Nature Agriculture

In Populus species, lignin can also be further modified by acylation with phydroxybenzoate. Zhao et al. used wild type , lignin p-hydroxybenzoate deficient, and p-hydroxybenzoate overproduction plants to investigate the role of this modification in the response of plants to gravitropic/mechanical stress. This increase is correlated with a significant induction of expression of a gene encoding aBAHD family acyltransferase, namely, p-hydroxybenzoyl CoA: monolignol p-hydroxybenzoyltransferase 1 whose gene product preferentially conjugates p-hydroxybenzoate to Slignin monomer sinapyl alcohol.Plant phenylpropanoids and their derivatives are essential for plant growth, stress responses, and health benefits for humans. A comprehensive understanding of the biosynthetic mechanisms and transcriptional regulatory network of phenylpropanoid metabolism in various plant species is central for developing biotechnological approaches to produce economically desirable traits and products. Additionally, advancements in synthetic biology and biosensor technology illuminate the potential of real-time control of phenylpropanoid metabolism in the future. Ferreira and Antunes reviewed current progress on synthetic biology and highlighted the application of biosensors for re-engineering and autonomously controlling plant phenylpropanoid metabolism. Lam et al. reviewed the understanding and bioengineering of the biosynthesis of tricin, a type of plant flavonoid that is an essential plant defense chemical and a promising nutraceutical. Sullivan et al. established a de novo hydroxycinnamoyl-malate ester biosynthetic pathway in alfalfa via heterologous expression of a red clover gene and enhanced alfalfa post-harvest protein protection.

A transcriptomic study of transgenic tomato plants by Zhao et al. defined a GATA transcription factor mediating the co-regulation of drought stress response and phenylpropanoid biosynthesis. Genetic, flower buckets wholesale biochemical and physiological studies from Lee et al. found that Arabidopsis needs optimal anthocyanin content for better growth under high nitrate and high salt conditions. A study by Roldan et al. using transgenic white clover with high levels of foliar condensed tannins discovered that condensed tannins bind to forage proteins to reduce anthropogenic greenhouse gas emission. Huber et al. chemoenzymatically synthesized a series of new phenylpropanoid derivatives and studied their structures and biological effects. Using qualitative and quantitative phytochemical analyses, Gampe et al. demonstrated that Ononis hairy root cultures produce isoflavonoids with less chemical divergence and in higher quantity, suggesting a promising system for large-scale isoflavonoid production. Systems biology and biotechnology have largely contributed to enhance our understanding on the molecular mechanisms underlying the biosynthesis of phenylpropanoids in plants, as well as to manipulate the phenylpropanoid metabolism to exploit its economic, medicinal and nutraceutical potential. Articles in this volume further contribute to these goals, covering different aspects and branches of the pathway. Novel insights and exciting biotechnological strategies involving the phenylpropanoid pathway are expected in the years to come.The actinobacterial genus Nocardia, the type genus of the family Nocardiaceae emend. Zhi et al., has a long and convoluted taxonomic history mainly due to an overreliance placed on morphological properties. The application of polyphasic taxonomic procedures led to marked improvements in the classifcation of nocardiae and related mycolic acid containing actinobacteria.

In general, the genus encompasses aerobic, Gram-stain-positive, acid-alcohol-positive, nonmotile, chemoorganotrophic actinobacteria which form rudimentary to extensively branched substrate hyphae that fragment into coccoid to rod-shaped elements, aerial hyphae may only be visible microscopically; the diamino acid of the peptidoglycan is meso-diaminopimelic acid , the characteristic whole-organism sugars are arabinose and galactose; diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides are the major polar lipids; the fatty acids consist of straight-chain, saturated, unsaturated and 10-methyl components; mycolic acids have 46-64 carbon atoms and up to four double bonds; the predominant respiratory quinone is a hexahydrogenated menaquinone with eight isoprene units where the two end ones are cyclized and the DNA G+C content ranges from 63-72 mol%. Many of the 119 Nocardia species with validly published names are recognized using combinations of genotypic and phenotypic properties. Most of these taxa are composed of strains isolated from natural habitats but the best-known species contain causal agents of serious suppurative and granulomatous diseases in humans and animals, especially mycetoma and nocardiosis . In contrast, Nocardia vaccinii produces galls on blueberry plants. Soil is probably the primary reservoir for Nocardia strains as they are found in diverse soil types, including acidic forest, arid, Cerrado, karst cave, rhizosphere and saline soils. However, they have also been isolated from marine habitats, especially from sponges, as well as from the gut of fungusgrowing termites and are increasingly being isolated from plant tissue, notably from nodules of actinorhizal plants suggesting that they may have a role in promoting plant growth and inhibiting phytopathogens. Two Nocardia strains isolated from Casuarina glauca nodules induced root nodule-like structures in the original host plant.

Nocardiae are an important source of novel antibiotics, as exemplifed by the production of amicoumacin B from Nocardia jinanensis, asterobactin from Nocardia asteroides, brasilicardin A from Nocardia brasiliensis, nocardicins from Nocardia uniformis subsp. tsuyamanensis and tubelactomicin A from Nocardia vinacea. A comparative survey of nocardial genomes showed that their biosynthetic potential to produce diverse novel natural products is comparable to that of better studied actinobacterial taxa, such as Amycolatopsis and Streptomyces, thereby making them an attractive source of new drug leads. These researchers showed that Nocardia strains from diverse sources, including clinical material, were equally spread across six phylogenetic clades and found that the genomes of the more pathogenic strains were, on average, slightly smaller than those of most of the other genomes and contained fewer BGCs . Similarly, information from the genome of Nocardia cyriacigeorgica shows evidence of adaptation from a saprophytic to a pathogenic lifestyle. The present study was designed to establish the taxonomic status of Nocardia strain ncl2T, isolated from a root nodule of an actinorhizal plant, and to determine its biotechnological and ecological potential. The strain was the subject of a genome-based taxonomic study which showed that it formed a new centre of evolutionary variation within the genus Nocardia, the name proposed for this organism is Nocardia alni sp. nov. with isolate ncl2T as the type strain. The genomes of N. alni and N. vaccinii strains contained natural product biosynthetic gene clusters predicted to synthesize novel specialised products, notably antibiotics and genes associated with the expression of plant growth promoting compounds. Statistical comparison between genomic features of the isolate and its taxogenomic neighbours were undertaken to establish any positive correlations between them. Antismash 5.0 predicts NP-BGCs based on the percentage of genes from the closest known bioclusters which show BLAST hits to the genomes of the strains under consideration. The genomes of strain ncl2T and N. vaccinii NBRC 15922T contained 36 and 29 well-defined bioclusters that are predicted to encode for a broad range of specialized metabolites albeit with low levels of gene identity, as shown in Table S2. The genomes of the strains are well equipped to synthesize non-ribosomal peptide syntethases, type I polyketides, ribosomally synthesized and post-translationally modified peptides, as well as betalactone and carotenoidlike terpene compounds. They have the genetic capacity to produce products most closely related to himastatin , an antitumor antibiotic produced by Streptomyces hygroscopicus, stefmycin D , which was initially produced by a Streptomyces strain and inhibits ras-oncogen expressed cells, and teicoplanin, a product of a Streptomyces strain that inhibits growth of Gram-positive bacteria, including Enterococcus faecalis and methicillinresistant Staphylococcus aureus strains. The strains also contain bioclusters predicted to synthesise arylpolyene-like compounds that are structurally and functionally similar to caretonoids and which show antimicrobial and antioxidant activity. They also have bioclusters predicted to encode for ectoine , flower harvest buckets a protective molecule which enables bacteria to survive extreme conditions. It can be concluded that strain ncl2T and N. vaccinii NBRC 15928T have genomes rich in NP-BGCs, notably ones predicted to express for putatively novel polyketide and non-ribosomal peptide compounds thereby providing further evidence that nocardiae are a potentially prolifc source of new bioactive compounds.

It is particularly interesting that these strains have the capacity to synthesise antifungal and antibiotics given their association with plant tissues. Clearly, nocardiae should feature more prominently in natural product discovery campaigns.Comparative genome mining of strain ncl2T and the type strains of N. jiangxiensis, N. miyunensis and N. vaccinii, its closest phylogenomic neighbours, revealed the presence of genes associated with direct and indirect mechanisms that promote plant growth. Nocardia casuarinae BMG51109T and N. pseudobrasiliensis DSM 44290T were included in these analyses to represent taxa isolated from plant and clinical sources, respectively. Microbes have a pivotal role in making phosphorus available to plants either enzymatically or by producing organic acids and siderophores and other molecules that solubilize inorganic phosphate.The genome of all of the strains, apart from that of the N. pseudobrasiliensis DSM 44290T, contained genes associated with phosphate regulation and metabolism . These included gene ppx–gppA, which is responsible for the solubilization of inorganic polyphosphate and gene pstS which encodes for phosphate binding protein PstS that is involved in the production of the phosphate ABC transporter. The pstS gene was not detected in the genome of the clinical isolate thereby suggesting a possible correlation between the environmental origin of the other strains, namely soil and plant tissues, and phosphate metabolism. The genome of all of the strains contained gene senX3 which is associated with the production of histidine kinase, a high afnity phosphate transporter which has a role in controlling the phosphate regulon. Phytohormones have a central role in plant growth, notably indole -3-acetic acid and ethylene; the levels of these and other auxins in plants can be regulated by soil microorganisms able to synthesize them. The genome of all of the strains contained genes encoding for indole-3-glycerol phosphate synthase, the precursor of IAA in the tryptophan biosynthetic pathway in plants. They also contained genes encoding for other components of this pathway, including anthranilate phosphoribosyl transferase , anthranilate synthase , and aminase. Similarly, gene trpF, which is associated with the synthesis of anthranilate phosphoribosyl transferase, was present in the genomes of all of the strains, apart from N. pseudobrasiliensis DSM 44290T. Genes pdxl and aad, which encode for pyridoxine 4-dehydrogenase and aryl-alcohol dehydrogenase and are involved in auxin signaling pathways, were found in the genomes of strain ncl2T, N. jiangxiensis NBRC 101359T, N. miyunensis NBRC 108239T and N. casurinae BMG51109T . In contrast, the genomes of all of the strains contained genes associated with tricarboxylic acid biosynthesis, as shown in Table S3. However, only the genome of strain ncl2T contains gene acc that encodes for 1-aminocylopropane-1-carboxylatedeaminase, an ACC deaminase which reduces toxicity due to high levels of ethylene in plants caused by plant growth promoting rhizobacteria. This enzyme also regulates ethylene levels produced by the plant by converting ACC to ammonia and α-ketobutyrate. Plant growth promoting microorganisms can also enhance plant growth by modulating biotic stress as they can decrease, neutralize or prevent infections caused by phytopathogens by synthesizing antibiotics and lytic enzymes. The genomes of all of the strains were equipped with genes associated with the production of chitinases and glucoamylases, as shown in Table S3. They also contained genes involved in the biosynthesis of antibiotics, as exemplifed by fabG, bacC2 and hdhA which express for 3-oxoacyl-[acyl-carrier-protein] reductase, bacitracin synthase and 7-alpha-hydroxysteroid dehydrogenase which play a role in the biosynthesis of pentalenolactone, bacitracin and clavulanic acid, respectively. Further, the genomes of all of the strains, apart from N. pseudobrasiliensis DSM 442990T, contained gene auaJ which encodes for the epoxidase LasC that is involved in the synthesis of lasalocid, a polyether antibiotic. In contrast, only strain ncl2T contained gene tcmO which expresses tetracenomycin polyketide synthesis 8-O-methyl transferase, a gene is associated with tetracenomycin biosynthesis. It can be concluded that while strain ncl2T is most closely related to the type strains of N. jiangxiensis, N. miyunensis and N. vaccinii, it can be distinguished from them as it forms a distinct branch in the phylogenomic tree, has a distinct fatty acid profle and shares low ANI and dDDH values with them. Genomic features, notably genome size and CDS numbers, show that the strain is most closely related to N. vaccinii NBRC15992T, but can be distinguished from the latter by a wealth of chemotaxonomic, genomic and phenotypic data. It is, therefore, proposed that strain ncl2T should be recognized as a new species within the genus Nocardia for which the name Nocardia alni sp. nov. is proposed.Aging can be modulated by genes and lifestyle. For instance, specific gene variants of insulin-like growth factor-1 receptor and forkhead box O3A are associated with longer lifespan in centenarians. In terms of lifestyle, one of the most studied interventions that delay aging is caloric restriction , which can increase lifespan in organisms ranging from yeasts to primates.

