Horticulture

EDD regularly obtains data on farmworkers and wages paid when employers pay unemployment insurance taxes. Employers who pay more than $100 in quarterly wages are required to register with the EDD and pay taxes of up to 6% on the first $7,000 of each worker’s earnings to cover the cost of unemployment insurance benefits for laid-off workers. We extracted all Social Security numbers reported by California agricultural employers in 2015 and tabulated all of the farm and non-farm jobs and earnings of these farmworkers. This allowed us to assign workers who had more than one job to their primary commodity, the North American Industry Classification System code of the employer, and the county where they had their maximum earnings. Figure 1 shows average employment in California crop agriculture since 2007. Average employment rose over 10%, but there was an important change in crop agriculture after 2007, when non-farm crop support employers — those who bring workers to crop farms, such as farm labor contractors — began to bring more workers to farms than were hired directly by crop farmers. There are several reasons why farmers may turn to FLCs for workers, package of blueberries including the ability of FLCs to assemble crews of workers at lower cost than farmers who hire workers directly.

According to EDD data, over the past decade crop farmers have hired a few more workers directly, animal agriculture has had stable average employment, and there has been a sharp increase in crop support employment , most of it with FLCs. The average employment of crop support establishments has been rising by 10,000 a year, so that in 2016 non-farm crop support firms brought an average 215,000 full-time equivalent workers to crop farms, more than the average 173,000 FTE workers that these farms hired directly . Average FTE employment in animal agriculture has been stable at about 29,000, while animal support employment fell slightly. The total number of farmworkers employed sometime during the year is larger than average employment because of seasonality and turnover. In 2015, employment peaked at 475,000 in July and reached a low of 350,000 in December, guaranteeing at least 475,000 unique farmworkers. The actual number of workers is higher because of turnover: some workers do only a few days or weeks of farm work and quit, and workers employed in the Coachella and Imperial Valleys during the winter and spring rarely migrate to the San Joaquin Valley for the summer harvest, so different workers are required in different areas. After making adjustments for what appeared to be false or shared SSNs, in 2015 there were 848,000 unique SSNs reported by agricultural establishments, twice the average FTE employment of 421,000 .

This suggests two workers per FTE job, a ratio that has been stable over the past decade. Average FTE employment and the number of unique farmworkers each rose 10% between 2007 and 2015. The average annual pay of FTE agricultural workers varies by commodity. In 2015, the average annual pay of a directly hired FTE crop worker was $32,500, that of an FTE animal worker was $35,900, and that of an FTE crop support worker was $27,500. Table 1 presents data on the number of establishments, average employment and average annual pay for California commodities where average employment was at least 10,000 employees. The four crop categories in the table accounted for almost all establishments and average employment in the NAICS code for crops ; dairies accounted for half of NAICS 112 animal employer establishments and two-thirds of animal employment and total wages. The four crop support services listed under NAICS 1151 in the table accounted for almost all of the establishments, average employment, and total wages in the crop support category. Farm employment is concentrated in a few commodities. Fruits and nuts accounted for 57% of average direct-hire crop employment in 2015, dairy for 64% of direct-hire animal employment, and FLCs for two-thirds of average crop support employment.

For a FTE worker, the implied average hourly earnings ranged from a low of $10.80 for FLC employees to $18.34 for other post-harvest activities such as cooling and cleaning crops after they are harvested. California’s minimum wage was $9 per hour in 2015. Since we have data on all workers who were employed in a commodity, we can calculate the difference between the earnings of an FTE worker and the earnings of an average worker. We assigned farmworkers to the commodity or NAICS code in which they had their highest earnings, and found that 705,000 workers had their maximum earnings from a farm employer; we call these workers primary farmworkers. Table 2 shows that these primary farmworkers averaged $17,434, or 58%, of what an FTE worker employed in agriculture would have earned. We assigned the 705,000 primary farmworkers to the NAICS code or commodity in which they had their maximum earnings in order to determine what share of FTE earnings in that commodity a typical worker received; for over 100,000 farmworkers, this was a non-farm NAICS or employer. Primary crop workers were those whose maximum earnings were from employers with NAICS 111, and they averaged $21,467, two-thirds of what an FTE crop worker would have earned . Those whose maximum earnings were in greenhouses and nurseries earned 84% of FTE earnings in this commodity, while those whose maximum earnings were in more seasonal fruits and nuts earned 57% as much. Primary workers in animal agriculture earned 86% of what an FTE animal worker would have earned, and dairy workers, who were almost two-thirds of primary animal agriculture employment, earned 87% of what an FTE dairy worker would have earned , likely reflecting more hours of work during the year. Support service workers outside of crops earned almost as much as an FTE worker, but not crop support workers, who earned only half of what an FTE crop support worker would receive. The seasonality and turnover in crop support means that primary workers employed by FLCs, the largest group of workers, earned only 44% as much as an FTE worker employed by FLCs . Since the implied hourly wage for an FTE worker employed by FLCs was only slightly above the state’s minimum wage, the low average earnings of primary FLC employees must arise from fewer hours of work. A worker employed 1,000 hours at $9.86 an hour would have earned the average amount of a primary FLC employee in 2015, $9,878. Most primary agricultural workers, 70%, had only one job in 2015; this was a farm job, since having a farm job was necessary to be selected. Over 85% of animal workers employed in sheep, hogs and poultry had just one job in 2015, but less than 60% of workers who were employed in strawberries and vegetables had only one job. About 70% of primary FLC employees had one job in 2015. A quarter of farmworkers, some 223,000, had two or more jobs, and 18% had three or more jobs. Half of the 51,500 primary FLC workers who had two or more jobs in 2015 had one farm and one non-farm job, nft hydroponic while two-thirds of the 11,300 post-harvest crop support workers had one farm and one non-farm job. Half of the primary FLC workers with two or more jobs got at least 75% of their annual pay from FLCs, just as half of the dairy workers with two or more jobs got at least 75% of their annual pay from dairies. This same pattern held for most commodities, viz, half or more of two-job workers with primary earnings from strawberries or vegetables got at least 75% of their annual pay from this same commodity.

A quarter of the 51,500 primary FLC workers with two or more jobs in 2015 had at least two farm jobs and one non-farm job. However, these 14,000 workers were only 5% of the 293,000 workers whose primary earnings were with FLCs and less than 2% of all farmworkers, suggesting that combining farm and non-farm jobs is relatively rare.The 848,000 workers with at least one farm employer in 2015 can be assigned to the county where they had their highest-earning job, which could be a farm or a non-farm job. Kern , Fresno and Monterey had 36% of the state’s farmworkers, and the eight counties that each had at least 30,000 farmworkers had over 60% of the total, including Tulare , Ventura , San Joaquin and Santa Barbara and Los Angeles . Workers are assigned to the county of their employer, so that an employee of an agribusiness operatingin several counties could be assigned to the headquarters county. The largest employer in most counties was an FLC : they employed 65% of primary workers in Kern County, 47% in Fresno County and 41% in Monterey County . In Kern County, the next largest employers were tree nuts and grape vineyards , each with 7% of primary workers. In Fresno, the next largest were employers engaged in post harvest activities with 13% of workers and grape vineyards with 8%. In Monterey, postharvest activities employed 15% of primary workers, vegetable farming 12% and strawberry farming 11%. In Tulare County, FLCs accounted for 54% of farmworkers, followed by 9% for post harvest activities and 7% for dairy . In Ventura County, 32% of farm workers were in strawberry farming, followed by 19% with FLCs and 16% with other berries . Los Angeles was the most unusual county. All workers had to have one farm employer to be included in the analysis, but the largest employers of farmworkers who had their highest earnings in Los Angeles County were employment services , with 12% of farmworkers; restaurants , 8%; nurseries , 4%; and strawberries, 3%. In Napa County, 34% of the 8,000 farmworkers were employed by grape vineyards, followed by 32% employed by farm management services ; 7% each were employed by FLCs and beverage manufacturers .In an effort to improve wine quality, many smaller high-end wineries employ laborers to hand sort individual berries after destemming to remove unwanted material such as raisins, diseased berries, unripe berries, and materials other than grapes such as leaves and stems. This can be costly, labor intensive, and it can slow down the process line. To reduce costs and increase throughput, many wineries have adopted optical sorting technology. Using this technology, MOG can be removed more efficiently, and parameters such as color, shape, and size can be used to sort individual berries. Depending on the type of sorter, processing speeds can range between 2 and 15 tons per hour. Furthermore, fewer workers are needed to operate an optical sorter than to hand sort the respective amount of fruit. In addition to saving time and money, optical sorters have the potential to decrease the impact of inconsistent ripening in grapes. One study successfully sorted Carlos Muscadine grapes into four different ripeness levels using light at two different wavelengths in the visible spectrum. The researchers found that with successive sorting levels, there was an increase in Brix and pH, along with a decrease in titratable acidity in grape samples. In the wines, an increase in tannin and pH and a decrease in titratable acidity was found with increasing sorting. In sensory analysis, the first and fourth sorting levels were found to be inferior compared to the middle two treatments. Even though this study used outdated equipment compared to today’s standards, it shows that white grapes can be sorted into different ripeness levels and this can affect the quality of the wine produced. A recent study used visible near-infrared spectroscopy to classify table grapes into different groups based on soluble solid and phenolic content. The researchers were able to differentiate berries of different classes with accuracy ranging from 77% to 94%. Another study found that wine made from optically sorted Chardonnay grapes had higher residual sugar, pH, and total phenols than the unsorted control. The wines were analyzed sensorially with descriptive analysis and the judges scored the sorted wines significantly higher in tropical fruit and sweetness. However, with only two significant attributes out of twenty, the wines were determined to be similar in character. Another study investigating the effect of mechanical harvesting and optical berry sorting on Pinot noir grapes found that, in general, wines made from optically sorted fruit were significantly lower in total phenol and tannin, potentially due to the removal of MOG during sorting.

Putting these numbers into practice will require understanding how management practices affect both bird and CBB densities.The availability of safe, high-quality food for the burgeoning world population continues to be a major challenge in light of the deterioration of natural resources coupled with climate change. To feed the estimated 10 billion people safely and sustainably by 2050, the world will need to produce significantly more food. It is anticipated that global demand for meat will increase by 70% from today, and planetary resources will be insufficient to meet the demand of the world population by 2050. Within this larger global challenge, aquatic sources provide nutritional protein-rich foods, including omega- 3-enriched sources of fatty acids and bio-available micro-nutrients. Stagnant levels of fish harvested from open water fisheries and the growing challenges with the sustainability of aquaculture systems are concerns. To adequately feed the growing global population by 2050, increases in seafood production of 100% are projected as a need. Decade-wise comparisons of global per capita consumption, capture fisheries production and aquaculture production from 2000 to 2020 based on FAO data are given in Fig. 1. Hence, plastic growing bag there is an imperative to establish alternative sources of fish and shellfish to effectively meet the growing global protein demand in the foreseeable future.

