3 CSPs, 35 OBPs, 74 ORs, and 40 IRs in B. dorsalis and 45 OBPs, 76 ORs, and 70 IRs in C. capitata , than two host-limted species .

A similar situation was observed in the GR family. There are also more GR genes in C. capitata and B. dorsalis than in the host-limited species P. utilis , C. vesuviana , and Z. cucurbitae . The increased numbers of these genes are associated with chemosensory-related gene family expansion via gene duplication and differentiation , which exertimportant roles in tephritid fly adaptation to other hosts and expansion of their host ranges. Obvious chemosensory-related gene expansions were also reported in Tribolium castaneum , Spodoptera frugiperda , and Heliconius melpomene . For example, the pea aphid Acyrthosiphon pisum , with broader host ranges, experienced obvious expansion of the OR, OBP, and GR gene families, with 87 ORs, 18 OBPs, and 78 GRs, compared to the soybean aphid Aphis glycines , with 47 ORs, 10 OBPs, and 61 GRs . Altering gene expression levels also helps tephritids respond to different host plants and realize host expansion. OR13a and OR82 expression are higher in antennae in B. dorsalis in response to 1-octen-3-ol and geranyl acetate, respectively, which are major volatile components of its host fruits, mango and almond fruit . For B. minax, increasing the expression levels of several GR genes regulate the taste process in response to different chemosensory stimuli of hosts .Once a tephritid adult identifies a potential novel host fruit for oviposition or feeding, the plant fruit must be suitable for larval development, which includes overcoming any secondary toxic chemicals in the novel host fruit . Therefore, detoxifcationand other digestion-related genes also play core roles in mediating the host plant expansion of tephritids. Common detoxifcation-related genes of insects include gene families of cytochrome P450s , glutathione S-transferases , UDP-glycosyltransferases , carboxyl/cholinesterases and ATP binding cassettes .

The cytochrome P450 family belonging to phase I enzymes includes various CYP subfamilies for different tephritid species . The GST superfamily consists of phase II enzymes divided into at least seven major subclasses: the delta, epsilon, omega, sigma, theta, zeta, and microsomal classes . The PGE phase II enzymes are a large family that can be divided into 13 clades, including the dietary detoxification class , the hormone/semiochemical processing group , and the neurodevelopmental group . The ABC transporter superfamily belonging to phase III enzymes can be subdivided into eight subfamilies, from ABC-A to ABC-H. The cytochrome p450 gene family of phase I mainly contributes to the catalysis of numerous oxidative reactions during endogenous and exogenous metabolism . The important roles of genes in this family are the metabolism of xenobiotics, plant allelochemicals , and even insecticides. GSTs are multifunctional genes of phase II enzymes that play a crucial role in the detoxification of endogenous and xenobiotic compounds, including plant secondary metabolites and pesticides. CCE families of phase II have been shown to be involved in the detoxification of plant-derived allelochemicals as well as insecticides . The ABC transporter genes of phase III encoding membrane-bound proteins typically function in the ATP-dependent transport of various substrates across biological membranes . The roles of ABC genes are mainly in handling xenobiotics such as plant phytotoxins and insecticides . These genes can participate in regulating detoxification of host plant secondary metabolites of tephritid flies by coding corresponding enzymes, which help to transform toxins entering the insect system into hydrophilic compounds that can be eliminated and in the adaptability of different hosts . The major digestive-related genes include gene families of cysteine proteases, proteases, lipase, glucosidase, and serine proteases . The serine proteases are members of the supergene family, including chymotrypsin, trypsin, thrombin, subtilisin, plasmin, and elastase. subclasses . Various digestive proteases exert important roles in the nutrition digestion of tephritid flies from novel host plants that they try to expand to. However, protease inhibitors of host plants are a widespread defense against herbivores such as tephritids. Therefore, genes coding various proteases react to protease inhibitors by regulating inhibitor-sensitive proteases or expressing proteases that are not targets of the inhibitors . When expanding to other novel hosts, large plastic pots tephritid flies must adapt to different chemical environments from their native hosts. Detoxification-related genes regulate the host expansion of tephritids via gene family expansion similar to chemosensory-related genes. The major gene families of detoxifcation GSTs, P450s, CCEs and ABC transporters are more numerous in polyphagous B. dorsalis and C. capitata than monophagous P. utilis and B. oleae . However, reports about digestive gene family expansion in tephritids are still rare. Overall, detoxifcation and the digestive-related gene family combined with chemosensory-related gene family amplifcation exhibit a close association with host range extension. This gene family expansion is helpful for the host plant expansion of fruit flies. Cases in other insects strengthen this idea. For example, Helicoverpa armigera and Helicoverpa zea are two species of caterpillars that have considerably broader host ranges than any other lepidopterans.