Selection also favors larger flowers in coastal perennial habitats, further supporting this hypothesis . Flowering time and plant size appear to be widely correlated among populations of M. guttatus. In a survey of annual populations, Kooyers et al. found that plant height and stem diameter are positively correlated with flowering time, suggesting that these traits have evolved together as part of a drought-escape strategy. Similarly,in an F2 mapping population between annual and coastal perennial genotypes, flowering time and stolon production map to several shared QTL, suggesting that vegetative growth through stolons is pleiotropically linked to later flowering . These broad patterns are reflected in this study and influence the strength and pattern of selection. In both annual ecotypes, later flowering is correlated with larger flowering stems across both years. Given that direct selection consistently favored larger flowering stems, this correlation either increased selection for later flowering or weakened selection for earlier flowering. Further, inland perennials experienced selection for later flowering through increased rosette production in 2012, supporting a link between delayed flowering and vegetative growth. Thus, in a perennial habitat, selection may generally favor later flowering either through fecundity selection for larger flowering stems or selection through rosette production. This is in contrast to annual habitats, black plastic plant pots wholesale which impose strong fecundity selection for early flowering before the onset of drought .

The strength and direction of phenotypic selection is highly variable over time , providing one potential explanation for the paradox of strong selection within populations and evolutionary stasis. Given the apparent rarity of stabilizing selection in wild populations, shifting patterns of directional selection may be evidence of a population “wobbling” around a fitness peak . Alternatively, selection may not be detected at all if the breadth of a fitness peak exceeds the phenotypic variance, so that the fitness landscape appears flat. In the native montane perennial ecotype, I only detected selection on flowering time and marginal selection for smaller flowering stems in 2012. Interestingly, selection on flowering time was weak and changed directions between years. Additional temporal replication could test whether this native montane perennial ecotype is near a fitness peak for later flowering. Throughout the Sierra Nevada, 2013 was a severe drought year due to low snowpack in the previous winter. Across all ecotypes, fecundity and survival was reduced in 2013 and LTREs confirm that 2013 contributed negatively to overall population growth. The earlier onset of drought-induced mortality in 2013 increased the strength of selection for larger flowers and flowering stems and caused selection to favor earlier flowering in robust annuals. In inland perennials, the death of all above-ground biomass due to drought imposed an annual life-cycle, changing the pattern of selection from traits that maximized rosette production in 2012 to traits that maximized fecundity in 2013. This resulted in a more “annual-like” pattern of selection for larger flowers and flowering stems. Although the time-scale of this study is limited, this suggests that dry years can exert selection for traits that maximize first-year fecundity even within a perennial habitat, supporting a potential path for the evolution of an annual life history strategy from a perennial ancestor.

By estimating integrated selection, this study highlights the complexity of fitness landscapes once multiple fitness components are considered over several years. Recent reviews have identified the use of single fitness components and lack of temporal replication as major limitations toward a general understanding of selection in natural populations . The few studies that have examined integrated selection have found that the strength and pattern of selection varies among fitness components and years, often altering qualitative conclusions . This study further supports the limited utility of selection estimates based on single fitness components, particularly for iteroparous taxa. For example, the relative importance of individual fitness components in inland perennials changed dramatically over two years. This growing appreciation of the complex interactions among fitness components and traits has motivated the development of methods to decompose the effects of specific environmental conditions and fitness components on long-term selection . Clearly, this is a promising approach for linking selection to specific environments and forecasting evolutionary responses to environmental change. Although previous empirical estimates of integrated selection have relied on long-term demographic data sets , this study demonstrates that integrated selection can be estimated even within an experimental framework or over a relatively short time period . By collecting data on multiple fitness components and estimating a few additional demographic parameters, researchers stand to gain a much richer understanding of natural selection.Species encounter divergent selective environments throughout their range and local adaptation to these environments is the primary mechanism driving speciation .

Adaptive divergence can generate reproductive isolation directly by decreasing the fitness of immigrant or hybrid genotypes and indirectly by shaping traits related to mate choice or reproductive compatibility. Yet, ecological speciation is not a necessary consequence of adaptive divergence, and speciation is best viewed as a continuum of increasingly strong and irreversible reproductive isolation in sexually reproducing species . Understanding the factors that promote or constrain progress towards speciation requires documenting the order in which specific reproductive barriers evolve as well as the mechanisms driving their evolution . Comparative approaches have found strong support for increasing genetic isolation with greater ecological divergence . Yet, ascribing causality with molecular signatures of gene flow is difficult because reduced gene flow may in fact promote adaptive divergence, rather than vice versa . A stronger test is to quantify how reproductive barriers themselves evolve with adaptive divergence . A few studies have implemented this comparative approach by regressing reproductive barriers onto adaptive divergence among taxon-pairs. For example, mating isolation increases with divergence in color pattern in cichlids while intrinsic post-zygotic isolation increases with body size divergence in fish . Across a broad swath of taxa, reproductive isolation is positively correlated with ecological divergence between taxon-pairs . However, to my knowledge, no study has quantified the strength of multiple reproductive barriers among population pairs that vary in their degree of adaptive divergence within a species . Thus, it is unknown how multiple barriers evolve with adaptive divergence within a system or whether the relative importance of different barriers changes as population pairs become more ecologically divergent. In a recent symposium, these questions were identified as “perhaps the most important contribution” towards advancing the field of ecological speciation . Comparing the evolution of multiple reproductive barriers along the speciation continuum is necessary to understand how total reproductive isolation evolves during adaptive divergence. Ecological reproductive barriers may be offset by other trait interactions that act as reproductive enhancers , particularly in the early stages of divergence . In Colias butterflies, for example, males from high-elevation populations prefer the lighter wing patterns of low-elevation females over the more thermally efficient dark patterns of high elevation females . Selection against immigrants and hybrids have been predicted to be the earliest and strongest reproductive barriers to evolve during adaptive divergence, whereas other ecological or intrinsic barriers are predicted to evolve later or more sporadically . Although most speciation studies find strong selection against immigrants , the evidence for ecological selection against hybrids is mixed, with many closely related taxa exhibiting heterosis . Further, black plastic plant pots bulk intrinsic post-zygotic barriers can evolve as a direct by-product of divergent selection and could be important in the early stages of divergence .

Finally, the relative importance of multiple barriers will likely vary throughout a species range, depending on the spatial distribution, opportunities for gene flow, strength of selection, and adaptive potential for a given population. Quantification of these barriers among closely-related population pairs that span a range of adaptive divergence is necessary to test these predictions . To clarify the evolution of reproductive barriers along a speciation continuum, I compared five reproductive barriers among 10 population pairs spanning a range of life history divergence in common monkeyflower, Mimulus guttatus DC . Western North American monkeyflowers are a model system for dissecting the evolution of ecological reproductive barriers .Speciation in this group is characterized by a budding process, in which small ecologically divergent populations give rise to new species , and this ecogeographic divergence is an important component of reproductive isolation . The M. guttatus species complex is particularly diverse, comprising annual and perennial taxa that occupy a wide range of edaphic, coastal, and montane habitats . Yet despite this extensive adaptive divergence, genomic variation in M. guttatus is partitioned geographically rather than among habitats or life history strategies, reflecting recent divergence and on-going gene flow . Thus, ecotypic divergence within M. guttatus provides an ideal system for testing the evolution of early reproductive barriers in an actively diverging complex. I used a common garden experiment to mimic the dispersal of immigrant genotypes from ten populations in central California into a focal population and quantified the ecological and intrinsic barriers to gene flow that would operate in sympatry. I used a regression approach to test whether the strength of individual barriers and total isolation were related to adaptive phenotypic divergence in life history traits. Finally, I compared the contributions of immigrant and hybrid fitness, flowering and pollinator isolation, and intrinsic crossing barriers to total reproductive isolation for each population and ecotype. Taken together, this work provides a comprehensive evaluation of the reproductive barriers operating along a continuum of adaptive divergence for this species.Mimulus guttatus is an herbaceous annual or short-lived perennial plant that grows in mesic habitats in western North America. Life history divergence in this system is driven by variation in the length of the growth season. Annual populations occur in seasonally-drying seeps or meadows, whereas perennial populations occur in habitats with year-round moisture such as lakes, streams, and coastal seeps. Annual and perennial genotypes differ in several life history traits that are linked to a chromosomal inversion region that occurs throughout the species range . In general, perennials flower later at a larger size, produce larger flowers and stems, and reproduce vegetatively through clonal rosettes . Yet within each of these broad life history strategies, populations exhibit extensive adaptive variation in morphological, phenological, and life history traits. Among annual populations, clinal variation in the length of the growth season is correlated with variation in drought escape traits including flowering time and size at first flower . Among perennial populations, coastal, inland, and montane habitats are associated with distinct ecotypes. Coastal perennials have evolved tolerance to salt spray , whereas montane perennials invest extensively in below-ground rhizomes and produce few flowers .Despite this extensive adaptive divergence, taxa within the M. guttatus species complex are at least partially interfertile and taxonomists have divided this complex into as few as 4 and as many as 20 species in different treatments . Although the most recent treatment by Nesom splits M. guttatus into multiple morphological species, recent genomic evidence has challenged this view . Thus, to be consistent with genomic evidence as well as previous work in this complex, I treat life history strategies in this study as ecotypes within M. guttatus sensu lato .Ecological speciation is often defined as the by-product of reciprocal local adaptation to divergent selective environments, and fitness-related reproductive barriers are an integral component of the ecological speciation framework . These barriers are predicted to evolve early in the speciation continuum and to be highly correlated with adaptive divergence, whereas other ecological or intrinsic barriers are predicted to evolve later or more sporadically . However, reviews of the local adaptation literature have found that reciprocal fitness trade-offs are relatively rare . Immigrant genotypes may enjoy a fitness advantage due to maternal effects , sexual selection , inbreeding depression , or local adaptation of pathogens or antagonists . Although these factors would swamp adaptive divergence if gene flow is high , divergent selective environments are often at least partially isolated geographically . Whether strong ecological isolation can evolve in the absence of reciprocal fitness trade-offs, particularly early in the divergence process, is an intriguing question. Indeed, ecology may play a much more general role in the speciation process than those conditions usually considered within the ecological speciation framework . In this study, I found little evidence for immigrant inviability relative to the focal population. The montane perennial ecotype produces few flowers and relies on overwinter survival and vegetative growth. Inland perennial populations had high overwinter survival and growth while also producing more flowers, resulting in a net fitness advantage.

Evolutionary responses can be predicted from the breeder’s equation, but this requires extensive long-term population data. For species that can be stored in a dormant state, such as plants, it is possible to contrast ancestral and descendent genotypes grown under common conditions, and Franks et al. were able to demonstrate evolution to earlier flowering in Brassica using stored seeds. Seedbanks and other collections that hold propagules, intentionally or incidentally, could thus provide important data for exploring evolutionary responses and testing whether species might be approaching limits in their adaptive responses. Sequencing of archived tissue of plants and animals already allows for the signature of selection to be sought directly in their DNA. New collections could systematically sample seeds through time or across populations, providing the potential to resurrect past populations and examine micro-evolutionary change. Evolutionary insights from herbarium specimens might be particularly useful for adapting agricultural practices with global change. Alongside the insights that collections data can provide on ecological and impacts of global change in natural systems, herbaria are additionally repositories of crop wild relatives . CWR are important sources of phenotypic and genetic information on pest and disease resistance that may be introgressed into crops. For example, comparative analyses of CWR might provide an opportunity to identify herbivore-resistance traits relevant to agricultural and ornamental species, such as glandular trichomes that act as physical defences against insects and can be detected on herbarium specimens with a microscope.

In addition, 30 planter pot herbivore damage on CWR herbarium specimens might help predict increases in pest pressure on crops, because closely related host species tend to be vulnerable to similar suites of pests and pathogens. Specimens in herbaria can also serve as records of past biotic threats and inform how we can avoid these threats in the future. For example, Yoshida et al. sequenced the genomes of Phytophthora infestans, the cause of potato late blight, infamous for its role in the Irish Potato Famine, from herbarium collections of infected potatoes and tomatoes. Using genomic tools, they found one strain of P. infestans linked to the potato blight in the nineteenth century, but that multiple strains moved globally in the twentieth century. Specimens may also offer insights into more recent effects of global change on crop species. In a recent study, we quantified historical insect damage on a crop species, the lowbush blueberry, Vaccinium angustifolium, growing in the wild to determine how pest pressure has changed with recent climate change. The low bush blueberry is an ecologically and economically important endemic species in northeastern North America, whose production has seen recent increases owing to awareness of the health benefits of blueberries. Collection records from the Harvard University Herbaria suggest that herbivore damage has increased in recent years, with evidence that increased herbivory is a result of winter climate warming. This highlights the need for increased monitoring of herbivore species on V. angustifolium and allows the development of proactive pest management practices that could be implemented before economic impacts are felt.Given the millions of plants and insect specimens that are becoming available online, it will increasingly be possible to assess changes in phenological synchrony, distributions, and occurrence over time across diverse taxa and large spatial areas.