Presently, 89% of the aquatic animals produced—equal to 157.4 million tons—are used for human consumption, considering the per capita consumption of 20.2 kg fish per year by 7.8 billion people. The rest is used mainly for non-food uses including fish oil and fish meal production. Future projections for capture fisheries and aquaculture production by 2050 are 98.3 and 140 million tons, respectively. Thus, increases in future fish production will rely mostly on aquaculture production, which is challenging in the context of sustainable production. For fish production to be maintained at a sustainable level, critical efforts will be required to provide larger volumes of feed to support aquaculture, to maintain quality for aquatic environments, to reduce pressure on wild aquatic organisms used for food and provide quality aquatic foods to consumers. These challenges prompt the development of alternative sources of aquatic food through cell cultivated approaches.Cell-cultivated seafood has gained attention as an alternative sustainable food production system, where animal cells are grown in vitro using cell culture techniques to form edible seafood products without the need for the animal. Cellular agriculture is one of the key transformative food production systems to help address the above challenges, which originated with the cultivation of goldfish in a study funded by NASA. Cell-cultivated fish production requires the large-scale cultivation of cells to generate large masses of seafood-relevant cells and tissues. These cells and tissues can be used to form unstructured products such as surimi or fish fingers using well-established food processing techniques, or they can be further cultured on three-dimensional biomaterial scaffolds to generate structured products akin to fish fillets.

The many advantages to producing seafood from cell cultures rather than using native fish includes improved freshness, food quality and avoiding nonedible components such as bones, skin, shells, and scales as wastes that can negatively impact the environment. Cell-cultivated seafood may also shorten food production cycle time and provide continuous production; cell cultures may require weeks to generate functional foods and may do so in a continuous manner . Fisheries and aquaculture are relatively sustainable food production systems compared to terrestrial livestock, however, due to overfishing, pressure on wild stocks, emerging diseases, antibiotic-resistant bacteria, global warming, and marine acidification with adverse impact on organisms’ physiology, loss of biodiversity and species migration, byproducts of production, microplastics, chemical contaminants in waters, and the lack of clean water , the seafood industry requires alternative and innovative production systems to overcome these current challenges.There are several gaps in research and development to be filled in order to progress cell-cultivated seafoods. Limited seafood cell lines: Producing seafood from fish cell cultures is an intriguing opportunity for cellular agriculture, yet few fish cell lines are currently available that have direct relevance to seafood production. Cell-cultivated seafood processes rely on native seafood sources for harvesting muscle and fat cells, which are then immortalized. Both cell isolation and the immortalization processes remain challenging.

For example, access to embryonic stages of many aquatic organisms as a source of stem cells is difficult. The number of cell sources has been expanding thanks to continuous research. Many of these sources, however, still need to be validated in a large-scale culture. Limited knowledge of seafood cell differentiation: There remains limited knowledge in terms of in vitro fish, crustacean, and mollusk muscle cell or fat cell proliferation, differentiation, and maturation. Omics-based methods, including genomics, proteomics, and metabolomics, are helping to elucidate factors involved in the different stages of differentiation to accelerate cell-cultivated seafood production. Further, a number of studies with fish have provided insights into growth factor requirements and growth conditions . Myogenic precursors from juvenile trout showed higher proliferation and differentiation rates than adult trout myogenic precursors, and insulin-like Growth Factor and IGF-2 stimulated the proliferation in primary cell cultures of myoblasts from rainbow trout . Gilthead sea bream myocytes were cultured to evaluate the role of IGFs in muscle growth and differentiation via the regulation of myogenic regulatory factors expression. At the beginning of the cell culture and during the proliferation, the IGF-2 expression was highest. Additionally, further evaluations indicated that myod2 and myf5 expression was increased by IGF-2, whereas IGF-1 increased mrf4 and myogenin expression. Lack of serum-free media: Serum-free media has been developed for mammalian cells, yet this remains a challenge for cell-cultivated seafood. Cell line development for seafood can require up to 20% serum, making cell based seafood production unsustainable and expensive. Reducing serum can result in changes in morphology or slower to no cell growth. Reduction of serum in fish cell cultures has been achieved using IGF-2, algal extracts, and protein hydrolysates, but elimination of serum without negative impact on growth remains a challenge . More research is required to develop serum alternatives for cellular aquaculture, such as specific plants or bacterial/algal-based products. Limited genetic tools: Exploring genetic modifications for seafood cells, wholesale grow bags to accelerate both understanding of cell proliferation and differentiation, as well as to develop cell lines, remains challenging due to the few genetic tools developed for seafood cells. Yet, optimization of immortalization and trans-differentiation processes through genetic modification, including CRISPR-Cas9 editing of fibroblasts that convert them into skeletal muscle or adipose cells, will address some of the cell sourcing challenges for cell-cultivated seafood. Induced pluripotent stem cells are available for adult zebrafish, with limits to other publicly available other seafood species. There remains limited knowledge of differentiation pathways in aquatic species other than zebrafish. Genetic tools in other, traditionally consumed, species need to be pursued. Given that these technologies still require genetic modification, consumer acceptance and reactions to the consumption of genetically modified cells must be evaluated. Scale Up Demonstration: Compared to mammalian cells, fish cells may be more suitable for bioreactor production due to their tolerance for hypoxic conditions, which reduces the need for active oxygenation; their increased tolerance for different pHs; and in some cases, their growth at lower temperature to reduce energy costs. However, long doubling times are problematic and scale up data remains to be demonstrated. Lack of available consumer-ready products: The inclusion of heme proteins in plant-based meat increased meat-like flavor and natural color. Similar approaches are needed for aquatic cell-cultured foods to address consumer perceptions.

The Peptide Atlas and Protein Map developed from Rohu is a useful source for identifying proteins involved in the quality and color of cell-cultivated seafood. Nutrition, flavor, texture, and quality of products and cultural relevance are important parameters that will need to be addressed for cell-cultivated seafood to achieve consumer acceptance as the field progresses. Flavor in conventional seafood is mainly due to the fatty acids, and some amino acids. Developing these flavors in the cultivated meat could be achieved by cell engineering to generate specific amino acids and fatty acids, manipulating cell culture media to contain more marine flavor-based compounds such as protein hydrolysates from marine plants, and adding flavor extracts to the final products.Developing cell-cultivated seafood starts by isolating embryonic stem cells, adult stem cells, or generating induced pluripotent cells from the species of interest . Despite efforts to establish cell lines from aquatic organisms , the challenge remains to isolate and immortalize viable cells . Tissue selection is the first step for sampling, in the case of fish samples for myogenic cells, this often involves using white muscles with significantly less fat content compared to red muscles, however, the spatial arrangement differs among species . In order to isolate cells, adult tissue selection for mollusks plays a crucial role in establishing primary cell culture methods. Mollusks, such as oysters, have diverse tissue types that can dictate the culture conditions and cell dissociation methods. Tissue from three main oyster species, Pacific , Eastern , and European Flat oyster , have been studied for drug, toxicity, and disease research, including embryo, heart, mantle, digestive gland, gill, ventricle, and adductor tissues . Among oyster tissues studied, heart tissue was most frequently selected as it had better potential in establishing a permanent cell line than oyster embryos. These previous studies indicate that the tissue of origin often dictates the success of oyster cell culture, along with culture conditions and decontamination treatments. A significant challenge for seafood cell isolation is contamination from other species, particularly for marine filter feeder bivalves such as oyster, mussel, clam and scallop. Protozoans , amoeba, motile zoospores, sporangia, yeast, endospores, and microalgae are common contaminants in marine invertebrate cell culture. Finding optimal antibiotics and anti-fungal conditions during the initial cell isolation step is also challenging because high concentrations candamage or kill the desired cells, and low concentrations may not effectively eliminate the contaminating microbes. In order to develop cells suitable for bioprocesses for seafoods, immortalized cells are required. Three methods of immortalization are generally pursued, spontaneous genetic processes, genetic modification approaches such as the expression of the catalytic subunit of telomerase , or genetic inactivation of p53/p14/Rb . Spontaneous immortalization has benefits and limitations. For example, spontaneously immortalized cells are not considered genetically modified , which allows companies access to European markets that restrict the use of GM foods. However, this immortalization process is not controlled, thus additional genetic changes are feasible. In addition, every cell type has its own susceptibility towards spontaneous immortalization. For example, fish cell lines have a higher susceptibility for spontaneous immortalization due to the high regenerative capacity of the adult stem cell population compared to mammals with more effective DNA repair mechanisms. For cell-cultivated seafood production, spontaneous immortalized cell lines from Atlantic mackerel were developed  and a skeletal muscle cell line was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 and myosin heavy chain immunostaining, respectively. Importantly, an adipocyte-like phenotype was demonstrated for these cells through lipid accumulation from the environment, confirmed via Oil Red O staining and quantification of neutral lipids, as an alternative path to adipogenesis utilizing adipose-derived cells. Limited antibody markers for fish derived cells, including adipocytes and myocytes, continue to make cell identification a challenge for the field.A simple basal medium with added artificial seawater or sterile seawater helped to provide osmolarity similar to marine habitats. For example, for oyster cell culture media, osmolarity was adjusted to 650–720 mmol/kg31. The most common medium used for many aquatic organisms in cell culture is L-15, which contains salts, amino acids, galactose, vitamins, and minerals. However, the L-15 medium contains no proline or taurine, which are present at high levels in the body fluids or tissues of aquatic organisms. Proline and taurine are likely essential components for cell proliferation in mammalian cells. Therefore, adding proline or taurine to oyster cell culture media by using oyster body fluid or tissue extracts could be necessary for supporting cell proliferation. In addition to basal media, many media supplements and growth factors such as fetal bovine serum , adult organism soft body fluid, embryo or gonad extract, fibroblast growth factor , insulin, and epidermal growth factor have been tested for cell proliferation but with inconsistent outcomes. Different cell culture media, supplements, and incubation temperatures used for bivalve cell culture are presented in Table 2. For oyster cell cultures, penicillin, streptomycin, and amphotericin B are the most commonly used antibiotics.

Several additional wild octoploid subspecies have since been used as parents in breeding, creating an admixed population of F. × ananassa individuals with genomes that are mosaics of phylogenetically and demographically diverse progenitor genomes. While several subgenome origin hypotheses have emerged from cytogenetic, phylogenetic, and comparative genetic mapping studies, a complete hypothesis for the origin and evolution of the octoploid genome was only recently proposed with the publication of the “Camarosa” reference genome. Through phylogenetic analyses of the transcriptomes of all described extant diploid species, including four subspecies of Fragaria vesca, the putative subgenome donors found in the octoploid were identified as F. vesca subsp. bracteata, Fragaria iinumae, Fragaria viridis, and Fragaria nipponica. Edger et al. provided strong support for earlier hypotheses that F. vesca and F. iinumae were two of the four subgenome donors. Until the octoploid reference genome was published, plastic nursery plant pot the origin of the other diploid subgenome donors had remained unclear, although multiple hypotheses had been proposed.