Great expansion of detoxification and digestive gene families was found in the two species. In addition to gene family amplification, detoxification and digestive genes also regulate host expansion of tephritid flies by activating various gene subfamilies, subclasses, or clades. To respond to various toxic environments, including secondary toxic chemicals of different hosts, B. dorsalis primarily triggered the delta subfamily of GSTs, CYP3 and CYP4 subclasses of P450s, A–C clades of CCEs, and ABC-A, ABC-B, and ABC-G subclasses of ABC transporters , C. ceratitis activated the epsilon subfamily of GST, CYP6 and CYP12 of P450s, B clade of CCEs , and P. utilis mainly triggered the delta, epsilon and microsomal subfamilies of GSTs, CYP4, and CYP9 of P450s, C clade of CCEs and ABC-G subclass of ABC transporters , but R. pomonella mainly launched CYP4 and CYP6 of P450s . For the digestive gene family, B. dorsalis and C. capitata primarily triggered aminopeptidase, trypsin and serine peptidase digestive genes, but B. oleae, which is a strictly monophagous species, triggered serine protease and nuclease digestive genes to respond to different host secondary chemical environments . Detoxifcation- or digestion-related genes also facilitate tephritid fly adaptation to different hosts by altering gene expression levels. Rhagoletis zephyria evolved fromRhagoletis pomonella and experienced host expansion from apple to snowberry plants . Increased expression levels were found in some detoxifcation-related genes, including cytochrome P450, glutathione S-transferases, and glycosyltransferase, in R. zephyria facing the apple host environment . Z. cucurbitae is the species that mainly attacks cucurbit plants, and the fly responds to different secondary chemical environments of Mucuna pruriens plants by reducing the expression levels of trypsin and chymotrypsin digestive genes .Although the importance of chemical stimuli is highly emphasized in the host expansion of tephritids, other nonchemical stimuli, such as the color of the novel host fruit, should not be ignored. Many insects locate their host plants primarily by color signals, including beetles, Altica engstroemi , Hylastes ater , and Arhopalus ferus . For tephritid flies, Neoceratitis cyanescens , B. minax , B. dorsalis , and Z. cucurbitae are typical examples of species that appear to select different hosts frst by fruit color rather than chemical signals. Z. cucurbitae realized its host expansion to a novel host, papaya , in Hawaii by strongly relying on the color location of fruits by vision . However, the underlyinggenetic mechanism has not been revealed for Z. cucurbitae. In fact, nonchemical stimuli, such as color, are associated with vision-related genes that allow the identifcation of different hosts . The genes responsible for color discrimination in Diptera are primarily related to opsin proteins in the photoreceptor cells of the eye . Six types of Rh opsin-expressed genes have been identifed as major genes involved in color recognition and photoreception in Diptera insects. The Rh1 and Rh2 opsin genes are associated with motion detection and direction, respectively . Rh3 and Rh4 are UV-sensitive opsin genes, Rh5 is a blue-sensitive gene and Rh6 is a green opsin gene . These opsin genes lead the photoreceptor of eyes to receive various chromophore pigments and then activate a series of visual transduction cascades to launch corresponding color identifcation behavior. In the genome of polyphagous C. capitata, the long wavelengthsensitive genes Rh1, Rh2, and Rh6 and the UV-sensitive genes Rh3 and Rh4 were found, while Rh2-4 and Rh6 were found in the phototransduction pathway of oligophagous B. minax . Moreover, the role of Rh6 in modulating green color discrimination was reported in C. capitata and B. minax . In B. minax, the function of Rh6, which is responsible for green spectral sensitivity, has been identified by knockdown of the gene B. minax in female adults, and B. minax flies significantly reduced their preference for green fruit after cutting Rh6 . Absence of a member of the bluesensitive opsin subfamily was found in both tephritid species C. capitata and B. minax, but Rh5 can be specifically expressed in Drosophila . Reports about vision-related genes directly involved in the host expansion of tephritids are still very few.Tephritid fruit fly hosts expand to other new host plants, and the phenology of the new host is another nonchemical stimulus that affects fly adaptation. The phenology of the host plant fruits includes the time of flowering, fruiting, or maturation .