The sampling of species within museums and herbaria, however, is non-random and often sparse, which can present distinct challenges depending on the response variable of interest and how robust the data are for answering particular questions. However, the depth of sampling within natural history collections is difficult to assess because natural history collections data are often dark—without searchable databases—despite efforts to rapidly digitize. Another obstacle is that data associated with museum specimens can have large uncertainty; for example, specimens collected before the advent of geographical information system technology often have only coarse scale location data that may prohibit local-scale analyses. When assessing phenological change, the most important challenges arise because of biases in collecting. Herbarium specimens are more likely to be collected near roads; rare species are, perhaps unsurprisingly, collected less frequently, and collections are more likely to be made in spring or summer months. Such biases can make specimen data difficult to work with. For example, roads might be warmer than the surrounding countryside, and observations of shifts in phenology through time might, in part, also reflect the increasing extent of the road network. Finally, sampling frequency may bias estimates of first events, because we are more likely to observe earlier events with greater sampling intensity. Such sampling biases can make it difficult to compare across species when sampling effort varies, for example, between common and rare species. New methods offer a solution to such challenges. For example, methods have been established for calibrating species distribution models according to known biases in presence-only data, and newly constructed statistical models allow robust estimates of the tail of a distribution—in the context of phenology, first flower, for example—even when sampling is uneven.

Shifting collection practices may also introduce biases. Herbivory measurements derived from herbarium specimens are probably underestimates in most cases because collectors try to avoid collecting damaged specimens. Even so, herbivory is prevalent on specimens and matches patterns derived from theory and observations from living plants. Importantly, biases introduced by collectors are not necessarily problematic if they do not vary across axes of interest. For example, if collectors are equally likely to collect relatively undamaged specimens across latitude, herbarium specimens might still provide insights into how herbivory varies with latitude. When there are concerns that collecting practices may have influenced observations—for example, perhaps collectors are more or less likely to collect damaged specimens over time—collector identity may be added to statistical models to partially control for such biases. A unique challenge to using herbarium specimens is that they are eaten by a suite of insects within museums. For plants, chewing herbivory created indoors, after a plant was collected, can be confused with damage created while plants were alive. We developed protocols that allowed us to reliably discriminate chewing damage created pre- and post- collection described in. Such approaches, however, require careful examination of specimens with a microscope and entomological knowledge to recognize diagnostic features of damage to plants, which is a barrier to large scale, rapid scoring of chewing damage on digitized specimens. Other types of insect damage, such as leaf mines, skeletonization and galls, are almost never a product of insects eating plants within museums, and might be scored more reliably, although their prevalence is lower.We have previously argued that herbaria should be repositioned as hubs for ecological research, and we provided suggestions for how to manage collections to promote ecological research on global change. Because the vast majority of research in collections has historically been on taxonomy and systematics, collections are rarely faced with providing data for ecologists and evolutionary biologists. Stronger relationships between researchers in the field, collections managers, and digital data providers would help ecologists to better address the challenging ecological questions of global change, and, importantly, could also increase funding opportunities for maintaining and building natural history collections, which are often under-funded and threatened by institutional priorities. Here, we provide three suggestions for how natural history collections and ecologists can work together to support global change research on species interactions. First, it would be helpful to detail the sampling protocol used to collect specimens. In many cases, collecting practices are haphazard, but if, for example, a curator collects a specimen expressly to document a gall, plastic growers pots this would change the inferences we can make from this specimen . Second, ecologists could engage with curators in projects that involve resampling areas and taxa that have long historical records, ideally at the same time of year and with the same research effort involved in previous collections. Third, specimens are most useful when researchers can associate collections with important predictor variables representing species traits or abiotic data related to global change. New opportunities exist to link specimens to the published literature and trait databases , BIEN as well as non-traditional data sources, such as written records and historical photographs. The better integration of new bio-informatics tools and digital databases within biological collections will help transition museums and herbaria into ecological data centres.Human-altered landscapes are expanding globally and are often associated with declining natural habitat, non-native species, fragmentation, and transformations in structure, inputs, climate, and connectivity.

These changes collectively have resulted in shifts in both spatial distributions and species diversity across many taxa including birds, mammals, reptiles, amphibians, invertebrates, and plants. One common driver of global change is urbanization, which in the extreme is associated with a reduction in biodiversity compared to habitats in their more natural state. However, in moderately urbanized areas, the effects of urban impacts on species distribution and diversity can vary greatly and depends on region, type of change, and taxonomic group, among other factors. Documenting the effects of urbanization compared to natural communities has proven problematic, making predictions of community change associated with urbanization difficult. Human-altered landscapes are often associated with many non-native species which add to species diversity but also can obscure changes in community dynamics. Thus, to assess accurately the complex impacts of land use change on ecological communities, one must look beyond species richness to investigate ecological processes themselves. Ecological processes are the links between organisms in a functioning ecosystem, and are critical in understanding how altered biodiversity can lead to changes in ecosystem functioning. Global environmental change has been found to have a wide variety of impacts on ecological processes in different systems. Pollinator-plant relationships in particular are found to be particularly vulnerable to land use change, resulting in decreases in interaction strength and frequency. Pollination services are crucial ecosystem processes in natural systems, but also in agricultural and urban areas. Bees provide the majority of animal-mediated pollination services on which it is estimated 87.5% of flowering plants depend. The value of pollination in agriculture is estimated at $200 billion worldwide , largely due to many foods that are essential for food security and a healthy human diet, including numerous fruits, vegetables, and nuts that require bee pollination. As urban areas expand, there has been increasing interest in urban agriculture to ensure food security and access to healthy foods for growing populations, and these systems also depend on pollination. For example, Kollin estimated that the economic value of urban fruit trees to be worth $10 million annually in San Jose, California. Despite the important role of pollinators and concerns about bee declines, there remain many uncertainties regarding the impact of land use change on pollinators. Urbanization has resulted in more interfaces with both natural and agricultural landscapes, creating new transitional zones of peri-urbanization. While there has been extensive pollinator research in agricultural and natural systems, less attention has focused on pollination in neighboring urban areas and how the changing landscape has impacted pollination. In addition, very few studies of urban areas have looked beyond changes in bee diversity to understand explicitly the effect of urbanization on pollinator-plant interactions . Here, we investigate the effect of land use change on pollinatorplant ecosystem processes. We make use of a ‘‘natural experimental design’’ in which urban, agricultural, and natural areas intersect. Bees visit flowers for both pollen and nectar resources, and floral visitation is a commonly used as an index of pollination services. However, depending on the flower, certain bee groups are much more effective pollinators than others. Thus, while visitation is important, it alone does not definitively indicate whether pollination services were received by the plant. When pollen is limited by other factors, consequences for plant fitness can include failure to set seed, production of smaller fruits, and even complete lack of reproduction. By looking at rates of bee visitation and comparing this with other measures of plant fitness, such as seed set, we can develop a more complete understanding of how shifts in bee distributions between areas that differ in land use are impacting pollination services. To study the impact of changing land use on pollinator-plant interactions, we focus on bee pollination of a widespread plant, yellow starthistle , a common weed found in natural, agricultural, and urban habitats. Using standardized observations of floral visitation and seed set measurements of yellow starthistle, we test the hypotheses that increasing urbanization decreases 1) rates of bee visitation, 2) viable seed set, and 3) the efficiency of pollination .

We used a drip-line system to irrigate plants every morning; the irrigation treatment included two levels: 2.2 L water/plant/day and 0.35 L water/plant/day . Mean volumetric water content for plants in the low irrigation treatment was 10% lower than that of plants in the high irrigation treatment . The volumetric water content for plants in the high irrigation treatment ranged from 38 – 51%, whereas that of plants in the low irrigation treatment ranged from 24 – 48%. All analyses are restricted to data collected from July 21 – August 21 during the height of flowering and pollinator visitation.To test for differences in seed set due to pollen deposition from high and low moisture plants, we used florescent powdered pigments to track bee transfer of pollen from male flowers of plants in high and low irrigation treatments to female flowers of plants in both treatment groups . Before allowing bees access to flowers in the morning, we applied powdered DayGlo© fluorescent pigments to male flowers as in Irrigation experiment I. Male flowers on plants in the high-irrigation treatment received one pigment color, male flowers on plants in the low-irrigation treatment received the other pigment color. Each day we switched the color assignments to mitigate for any color preferences exhibited by bees. As in Irrigation experiment I, the number of pigment particles acted as a proxy for the number of pollen grains transported by bees to the stigmas of the female flowers . We measured pollen and pigment deposition as in Irrigation experiment I and seed set as in the Temperature x irrigation experiment. Mean total pigment particle count on bee pollinated stigmas was highly correlated with mean total pollen grain deposition .

To determine the effect of soil moisture from plants donating pollen on seed set, we used a general linear model with one fixed factor, blueberry in container proportion of deposited pigment from plants grown under low irrigation. For all analyses in Irrigation experiment III, plant is the experimental unit of analysis. All but five plants were given at least one chance to set fruit from a pollinated female flower. For plants that set more than one fruit , we used mean values for total seed mass.Reproductive allocation in bee-pollinated squash plants was affected by the manipulation of temperature and soil moisture, but these factors generally had independent effects on floral traits . When effects on allocation were evident, low soil moisture levels were always inhibitory. For both flower sexes, flower size increased with increasing soil moisture and decreased under warmed conditions . Nectar volume increased with increasing soil moisture and increased temperature for male flowers, but this effect was not significant in female flowers . In addition, nectar concentration increased with soil moisture in ambient temperature male flowers but not in female flowers . There were no effects of temperature or soil moisture on the proportion of viable pollen grains in male flowers . Lastly, per plant flower production was unaffected by the manipulation of temperature and soil moisture, except for the potential availability of non-self flowers . The main pollinators observed visiting squash flowers in this experiment were honey bees and squash bees pruinosa. Together these two species accounted for 97% of all floral visits. In general, pollinator visitation rates did not differ with respect to variation in temperature and soil moisture experienced by squash plants , but this experiment was not designed to examine pollinator choice.

The one exception with respect to visitation was that we observed that the cumulative pollen collecting per min for Apis mellifera was higher in warmed plants . There was also a significant interaction between soil moisture and temperature for visits per female flower per minute for Apis and Eucera ; however, these results were not significant when broken down between warmed and ambient treatments. Fruit set and seed set of squash plants increased with increasing soil moisture . Aside from soil moisture, no other measured variable, including pollination type, affected the likelihood of fruit set . In contrast, seed set increased with increasing soil moisture but only for bee-pollinated plants . The seed set of fruits that resulted from hand-pollination did not depend on soil moisture and was almost 27% higher compared to that from fruits that resulted from bee pollination . Based on seed set measurements, the pollen limitation index increased with decreasing soil moisture . There was no effect of temperature on pollen limitation .We assessed the degree to which pollinators influence how experimental warming and drought affect the reproduction of cultivated squash . For those floral traits that responded to the experimental manipulations, low soil moisture levels always resulted in inhibitory effects: smaller flower size, reduced nectar volume and nectar concentration , and a reduced availability of non-self, male flowers. By comparing responses of squash that were either pollinated by bees or by hand, we observed that be pollinated plants experienced increasing levels of pollen limitation as a function of decreasing soil moisture. This result illustrated that pollinators mediate how soil moisture variation affects the outcome of plant reproduction. Additional soil moisture manipulations indicated that drought-induced pollen limitation results, at least in part, from decreased pollen competition resulting from low-levels of stigmatic pollen deposition, reduced viability of pollen produced by plants grown under low soil moisture conditions, and a reduced capacity of self pollinated fruits to respond to increasing soil moisture.

In the present study levels of warming were realistic based on current climate projections , but mean summer temperatures at our coastal study site were mild and even experimentally warmed conditions likely did not result in prolonged physiological stress for squash plants, which are often grown in areas with higher mean summer temperatures compared to those at our study site . In contrast to previous studies that have documented reduced reproductive allocation to warming , two of the three significant responses to temperature in the Temperature x irrigation experiment represented increases in performance . Our study is not unique in this respect; Hoover et al. also report elevated nectar production by pumpkins experiencing experimental warming. Aside from flower size, which decreased under warmed conditions, all of the measured aspects of reproductive allocation in this study either did not respond to the elevated temperature treatment or apparently benefitted from it. The lack of interactive effects observed in Temperature x irrigation experiment may have thus been partly due to a temperature treatment that did not physiologically challenge plants, but other studies also report that the effects of temperature and soil moisture can act independently of one another . In contrast to the effects of temperature, we found that soil moisture variation affected more reproductive traits and that these responses were all negative . Observed effects mirror those reported in previous studies. Known effects of drought, for example, include reductions in flower size and nectar volume . Changes in floral traits caused by drought stress can also reduce visitation by pollinators , but this trend seems to exhibit context specificity and is not universal. Although the responses observed in our system might be expected to reduce visitation by pollinators, our experiment was not designed to measure pollinator choice as open flowers on a given day were all from the same treatment group. Pollinator visitation is the focus of Chapter 2, in which we explicitly test the consequences of floral choice made by specialist and generalist pollinators in an experimental setting where bees can simultaneously visit squash flowers produced by plants grown under different levels of soil moisture. An additional reproductive trait that varied with soil moisture was the availability of non-self, male flowers. Previous studies have also reported changes in flower production in response to drought stress. Al-Ghzawi et al. , Philips et al. , Descamps et al. , for example, all report examples of drought-induced reductions in flower production, plastic planters bulk but this finding is not universal and likely depends on the timing of floral development relative to the onset of drought stress . To examine the role of pollinators in mediating how soil moisture variation affects plant reproductive success, we compared seed set for plants pollinated by bees versus those pollinated by hand and found that bee-pollinated plants grown under low soil moisture levels were pollen limited. To our knowledge, drought-induced pollen limitation has not previously been reported in the literature but could be common given well-known effects of non-lethal, drought stress on various aspects of plant reproductive allocation . Although pollen limitation commonly results from inadequate visitation by pollinators, this mechanism seems unlikely to explain our result given that bee visitation did not increase with increasing soil moisture level. Differential pollen production can also be ruled out as an underlying mechanism for drought-induced pollen limitation given that the amount of pollen produced was independent of soil moisture.