Liston et al. then reasoned that Edger et al. may have misidentified two of the progenitors due to bias from excluding in-paralogs in their phylogenetic analyses. To address this concern, Edger et al. developed a chromosome-scale assembly of the F. iinumae genome and reanalyzed the original data with in-paralogs. The revised analysis supported the original model that the genome of octoploid strawberry originated through successive stages of polyploidization involving four progenitor species: diploid × diploid → tetraploid × diploid → hexaploid × diploid → octoploid ancestor. In addition, the chromosome-scale genome assembly showed that the diploid subgenomes were not static building blocks walled off from one another. Rather they have dynamically evolved through homoeologous exchanges, which are well-known in neopolyploids. Homoeologous exchanges in octoploid strawberry were found to be highly biased toward the F. vesca subsp. bracteata subgenome replacing substantial portions of the other subgenomes. However, homoeologous exchanges are not unidirectional. Although the chromosomes are architectural mosaics of the four diploid subgenome donors and their octoploid descendants, F. × ananassa is strongly allo-octoploid. Because the F. × ananassa chromosomes are complex admixtures of genes with different phylogenetic histories via homoeologous exchanges, Edger et al. developed a nomenclature that precludes oversimplified oneto-one assignments to a specific diploid progenitor.

The F. × ananassa genome has not only been reshaped by polyploidization events, especially homeologous exchanges, gene-conversion, and selection , but by repeated interspecific hybridization in breeding that has resulted in the introgression of alleles from phylogenetically and demographically diverse F. chiloensis and F. virginiana ecotypes. At this point in time, the decades long debate among geneticists and evolutionary biologists about the origin of the F. × ananassa genome seems to have reached an initial zenith. Remaining disagreements might only be settled when chromosome-scale assemblies of the other hypothesized diploid progenitors are assembled and analyzed. Aside from the question of subgenome origin, what other evolutionary questions might be worthy of exploration at this juncture? First, while the four extant relatives of the diploid progenitors have been putatively identified, the history and timing of the intermediate polyploids remain poorly understood. When and where were the tetraploid and hexaploid ancestors formed? Are any of the known wild polyploids endemic to Asia descendants from these intermediate polyploids? Which subgenome is dominant in these polyploids? Second, a single dominant subgenome was uncovered in Fragaria × ananassa that controls many important traits including fruit quality. Just how deterministic is subgenome dominance? In other words, is it possible to resynthesize the octoploid with a different degree of subgenome dominance, or with a different subgenome becoming dominant?

The answer to this question could have implications for genetic improvement of the cultivated species.Genotyping advances in strawberry have naturally followed advances in humans, model organisms and row crops. The development of the Affymetrix Axiom® iStraw90 single-nucleotide polymophism genotyping array was a significant advance that enabled the facile production and exchange of genotypic information across laboratories with high reliability, minor amounts of missing data, and negligible genotyping errors. The ease-of-use, speed of analysis, simplicity of data management, and outstanding reproducibility of SNP genotyping arrays have been important factors in their continued adoption in strawberry and other plant species with complex genomes. Underlying computational challenges associated with genotyping by sequencing and other nextgeneration sequencing facilitated approaches have limited their widespread application in octoploid strawberry thus far. The challenges are similar across species, but obviously exacerbated in allogamous polyploids: uneven and inadequate sequencing depth, copy number uncertainty, heterozygote miscalling, missing data, sequencing errors, etc., all of which challenge the integration of DNA variant information across studies. As with the other DNA marker genotyping approaches reviewed here, the first GBS study in octoploid strawberry utilized the diploid F. vesca reference genome in combination with a phylogenetic approach for aligning, classifying, and calling DNA variants. Recently, Hardigan et al. whole-genome shotgun sequenced 88 F. × ananassa, 23 F. chiloensis, and 22 F. virginiana germplasm accessions. Strikingly, 80% of the short-read DNA sequences uniquely mapped to single subgenomes in the octoploid reference. Approximately, 90M putative DNA variants were identified among F. × ananassa, F. chiloensis, and F. virginiana individuals, whereas 45M putative DNA variants were identified among F. × ananassa individuals. An ultra-dense framework was then developed of genetically mapped DNA variants across the octoploid genome by WGS sequencing 182 full-sib individuals from a cross between F. × ananassa “Camarosa” and F. chiloensis subsp. lucida “Del Norte”. Large expanses of homozygosity within the commercial hybrid parent prevented complete end-toend mapping of all 28 octoploid chromosomes in F. × ananassa as was accomplished with the wild parent, further demonstrating the value of dense NGS data for understanding sources of genotyping and mapping challenges in the octoploids.

As these WGS-GBS and GBS mapping results demonstrate, several NGS-based genotyping approaches should work well in combination with the octoploid reference genome. In summary, while the complexity of the octoploid genome has historically complicated DNA variant genotyping and genetic mapping in strawberry, the chief technical challenges were addressed with: the development of a high-quality octoploid genome assembly; WGS resequencing of numerous octoploid individuals that shed light on the extent of intra- and inter-homoeologous nucleotide variation; identification and physical mapping of DNA variants across the octoploid genome; and comparative genetic mapping of the wild octoploid progenitors of F. × ananassa using SNPs anchored to the octoploid reference genome. DNA variants genotyped with different platforms and approaches predating the octoploid reference genome were independent and disconnected, seedling starter pot resulting in the proliferation of linkage group nomenclatures, absence of a universal linkage group nomenclature, uncertainty in the completeness of genome coverage, and inability to cross-reference physical and genetic mapping information across studies, populations, and laboratories. The DNA marker sequences from many of the previously published mapping experiments were either not readily available or too short or nonspecific to enable unambiguous mapping to the octoploid reference genome. The one exception was the genetically mapped double digest restriction-associated DNA sequence markers described by Davik et al., which were used by Edger et al. for scaffolding the octoploid reference genome. Most F. vesca DNA probe sequences used to assay SNPs on the iStraw35 and iStraw90 SNP arrays were too short and nonspecific to unambiguously determine their physical marker locations in the octoploid genome. Hence, genotypes produced with these SNP arrays could not always be effectively utilized for genome-wide association studies or other applications requiring subgenome resolution. Moreover, none of the previously published iStraw90 based genetic mapping studies have shared SNP marker genetic locations, complete genetic maps, or other critical enabling information needed to identify corresponding linkage groups across laboratories. These long-standing issues were resolved with the development of a new 850,000-SNP genotyping array populated exclusively with DNA variants and reference DNA sequences that unambiguously mapped to single homoeologous chromosomes in the octoploid reference genome. Using the 850,000 SNP array, a second array with 50,000 subgenome specific SNPs, including 5819 genetically mapped SNPs from the iStraw35 array was developed facilitating the integration of genetic and physical mapping information across studies. These new arrays provide telomere-to-telomere coverage and target common DNA variants within and among domesticated populations. Although the full set of iStraw SNP probe DNA sequences could not be unambiguously aligned to a single octoploid subgenome, the true physical position for 97% of the retained iStraw probes were identified using linkage disequilibrium with the newly developed SNPs probes anchored to the octoploid reference genome.

Comparative mapping of SNPs in several wild and domesticated populations facilitated the integration of earlier linkage group nomenclatures and the development of a universal linkage group nomenclature substantiated by the observation of genome-wide synteny among diverse octoploid genetic backgrounds. These recent advances in genotyping and mapping are expected to have tremendous and immediate impacts on applied research in genetics and breeding of strawberry. But other research questions arise which have bearing on the utility of these new tools and resources, particularly with regard to diversity among genomes that is currently undescribed. For example, what large-scale structural variations exist in octoploid Fragaria germplasm? Recent advances in long read sequencing platforms resulted in significant decreases in costs and increases in read lengths and should soon permit inexpensive assessments of structural variants across the cultivated strawberry pangenome. On a smaller scale, what percentage of genes in cultivated strawberry exhibit presence–absence variation? Recent pangenome studies in plants have revealed that a significant proportion of gene content exhibits presence–absence variation. For example, nearly 20% of the genes in Brassica oleracea are found in only certain genotypes and are enriched with functions encoding major agronomic traits. This suggests that genes in strawberry will be missed when utilizing a single octoploid reference genome and genotyping resources based on that genome alone. To construct a useful pangenome, how many individuals need to be included to capture most variation in gene content? These questions will soon be addressed as additional octoploid genomes become available.For many years genome-assisted breeding in strawberry lagged behind agronomic crops and even many specialty crops. However, surveys conducted by the RosBREED consortium and funded by the NIFA Specialty Crop Research Initiative have documented the rapid rise in the use of DNA information in strawberry breeding in the last decade. In 2010, only 43% of surveyed strawberry breeders had employed DNA markers or other genomics-based tools. By early 2019, data on 12 of the 14 active strawberry breeding programs in the U.S. indicated that all but one of these 12 programs had used DNA information for at least one of four purposes. The most common application was for verifying the identity or better understanding the lineage of plant materials used in the program. Two-thirds of the programs had used DNA markers or other genomics-based tools to choose parents and plan crosses, and seven of the 12 had used DNA information for seedling selection. Two-thirds of the programs were involved in upstream research of direct relevance to their programs, e.g., creating or validating DNA tests of particular applicability for their plant materials and breeding goals. Some of these were onetime or infrequent applications; however, seven of the 12 programs reported using at least one application of DNA information “on an ongoing, routine basis” . Among the many breeding-relevant loci discovered in the cultivated strawberry genome, flowering, and fruit quality loci have been prominent, as would be expected in a high-value fruit commodity. These, include discovery of the locus controlling day-neutrality or PF and its sub-genome localization as well as multiple loci controlling volatile compounds such as gamma decalactone, mesifurane, and methyl anthranilate. For uncovering disease resistance loci, quantitative trait locus mapping has been the most prominent approach. While traditional biparental populations have been effective for QTL discovery, pedigree-based analysis in multiparental populations using FlexQTL™ has been increasingly applied, as pedigree breeding and maintenance of clones across generations are common in strawberry. Pedigree-based analysis in complex family structures has allowed the simultaneous detection of multiple QTL alleles and the quantification of their phenotypic effects across diverse genetic backgrounds, as demonstrated for the FaRPc2 locus. The use of DNA tests in breeding has been greatly enhanced by RosBREED efforts in marker development and validation. Assays for SNP detection such as kompetitive allele-specific polymerase chain reaction and high-resolution melting have become the tests of choice for breeding applications due to an abundance of SNP information from array genotyping, accuracy and ease of scoring, and resilience to crude strawberry DNA extracts.