The Center’s latest research brief, Farming the College Market: Results of a Consumer Study at UC Santa Cruz, by CASFS social issues associate specialist Jan Perez and social issues specialist Patricia Allen, examines student, staff, and faculty’s food-related concerns, interests, and level of support for specific food criteria, including whether they would be willing to pay more for food produced in an organic and “socially just” manner. Write the authors, “Since the success of farm-to-college programs involves their ability to meet the needs of campus consumers, we undertook a study of our local campus . . . to learn about the perspectives and preferences of campus food consumers.” Perez worked with members of the campus’s Food Systems Working Group, including UCSC Dining Services, Community Agroecology Network, and Students for Organic Solutions, to develop a web-based survey designed to find out what the UCSC community thinks about food system issues. Those responding to the survey included students , staff , and faculty . Based on the online survey, the study found that food issues are important to the UCSC community, particularly regarding concerns for the environment and for people. Key points from this study include: There is significant interest in campus food that is nutritious, safe, supports workers, and is environmentally sound; interest in local food and GE-free food is lower. People are interested in sustainably produced food and a majority of people already purchase food with labels based on these criteria. Many people are willing to pay more for food that meets social justice criteria. A campus community is likely to be receptive to education and discussion about food-system issues. Since people had a great interest in nutrition and food safety, square pots for plants framing discussions of food-system issues in terms of health will meet people’s needs as well as capture their attention for education on other food-system issues, such as working conditions and the environment.

The authors conclude that, “[Although] It would not be appropriate to extrapolate too much from a study of one campus . . . the results of the UC Santa Cruz study support the idea that colleges and universities are excellent choices for developing farm-to-institution programs and for popular education on food-system issues.” A similar survey was recently distributed to a nationwide audience as part of CASFS research on farm-to-institution programs .Finding a non-fumigant alternative to the soil fumigant methyl bromide has been identified as a top priority by the California Strawberry Commission, and by growers who are facing the phase out of this ozone-depleting pesticide. However, most current state and federal research is focused on alternative fumigants rather than non-fumigant techniques to control soil diseases, weed seeds, and harmful nematodes. Preliminary research conducted at the UCSC Farm has shown promising results with an alternative approach that starves pathogens and weeds of oxygen. Researchers introduce a carbon source such as chopped cover crops, wheat bran, or molasses to the strawberry bed, then irrigate and tarp the beds to create temporary anaerobic conditions. This technique, known as anaerobic soil disinfestation , has been tested for the past several seasons at the Farm and has been shown to control the soil pathogen Verticillium dahliae, a major diseases of strawberries. A new study, funded by a grant from the US Department of Agriculture, will expand this initial work to examine the efficacy of various carbon sources, irrigation techniques, tarp types, and tarping periods to create sufficient anaerobic conditions to control weed seed germination and V. dahliae. The study, conducted by environmental studies professor Carol Shennan, UCSC researcher Joji Muramoto, and colleagues from California and Florida, will also look at using the technique to control diseases, pests, and weeds in Florida cropping systems, which also rely on methyl bromide. The three-year study will take place on conventional farms so that results can be compared to those obtained with standard methyl bromide fumigation.