Given that we observed an apparent reduction in the availability of non-self, male flowers as a function of low soil moisture levels, the transfer of self pollen might have been higher for plants grown under low soil moisture conditions. However, we found no evidence for this type of effect through the use of pigments to estimate patterns of pollinator movement. Our results suggest that drought-induced pollen limitation in part stemmed from reduced pollen competition on stigmas that received pollen from bees. Winsor , for example, found that seed set and offspring vigor in C. pepo increased with increasing pollen load and concluded that more intense pollen competition was the underlying cause of this effect. Like Winsor , we observed that seed set increased with increasing pollen deposition . In the Temperature x irrigation experiment, the greater than two-fold higher pollen loads on hand pollinated stigmas, compared to bee-pollinated stigmas, would have heightened pollen competition and allowed only the fastest-growing pollen tubes to achieve fertilization. Consistent with this hypothesis is the fact that the seed set of hand-pollinated plants was independent of the soil moisture levels that these plants experienced while growing. In contrast, the smaller pollen loads deposited by bees would have led to relatively lower levels of pollen competition and a greater opportunity for self pollen or pollen of reduced viability to achieve fertilization. Relaxed pollen competition in bee-pollinated plants could help to explain why we observed drought-induced pollen limitation, but for this mechanism to reduce seed set pollen deposited on the stigmas of must have been of inferior quality. Two lines of evidence support this hypothesis. First, we observed a negative relationship between seed set and the amount of pigment from the male flowers of plants grown under low soil moisture conditions . Moreover, we found that seed set increased with increasing levels of soil moisture experienced by plants contributing non-self pollen . These results taken together suggest that the pollen viability assays used in the Temperature x irrigation experiment may not have been sensitive enough to measure more subtle aspects of pollen viability as measured here. Previous studies that did find significant effects of drought on pollen viability used different assays than the one performed here. Second, we observed that the seed set produced by flowers pollinated with non-self pollen increased with soil moisture, whereas the seed set resulting from flowers pollinated with self pollen did not respond to increasing soil moisture. Although the reduced availability of non-self, male flowers for plants grown under low soil moisture conditions was on some level an artifact of our experimental design , it would seem common for plant species to grow in discrete enough patches for the availability of self pollen versus non-self pollen might influence the outcome of pollination. For example, buffalo gourd, which is the local, native congener of C. pepo, grows in patches of up to five individuals with hundreds of meters separating patches . In conclusion, our results reveal that pollinators can mediate effects of warming and drought on plant reproduction by increasing pollen limitation as a result of decreased pollen competition caused by low-levels of stigmatic pollen deposition, reduced viability of pollen produced by plants grown under low soil moisture conditions, and a reduced capacity of self-pollinated fruits to increase seed set in response to increasing soil moisture. Although this study focuses on only a single plant species, drought-induced pollen limitation could be a common phenomenon based on the frequency with which drought stress affects reproductive allocation in other plant species. Water stress, for example, commonly lowers pollen viability .

Paper chromatography , thin layer chromatography techniques, high speed counter current chromatography , capillary electrophoresis , and supercritical fluid chromatographic techniques are also used to identify the phenolic compounds.With organic agriculture poised to represent 10 to 20 percent of California cropland by 2024, the federal government has tapped UC Santa Cruz to lead a research program that will give organic farmers the same kind of boost the university has given conventional farmers for decades. The U.S. Department of Agriculture has awarded UCSC’s Environmental Studies Department a $571,000 grant over four years to bolster scientific knowledge about organic systems and to strengthen the Central Coast network of organic farmers and agricultural researchers. Researchers and staff from the Center for Agroecology and Sustainable Food Systems will play key roles in this effort. In collaboration with farmers, agroecology researchers at UCSC have pioneered organic production methods for strawberries and other important regional crops. This project will build on those successes and prepare the organic industry for continued rapid growth by developing baseline nutrient management tools and addressing stubborn challenges, such as soil pathogens and pest management. “Conventional farmers have decades worth of research to draw on, while organic growers have very little scientific data to rely on,” said environmental studies professor Carol Shennan, the Center’s director and one of four UCSC leaders of the project. “Organic production is a complex system that integrates soil fertility, crop rotation, water management, growing berries in containers and pest and disease control. It requires a systems approach, but agricultural research has historically tended to focus on narrow, single-issue problems.”

The grant will fund a series of coordinated experiments at multiple locations in the Monterey Bay region designed to give farmers hands-on information. The results will be dispersed throughout the farming community with the help of organizations such as California Certified Organic Farmers, the Organic Farming Research Foundation, the Community Alliance with Family Farmers, and the Agriculture and Land-Based Training Association. UCSC’s research team will be made up of Shennan, environmental studies professor Stephen Gliessman, research associate Joji Muramoto, and entomologist Sean Swezey, the Center for Agroecology and Sustainable Food System’s associate director. “Over the years, we’ve done research that farmers want, and they’ve had a role in directing it,” said Gliessman, the Alfred E. Heller Professor of Agroecology. “Our job is to take their problems and do the research they need to solve those problems.” Gliessman recalls the skepticism that greeted early collaborations. “When we started this work 17 years ago with Jim Cochran of Swanton Berry Farms, people said, ‘You’re crazy. You aren’t going to grow strawberries organically.’ Now, the USDA is saying, ‘This is important. It has to be done.’ It’s the farmers who took the risk.” Cochran will be joined by other experienced, innovative organic growers who will participate in the study, including Daniel Schmida of Sandpiper Farms and Steve Pedersen of High Ground Organics. Landowner Robert Stephens has also set aside a portion of his acreage at Elkhorn Ranch for use in the study. In addition, research will be conducted on the 25-acre UCSC Farm. Representatives from UC Cooperative Extension offices in Santa Cruz, Monterey, San Benito, San Luis Obispo, Santa Barbara, and Ventura Counties will participate, as well as a scientist from the USDA’s Agricultural Research Station in Salinas and an agricultural economist from UC Davis. The grant will enable researchers to take ongoing studies to a new level and test the limits of monoculture farming, said Gliessman. “We want to redesign the system to better resist disease,” he said. “It may turn out to look very different from what we’re used to. You probably won’t see acre after acre of organic strawberries.

You may see a patchwork of strawberries and other crops, because monoculture brings problems. We need to build on the strengths of diversity.”By conducting replicated trials on organic farms, researchers will assess the effects of crop rotations and different fertility, disease, and pest management strategies on yields, soil quality, weeds, pests, and soil pathogens. Experiments will focus on – • Testing biological alternatives to methyl bromide to suppress Verticillium dahliae, a soil pathogen that poses the greatest threat for organic strawberry production in the state. Anaerobic decomposition of cover crop residues and bio-fumigation with Brassicas will be evaluated. Given the upcoming ban on methyl bromide, such experiments may be of value to conventional farmers, as well. This part of the study will include testing a technique pioneered in the Netherlands to control a number of soilborne diseases. Blocks of both a cover crop mix and mustard will be incorporated into the soil, then covered with a tarp as the crops decompose. Following the tarping, researchers will measure levels of V. dahliae in the soil to assess this technique’s potential for disease suppression. • Developing tools to help organic farmers monitor changes in soil nutrient levels, or “what goes in and what comes out,” as Shennan put it. Researchers will analyze the nutrient value of soil amendments, including cover crops, commercial composts, and fish emulsion fertilizer, and document what nutrients are removed when the crop is harvested. They will also develop plant tissue nitrogen tests so farmers can assess how well their fertility management is working, said Shennan. Building a database of their findings will give farmers a powerful resource to draw on that will supplement their own soil tests, she said. This issue is especially timely on the Central Coast, where non-point source pollution of waterways is subject to intense public and regulatory scrutiny. Demonstrating organic practices that help minimize leaching and runoff will be important for the region’s growers.

The fertility management work will build on several years of water monitoring data collected by researchers from the Center for Agroecology and Sustainable Food Systems as part of the Central Coast project . • Applicability of three models to simulate how management changes could impact crop harvests, soil nitrogen availability, and the movement of nutrients under a range of weather conditions and for different types of soils. If any model works well for predicting farming outcomes in this area, it could be used to help farmers make decisions about crop rotations, cover crop use, and management of fertility inputs, said Shennan. • Use of organic pest control to combat pests that prey on strawberries. Vacuum devices and an alfalfa trap crop will be tested against the western tarnished plant bug , and researchers will evaluate the movement and effectiveness of beneficial insects introduced into or near the trap crop vegetation. Researchers will also assess the value of native-plant hedgerows in attracting beneficial insects that prey on crop predators. This work will expand on research efforts showing that alfalfa can be an effective trap crop for WTPB and can be managed to reduce energy use associated with crop vacuuming, as well as enhance populations of beneficial insects. For results from an earlier phase of this study, see “Trap crops show potential to reduce pest damage, save time and energy in organic strawberry production,” The Cultivar, Vol. 22 No. 1, Spring/Summer 2004. Results from the various studies will be passed on to growers via a combination of meetings, trainings, field days, listening sessions, focus groups, blueberry containers and other exchange opportunities designed to promote feedback between researchers and growers.The scope of the project reflects the importance of organic agriculture in the economy and UCSC’s role supporting organic farmers, said Shennan. The Central Coast is well known for its high concentration of organic farms, and many producers have benefited from partnerships with UCSC researchers and UC Cooperative Extension specialists. In Santa Cruz and Monterey Counties, more than $140 million, or 6 percent, of the region’s $2 billion vegetable production in 2001 was certified organic, according to Shennan, and the two counties generated more than $400 million gross value in strawberries. The California organic agriculture industry has grown quickly, producing sales of $340 million in 2003, according to the California Department of Agriculture Organic Program. The state produces nearly half of the total organic vegetables certified in the United States; strawberries are the most lucrative organic commodity in the state on a per acre basis, valued at $17.5 million. “Organic farmers face the same production challenges as conventional growers, but the research community has overlooked their needs,” said Shennan. “With one of the oldest university-based organic research and training programs in the world and one of the pioneering academic programs in agroecology, UCSC is in a good position to help fill in the gaps of scientific knowledge.”Most consumers feel that they don’t know enough about how their food is grown and processed, how it reaches them, or what’s involved in food marketing. They’d like to see a system of eco-labels that would provide information on such criteria as whether the workers receive a living wage, whether the animals were treated humanely, and whether the food was locally grown.

These are some of the conclusions of a study by social issues researchers Phil Howard and Jan Perez of the Center for Agroecology and Sustainable Food Systems. The study asked consumers what aspects of food production, processing, transportation, and retailing they were most interested in knowing more about, and how they wanted to get that information. “Our goal was to give consumers a voice they might not have, and the first step was to find out what they want to know,” said Howard, a second-year postdoctoral researcher. “Food retailers, processors, and growers should all start looking closely at these issues because people are interested in supporting them through their purchases.” In 2004 Howard and Perez conducted five focus groups and mailed a 26-question survey to 1,000 randomly selected households in San Mateo, Santa Clara, Santa Cruz, San Benito, and Monterey Counties; the survey response rate was 48 percent. The study was funded by a U.S. Department of Agriculture grant to foster sustainable agriculture on the Central Coast as part of the Center’s Central Coast Research Project.Eight food system-related topics were identified as themes that interested the focus groups; these topics were then presented in the written survey. Not surprisingly, the scores indicated that survey respondents were most interested in safety and nutrition; nearly all respondents ranked these topics near the top of a scale from 1 to 10 . A number of surveys have consistently shown these to be important concerns, even for those with few other food-related interests. One focus group participant highlighted this fact when stating, “Who knows what the heck is in half the stuff we buy, I mean I don’t … Frankly, I don’t care as long as it doesn’t get me sick.” This was a minority view, however, as most focus group participants also had a number of concerns beyond their personal health. The survey results supported this broader concern. Treatment of animals involved in food production, environmental impacts, and working conditions all received an average score of greater than 7. In the focus groups, the treatment of animals elicited the most emotion. Several participants had toured slaughterhouses and said this experience had a lasting effect on them. Others had changed their consumption habits after learning of the way some animals are treated, such as veal calves. For example, a focus group participant discussing the inhumane aspects of confinement animal production asked, “then are you eating growth hormone and … or whatever you’re putting in them, and what does that do? I mean, in the long run you know, what’s that doing to you?” On the issue of environmental impacts, focus group participants most frequently expressed concerns related to pesticides and genetic engineering. Some participants were also concerned about irradiation and the impacts of food packaging or food waste. Several participants noted that environmental impacts were much more important to them when compared to other concerns about the food system. On the topic of working conditions and wages, focus group participants were interested in the treatment of farm workers, such as the backbreaking labor performed for very low pay, and the exploitation of migrant workers. Workers involved in other aspects of the food system, such as processing or retail, were not discussed as frequently. When asked specifically to list criteria they would like to see improved for workers involved in the food system, focus group participants mentioned higher wages, protection from pesticide exposure, health care, education, adequate food, limited working hours, and adequate housing.