These results suggest that S. torvum rapidly induces defense responses against A2-O, which inhibits the maturation of A2- O and gall formation. In contrast, A2-J inhibits or evades the induction of defense responses, continues development, and induces gall formation.RNA-seq analysis was performed to understand the differences in transcriptional regulation of the S. torvum response to infection by nematodes that induce an immune response or that are successful parasites. Eleven-day-old S. torvum seedlings grown on MS-Gelrite plates were inoculated with 200–300 surface-sterilized J2s of A2-J or A2-O, or treated with SDW in vitro. Since there were clear morphological differences between the root tips infected with A2-J and A2-O after four days , it should follow that the success or failure of infection is determined within a few days post inoculation. We therefore decided to analyze the transcriptome at 1–3 DPI, corresponding to the early stages of infection. In addition, to detect gene expression in cells directly affected by the nematodes, we carefully collected infected root tips under a stereomicroscope . Root tips were cut with precision forceps and flash-frozen with liquid nitrogen to preclude the induction of wound responses. More than 50 root tips were pooled for each treatment, bucket flower and four biological replicates were used for the RNA-seq based transcriptome analyses.

We also carried out RNA-seq of whole roots and shoots of S. torvum infected with A2-J or A2-O, or mock treatment to improve the completeness of de novo transcriptome assembly. As a result, we obtained 218,024,788 paired-end reads from root tips and 341,297,551 single-end reads from whole shoots and roots after quality filtering . After removing the reads derived from nematodes, we performed de novo assembly using multiple assemblers with a variety of k-mer sizes . The final assembly had 88,596 contigs with an N50 of 1,298 bp, an average size of 800.62 bp, and a total length of 70,931,593 bp . We assessed the accuracy and completeness of the final assembly using BUSCO. The assembly included an estimated > 95 % of the assessed dataset, improving the current status of the transcriptome assembly of S. torvum and provided a high-quality transcriptome assembly of S. torvum for further analyses. Differential expression analysis showed that 1,220 genes were significantly up-regulated and 261 genes were down-regulated upon infection with A2-J, while 2,535 genes were up-regulated and 802 genes were down-regulated by infection with A2-O at at least one-time point during root tip infection, compared to the mock treatment . 1,029 genes were upregulated, and 180 genes were down-regulated at at least onetime point in both A2-J and A2-O infected plants .

Previous studies showed that the expression of genes associated with the salicylic acid , jasmonic acid , and ethylene signaling pathways are induced in resistant plants infected with PPNs , so we investigated the expression of marker genes for hormone biosynthesis, hormone signaling , and defense responses . Importantly, at 1 DPI, A2-J did not induce any statistically significant changes in the expression of any genes, whereas A2-O induced 204 genes, suggesting that infection with A2-O rapidly induces the expression of early responsive genes, which is prevented or avoided in A2-J infection. Since the speed of a defense response is one of the most important factors for successful immunity against pathogens, we hypothesized there must be important defense components among the 204 up-regulated genes. We therefore performed a GO enrichment analysis to identify significantly represented GO terms amongst the 204 up-regulated genes . The list of enriched GO terms was further reduced using “Reduce to most specific terms” option in Blast2GO to remove general GO terms and obtain only the most specific terms . Some GO terms that were significantly enriched among the 204 genes were related to the biosynthesis of isoprenoids ”, “sesquiterpene biosynthetic process ”, and “terpenoid biosynthetic process ”. To follow up on this result, we checked the expression of all the genes up-regulated by A2-O that are related to isoprenoid biosynthesis and found that A2-O infection induced the expression of genes encoding sesquiterpene synthases, such as viridiflorene synthase, vetispiradiene synthase, germacrene C synthase-like protein, and 5-epiaristolochene synthase.

Several other enzymes involved in isoprenoid biosynthesis, such as xanthoxin dehydrogenaselike protein and UDP-glycosyltransferase 91C1 were also up-regulated . Sesquiterpene synthases convert farnesyl diphosphate to sesquiterpenes such as germacrene C, 5-epiaristolochene, viridiflorene, and vetispiradiene. Because some isoprenoids have nematicidal activity , it is possible that the sesquiterpenes produced by S. torvum in response to infection with A2-O are nematicidal and contribute to suppressing A2-O infection. Other GO terms significantly enriched among the 204 up-regulated genes were related to oxidative stress ” and “response to oxidative stress ”. The most up-regulated genes by A2-Oin the GO term group were class III peroxidases, which are involved in lignification, cell elongation, seed germination, and response to abiotic and biotic stresses . The transcriptional up-regulation of the class III peroxidases is consistent with the fact that resistant tomato lines more strongly elevate peroxidase activity during RKN infection than susceptible lines . To identify the expression pattern of genes that are specific to A2-J or A2-O infection and common in both pathotypes, we clustered genes according to their transcript profiles by PCA with SOM clustering. SOM clustering grouped 6,502 genes into nine clusters based on their differential gene expression profiles after mock treatment or infection with either A2-J or A2-O . 429 genes in Cluster 2 and 554 genes in Cluster 4 were specifically up-regulated after infection with A2-J. In contrast, 1,769 and 600 genes in Cluster 8 and 9, respectively, were specifically up-regulated after infection with A2-O. 1,000 genes in Cluster 7 were up-regulated after infection with either A2-J or A2-O . We once again used GO enrichment to identify functional terms enriched in the genes in each cluster by further filtering enriched GO terms using the “Reduce to most specific terms” option in Blast2GO. In Cluster 4 , significantly enriched GO terms were related to cell wall remodeling, including “cell wall modification ”,“cell wall organization or biogenesis ”, and “pectin catabolic process ” . This is consistent with the observation that the expansion of giant cells is associated with an increase in cell wall thickness . GO terms associated with the significantly A2-J up-regulated genes include enzymes such as cellulose synthase-like protein, xyloglucan endotransglucosylase/hydrolase protein, and a non-catalytic subunit of a polygalacturonase isozyme . The transcriptional up-regulation of these enzymes is consistent with the presence of the common polysaccharides pectic homogalacturonan, xyloglucan, cut flower bucket and pectic arabinan in the cell walls of giant cells . Similarly, A2-J infection also activated the expression of COBRA-like protein, expansin, and LRR-RLK PXC1, which play important roles in cellulose deposition, loosening of cell walls, and secondary wall formation . Another GO term significantly enriched in Cluster 4 was “transmembrane transport ” . Other significantly up-regulated genes encode sugar transporter ERD6-like protein and amino acid transporter family protein . These transporters may promote the uptake of nutrients into giant cells or alter transportation through cells surrounding giant cells . We also performed GO enrichment analyses for Cluster 2, but no GO terms were enriched. In addition to GO enrichment analyses, we looked for interesting genes whose expression were dramatically upregulated in Clusters 2 and 4. These clusters included genes encoding chalcone synthase and a spermidine synthase that were specifically and highly expressed after infection with A2- J . Chalcone synthase is the first enzyme of the flavonoid biosynthetic pathway , and spermidine synthase is a key enzyme involved in polyamine biosynthesis . In summary, A2-J infection significantly and specifically up-regulates genes related to cell wall modification In Cluster 8 , GO terms that were significantly enriched were related to defense responses, including “defense response to fungus ”, “defense response to bacterium ”, “killing of cells of other organism ”, and “regulation of salicylic acid biosynthetic process ”. In addition, GO terms involved in lignin biosynthesis, including “lignin biosynthetic process ” was also overrepresented in Cluster 8 . In Cluster 9, the significantly enriched GO terms were related to biosynthesis of isoprenoids ”, “terpenoid biosynthetic process ”, and “farnesyl diphosphate catabolic process ” .

We also found that the genes that are highly expressed after infection with A2-O in Cluster 8 and 9 include defense-related genes encoding chitinase, β-1,3-glucanase, and serine protease inhibitor, sesquiterpene synthase, fatty acid desaturase 2 , ferulic acid 5-hydroxylase which is involved in lignin biosynthesis, berberine bridge enzyme -like protein, which is involved in oxidation of cinnamyl alcohol . Fatty acids are major and essential components of all plant cells and are also precursors for a variety of plant metabolites, including signaling molecules and phytoalexins . FAD2 encodes 1 12-desaturase that catalyzes the conversion of oleic acid to linoleic acid . The Arabidopsis genome has only a single FAD2 gene , but most other plant species carry multiple FAD2 homologs . The duplication of FAD2 genes in plants would have enabled the functional diversification of these enzymes, leading to divergent catalytic activities and the synthesis of novel metabolites. For example, recent studies have shown that tomato has non-canonical FAD2 family proteins that lack 1 12-desaturase activity . In particular, ACET1a/b and FAD2-9 are noncanonical FAD2 involved in the biosynthesis pathway from linoleic acid to a phytoalexin, falcarindiol . Falcarindiol has not only anti-bacterial and anti-fungal activities but also nematicidal activity to M. incognita and pinewood nematode Bursaphelenchus xylophilus . Infection with A2-O rapidly induced the expression of ACET1a/b and FAD2-9 , suggesting that infection with A2-O rapidly activates a biosynthesis pathway similar to the falcarindiol pathway, but the production of falcarindiol by S. torvum needs to be experimentally confirmed in the future. The GO enrichment analysis of Cluster 8 revealed that “lignin biosynthetic process ” was significantly enriched , and that the expression of F5H in Cluster 8 was very high andspecifically induced after infection with A2-O . These results suggest that the infection with A2-O transcriptionally activates lignin biosynthesis. Lignin is a phenylpropanoid polymer that is deposited predominantly in the secondary cell wall, making the cell wall rigid and impervious to water . Lignin polymer is synthesized via oxidative combinational coupling of lignin monomers , namely p-coumaryl alcohol, sinapyl alcohol, and coniferyl alcohol. The lignin subunits constituted by these monolignols are p-hydroxyphenyl , syringyl ,and guaiacyl groups, respectively. All of the monolignols are synthesized from phenylalanine through the general phenylpropanoid and monolignol-specific pathways . Normally, lignin deposition occurs in the root endodermis of the differentiation zone and constitutes the Casparian strip, which functions as a physical barrier that prevents free diffusion of solutes and ions between the xylem and the soil . However, biosynthesis and deposition of lignin can be induced in response to biotic stresses , which prompted a closer examination of the expression patterns of genes involved in the lignin biosynthetic pathway whose expression was up-regulated by infection with either A2-J or A2-O . Infection with A2-O induced the expression of genes encoding phenylalanine ammonia-lyase , cinnamate 4-hydroxylase , 4-coumaroyl-CoA ligase , p-hydroxycinnamoylCoA:shikimate p-hydroxycinnamoyl transferase , caffeoyl-CoA O-methyltransferase , cinnamoylCoA reductase , F5H, caffeic acid O-methyltransferase , and cinnamyl alcohol dehydrogenase . Infection with A2-J also induced the expression of some of these genes, but to a much lesser extent than A2-O . Phloroglucinol staining of infected roots allowed us to visualize the intensity and location of lignin accumulation . Infection with A2-O, but not with mock treatment, induced ectopic accumulation of lignin in root tips. With A2-J infection, the area of the root proximal to gall tissue was very slightly stained with phloroglucinol, but the gall itself had little or no detectable phloroglucinol staining. These differences in lignin staining intensity may reflect differences in the expression of lignin biosynthetic genes after infection with A2-J and A2-O . There was no enrichment of specific GO terms in Cluster 7 . However, we found that both A2-J and A2-O strongly activate the expression of suberin biosynthetic genes, including aliphatic suberin feruloyl transferase , cytochrome P450 86A1 , cytochrome P450 86B1 , glycerol- 3-phosphate acyltransferase 5 , and β−ketoacyl−CoA synthase , but A2-O induced slightly higher expression of these genes than A2-J. Suberin is a cell wall component that restricts water loss, nutrient elution, and pathogen infection . It is normally deposited in the cell walls of endodermal cells, but not in the root tip , and several reports showed that suberin synthesis is induced in wounded tissues .