Following an initial year of field trials in Watsonville and Oxnard, the research team will consult with local growers to determine which ASD options hold promise for larger-scale commercial application.Patricia Allen has been named director of UCSC’s Center for Agroecology and Sustainable Food Systems by Social Sciences Dean Sheldon Kamieniecki. Allen, whose appointment took effect July 1, 2007, had been serving as acting director since January 2007. She joined the campus’s Agroecology Program in 1984. She takes the helm from Carol Shennan, CASFS director for the past 10 years. Following her 2007 sabbatical, Shennan will continue at UCSC as a professor in the Environmental Studies Department. Allen is one of the nation’s leading scholars on the social aspects of sustainable food systems. Her work addresses issues such as labor, gender, and access to food. She is the author of Together at the Table: Sustainability and Sustenance in the American Agrifood System and editor of Food for the Future: Conditions and Contradictions of Sustainability . Allen earned a B.S. in political economy of natural resources from UC Berkeley, an M.S. in international agricultural development from UC Davis, and a Ph.D. in sociology from UCSC in 1998.In the world of sustainable agriculture, it doesn’t get any better than the “Sustie” award, and the UCSC Apprenticeship in Ecological Horticulture took home the top honor at this year’s Ecological Farming Conference. Established in 1988, the “Sustie” award is presented each year by the Ecological Farming Association to “stewards of sustainable agriculture” who have made a significant contribution to the well-being of farming and the planet. Past recipients include chef Alice Waters, publisher Robert Rodale, and several graduates of the apprenticeship itself. UCSC Farm manager Jim Leap and apprenticeship coordinator Diane Nichols accepted the Sustie on behalf of the apprenticeship during the conference’s awards banquet on January 26 at the Asilomar Conference Grounds in Pacific Grove. “There are more than 25 extremely motivated and dedicated individuals who are instrumental in making the training what it is each year,” said Leap. “All of us work collectively to teach and train and run the UCSC Farm, and it is all of us who will be sharing in the acknowledgment that this award represents.” The apprenticeship, which is celebrating its 40th anniversary this year, is the nation’s premier hands-on training program in organic farming and gardening. Widely regarded as one of the most significant influences in the growth of sustainable agriculture, the six-month full-time program has prepared more than 1,200 graduates who have spread their expertise throughout the world. “There’s simply nothing that compares to the apprenticeship for the depth of its program or the breadth of its impact,” said Sheldon Kamieniecki, dean of the Division of Social Sciences at UCSC, who attended the awards banquet. Graduates of the apprenticeship go on to operate commercial farms and market gardens, run community and school gardens, and work at the forefront of international development, food policy, and social justice programs. The impact of the apprenticeship is apparent in the number of graduates who have received Sustie awards, including Cathrine Sneed of The Garden Project in San Francisco; Wendy Johnson, garden manager at Green Gulch Farm in Marin County; Jim Nelson of Camp Joy Gardens in Boulder Creek; Gloria and Steven Decater of Live Power Community Farm in Covelo, CA; Orin Martin, manager of the Alan Chadwick Garden at UCSC; and Kay Thornley, who helped launch UCSC’s Agroecology Program.