However, the spinach leaf disks stored under constant darkness at 22°C for 6 days completely disintegrated and therefore could not be moved for photographic imaging. Lettuce and spinach leaf disks stored in constant darkness at 4°C largely maintained dark green coloration at 6 days and were lighter green at 9 days, similar to that of the disks stored under light/dark cycles at 22°C . However, after 6 days of storage at 4°C, the lettuce leaf disks also displayed browning around the vascular tissues . Overall, the image analysis shown in Figure 1 suggests that postharvest storage under cycles of 12-hour light and 12-hour darkness may enable kale, cabbage, lettuce, and spinach leaf tissues to maintain physiological functioning for longer durations after harvest. The reduction in green color and appearance of brown discoloration suggests that postharvest storage in constant light or constant darkness may accelerate loss of tissue viability. To further characterize kale, cabbage, lettuce and spinach leaf health and viability during postharvest storage, we quantified chlorophyll content in leaf samples after storage under cycles of 12-hour light/12-hour darkness to leaf tissues stored under constant light or constant darkness. Three sets of comparative data are shown . Because our primary focus was to determine whether light/dark cycles were advantageous relative to constant light or constant dark storage conditions, we first conducted two-way comparative statistical analyses between data derived from the samples stored under light/dark cycles relative to comparable samples stored under the alternative condition. Figure 2 presents statistical analysis of storage-dependent differences in chlorophyll levels relative to dry weight at each time point.

Additional file 1: Figure S3 shows similar analyses but of storage-dependent differences in chlorophyll levels relative to fresh weight. Finally, container growing raspberries to evaluate whether there were significant changes in chlorophyll levels of each plant type over time, statistical analyses of differences in chlorophyll content at the beginning and end of the experiments for kale, cabbage, lettuce, and spinach are shown in Additional file 1: Figure S4. Consistent with the loss of green coloration in the representative leaf disk samples shown in Figure 1A, postharvest storage of kale leaf disks in either constant light or constant dark led to significantly greater losses in kale chlorophyll content within 3 or 6 days of postharvest storage compared to storage under 12-hour light/12-hour dark cycles . Kale leaf disks stored for 15 days under constant light lost 97% and 93% of their original chlorophyll content relative to dry and fresh weight, respectively ; kale leaf disks stored under constant darkness lost 88% and 89% of their chlorophyll content relative to total dry and fresh weight, respectively . In contrast, 15 days of storage under cycles of 12-hour light/12-hour darkness led to loss of only 36% and 9% of the kale leaf disk chlorophyll relative to dry and fresh weight, respectively . Kale leaf disks stored at 4°C under constant darkness, however, performed statistically better than those stored under cycles of light/dark , with no significant decreases in chlorophyll content relative to total dry or fresh weight over the full 15 days of the experiment . Postharvest storage of cabbage leaf disks under light/ dark cycles resulted in significantly higher chlorophyll levels than storage under constant dark either at 22°C or 4°C at all time points examined . Indeed, cabbage leaf disks began with only modest chlorophyll levels . However, when the cabbage leaf disks were stored under either constant light or light/dark cycles, chlorophyll content increased over time with significantly higher levels remaining even after three weeks of storage . Light-induced synthesis is likely responsible for the elevated chlorophyll levels and the enhanced green coloration observed in the cabbage leaf images of the samples stored under light/dark cycles or constant light but absent in the cabbage leaf disks stored under constant darkness either at 22°C or 4°C .

Storage under light/dark cycles was also more successful than constant light exposure in maintaining higher chlorophyll levels after long-term storage of 3 weeks . Over time, storage under constant light may be counterproductive; whereas cabbage leaf disks stored for 7 days under constant light had significantly higher chlorophyll content than leaf disks stored under light/dark cycles , by three weeks of storage, the leaf disks stored under light/dark cycles retained at least 2-fold more chlorophyll than samples stored under constant light . These results indicate that light during postharvest storage can have a profound effect on chlorophyll levels in cabbage, consistent with previous reports, and that diurnal cycling of light and darkness prolongs this benefit during longer term storage. Storage under cycles of 12-hour light/12-hour darkness also promoted chlorophyll retention in lettuce leaf disks, comparable to that of lettuce leaf disks stored under refrigeration; chlorophyll levels were statistically indistinguishable between lettuce leaf disks stored under light/dark cycles versus those refrigerated under constant darkness conditions after 3, 6 or 9 days of storage . Postharvest storage of lettuce leaf disks either at 22°C under light/dark cycles or under refrigeration resulted in no significant change in chlorophyll content over the course of the 9-day experiment, whereas the lettuce leaf disks stored under either constant light or constant darkness, lost more than 50% of their starting chlorophyll content . Chlorophyll content of spinach leaf disks was not significantly affected by treatment conditions for the first 3 days of postharvest storage . However, the spinach leaf disks stored at 22°C in constant darkness disintegrated by 6 days and were therefore unable to be further analyzed. Spinach leaf disks stored under light/dark cycles had similar chlorophyll content to those stored under constant light with relatively stable chlorophyll retention until day 9 when chlorophyll levels in both samples decreased significantly from initial levels . In contrast, refrigeration led to stable chlorophyll levels in the spinach leaf disks over the course of the experiment . These results indicate that chlorophyll content of postharvest green leafy vegetables varies depending upon the storage conditions and suggests that storage under 12-hour cycles of light and darkness, known to maintain the plant circadian clock, can improve kale, cabbage and lettuce chlorophyll content maintenance relative to storage in constant light or constant dark. Perhaps surprisingly light/dark cycles during postharvest storage may be at least as beneficial as refrigeration with respect to chlorophyll content for cabbage and lettuce.Over time during postharvest storage, plant tissues typically show visible signs of tissue disintegration . To determine if maintaining light/dark cycles during storage of post harvest leafy vegetables could prolong tissue integrity, we compared electrolyte leakage from kale, cabbage, lettuce, and spinach leaf disks stored over time under cycles of 12-hour light/12-hour darkness to leaf disks stored under constant light or constant darkness at 22°C or constant darkness at 4°C. Figure 3 shows that postharvest storage under light/dark cycles and refrigeration were comparable, with respect to leaf tissue integrity maintenance of kale, cabbage and lettuce, as measured by electrolyte leakage, . When directly comparing light/dark storage to other conditions, a statistically significant benefit to diurnal stimuli during storage was apparent relative to constant light for kale , constant darkness for cabbage and lettuce , and constant darkness and constant light for spinach . Postharvest storage under constant dark was detrimental to kale, cabbage, and lettuce tissue integrity, with at least 4-fold increases in electrolyte leakage, whereas storage under light/dark cycles at 22°C or refrigeration resulted in no significant increase in electrolyte leakage over the course of the experiment .

Constant light treatment also led to significant increases in electrolyte leakage from kale and lettuce leaf disks, blueberries in pots but not cabbage leaf disks, over the storage periods examined . Overall the results shown in Figure 3 and Additional file 1: Figure S5 provide evidence that daily cycles of light and darkness during postharvest storage resulted in superior leaf tissue integrity maintenance largely comparable to refrigeration, whereas either constant light or constant dark storage conditions were detrimental.Our results indicate that storage of kale, cabbage, lettuce, and spinach in light/dark cycles can improve the postharvest longevity of chlorophyll levels and tissue integrity. Next we were interested in determining whether plant maintenance under daily cycles of light and darkness affects human-health relevant metabolite content. In particular, we sought to examine whether kale and cabbage stored under light/dark cycles maintain their glucosinolate content longer than when stored under constant light, constant darkness, or refrigeration. Figure 4A shows total glucosinolate levels in kale leaf disks after 0, 3, 6 and 15 days of postharvest storage under different conditions. Individual glucosinolate levels are shown in Additional file 1: Figure S1. Total glucosinolate levels were comparable between kale leaf disks stored at 22°C under light/dark cycles and leaf disks stored at 4°C in the dark ; after 15 days of postharvest storage under these conditions, total glucosinolate levels decreased by less than 35% . In comparison to light/dark storage conditions, constant light or constant darkness exposure during storage resulted in significantly reduced glucosinolate content in the kale leaf disks . By 15 days of postharvest storage under constant light or constant darkness at 22°C, the kale leaf disks lost over 80% and 99% of initial levels, respectively. Daily cycles of light and darkness also promoted maintenance of glucosinolate content during postharvest storage of cabbage . Total glucosinolate content in the cabbage leaf disks stored under light/dark cycles remained stable with no significant fluctuation in levels over the 21 days of analysis . In comparison to total glucosinolate levels in light/dark-stored cabbage, the glucosinolate levels were significantly lower by 7 days when cabbage leaf disks were stored under constant darkness and by 21 days when stored under constant light . Total glucosinolate levels declined by 70% and 88%, respectively, in cabbage disks stored at 22°C under constant light or constant darkness. Remarkably, glucosinolate levels of the cabbage leaf disks stored at 4°C also showed a significant decrease by 21 days, with a loss of 50% of the initial glucosinolate levels , indicating that storage under cycle of light/darkness led to enhanced retention of this valuable phytochemical even relative to refrigeration.In this work, we examined whether kale, cabbage, lettuce and spinach leaf tissue maintain the ability to respond to light/dark cycles during postharvest storage and whether under these conditions that better mimic the natural light cycles of the environment tissue deterioration would be reduced. Our goal was to expose plant tissues to diurnal conditions known to maintain the functioning of the circadian clock and thereby capitalize on physiological enhancements conferred by robust circadian rhythms. Plants grown under light/dark cycles that match the endogenous cycling of their internal circadian clock have a growth and reproductive advantage over plants exposed to light/dark cycles that do not match their internal oscillator. Furthermore, phasing of circadian rhythms so as to be synchronized with the external environment promotes biotic stress resistance. We found that storing green leafy vegetables in cycles of 12 hours of light followed by 12 hours of darkness improved several postharvest performance markers compared to postharvest storage of the leaf tissues under constant light or constant darkness. Similarly, a modest reduction in senescence was noted for post-harvest broccoli stored under natural light/dark cycles. Perhaps surprisingly, we found that storage in light/dark cycles resulted in several aspects of postharvest performance being comparable to storage under refrigeration, a commonly practiced method of postharvest storage thought to slow down cellular breakdown. The longevity of kale and lettuce leaf color, chlorophyll levels, and tissue integrity, which are important contributors to the appeal of green leafy vegetables to consumers, were largely indistinguishable whether the kale and lettuce leaf samples were stored at 22°C under light/dark cycles or were stored under refrigeration in constant darkness . Spinach leaf samples also maintained green coloration and chlorophyll levels under light/dark cycles at 22°C as well as when refrigerated, but refrigeration was more successful at preventing spinach leaf tissue breakdown. Significant improvement of green coloration and chlorophyll content was seen when cabbage leaves were stored under light/dark cycles at 22°C compared to refrigeration, demonstrating that light may not only be important for clock entrainment but also can provide the additional benefit of promoting continued photosynthesis during postharvest storage. Promotion of photosynthesis and/or chlorophyll levels was previously observed in post-harvest crops stored under light. However, constant light during postharvest storage can also cause detrimental physiological activity, such as respiration leading to browning and transpiration contributing to weight loss.

The year 1980 was selected as a break point for these data as it has previously been identified by the Intergovernmental Panel on Climate Change as the year after which global land temperatures rose consistently above the 1961–1990 normal . We then tested for significant differences in mean estimated frost risk between historical and recent time periods across all taxa by conducting a comparison of the pre- 1980 and the 1980–2015 frost risk estimated for each species using a Wilcoxon paired rank test. It should be noted that some late-spring and summer-flowering species were not exposed to frost within the duration of this study, or were exposed to frost only rarely. However, to avoid the inherent biases that would result from selectively including only those species that were exposed to frost , all species were included in this analysis regardless of frost exposure. The number of specimens of each species that were collected prior to 1980 often differed from the number collected post-1979, as did the mean frost date normals of the locations from which those specimens were collected. This difference in sampling had the potential to bias estimates of the change in frost risk between these two periods. We therefore conducted a multiple regression to test whether significant differences in frost risk between pre-1980 and 1980–2015 samples persisted after controlling for variation in the mean flowering dates among species, the number of specimens of each species that were collected pre-1980 and post-1979, and the mean normal frost dates of the locations from which specimens were collected pre-1980 and post-1979. In this analysis, frost risk was the dependent variable, plant pots with drainage while species-specific mean flowering dates, species-specific mean frost date normals , and the time period of collection were treated as independent variables.