This experiment was conducted at least twice. Calculation of the EC50 values for potassium phosphite of each isolate was determined as described in Adaskaveg et al. . Briefly, the EC50 values were determined by fitting regression lines of the natural log-transformed phosphite concentration against the logit-transformed percent inhibition as compared with the control for each isolate. Regression equations were then solved for concentration at 50% inhibition using Microsoft Excel . The EC50 values for mefenoxam , fluopicolide , and oxathiapiprolin , currently used to control diseases caused by oomycete pathogens, were determined using the spiral gradient dilution method described by Förster et al. . Mefenoxam, oxathiapiprolin, and fluopicolide were dissolved in sterile water to make the corresponding stock solutions at 50, 5, and 100 µg/ml, respectively. Each fungicide was radially applied to a 15-cm 10% cV8A plate using a spiral plater . Isolates were grown for 7 days in the dark at 22°C on 10% cV8A media. Cultures were then cut into strips and placed between sterile hydrophilic cellophane strips on new 10% cV8A plates. Plates were incubated for another 7 days in the dark at 22°C or until the mycelium had evenly covered the strips. Mycelium-covered cellophane strips were then radially applied across the fungicide concentration gradient on the spiral gradient dilution fungicide plate.

The ranges of concentrations used were between 0.0025 to 0.6837 µg/ml , 0.0038 to 1.4054 µg/ml , procona London container and 0.0002 to 0.0572 µg/ml for mefenoxam, fluopicolide, and oxathiapiprolin, respectively. For each isolate, duplicated strips were placed at the opposite location on each spiral gradient dilution fungicide plate . Controls consisted of cV8A plates spirally plated with water, to which cellophane strips were placed as described above. Plates were incubated in the dark at 22°C. Two independent experiments were conducted. Two days after incubation, the mycelial growth of each isolate in each of the control plates was measured. The location where mycelial growth was inhibited by 50%, compared with growth on the control plates, was determined. This distance was used to determine the local fungicide concentration on the agar at the location of 50% inhibition using the SGE software . The EC50 values for each fungicide were calculated using the spiral gradient endpoint software as described in Förster et al. .Nine-month-old avocado rootstocks liners were inoculated with P. cinnamomi colonized millet seed. Millet inoculum was prepared according to the method of Drenth and Sendall . Briefly, 175 ml distilled water was added to 144 g of millet seed. Excess water was poured off and moistened millet was autoclaved in a 250 ml volumetric flask.

Autoclaving was repeated the following day. For each isolate, ten plugs of 6-mm-diameter were cut from the margin of a 4-day-old colony of P. cinnamomi plate and were added to the flask containing the sterilized millet seeds. Inoculated millet was incubated in the dark at 25°C for 21 days. The millet was then harvested, weighed, and homogenized in a plastic bag. A total of 4.4 g of inoculum per plant was used for inoculation by placing the inoculated millet around the root collar. Combination isolate inoculum was produced by thoroughly mixing equal amounts of each corresponding isolate inoculated millet seeds together. Dusaâ and PS.54 ungrafted clonal rootstocks were inoculated with individual isolates, a combination of Northern California isolates , a combination of Southern California isolates , and a combination of all isolates . Control plants were treated with uninoculated millet seeds that were sterilized and prepared in the same manner as the inoculated millet seeds. The experiment was conducted in a greenhouse using a randomized complete block design with five replicates per treatment. The average maximum temperature in the greenhouse was 28°C with an average minimum temperature of 14°C. To confirm that plants were successfully infected, roots samples were excised from harvested plants and 10 to 20 segments per plant were surface sterilized in 70% ethanol and plated onto the PARPH-V8 Phytophthora-selective media identified by Paglaccia et al. .

The A2 clade I contained P. cinnamomi isolates collected from 1989 to 2010, whereas the A2 clade II only contained a unique set of isolates collected in 2009 and 2010 from Southern California avocado growing regions. This study reported significant differences in vegetative growth, fungicide sensitivity, and virulence among all the isolates tested. Moreover, Southern isolates were less sensitive to potassium phosphite and have slower growth rate at 22℃ whereas Northern isolates were more sensitive to potassium phosphite and have higher growth rates. This trend observed suggests a correlation between mycelial growth rate at 22℃and potassium phosphite sensitivity with specific group of isolates. The group of Northern isolates corresponding to the A2 clade I mating type group exhibited higher mycelial growth rates than the Southern group of isolates at 22°C independent of the nutrient media used . Consistent with the results of Zentmyer et al. , there was a significant effect of nutrient media and mycelial growth rate of the P. cinnamomi study isolates, however this effect did not alter the split of the Northern and Southern isolates by their growth rate. The higher sensitivity of the A2 clade I isolates, collected from Northern California, to higher temperatures when compared with the Southern isolates might be explained by the fact that the median temperature is higher in Southern than in Northern California throughout the year.Previous studies reported phenotypic differences in colony morphology, growth rate, and optimum growth temperature among P. cinnamomi isolates, however the majority of these studies compared isolates from different mating types, origin , and host plants . Only a few studies have included P. cinnamomi associated with avocado when assessing phenotypic variability . In the majority of these studies, the authors could not assign a phenotypic trait to a specific group of isolates. Others have reported varying results on the linkage between genotype groups and colony morphology. Dobrowolski et al. reported that colony morphology of Australian P. cinnamomi isolates grouped with a particularly genotype. In contrast to these results, colony morphology among the twelve isolates tested in this study did not vary significantly. Lopez-Herrera and Perez-Jimenez reported significant differences on colony morphology among P. cinnamomi A2 isolates collected from avocado trees in Spain, but these isolates did not exhibit significant differences on mycelial growth rates. California produces 95% of the avocado crop for the U.S.A and PRR caused by P. cinnamomi is responsible for commercial losses totaling $40 million annually statewide. Mefenoxam and phosphite applications are widely used to prevent and combat this disease , however, there is a notably preference for phosphite products over mefenoxam among the growers and this is particularly true for California growers. This preference could explain why mefenoxam-resistant isolates were not found in this study. Moreover, the range of the EC50 values for mefenoxam was consistent with previous values reported for P. cinnamomi in the U.S.A . Duan et al. reported minor variation in sensitivity to mefenoxam among P. cinnamomi isolates collected from diseased ornamental plants in South Carolina. The EC50 values of the majority of these isolates were less than 0.1 µg/ml. Hu et al. found that there were more variations in mefenoxam sensitivity among P. cinnamomi isolates collected from different host species than from the same host.

In this study, cut flower transport bucket the authors classified isolates as sensitive to mefenoxam when their EC50 values ranged from 0.01 to 0.02 µg/ml and as intermediate when EC50 values ranged from 0.03 to 0.08 µg/ml. The EC50 values for mefenoxam in this study were also less than 0.1 µg/ml suggesting that the repeated use of this fungicide to control PRR in nurseries and avocado orchards does not appear to have reduced the mefenoxam sensitivity of P. cinnamomi isolates. In contrast to mefenoxam, we detected a significant variability among the isolates in potassium phosphite sensitivity but we did not find potassium phosphite-resistant isolates. The Northern isolates corresponding to the A2 clade I group had EC50 values of < 34.1 µg/ml, whereas the Southern isolates had EC50 values of > 98.9 µg/ml. It is worrisome that the Southern group of isolates including the A2 clade II isolates that exhibited more virulence in the moderate resistant Dusaâ, which is the current industry standard rootstock among California avocado growers, were less sensitive to potassium phosphite. Their higher EC50 values could represent a selection from higher doses of potassium phosphite being necessary to suppress and control avocado root rot where these isolates are present. A more detailed study with a larger number of isolates and history of phosphonate applications in the field are needed it to test this hypothesis. Dobrowolski et al. demonstrated that P. cinnamomi isolates exposed to long periods of phosphite treatment in avocado orchards in Australia exhibited reduced sensitivity to phosphite when evaluated on avocado, lupin, and eucalyptus suggesting the onset of resistance to this fungicide. Similar results have been reported for P. cinnamomi isolates from avocado orchards in South Africa . This study reports for the first time, the presence of P. cinnamomi isolates, collected from PRR diseased avocado roots in California, that are less sensitive to potassium phosphate. To help delay the development of phosphite-resistant P cinnamomi isolates, care should be taken to alternate or mix phosphite products with other effective fungicides with different mode of action to control avocado PRR. Phosphite and mefenoxam rotation with alternative fungicides is commonly used to prevent or reduce the emergence of Phytophthora resistant isolates , however there are no other fungicides tested or registered to control P. cinnamomi in avocado. Fluopicolide and oxathiapiprolin are two new oomycete-targeted fungicides that have been tested for several Phytophthora spp., but not for P. cinnamomi . In this study, we report for the first time that the EC50 values for mycelial growth inhibition of P. cinnamomi avocado isolates are within the range of the EC50 values reported for other Phytophthora spp. using these two fungicides . Gray et al. recently reported the Fluopicolide and oxathiapiprolin EC50 values for several Phytophthora species associated with citrus on California. Fluopicolide EC50 values for P. citrophthora, P. syringae, P. nicotianae, and P. hibernalis ranged from 0.031 to 0.087, 0.02 to 0.0461, 0.039 to 0.095, and 0.017 to 0.018 µg/ml, respectively. Of the four fungicides evaluated in this study, oxathiapiprolin had the lowest EC50 values . This range is similar to EC50 values reported for other Phytophthora spp. including P. sojae, another member of the phylogenetic clade 7, which includes P. cinnamomi . In agreement to our study, Gray et al. , also found that this fungicide had the lowest EC50 values among five different fungicides tested. The authors reported oxathiapiprolin EC50 values for P. citrophthora, P. syringae, P. nicotianae, and P. hibernalis ranged from 0.0002 to 0.0015, 0.0002 to 0.0003, 0.0003 to 0.001, and < 0.0003 µg/ml, respectively. Interestingly, in contrast to potassium phosphite sensitivity, the Southern isolates in this study exhibited lower EC50 values than the Northern A2 clade I isolates. These results suggest the potential to rotate oxathiapiprolin with phosphonates and mefenoxam for controlling avocado PRR in California reducing the risk of the emergence of phosphonate-resistant P. cinnamomi isolates. Significant variation in pathogenicity and virulence among P. cinnamomi isolates collected from different host plants have been previously reported . Few cases have reported no differences in virulence among P. cinnamomi isolates collected from different hosts and unique host including one study in avocado . In this study we found significant differences in virulence among three representative P. cinnamomi isolates from the two A2 clonal groups identified by Paglaccia et al. when we inoculated a moderate resistant avocado rootstock but not the most susceptible one. The use of highly susceptible plants to distinguish between isolates with different levels of virulence is challenging and could explain why we did not detect significant differences in virulence among our isolates when we used a susceptible avocado rootstock . Although the mixture of the A2 clade II isolates were more virulent than the mixture of the A2 clade I isolates , we observed that the mixture of all the isolates together exhibited the same disease severity as the mixture of the less aggressive ones . This observation might be explained by the level of competition between these two groups of might reflect the induction of specific plant defense responses triggered by these two distinct groups of isolates, a more comprehensive study is required to test these two possibilities.