Its success is also evident in the number of similar college-based farm-training programs sprouting up at the University of Georgia, Michigan State University, Washington State University, the University of Montana, and other campuses.Perhaps an overview statement might be helpful—let’s define terms. The French intensive, raised bed style of gardening is a handworked system featuring deep cultivation . This technique’s primary effect is on the physical properties of a soil: the aim is to rapidly improve soil structure and fertility. Improved physical properties can positively influence the biological and chemical properties of a soil as well. The main idea is to create a well-drained, well-aerated, large square plant pots fertile soil structure by digging deeply and placing nutrients at specific levels. This gives rise to a profile that enables plant roots to probe/penetrate throughout the bed with ease, especially in a downward direction. Such an arrangement has a continuous system of large and intermediate pore spaces from the surface to the subsoil. Plants’ needs for air, water and nutrients are best met with such a continuous system of pores. The French intensive system is not appropriate in all soils and in all climatic situations. For instance, on deep, improved soils, it’s superfluous, even deleterious. On sandy soils and in hot, windy situations it can “burn up” precious organic matter and cause water losses both through surface evaporation and excessive drainage. As is so often the case in life, there are no panaceas, but we tend to be creatures of habits, creatures of dosages; that is, we want to do the same thing in the same way, with the same amount, repeatedly. The judicious use of deep digging for a few years to develop a soil, followed by lighter, less disruptive surface cultivation and perhaps periodic renewal via deep digging again might be more prudent. Caveat emptor: Digging is a radical act, potentially destructive of soil structure and biological processes Do it skillfully and as infrequently as possible! Conventional wisdom often states that it can take 1,000–2,000 years for 1 foot of topsoil to develop in place. With French intensive it is possible to simulate the creation of 1 foot of topsoil in 3–5 years .Often when people hear French intensive, they automatically think of raised beds. In fact, the beds may be raised slightly or in an exaggerated sense , flat, or even sunken. The degree of “loft” is a function of climate, soil type, and seasonal weather. On a transect from Seattle to Santa Cruz to Santa Fe, the response might be: 1) Seattle, with its high annual rainfall and cool temperatures, can have dark soils with high organic matter and high clay content, and a tendency to remain cold and wet. Thus a raised bed would yield better growth, allowing the soil to warm more quickly. Santa Cruz, with its mild Mediterranean climate, dry summers and wet winters would feature a slightly raised bed during the rainy season and an almost flat bed in summer. Santa Fe might yield a flat or even sunken bed for water catchment, to minimize water loss and afford protection from wind. Permanent beds, be they raised or flat, substantially reduce soil compaction. The bed equals the zone of maximum fertility—you could say “Don’t tread on me,” or only minimally and lightly. The path equals the zone of degradation, with much foot traffic and resultant compaction. Permanent beds foster maintenance of ideal soil structure. While compaction is a primary problem in mechanized agriculture, it can be virtually eliminated in handworked permanent bed systems. In agriculture, it can be said that the back of the tractor is simply undoing the work of the front of the tractor. Some common causes of soil compaction are: ploughing—a “plow pan” develops just below the depth of tillage; 2) machine and foot traffic —the bigger the machine, the greater the number of passes, the greater the compaction; 3) the pounding action of rain drops on open soil, which can destroy surface soil aggregates and lead to crusting and erosion. Natural forces also cause compaction—over time, the fine particles of clay leach downward, accumulate in layers, and create subsurface compaction or a hard pan. Compaction can be measured by an increase in bulk density. It includes pore space as well as solids. It is distinct from particle density, which simply measures the weight of a soil as if there were no pore spaces. Permanent beds also focus efficient placement of fertilizers/nutrients only where plants will be growing.The Apprenticeship in Ecological Horticulture is one step closer to finally building permanent apprentice housing on the UCSC Farm, thanks to long-time Friends’ member Olivia Boyce-Abel. When the campus approved the plans for eight 4-room cabins, the price tag that came with it was $487,000. Boyce-Abel not only pledged $40,000 from her obaboa Foundation for the project but also offered to help inspire other former apprentices and program supporters to give. She has put out a challenge that so far has more than matched her $40,000, with a total of $85,000 raised to date.