Pre- 1980 values for each species were distinguished from post-1979 values using a categorical variable coded as 0 for pre-1980 values, and 1 for post-1979 values .To determine whether clades differ with respect to mean frost risk or either the magnitude or direction of the change in their frost risk in response to recent climate change, we estimated Bloomberg’s K, a metric of phylogenetic conservatism for both the pre-1980 frost risk for each species and the magnitude and direction of changes in frost risk post-1979. Phylogenetic distances used in this study were calculated using angiosperm nodes aged according to the V.Phylomaker package in R , which was also used to estimate Blomberg’s K. Note that all genera used in this study were present in this tree, and that all genera were considered to be polytomies.To evaluate the relationship between frost risk and short-term climate deviations, we first calculated the mean frost risk for all species using a 20-year moving window average calculated at 1-year increments from 1920 through 2015. To eliminate those taxa for which sample density was too low to produce robust estimates of frost risk throughout the entire time series, only those taxa that were represented by a minimum of 40 specimens within all 20-year windows were included in this analysis. A total of 268 species distributed throughout North America met this criterion. Overall frost risk within each 20-year window was then assessed by calculating the mean estimated frost risk across these 268 species within that time period. For each specimen that fell within a given 20-year window, we also calculated its annual deviation from the local normal date of last frost at the location in which it was collected . For each species, we then calculated both the frost risk and the mean annual deviation from normal dates of last frost among all the specimens represented within each 20-year window. Within each successive 20-year window, we then calculated the mean frost risk and mean annual deviation from normal dates of last frost among all species.

We predicted that, among the 20-year windows, as the mean deviation between current and historical last frost dates increases , the mean frost risk of the species in each successive 20-year window would also decline. To test for the effects of annual deviations in local dates of last frost on frost risk, we conducted a linear regression of the mean frost risk within each 20-year window on the mean annual deviation in the date of last frost within that 20-year window. To determine whether the relationship between frost risk and short-term climate deviations persisted across regions of North America characterized by both early and late dates of last frost, we separated taxa into two groups: those for which the mean last frost date normal of the collection sites was earlier than the median last frost date normals across the entire dataset and those for which the mean late frost date normal of the collection sites was later than the median last frost date normal. Having separated taxa that were predominantly sampled from locations characterized by early versus late last frost dates, we conducted the above analyses separately on each group of taxa.Across all 1,653 species that were well documented both pre-1980 and post-1979, mean frost risk has declined significantly since 1979, from 12.1% to 8.7% , representing a ~28% reduction in frost risk. Moreover, the number of species that experience minimal frost risk has increased from 234 species pre-1980 to 291 species post-1979 . Of the 66.1% of species that exhibited a reduction in frost risk post-1979, the mean frost risk declined from 16.0% to 9.7%. Conversely, of the 24.5% of species that exhibited a post-1979 increase in frost risk over this pre-1980 frost risk, mean frost risk increased from 6.3% to 9.3% , with only 77 species exhibiting a net increase in absolute frost risk greater than 5% . Median rates of directional change in dates of last frost over time were 3.3 times greater than rates of directional changes in flowering time among species . Consistently, while both frost dates and mean flowering dates among most species were found to occur earlier post-1979 than pre-1980, differences in the mean date of last frost between reference periods were of greater magnitude than those in flowering phenology .

Temporal changes in frost risk during this period are clearly associated with annual deviations from normal dates of last frost . From 1970 to 2015, as the annual dates of last frost have generally become progressively earlier than the 1901–1990 mean last frost date, the mean frost risk has declined. Moreover, among 20-year moving windows from 1920 to 2015, advances in the frost-free date explain 65% of the observed variation in mean frost risk . When examined separately, this pattern remained consistent among taxa occupying regions with late frosts and those occupying regions with early frosts, although late-frost regions experienced slightly greater reductions in frost risk during years experiencing earlier frosts . Analysis of 801 weather stations across North America revealed that rates of change over time for the date of last occurrence of 0°C temperatures were highly correlated to changes in last occurrences for −1°C, −2°C, −3°C, and −4°C temperatures . The magnitude of change over time in last 0°C dates closely matched that of changes for last −1°C and −2°C dates of occurrence . In turn, directional changes in dates of last frost defined using −3°C, and −4°C were of moderately lesser magnitude than changes in 0°C dates of last frost . Furthermore, advances in the date of last frost from 1920 to 2015 have typically outpaced advances in flowering phenology, despite advances in both frost dates and mean flowering dates among most species. We recognize that, for any given species, the frost risk during any particular time period could have been overestimated if the species was disproportionately sampled from sites where phenological advancement exceeded the advancement of the date of last frost, or underestimated if the species was disproportionately sampled from sites where the advancement of the date of last frost exceeded phenological advancement. However, the fact that these results remained consistent across so many taxa, and remained similar even when taxa occupying relatively warm regions and those occupying relatively cold regions were examined separately, indicates that such biases did not contribute significantly to the observed temporal changes in frost risk. Moreover, by constructing models in which we controlled statistically for spatial variation in the climate normals of each species’ specimens sampled before 1980 versus after 1979, plastic plants pots we further minimized this possibility. Thus, although it is well documented that early-flowering species, which flower in closest proximity to the last spring frost, are typically more phenologically sensitive to local temperature and show more rapid advances under warming conditions than late-flowering species , this study indicates that early-flowering species do not typically advance more rapidly than the date of last frost throughout North America. This pattern is relatively consistent among taxa, with less than 25% of sampled taxa exhibiting increases in frost risk post-1980. However, these results also indicate that, while significant phylogenetic signal for exposure to frost exists among the taxa sampled here, the degree to which frost risk is conserved among closely related taxa is minimal. Instead, similarities in frost risk among related taxa are more likely to be the result of convergent evolution in traits that are correlated with attributes that lead to exposure to frost rather than to strong phylogenetic conservatism . Thus, it does not appear that there are systematic differences among angiosperm clades with respect to the effect of recent warming on frost risk. Similarly, while a higher proportion of invasive species was observed in relatively warm regions , no systematic differences in frost risk were apparent between native and invasive taxa after controlling for differences in long-term frost dates across their ranges . Thus, although previous studies have indicated that invasive species often exhibit more responsive phenologies and weaker chilling requirements than their native counterparts, this study indicates that more successful avoidance of frost risk is unlikely to be a systematic factor that determines the invasive ability of most exotics. Dates of last frost have typically advanced more rapidly than dates of flowering for most of the 1,653 North American species sampled in this study , and temporal advances in the mean date of last frost are closely associated to reductions in frost risk . These results are supported by previous examinations of exposure to frost by growing leaves among North American tree species by Zohner et al. who also found reduced frost exposure in association with recent warming. Collectively, these results demonstrate that the risk of frost exposure and damage to both reproductive and vegetative tissues are systematically decreasing across North America. The ecological implications of these reductions in frost risk may be complicated by variation among species in their resilience to frost-related damage and in the degree to which frost damage to floral tissues may reduce their reproductive success, both of which remain largely unknown for most species. We were not able to directly quantify changes in frost risk defined using temperature thresholds other than 0°C because, to our knowledge, no gridded products provide dates of last frost defined using a lower temperature threshold at high spatial resolutions and spanning our study period . However, weather station data revealed that rates of temporal change in dates of last frost defined using different temperature thresholds were highly correlated and of similar magnitude. Temporal changes in dates of last frost defined using thresholds of −3°C and −4°C were moderately lower in magnitude than rates of change for 0°C dates of last frost . However, given that rates of change over time in dates of last frost at 0°C were more than three times greater than those in flowering time , the moderately slower rate of change of −3°C and −4°C dates of last frost likely outpaced changes in flowering time as well. Therefore, although the identity and exact proportion of taxa experiencing changes in frost risk over time might vary when using freezing temperatures more severe than 0°C to define frost, the general trend toward lower frost risk over time is likely to be robust to different definitions of the temperatures that is likely to induce tissue damage. In addition to direct effects of frost damage on vulnerable vegetative or reproductive tissues, the effects of these changes in frost risk on the survival and local abundances of many species will likely be mediated by a variety of factors.

The root samples collected for this study were sour orange and trifoliate genotypes, but samples were grouped by organ to perform differential expression and subsequent analyses. To our knowledge, there are no comparative studies of citrus root transcriptomes between genotypes. In plants, the root system is critical for plant growth and development. It serves the functions of anchorage, nutrient and water uptake, and is the main boundary between the plant and its soil environment. Root growth relies on a specific set of signals that involves hormone signaling, availability of nutrients and carbon supply. There is a large degree of genotypic variation in crop plant root systems that can influence the plants growth and production including root length, root density, root angle, lateral root number, and root:shoot ratio. These parameters can impact the plant’s size, tolerance to biotic and abiotic stressors, and ability to uptake water and nutrients. For this reason, grafting, a process which connects the roots of one plant to the scion of another, has been widely using in plant breeding programs in order to improve vigor, alter plant architecture, enhance tolerance to disease and abiotic stress, and contribute to the quality of crops. In citrus, rootstocks are bred for a variety of traits that are imparted to scions, such as tree size, yield, tolerance to salt, cold, and drought, tolerance to various pests and diseases, and improved fruit quality. Many studies in citrus have been conducted to assess the impacts of genetically differing rootstocks on these traits. However, growing raspberries in pots the study of molecular mechanisms behind root attributes lags far behind above ground tissues in plants, especially in perennial crops.

Understanding the genetics of how root systems develop, and the regulatory controls of these processes will help optimize the improvement of yield and quality in citrus. Root system length, growth, and architecture control the ability of plants to respond to various stress conditions. The development of the root system and its architecture is determined by genetic factors interacting with numerous environmental factors. Plants must adapt to their environment by controlling their physiological reactions and morphogenesis. This can create complex root system architectures. For example, different root types can produce lateral roots that significantly extend the elaborate root system and allow the plant to search the soil for water and nutrient-rich areas. The increase in lateral root formation allows plants to more easily uptake these essential molecules in order to survive in unfavorable conditions. Rootstock genotypes exhibiting higher abilities to adapt to stress and create more extensive root systems improve nutritional status and water uptake, which can increase marketable yield. Studies in Arabidopsis, rice, and corn have identified several genes that influence root development and root system architecture. In citrus, transcriptomic studies have been performed to understand the effect of rootstocks on growth and in response to cold, nutrient deficiency, and fungal inoculation. Besides these studies, little effort has been invested into studying the effects of citrus rootstocks at the molecular level, especially genotype-specific effects and their relation to fruit quality. Plants have evolved to cope with a constantly changing environment, modifying the root system architecture in response to nutrient availability and soil microorganisms. This flexibility requires fine tuning of gene expression.

Among the molecules that control root development, small RNAs play a vital role in regulating genes at the post transcriptional level in plants. The most well-studied class of sRNAs are microRNAs , which are approximately 21 nucleotides in length and are produced from noncoding transcripts. Mature miRNAs have been shown to negatively regulate gene expression at the post-transcriptional level by specific binding and subsequent cleavage of their target mRNAs, or by the repression of target translation. Increasing evidence demonstrates that plant miRNAs play critical roles in almost all biological and metabolic processes. A review of miRNAs in roots indicated that they participate in root development, the modulation of root architecture, and root biotic interactions. This review focuses on numerous studies using Arabidopsis and legume plants as models. In citrus, miRNAs profiling has revealed their involvement in adaptation to nutrient deficiency, drought and salinity stress, and pathogen infection. However, no information, to date, is available about the role of miRNAs underlying differences in fruit quality observed between citrus rootstocks. In the present study, trees grafted onto four rootstocks were chosen from a rootstock trial at the University of California, Riverside to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich 16-6 trifoliate orange. Generally speaking, rough lemon rootstocks produce the highest yield and fruit size, but this fruit is often of lower quality . Trifoliate orange rootstock, when well adapted, produces high quality fruit, with high yield on smaller trees. Carrizo citrange rootstocks produce intermediate yield with good fruit quality. Sweet orange rootstocks produce good quality fruit but are very susceptible to various citrus diseases, and therefore are rarely used as rootstocks for commercial trees.