Subsequently, the biochemical data from both V7-berries and V9-berries were correlated to the WGCNA modules, and only 2 modules, M21 and M30, displayed substantial correlations with berry polyphenols, containing 5349 and 4559 genes, respectively . The M21 module was positively linked with TAC , but negatively associated with tannins, catechin, and quercetin glycosides . On the contrary, the M30 module exhibited a positive correlation with tannins, catechin, and quercetin glycosides , but was negatively linked with TAC . The DEGs obtained from the two pipelines were assigned to both M21 and M30, yielding 604 and 1362 genes, respectively . Interestingly, the number of DEGs in each module, M21 and M30, was roughly equal to the down- and upregulated genes, respectively . To identify flavonoids/tannins-related genes that might result in such astringency diversity between V7-berries and V9-berries, hub genes were searched in the DEGs list of both modules . Only 8 hub genes were identified based on their transcript abundances in V9- berries and predicted functions. However, based on our previous work , 30 litre plant pots we found another 11 genes that are significantly expressed but with a log2FoldChange less than 1.5, and they were included in our further analysis .

The enrichment analysis of GO showed considerable enrichment in the BP GO terms for secondary metabolite biosynthetic process , flavonoid biosynthetic/metabolic process , L-phenylalanine metabolic/ catabolic process , phenylpropanoid metabolic process , phenylpropanoid biosynthetic process , chorismate biosynthetic/metabolic process , cinnamic acid biosynthetic/ metabolic process , anthocyanincontaining compound biosynthetic/metabolic process . The KEGG pathway analysis confirmed the BP GO terms, exhibiting enrichment for the biosynthesis of secondary metabolites , phenylpropanoid biosynthesis , flavonoid biosynthesis , and glutathione metabolism .To precisely elucidate their significance in the tannins/astringency diversity between V7-berries and V9-berries, we studied the expression levels of the 19 hub genes associated with the shikimic and flavonoids pathway. Except for the PAL1_1 gene , the analysis of their relative expression by real-time quantitative PCR showed a significant correlation with the Transcripts Per Million values for genes of interest, validating the transcriptomic data from both V7- and V9-berries . In general, all genes showed higher expression levels in V9-berries compared to V7-berries, but with different degrees of induction. Forinstance, the two genes involved in the shikimic acid pathway, chorismate synthase , and chorismate mutase , showed visibly higher accumulation abundance in V9-berries at the third harvesting time with approximately 6-fold and 3-fold increases, respectively, compared to V7-berries.

Similarly, the upstream structural genes in the phenylpropanoids pathway, including phenylalanine ammonia lyase , trans-4- coumarate biosynthesis , and 4-coumaroyl:CoAligase 2 , were significantly induced by approximately 2- to 9-fold in V9-berries. Regarding flavonoids/PAs biosynthesis, chalcone synthase is considered a key enzyme in this pathway, converting p-coumaroyl-CoA to naringenin chalcone, which is later turned into naringenin by chalcone isomerase . Both genes were highly expressed in V9-berries . Naringenin is subsequently converted by flavonoid 3’-monooxygenase to dihydromyricetin and dihydroquercetin, which are further transformed by dihydroflavonol 4-reductase into leucodelphinidin and leucocyanidin, respectively . The expression levels of F3H and DFR also showed a commensurate induction with the upstream genes in V9-berries relative to V7-berries. Subsequently, leucoanthocyanidin dioxygenase and leucoanthocyanidin reductase catalyse the conversion of leucodelphinidin to delphinidin and -gallocatechin, respectively, as well as leucocyanidin to cyanidin and catechin, respectively. These three genes also exhibited a significant increase in V9-berries. Finally, the expression of genes encoding glutathione Stransferases , one of the most essential anthocyanin transporters, was significantly higher in V9-berries compared to V7-berries, with approximately 3- to 9.2-fold changes .

Our data showed that the expression of flavonoids/PAs related-genes was highly increased in V9-berries at the third harvest time compared to V7- berries, resulting in the accumulation of more PAs in V9-berries.Developing and producing table grapes with high quality is of utmost importance for the success of grapevine breeding programs. Scarlet Royal table grape , variety , is one such success story, producing premium fruit quality and becoming one of the major red varieties in California. However, under certain unknown circumstances, the berry quality of Scarlet Royal grapes can be affected by undesirable astringent taste, which can negatively impact marketability and consumer acceptance. Research on the relationship between astringency and phenolic composition in table grapes is still scarce, especially on the transcriptomic level. In this study, we aimed to understand the molecular events involved in the development of berry astringency, which is a complex set of sensations resulting from the shrinking, drying, drawing, or puckering of the mouth epithelium . We focused on Scarlet Royal berries from two different vineyards with contrasting astringency and analyzed the changes in phenolic-related compounds at six different time points from veraison until the last harvesting time. Our panel test revealed that the V9-berries were perceived as more astringent, a characteristic that could be attributed to their elevated levels of tannins . Differences observed between the two vineyards under study indicate that V7 vines yield more compared to V9. This difference may be attributed to the lower cluster count in V9, a factor known to potentially contribute to astringency, as suggested by Cañon et al., 2014 in wine grapes. Additionally, petiole analysis revealed higher levels of nitrogen and potassium in V9 vines compared to V7. Vine nutrient levels contribute to the final berry quality at harvest . These factors may also contribute to the higher levels of tannins detected in V9 berries; however, further research is needed to confirm this theory. It’s worth noting that weather conditions can play a role in inducing astringency. Nevertheless, the two vineyards are located in close proximity to each other, and weather data collected from the same station in the Delano area indicates similar conditions. Therefore, it is unlikely that astringency or higher phenolic compounds are induced by weather factors. In fact, several studies, mainly in wine, have pointed to PAs as a determining factor for astringency intensity . For example, Vidal et al. reported that the total amount of tannins is the most plausible factor for sensory astringency, with flavan-3-ols dimers, trimers, and non-galloylated tetramers contributing to the astringency sensation. The PAs are a group of oligomers and polymers of flavan-3-ols and are the naturally occurring and predominant type of tannins in grapes and wine . Another study on aronia berry juice confirmed PAs as the key astringent compounds using sensory evaluation and phenolic profile approaches along with in-vitro models . The study found that PAs with higher degrees of polymerization were responsible for the strong astringent mouthfeel . The composition of phenolic substances, especially PAs, 25 liter pot plastic seems to play a crucial role in determining berry astringency, and further exploration of this relationship is warranted in fresh fruits of different species and cultivars. Understanding the molecular basis of astringency development in Scarlet Royal berries can provide valuable insights for improving grape breeding programs and enhancing the overall quality of table grapes.The relationship between astringency and the berry polyphenols content has not been explored yet in table grape at the molecular level. To the best of our knowledge, the present study provides the first transcriptome profiling along with the changes of polyphenols in grape berries of the same variety but having astringency diversity .

The transcriptome profile of both V7-berries and V9-berries underlined the remarkable transcriptional shift during berry ripening at different vineyards . Commonly, berry transcriptome profiles may widely vary based on many factors, including genotypic variations among varieties/species and developmental stages , as well as environmental circumstances. In our case of study, stemmed differences from the developmental stages and genotypic variations were eliminated, andhence the difference of the vineyard locations was the main source of variance with 97% of variance . The identified DEGs output of V9-berries compared to V7-berries is highly explanatory, including polyphenolic-related genes that are robustly expressed and co-regulated with astringency development, particularly in the V9-berries. The enrichment of the up-regulated genes with BP GO terms related to the biosynthesis of secondary metabolites, phenylpropanoid, and nitrogen compound metabolic process , commensurate with the higher amounts of N found in V9- berries . In fact, not only the levels of N fertilization but also its different forms highly affected the composition of phenolic compounds in leaves and wine . However, the synergistic/antagonistic effects of other macro- and micro-nutrients should also be considered. Our results highlighted the negative impact of above-normal amounts of macronutrients, mainly N, and to a lesser extent P and K, on the desirable attributes of grape berries. Otherwise, the positive effect of Ca, Mg, and Mn were achieved as their levels were maintained within the normal range . These data should be also seen in the light of the highly enriched BP GO terms in the down regulated genes . Particularly, those for hormonal signaling pathways such as auxin, abscisic acid, strigolactones, as well as the KEGG pathway for the carotenoid biosynthetic pathway .The transcriptome profiling identified the common and unique molecular events featuring the development of tannins/astringency in grape berries. It is well-documented that the synthesis of PAs in grapevines is achieved via three sequential pathways: the shikimate pathway, the phenylpropanoid pathway, and ultimately the flavonoid pathway . Our results revealed that the expression levels of flavonoids/PAs-related genes were highly induced in V9- berries at the third harvesting time compared to V7-berries. The 19 selected genes were involved in the three pathways: the shikimate pathway, phenylpropanoids pathway, and flavonoids pathway. The shikimate pathway is an alternative route to produce aromatic compounds, including phenylalanine, tyrosine, and tryptophan, which serve as precursors for various metabolites, such as phenolic compounds . The up-regulation of genes like chorismate synthase and chorismate mutase in V9-berries may lead to the accumulation of phenylalanine, which is a critical precursor for the phenylpropanoid pathway. The latter pathway is responsible for synthesizing several end products, including PAs, anthocyanins, lignin, lignans, hydroxycinnamic acid esters, and hydroxycinnamic acid amides . Under the conditions of the V9 vineyard, several PAs/flavonoids structural genes such as PAL, C4H, 4CL, CHS, CHI, F3H, LDOX, LAR, and ANR were induced in V9-berries, leading to the accumulation of PAs in the berries . This process is facilitated by GSTs and transported by multidrug and toxic compound extrusion transporters. The activation of the PAs biosynthetic pathway in V9- berries may lead to a reduction in the necessary substrates for anthocyanin synthesis, resulting in low red color intensity in V9- berries compared to V7-berries. Additionally, the accumulation of PAs is associated with the development of astringency taste in V9-berries. Our study provides valuable insights into the molecular events underlying astringency development in Scarlet Royal berries. By integrating transcriptome profiling with polyphenolic composition analysis, the research shed light on the co-regulation of genes involved in the shikimate, phenylpropanoid, and flavonoid pathways, leading to the synthesis of PAs and ultimately influencing astringency. The findings from this research have implications for grapevine breeding programs and the production of high-quality table grapes. Understanding the molecular mechanisms underlying astringency development can help breeders in selecting and developing grape varieties with desirable attributes. Additionally, the knowledge gained from this study can inform vineyard management practices, such as nutrient fertilization, to optimize polyphenolic composition and berry quality. In conclusion, the data presented in this study indicates that berry astringency is strongly correlated with a high tannin content, likely resulting from the activation of nineteen genes within the phenylpropanoid pathway. The activation of these genes shifts the flavonoid biosynthesis pathway towards proanthocyanins, leading to increased tannin accumulation in the berries. It appears that triggering these events is associated with nutritional imbalances and a lower number of clusters per vine, as confirmed by petiole nutrient levels and the observed lower berry soluble solids and higher titratable acidity levels. The identification of these genes holds significant value for table grape genetic improvement programs. The nutrient imbalance theory derived from this research could be applied worldwide to optimize grapevine fertilization programs. Furthermore, it paves the way for further research in this area, with a particular focus on vine nutrients, crop load management, and berry astringency, thereby contributing to advancements in the field of table grape cultivation.The names of the repository/repositories and accession number can be found in the article/Supplementary Material.The cultivated garden strawberry , an allo-octoploid , has a unique natural and domestication history, originating as an interspecific hybrid between wild octoploid progenitor species approximately 300 years before present.

New migrants’ short United States experience translates into a less-developed network to assist their mobility and employment. As a result, they hold the worst and lowest-paid jobs, and are usually the last to be hired and the first to be fired as the peak harvest seasons run their course. In Santa Maria’s strawberry harvest, for instance, they typically occupy the crest of the high employment season and move on to other production sites before the season is completely over. Many find daily employment with a variety of employers but only to fill momentary gaps, to aid short-handed crews, or to meet special urgent contracts. Most, in effect, secure employment through farm-labor contractors. New migrants, in contrast with traditional migrants, are much more mobile and versatile. They travel up and down the California geography, and in and out of Oregon and Washington, following a variety of crops. Some chase the berry harvest, starting on the Mexican border in February and ending up in the state of Washington by mid-June, always striving to remain on the crest of the peak harvest season when piece-rate earnings are at their best. Others become involved in other highly seasonal crops such as cherries, asparagus, 30 plant pot pickle cucumbers, raisin grapes, oranges, and apples which are spread out throughout both the west coast geography and the agricultural calendar.

In the Santa Maria Valley, new migrants are especially present during the peak strawberry season, April-June, and again in September to participate in the short but intense wine-grape harvest. Since most new migrants come from highly impoverished rural regions of Mexico, they travel in large family groups without children or other unemployable dependents who could slow them down or hinder full employment during the course of their trek. It is not uncommon for these families to leave children and other dependents behind in shanty towns and camps located on the Mexican side of the border while the most productive and employable members seek jobs and wages in the United States. Many of the interviewed new migrants enter California in February and return to their home communities in southern Mexico by mid-November. During their stay in the Santa Maria Valley they typically crowd into local hotels and small unfurnished apartments. In 1991 and 1993 we observed as many as eight workers sharing a double hotel room and up to sixteen individuals jammed into a small one-bedroom apartment. The incidence of undocumented workers among both traditional and new migrants is quite high. Data collected in 1993 reveals that as many as fifty to sixty percent of traditional migrants are undocumented. Moreover, those who do possess appropriate authorization to work in the United States only received it recently through IRCA’s SAW program. In contrast, only ten to fifteen percent of the new immigrants are documented.

All, nonetheless, hold some sort of paperwork which is required to obtain employment. The high incidence of unauthorized/undocumented workers, compounded with the fact that many actually hold counterfeit documents, makes migrants into a population that resists enumeration and identification, a circumstance that is further complicated by migrants’ easy mobility and unconventional housing arrangements. Finally, with respect to census-taking efforts, it is important to emphasize that there are few migrants in the Santa Maria Valley by April 1. At that time, the vast majority of migrants are either just starting off on their annual treks from their home communities in Mexico or are busy harvesting strawberries in other southern producing areas such as Baja California, northern San Diego County, and the Oxnard- Ventura plain where the fruit matures earlier. As indicated above, the thick of the migrant presence in Santa Maria does not occur until strawberries reach their peak in mid-to-late April. Approximately one thousand individuals who form part of Santa Maria’s sizable agricultural labor force maintain a permanent home base in the U.S.-Mexico border area, either in small colonias in or near El Centro, Calexico, and Yuma or in Mexico itself, especially in or near the city of Mexicali. From these communities they regularly commute to the Santa Maria Valley where they participate in the lettuce harvest which extends from spring to fall. Most of the border area commuters are employed by a few large corporations that have come to monopolize the lettuce industry by, among other means, maintaining production sites in different parts of the state: in Santa Maria and other coastal valleys in the summer and in Imperial Valley in the winter.

Some of these companies regularly transport farm equipment, vacuum cooling plants, and office facilities over great distances, from summer to winter producing sites and back. Large lettuce producers have also developed a highly specialized and stable labor force that travels from site to site as needed. These are the lechugueros or specialized lettuce harvesters , who maintain a permanent home base in the border area, near winter production sites, from where they migrate seasonally to summer production sites such as the Santa Maria Valley. Lechugueros explain that in the border area they find affordable housing, lower cost of living, and a more favorable sociocultural environment. Few of them are originally from the border itself but after the Bracero Program was cancelled settled there as an ideal location from which to access U.S. employment. Many of them can be traced back to the traditional sending communities of Central Mexico. Professional lechugueros are a breed apart among California’s agricultural laborers in that they enjoy nearly year-round employment, always with the same employer, by following the crop from production site to production site. They also earn relatively high wages, $1,200 to $1,500 monthly with some benefits. At home on the border and during the winter months, while lechugueros are busy with the winter harvest, other family members also find part-time, intermittent employment in local agricultural endeavors. During the summer, however, only lechugueros migrate to the distant production sites, leaving behind dependents and other family members who, nonetheless, continue to seek part-time occasional farm jobs near home. Migrants return home occasionally, every two or three weeks, for a few days to visit family and friends, and to rest. During their stay in the Santa Maria Valley, lechugueros rent apartments which they share with other commuters to cut down expenses. A few experienced commuters have installed small, grow raspberries in a pot dilapidated trailers in the area which they use as a temporary second home. Most of the lechugueros we interviewed in 1991 and 1993 are documented and have been so for some time. A few who do not possess either “green cards” or citizenship use commuter border passes issued to them years ago by INS which entitle them to work in the United States while living on the Mexican side of the border. Those who, before 1986, lacked adequate documentation were subsequently able to legalize by accessing IRCA’s SAW program with the encouragement, sponsorship, and assistance of their employers. Because lechugueros, relatively speaking, lead fairly conventional lives, they are much less apprehensive about being identified and counted and, as a result, offer little resistance to census takers and other surveyors. Nonetheless, those who maintain a permanent home base on the Mexican side of the border are likely to be missed during the census count, while those who live on the U.S. side risk being counted twice owing to the circumstance that in the early spring they typically occupy two homes. Above we have described and categorized the bulk of the work force employed by Santa Maria Valley agriculture. The four types of workers we have described thus far have established some degree of routine or recurrent behavior over the past years according to opportunities created by their farm jobs and/or specific arrangements they have made with the larger family group to which they belong. The four described categories account for approximately eighty-five percent of the farm workers employed by Santa Maria farms during the course of a complete agricultural cycle. There is, in addition, an undetermined number of single unattached workers, mostly young males, who spontaneously show up in the valley during peak employment seasons and who remain there only as long as employment is available; otherwise they quickly move on to other work locations. Although it is difficult to ascertain the exact number of these transients, we estimate there is a constant pool of 300 to 500 such workers in the Santa Maria Valley at any given time.

Overall, up to two or three thousand individuals may pass through Santa Maria during the course of the year; some remain in the valley only for a few days while others may stay there for several months. Interviews conducted in 1991 and 1993 reveal that some of these workers follow a preconceived itinerary designed to land short-term jobs in especially demanding crops through a network of farm labor contractors . Indeed, a few of the interviewed transients were originally recruited by farm-labor contractors in the border area and in their home communities deep in Mexico to perform a specific job in California and, once completed, they were referred to other labor contractors in other work locations. Most, however, are on their own and seek out farm-labor contractors upon arriving at a new location to inquire for work and shelter. Single unattached transients land the worst possible jobs and receive rock-bottom wages, often at rates below the minimum wage. They are usually employed as day laborers and they rarely know for how long or for how much. In a good week a transient worker can yield as much as $200 but typically monthly earnings rarely exceed $400. They, moreover, experience long and frequent periods of unemployment between jobs. Despite their erratic and transient lives, these workers continue to be strongly attached to their families in their home communities in Mexico to whom they send part of their wages whenever they can spare them. Many, in effect, aspire to return home before Christmas with presents, new clothes, and $1,000 cash in the pocket. It is, however, not uncommon for transients to remain in the U.S. for several consecutive seasons, only to return home when they are broke, homesick, and/or ill. As would be expected, few transient workers are documented but most have acquired fake documents. While in Santa Maria, transients find shelter either in one of the few labor camps still open, or in garages, tool sheds, shacks and trailers offered by their employers at a price. Many camp out in the fields, in boxcars, or in their cars. Only when the weather forces them will they choose to stay in one of the local hotels that caters to migrant farm workers. They are, as a result, the most difficult workers to track down, find, interview, and enumerate. We came across them, almost fortuitously, when we examined and interviewed members of strawberry, wine grape, and celery harvest crews. In this report we have described the forces that attract immigrant and migrant farm workers from Mexico to the Santa Maria Valley , and we have described a variety of forms and behaviors of that immigrant and migrant labor force. Moreover, in the process we have identified some of the circumstances that hinder an accurate accounting of this population. In this section, we draw upon the information we have gathered in order to propose strategies to improve the identification, description, and enumeration of immigrant and migrant farm workers in California. One unassailable conclusion derived from our field research in the Santa Maria Valley is that a considerable number of immigrant and migrant farm workers prefer not to be identified and, hence, will actively avoid and frustrate efforts designed to enumerate them. They will, moreover, often provide erroneous, false, and incomplete information when they are pressed by surveyors. Some farm workers are reluctant to cooperate because they do not possess appropriate authorization to be and to work in the United States; others, although authorized, resist to protect family members and friends who are not; and many, although they have nothing to hide, have a deeply imbedded mistrust of any official government effort to identify, describe, and enumerate them. Many have at one time or another lived and worked illegally in the United States, have experienced apprehension, deportation, and harassment, and hence maintain a cautious, suspicious attitude towards all government officials, especially those who want to know more about them. Active resistance to identification and description compounds the well-known difficulties encountered in attempts to enumerate immigrant and migrant farm workers due to, among other circumstances, their frequent mobility, their unconventional housing arrangements, and language barriers.