An RNA-seq approach was used to assess differences in gene expression between rootstocks that produce fruit with varying quality traits with the aim of identifying genes that could potentially play a role in improvement of fruit quality. Moreover, miRNA expression profiles were obtained for each of the rootstocks to identify potential regulatory mechanisms associated with their target genes.Chronic metabolic diseases are associated with elevated FFAs and prolonged postprandial hyperlipidemia particularly with TG-rich lipoproteins 10 . High-fat meal-induced postprandial lipidemia is often associated with enhanced concentrations of proinflammatory marker proteins in the plasma . However, the mechanism by which postprandial hyperlipidemia affects inflammatory responses is unclear. Our previous studies revealed that saturated FAs can activate Toll-like receptor 4 and nucleotide-binding oligomerization domain receptor 2-mediated signaling pathways, whereas the n-3 FA DHA and certain plant polyphenols that are abundant in fruits and vegetables can inhibit TLR4 and nucleotide-binding oligomerization domain receptor 2-mediated inflammatory signaling pathways . Animal studies demonstrated that TLR4- or TLR2-deficient mice were protected from high-saturated fatdiet-induced inflammation and insulin resistance, suggesting that inflammation and insulin resistance induced by dietary saturated fat are at least in part mediated through the activation of TLR4 and TLR2 . In addition, activation of TLR2 by FFAs released from TGRLs derived from human subjects who consumed a high fat meal leads to inflammasome-mediated secretion of IL-1b in isolated primary blood monocytes or whole blood . FAs derived from dietary saturated fat from people who consume a high-saturated fat meal are transported in TGRLs as TGs in the postprandial state. The predominant FAs released from this TGRL after LPL treatment are saturated FAs . Our previous mechanistic study revealed that both exogenous palmitic acid and endogenous FFAs released from TGRLs derived from the human subjects who consumed a high-fat meal directly activate TLR2 and induce inflammasome-mediated secretion of IL-1b in isolated primary blood monocytes or whole blood. These results suggest that the plasma concentration of FFAs is an important determinant in modulating TLR-mediated cytokine production in blood. In a microenvironment where secreted LPL can hydrolyze TGs in plasma TGRLs to release FFAs, elevated plasma FFAs can enhance the propensity of monocyte activation. Monocytes possess constitutively active caspase-1 and thus can release IL-1b if the expression of pro-IL-1b is induced by the activation of TLRs . Thus, saturated FAs in plasma FFAs may be sufficient to induce inflammasome mediated IL-1b release, a hallmark of monocyte activation. Activated monocytes transmigrate into peripheral tissues and are differentiated into macrophages that generate inflammatory signals. Therefore, plant pot with drainage alleviation of monocyte activation by dietary constituents may be an effective strategy to suppress the triggering of inflammation that leads to enhanced inflammation in peripheral tissues. In this study, we determined whether changes in plasma FFA concentrations induced by a single moderately high-fat breakfast modulate plasma cytokine concentrations and the propensity of monocyte activation in blood and whether supplementation with blueberry powder that contains anti-inflammatory polyphenols inhibits these processes.This investigation conforms to the principles outlined in the Declaration of Helsinki and was approved by the Institutional Review Board for Human Subjects at the University of California Davis.

Written informed consent was obtained from all subjects who were recruited from the greater Sacramento, California area. The health screening and study visits were conducted at the Western Human Nutrition Research Center in Davis, California. This trial was registered at clinicaltrials.gov as NCT01594008. Inclusion criteria included: 1) age between 18 and 60 y and 2) a normal BMI . Exclusion criteria included: 1) total blood cholesterol >240 mg/dL, 2) TGs >300 mg/dL, 3) hemoglobin <11.5 mg/dL, 4) abnormal results in clinical chemistry and hematology panels, 5) inflammatory or metabolic diseases, 6) use of nonsteroidal anti-inflammatory drugs including asthma and allergy medications, 7) unwillingness to discontinue use of dietary supplements before and during the study period, and 8) vegetarianism. In total, 27 subjects participated in the study ; 4 subjects did not complete the study .This study was designed as a placebo-controlled crossover study. A schematic of the study design is depicted in Figure 2. Subjects were blinded to the dose of BB powder they were given. Researchers handling samples and data did not know what dose of BB powder subjects received on test day 2 or 3. The frozen BB yogurt supplemented with either placebo control or BB powder was coded by letter, and samples were coded by test day. The provision of the placebo control powder on the first day to all subjects was done to mitigate potential carryover effects of the BB powder. The registered dietitian coded the different treatments . The randomization scheme was generated by using the website Randomization.com s ‘‘second generator.’’ The registered dietitian created a randomized list of the 2 and 4 servings codes and assigned subjects sequentially as subject numbers were assigned. If a subject dropped out after being randomized, the next subject was assigned to the treatment schedule of the dropped subject. Subjects were instructed by a registered dietitian to follow a low polyphenol and low-omega-3 FA diet and limit consumption of fruits, vegetables, soy, fatty fish , whole grains, nuts, coffee, tea, and chocolate starting 3 d before each test day. The night before each test day, subjects consumed a standardized dinner provided by the WHNRC that included a burrito as well as yogurt and lemonade to minimize masking effects of previous diet and variations in fasting levels of endpoints caused by the different dinners consumed by the subjects . On each test day, subjects arrived at the WHNRC after a 12-h overnight fast. Subjects had their body temperature, blood pressure, and weight measured and then had blood drawn. Subjects consumed the breakfast meal with either placebo control or BB powder prepared in yogurt as described below under the test meals section. Subjects were given 20 min to consume the entire breakfast, after which they were instructed not to eat or drink anything other than water and were allowed to return to their normal daily activities. Subjects returned to the WHNRC 3.5 h after consumption of the test meal for a postprandial blood draw. Postprandial peaks of the plasma concentration of TGs occur on average 3.5 h after the consumption of a high-fat breakfast . Following the postprandial blood draw, subjects were allowed to return to their normal dietary habits until 3 d before their next test day. Subjects consumed the placebo control powder on their first test day. On the test days 2 and 3, subjects consumed varying amounts of the BB powder that were equivalent to 2 or 4 servings of fresh BBs in a random order.The MHF breakfast contained 650 kcal and met the following nutrient specifications: 40% kcal derived from fat, primarily from food sources containing long chain FAs ; 15% kcal derived from protein; and 45% kcal derived from carbohydrates . The MHF breakfast consisted of a bagel sandwich and sucrose-sweetened yogurt, which served as the vehicle for administering the BB or placebo control powder. Table 1 presents the nutrition information separately for the base MHF meal with and without the addition of the placebo control/BB powder,powder for each test meal. The placebo control and BB powder mixed in yogurt were prepared in advance and frozen, and each of them was served in a separate cup, so that each subject consumed all of the BB or placebo control powder.

Bees are capable of passing through a variety of landscape structures, and bumble bees, while generally exhibiting high site fidelity, have been found to cross both natural and artificial barriers with little difficulty. Therefore, it is likely that anthropogenic land use change is not a barrier to movement. As agricultural and urban areas continue to be the largest and fasting growing forms of land use conversion, it is critical to understand the impacts of these landscape-scale changes on species distributions in order to predict and plan for ecological impacts as global change continues. Relationships between community metrics and land use change alone to do not reveal the complex interplay of land use change and seasonality. Variability in patterns due to seasonality has not been taken into account in many pollinator studies in anthropogenic landscapes, despite the known clear linkage between pollinators and floral resources. Bringing attention to how anthropogenic landscapes experience different temporal dynamics is relevant to the numerous conservation and restoration projects being done in anthropogenic landscapes. There are many similarities between urban and agricultural landscapes, and projects being conducted within them to attract pollinators could benefit from a crossover understanding of the impacts of anthropogenic land use change. In addition to contributing to a better understanding of how change in landscape use, particularly urbanization, affects bee community population dynamics, this study illustrates the importance of observing temporal dynamics in urban ecology studies.Increasing temperatures as a result of global climate change have led to shifts in phenology for many species, drainage collection pot and widespread debate over the consequences of critical interaction mismatches. However, not all phenological change is the direct result of changing climate.

Land use change, such as urbanization and agricultural expansion, includes the deliberate introduction of novel plants into communities. These plants, both exotic ornamentals and crops, are often accompanied by watering and supplemental nutrient inputs that extend survival potential in the targeted landscape, leading to different flowering seasons. Therefore, on a plant community-scale, different land use types can be expected to experience distinct patterns of phenological change. Land surface phenology is the timing of overall surface vegetation growth, and differs from traditional definitions of vegetation phenology , which are being used to product 250-m spatial resolution products every 16 days . In some cases, the changes in land surface phenologies between years have given insight into the history of regions that have experienced socio-economic and geopolitical transitions, such as changes in irrigation regimes in central Asia after the fall of the Soviet Union, or the expansion of urbanization in parts of Mexico. Differences in phenology between neighboring land use types within a year also provide insights, such as vegetation phenology of urban landscapes found to be out of synchrony with patterns of phenology in the surrounding desert. As a result of land use change, such phenological differences in vegetation could lead to phenological differences in other groups of organisms, especially if plants exert bottom-up control on the organisms that interact with them. Primary productivity can be linked to biodiversity. Therefore, there exists great potential to use remote sensing of primary productivity data as a way to predict biodiversity. However, early efforts to apply this technique were less powerful than expected.

Some evidence suggests that remote sensing data can predict biodiversity; for example, peak vegetation indices in multiple studies are correlated with higher avian diversity, although in other systems the relationship is less clear. Linking remote sensing data with biodiversity has been limited despite its great potential. Part of the problem may be that, in many comparisons, vegetation indices are treated statically, rather than as temporally dynamic. Use of a multi-season within year vegetation index was found to be a much more accurate predictor of biodiversity. Additionally, these indices may be more able to uncover patterns across landscape types with more dramatic differences in vegetation, such as human-altered landscapes. Also, biodiversity may be too broad, and instead, we should focus on those organisms that exhibit tight linkages with plant communities. In this paper we focus on bees, a key pollinator group with close vegetation associations, since bees strongly depend on flowers for both nectar and pollen. Bees provide the majority of animal-mediated pollination services on which an estimated 87.5% of flowering plants depend. The value of pollination in agriculture is estimated at $200 billion worldwide , due to many foods that are essential for food security and a healthy human diet, including numerous fruits, vegetables, and nuts that require bee pollination. Bees are closely linked to floral availability in their environment. However, the temporal dynamics of floral resources can vary between land use types. In California grasslands, there is typically a large burst of blooming in the spring, which tapers off in the summer. Urban areas often have ornamentals enhanced with external inputs that results in a steady patterns with only minor changes throughout the year, while agricultural landscapes have booms and busts of flowering that follow the pattern of local crops.

For this project, we explore how vegetation phenology varies in a human-altered California grassland landscape, and whether or not these changes in phenology correlate with those of the bee community that depends on floral resources. Specifically, we ask: 1) Do humanaltered landscapes in California grasslands experience phenological diversity that is out of synchrony with surrounding natural areas and 2) Do these spatio-temporal patterns correlate with bee distribution data?The bee community was sampled at multiple time points from 2010-2012. Within this region, sites were selected to be at least 1 km away from all others, based on maximum assumed bee foraging ranges. Although certain bee species have been recorded foraging as far as 1400 m, most bees have nesting and foraging habitat within a few hundred meters of each other. At each site we laid out a standardized pan trapping transect of fifteen 12 ounce bowls spaced 5 meters apart in alternating colors of fluorescent blue, white, and yellow. Bowls were filled to the brim with soapy water . In 2010, transects were set up for a 4 hour period between 10:30am to 2:30pm , with 4 sites sampled per day, and all sites sampled on consecutive days. These 2010 transects were run twice, once in the early summer, and once in the late summer. In 2011 and 2012, sampling was altered for transects to be set up for a 24 hour period, so that more sites could be run simultaneously and collections made more often. All 24 sites were sampled within two consecutive collecting windows of 24 hours, and were run four times each year: early spring, late spring, early summer, and late summer. The goal of collection was to sample the bee community that was flying through the site searching for resources. Because we were interested in landscape-level effects, we tried to control local variables as much as possible. All sites were selected in easily accessible, open areas that received full sun. Natural areas were in grassland habitat, so we selected agricultural sites that were either weedy field margin edges or fallow fields, square plastic pot and urban sites that were vacant lots or green ways. The human-altered sites were deliberately selected to not be adjacent to any mass-flowering crops or gardens. Bees were collected from the pan traps by using a metal strainer, rinsed with water, frozen overnight or longer, and then pinned and labeled. Specimens were sorted to the genus level, and then to the species level with the assistance of Dr. Robbin Thorp . The only exception to identification at the species level were bees of the genus Lasioglossum, due to their overwhelming abundance, limited availability of taxonomic expertise for this group, and lack of known ecological diversity. Voucher specimens and the majority of the total collection will be deposited at the Essig Museum of Entomology at UC Berkeley.For most models of total bee abundance, there was a significant impact of land use type, vegetation index value, and an interaction between the two . AIC values decreased, indicating better model fit, as the time lag of vegetation data increased to 48 days prior to the collecting event. Similar patterns were not found for species richness, and there was little to no relationship betweeen vegetation indices and species richness. In general the relationship between higher vegetation indices and higher bee abundance was positive. However, a time lag of 16 days between the remote sensing data and the closest sampling date of bee collection exhibited differences in patterns between land use types. For natural areas, there appeared to be a positive correlation slope between EVI and higher bee abundance.

The slope for bee abundance in urban sites was close to zero, and was a negative slope in agricultural sites . No other scenarios of land use type, bee abundance, and vegetation index was significantly negative. Urban sites had a high amount of overlap between seasons in the correlations between vegetation indices and total abundance. Agricultural sites also had overlap between seasons, but overall variation was higher than with urban sites. Natural sites had little overlap between months and correlations were more dispersed, particularly for early and late spring .This study demonstrates the role of land use change in leading to shifts in phenology, a result often attributed to climate change. We found major differences in land surface phenology and bee community spatio-temporal distributions between urban, agricultural, and natural land use types. The phenology of land surface vegetation human-altered landscapes are is of synchrony with surrounding California grassland natural areas. In addition to different patterns of phenological change, different land use types exhibit variability in the ranges and standard deviations of their land surface vegetation phenology. Further, these patterns correlate with spatio-temporal bee distribution data. In California grasslands, floral availability is largely driven by temperature and rainfall, resulting in a large burst of blooms in the spring, and by the end of the summer, there are few floral resources available. However, in human-altered landscapes, landscaping and water irrigation patterns are likely even stronger influences. Vegetation in urban areas is highly diverse, and selected for aesthetic and convenience reasons. Urban areas, while having less green space, often have many exotic ornamental plants which are supplemented with water and nutrient inputs that allow for an extended flowering season. As a result, urban areas are characterized by low, but constant, floral resources throughout the year. Agricultural areas have large patches of dense, often homogenous, floral resources that will fluctuate greatly from early spring to the end of the summer. In our system, stone fruit orchards are in flower in the spring, but throughout the summer there are other crops in flower such as alfalfa, tomato, corn, and bell peppers. This asynchrony in land surface phenology between neighboring land use types is similar to what Buyantuyev and Wu found in the desert landscapes of the southwestern United States. They found the timing of highest peaks of vegetation indices to be different between land use types, which they attributed to a decoupling in the urban sites from the local climatic drivers. It is likely that a similar scenario occurs in California, with grasslands juxtaposed against urban and agricultural areas that have different vegetation types and additional inputs. Beyond the different timing in vegetation indices, it is important to note the significantly different standard deviation and ranges of pixels of the same land-use type. Natural land use types were quite similar to one another given their relatively small standard deviations and ranges over time. However, in the human-altered landscapes, particularly agricultural, there was much higher variation. In other words, even though natural areas can be considered patchy, they are not nearly as patchy as urban and agricultural areas. One reason for the enhanced patchiness is that in urban and agricultural landscapes, there are many different land owners and management decisions being made on a relatively micro-scale, resulting in a wide diversity of vegetation types being selectively planted and cared for in different ways across the landscape. Instead of vegetation type shifting on the scale of a few kilometers, it might actually differ on the scale of a few meters. Such extreme patchiness of vegetation can have many implications for organisms dependent on floral resources. Additionally, varying patterns in vegetation in urban landscapes have been found to be closely tied to socio-economic factors, suggesting an important factor to consider when exploring biodiversity in human-altered landscapes.

Large farms may benefit by blocking major gopher access routes with any available solid material, but I don’t believe there is a viable material that will completely head off an infestation.Although not appropriate for all operations, flood irrigation can be extremely effective not only at killing gophers under the water but also at driving the survivors to field edges where they can be trapped. Flooding burrows with a hose can sometimes be effective in a small operation, providing that it is done at a fresh burrow. Gas cartridges with sulfur and sodium nitrate as active ingredients are still allowed by organic certifiers and can be effective if used on new burrows . They cost $1–$2 per cartridge and have an approximately fifty percent success rate. After inserting a gas cartridge in a burrow, be sure to cover the opening to prevent the gas from escaping. One company sells a blower that is supposed to move the gas beyond blocks in the tunnel system. I feel this may dilute the strength of the gas, although no scientific evidence yet exists to validate this idea. The “Rodenator Pro” is a device that injects a mixture of propane gas and oxygen into a gopher burrow and then ignites it with a spark, destroying the tunnel system. I recommend occasional use of the Rodenator in vineyards and orchards to remove permanent tunnels that run along perennial crop rows. It’s not appropriate for annual vegetable operations as it can damage crops and is unwieldy in row crop settings. Some growers use trapping as a main strategy and the Rodenator for areas where gophers and ground squirrels have settled. After poison baits, round plastic plant pot which are no longer allowed under organic certification rules, trapping is the most effective way to control gophers.

The best sites for setting traps are where there has been recent activity, marked by fresh mounds of moist, dark soil in the morning or by holes that have been recently plugged. Three trap designs currently dominate the market. The most common in California is the Macabee trap, invented by Zephyr Macabee in 1900 to protect his Santa Clara Valley almond orchards. The Macabee is a “pincher” type trap that impales two wires in the body of the gopher when it bumps into a trigger. The usual set for these traps is to locate and dig down to the main, larger burrow and insert two traps facing away from each other, connected by a wire. After the set is completely buried the wire is left on the surface and flagged to help find the traps. There are different opinions about letting light and air into the tunnel where the trap is located. Some say light and air will encourage the gopher to pack the trap area with soil and not set the trap off, and some say it is the light that draws the gopher to the trap. I’ve found that both methods work and that adding some vegetative bait helps as well. I’ve seen some modifications where the Macabee trap is inserted into a section of two-inch ABS drain pipe about eight inches long, either left open or closed at one end . This addition seems to help catch the gopher even if it is pushing soil ahead of it. Another older, standard trap design that is still popular is the box trap. This small wooden box is open on the bottom and at one end, and houses a trigger and metal “choker” loop or cable that grabs the gopher as it enters. A more modern version called the “Black Hole” is made of plastic tubing and a cable choker. These traps work by fooling the gopher into thinking it is still in the tunnel. The gopher is lured to the end of the trap where a small opening allows light and air in and the gopher gets caught trying to close the opening.

Box traps are also placed in the main tunnel in pairs, although I’ve seen a single trap work when placed in the mound’s entrance tunnel. Although both Macabee and box-type traps are effective, I’ve had the most success using the Cinch trap from Oregon. This is also an older trap that for many years was used only by professionals and can be slightly hazardous and difficult to set due to its double trigger and strong spring. Like the box or Macabee traps, Cinch traps can be used in pairs, but can be set more quickly and can be even more effective than the other trap types when placed singly in the burrow entrance. The method I use is to open the burrow at the freshest mound and insert the round, extended jaws of the trap into the burrow entrance. I use a stake that is sized to open the burrow as wide as the trap’s jaws and then use the same stake to mark the trapping site. The gopher is caught when it comes to the surface to close the opening in the mound.Pawpaw is the only member of the neotropical family Annonaceae that is native to North America with wild populations found in forested lowland areas in Canada and the eastern United States, as well as the southern and midwestern United States. This fruit tree is considered a vulnerable or even an endangered species in several regions, including in New York state. Pawpaw trees have a pyramidal growth habit and may reach up to 10 m in height. They have a suckering habit with numerous saplings often surrounding adult trees. Individual trunks live for approximately 40 years, but the root system can continue to generate new growth. Clonal saplings, also known as root suckers, originate from the root systems of mature trees.

Pawpaw flowers are protogynous, and cross pollination from a genetically distinct tree, with a few cultivar exceptions is required to produce genetically heterogeneous seedlings arising from dropped seeds. Pawpaw fruits are the largest edible fruit indigenous to North America. Blossoms emerge in May and have a pungent scent that is attractive to detritivore insects, including flies and beetles. The pawpaw flower has one to nine ovaries with each fertilized ovary gives rise to a large, many-seeded berry, fruit. Pawpaw fruits have a thin, pale green skin, and are borne singly or in clusters. The fruit is ripe between mid-September and October, depending on the growing site and the genotype. Ripe fruits possess a soft pulp that maybe smooth, grainy, or gelatinous. Pawpaw trees reach their maximum fruit production after approximately seven years. In several regions of the United States, there is an increased interest in pawpaw cultivation. For example, several groups of Indigenous Peoples in the territories surrounding the Great Lakes Region, including members of the Haudenosaunee Confederacy, are planting pawpaw as a traditional food crop. An experimental orchard of pawpaw cultivars and advanced open-pollinated breeding lines was established at Cornell University in 1999. Virus-like symptoms consisting of tree stunting or foliar mosaic, patchy discolorations, chlorosis, mottling, and distortions were observed early on in some trees. In contrast, leaves of asymptomatic trees were uniformly dark green in color. Virus-like symptoms were apparent on trees and saplings, eventually suggesting the occurrence of a soil-borne agent. Symptoms were rarely present throughout an entire tree; instead, they were noticed along several adjacent branches with leaves on top of the scaffold of symptomatic trees often remaining asymptomatic, suggesting that some trees may have recovered from symptoms. Based on these observations, symptoms were assumed to be of virus origin. In this study, we hypothesized that one or several viruses, perhaps soil-borne viruses for which plant recovery from symptoms is known, are present in symptomatic pawpaw trees. We used high-throughput sequencing to characterize the virome of pawpaw trees. The HTS results were validated by RT-PCR in combination with Sanger sequencing and by serological assays.An experimental orchard of pawpaw cultivars and advanced open-pollinated breeding lines established at Cornell University in Lansing, New York in 1999 was selected for this study. Most trees and saplings exhibited virus-like symptoms in the experimental orchard. Prominent symptoms were tree stunting , leaf distortions, mosaic, vein clearing, patchy chlorosis, extended chlorosis, and mottling . Symptoms were often unevenly distributed in the tree canopy with asymptomatic leaves on some branches and in the upper tree scaffold. Monitoring pawpaw trees for virus-like symptoms in May 2022 revealed that 89% of them were symptomatic. Trees from the 10 commercial pawpaw cultivars and 18 advanced open-pollinated seedling lines were symptomatic, 25 liter round pot suggesting no association between virus-like symptom presence and pawpaw genotype. Similar symptoms were apparent on trees and saplings surrounding the symptomatic trees with most saplings underneath symptomatic trees being symptomatic . A few asymptomatic trees were identified. These were spatially located at the edge of the experimental orchard at the south end of rows 2 and 3 , and north end of rows 1, 3, and 4 .

Saplings beneath the asymptomatic trees were predominantly asymptomatic.We identified and characterized TRSV and ToRSV by HTS in pawpaw trees exhibiting stunting and/or foliar patchy discolorations, chlorosis, mottling, vein clearing, and distortions in an experimental orchard at Cornell University. The presence of TRSV and ToRSV was validated in the trees from which nucleic acids were isolated for HTS and in additional trees from the same experimental orchard by RT-PCR and DAS-ELISA. Although we did not attempt to demonstrate causality, it is reasonable to presume that TRSV in single infection or TRSV and ToRSV in dual infections are responsible for the symptoms observed in theexperimental pawpaw orchard at Cornell University, given the high association between the occurrence of one or both viruses and disease symptoms in the pawpaw trees tested, as well as the history of the pawpaw trees. Additionally, TRSV was found by RT-PCR and DAS-ELISA in symptomatic pawpaw trees in a commercial orchard in Maryland. TRSV and ToRSV are both transmitted non-persistently by ectoparasitic dagger nematode vectors of the Xiphinema americanum group. The almost even distribution of symptomatic trees throughout the experimental orchard at Cornell University, except at the edge at the north and south sides although some of these trees were infected with TRSV, suggested that most of the trees were likely infected with TRSV when the orchard was established. If X. americanum nematodes would be involved in the spatiotemporal distribution of this virus in the experimental orchard, patchy aggregations of symptomatic trees would be expected. This was not the case because the distribution of symptomatic trees was non-clustered. In addition, the fact that some of the asymptomatic trees were infected with TRSV suggested that the planting material rather than dagger nematode vector-mediated virus transmissions likely explains the widespread distribution of TRSV in the experimental orchard. Nonetheless, we cannot rule out the presence of X. americanum in the experimental orchard and their involvement in short distance spread of TRSV or even ToRSV. Soil samples would need to be collected at various sites and tested for X. americanum to ascertain the occurrence of this nematode vector in the experimental pawpaw orchard. Validating efforts of HTS results revealed TRSV in every leaf sample tested from symptomatic and asymptomatic trees in the experimental pawpaw orchard at Cornell University . This finding supports the idea that trees were likely infected with TRSV when the orchard was established. It is possible that the TRSV infection was latent initially, and disease symptoms became progressively apparent after the orchard was established. Viruses switching from latency to a disease symptom-causing state are occasionally observed in infected perennial crops due to various factors including environmental stimuli, developmental growth stage, and mixed virus infections. Additionally, it can be speculated that, given TRSV is pollen and seed transmitted in several crops such as soybean and Pelargonium hybrids among others, the pawpaw seedlings used as rootstock for producing the experimental trees were initially infected. The fact that TRSV was identified in different grafted pawpaw cultivars in distant orchards in New York and Maryland adds credence to the rootstock seedling origin of the virus. Similarly, the detection of TRSV in flowers collected in May 2022 from infected trees in the experimental orchard in New York, and in fruitlets sampled in the commercial orchard in Maryland in June 2022, adds plausibility to the virus transmissibility via pollen and/or seed in pawpaw; however, these hypotheses need to be experimentally tested for validation. The detection of ToRSV was sporadic in the experimental orchard at Cornell University, suggesting its possible presence in some but not all the propagation material used to produce the pawpaw trees.