The transfer and extension of ideas and concepts in order to bring new topics under the compass of existing theory has obvious scientific merit . It also has pitfalls. The failings of early “evolutionist” models of social evolution and their archaeological adaptations, as well as social Darwinist interpretations, are well-rehearsed subjects in anthropology. Contemporary anxieties about the use of neo-Darwinian theory in anthropology are more narrowly and analytically focused, and sometimes not so easy to set aside. A recent example would be debate over the claim by Rindos that his co-evolutionary account of plant domestication had successfully banished human intent from an explanatory role in this process . In the present volume we take for granted the relevance to agricultural origins of neoDarwinian and behavioral ecology theory. We reject without explicit argument the sub-stantivist claim of economic anthropology that none of the tools of formalist, microeconomics has any purchase outside of modern capitalist economies . To the contrary, we believe it evident that the basic concepts of HBE are fundamental to the analysis of any economy. Close attention to their use in HBE we believe will stimulate new applications and models specifically designed to analyze mixed economies and food production. We are more receptive to the argument that specific foraging models, developed as they were for foragers, may be only partially appropriate to the analysis of emergent food producers. For instance, raspberry plant container the diet breadth model assumes random encounter with resources, a condition increasingly likely to be violated as foragers become involved in the manipulation of individual species.

In as much as all models simplify reality and thus violate at least some conditions of their application, the unavoidable judgment is this: does the failure to fit this particular assumption completely vitiate the heuristic or analytical value of the model? With the specific cautions cited in individual papers, we believe the combined weight of the case studies developed in this volume add up to a strong presumption in favor of the utility of foraging theory, even as the foragers being analyzed direct more and more of their effort toward agricultural activities. We envision three levels where HBE might be applied to the question of agricultural origins. Extant models, although designed for the analysis of foraging, might be applied in the analysis of agricultural origins with little or no alteration in their structure and assumptions. This is the procedure of most authors in this volume. Extant models might be modified so to more directly address questions or situations specific to non-foraging aspects of economy, including cultivation and agricultural production. The modification of central place foraging models to analyze the question of field processing is an especially good example of this. Finally, entirely new models, inspired directly by the problem of explaining human subsistence transitions, might be devised using fundamental behavioral ecology concepts such as opportunity cost or discounting. We think of these options as adopt, adapt, or invent, respectively.

While options and hold great potential for novel and perhaps quite interesting analyses, it appears from the papers assembled here that there is much to be accomplished with the simple adoption of existing models.The fresh market berry industry in Santa Cruz and Monterey counties is an excellent example of transformation in the business of agriculture over the last 50 years. Located along the Central Coast of California, the two counties span the fertile Pajaro and Salinas valleys, and are well known for their amenable climate and production conditions, their diverse crop mix and grower demographics, and their developed agricultural infrastructure and support industries. The majority of the berry sector is comprised of strawberries , raspberries and blackberries , with blueberries and other miscellaneous berries produced on a much more limited basis. Substantial research-based literature and historical information is available for Central Coast strawberries; however, despite the area’s move towards greater production of raspberries and blackberries, less information exists for these crops. We seek here to provide a more complete portrayal and historical context for the berry industry in the Santa Cruz and Monterey area, which is the origin of the berry industry in California. While the berry industry has been very successful in recent decades, it now faces new challenges, such as invasive pests and the phaseout of the soil fumigant methyl bromide. This article draws on previous and more recent research to discuss some of the influences that have contributed to the berry industry’s dramatic expansion in Santa Cruz and Monterey counties, including selected innovations in agricultural practices and heightened consumer demand. During the 1960s and 1970s, the number of acres planted to berries, tons produced and value of production fluctuated. The fluctuations can be partly explained by farm management: in the past growers often rotated berry and vegetable crops to assist with soil and pest management, thereby influencing these statistics. However, annual crop reports from the county agricultural commissioners show that since the 1980s, berries have become increasingly important to each county’s overall value of production, and by 2014 accounted for 64% and 17% of the total value of all agricultural products in Santa Cruz and Monterey counties, respectively .

The industry’s growth can be explained by a shift of some acreage out of tree fruits and field crops , among others, into berries, and by additional acreage put into agricultural production. Strawberries are the undisputed leader in the berry sector and in 2014 represented 58% and 94% of the value of all berry production in Santa Cruz and Monterey counties, respectively , and 50% and 93% of all berry acreage . Table 2 documents the remarkable expansion of the strawberry industry over time in both counties with respect to acreage, tons produced and value of production. Between 1960 and 2014, acreage more than tripled and production increased tenfold. The value of production, in real dollars, increased by 424% in Monterey County and by 593% in Santa Cruz County, reaching an astonishing combined value of nearly $1 billion in both 2010 and 2014. The gains in all statistical categories in Monterey County were enabled in part by an expansion of production into the southern reaches of the county where more and larger blocks of farmland are available, container raspberries and where land rents are lower than in Santa Cruz and northern Monterey counties. However, from 2010 to 2014 Monterey County’s tonnage and production values declined, possibly because the area has recently experienced a shortage of labor to harvest fresh market crops. Tonnage was also lower in Santa Cruz County, but production values increased. This may be because of the county’s greater emphasis on local agriculture, organic production and direct market sales, which are often associated with higher crop values. For raspberries, the acreage, tons produced and value of production grew steadily and most strikingly in Santa Cruz County , where production conditions for caneberries are optimal. For example, caneberry fields in Santa Cruz County are situated in areas that have well-drained soils and are protected from damaging winds. Also, fields are planted to take advantage of the growth and yield gains associated with southern exposures. Moreover, field-to-cooler travel distances are shorter in Santa Cruz County, which is critical for safeguarding the quality and marketability of these highly perishable crops. By 2014, raspberries represented 33% of the county’s total value of production for all berries. In contrast, Monterey County raspberry production accounted for only 6% of the county’s total berry value. Blackberries have not been consistently reported as a separate category in archived statistical analyses, but instead were often included under the terms “bush- or miscellaneous berries”. Therefore, similar data for blackberry acreage and value of production cannot be reported here. However, between 1990 and 2010, Santa Cruz County agricultural commissioner crop reports reported an upward trend for the broad category with respect to acreage planted and value of production . In 2010, blackberries were promoted to a position of prominence in the report and shown as a separate statistic; at the same time, the miscellaneous berry category was shown to be very small indeed. Between 2010 and 2014, however, blackberry acreage and value of production leveled off and have shown only modest gains . This may be because there has been less emphasis on production and market research and promotion for blackberries than for strawberries or raspberries. No comparable data are available for Monterey County. The two counties have contributed significantly to California’s total berry sector: in 2014, area strawberry acreage represented 35% of the statewide total, 37% of the total tons produced and 38% of the total value of production . Area raspberry acreage represented 43% of the statewide total, 42% of the total tons produced and 39% of the total value of production. Comparable statewide statistics are not available for blackberries. County agricultural commissioners’ reports show that the majority of all berries produced in the two counties — up to 98% — are sold as fresh market fruit . In years with adverse production conditions or low prices, a higher percentage of the crop may be diverted to the freezer or processed products market. Fresh market fruit is handled and sold primarily through local grower-shippers; a much smaller share is sold directly to consumers through farmers markets, community supported agriculture operations, farm stands and other direct and intermediated market channels such as restaurants, independent grocers and schools.

Arguably the most momentous shift in cultural practices for strawberries was the introduction of preplant soil fumigants, beginning with chloropicrin in the 1950s and methyl bromide in the 1960s. Fumigation is a soil disinfestation practice that improves plant productivity and helps with the management of arthropods, nematodes, weeds, soilborne fungi and other plant pathogens. Some of the most difficult to control pathogens include Verticillium dahliae, Fusarium spp. and Macrophomina phaesolina. Without soil fumigation, these pathogens have the potential to completely destroy strawberry plantings. Early on, when CP and MB were mixed and applied together, the synergistic effects allowed strawberries to be produced as an annual rather than a biennial crop, and to be grown continuously on the same land without rotation to another crop, resulting in an increase in annual strawberry acreage. The use of fumigants also led to higher and more predictable yields and fruit quality, and further enabled the development of more stable markets for strawberries . Yields for strawberries statewide increased from a range of 2 to 4 tons per acre prior to the introduction of soil fumigants to 16 tons per acre by 1969 . Additional cultural improvements included the development of both UC and proprietary strawberry varieties uniquely adapted to coastal production conditions. Varieties were bred, for example, for disease resistance, yield and market potential. Notable UC-bred strawberry varieties include Tufts , Pajaro, Douglas, Chandler, and Selva , Camarosa and Seascape , and Aromas, Albion and Monterey . Irrigation practices also evolved, shifting from furrow irrigation in the 1960s to drip irrigation in the 1980s, which led to further improvements in plant disease management and greater water use efficiency. These and other enhancements meant that by 2012, yields could exceed 35 tons per acre . More recently, the strawberry industry has focused on “fine-tuning” fertility and water management for more efficient resource use, along with additional yield and fruit quality improvements . The Santa Cruz–Monterey area is also recognized for its early experience with conversion of conventional strawberry production to organic management . Organic strawberry production was shown to result in lower yields, which, when offset by premium prices could potentially offer higher net returns to growers. The importance of crop rotation for disease management was not addressed in the initial study by Gliessman et al. but has since been the focus of additional research, as have more complete analyses of the economics of organic strawberry production . Growers and area researchers continue to collaborate and advance organic strawberry production techniques. Most notably, a long-term research commitment has been made to determine organically acceptable disease management practices such as anaerobic soil disinfestation , the use of commercially available soil-applied biological organisms and the incorporation of soil amendments such as mustard seed and its derivatives. The area is now seen as a global leader in organic strawberry research, and in 2012 the first organic strawberry production manual was published by UC Agriculture and Natural Resources . Statistics documenting expansion of the organic strawberry industry over time are not available on a county-by-county basis, but statistics for California show prodigious growth in acreage and value of production: from $9.7 million in 2000 to $93.6 million in 2012, a 621% increase in real dollars .

Similarly, a comparison of herbivory damage on herbarium specimens of non-native plants in urban versus rural environments might provide insights into one pathway towards species invasion. Many non-native plant species are introduced into urban areas, and urbanization may provide warm, enemy-free space where they can establish and subsequently expand. Though the role of urbanization in natural enemy release and subsequent invasions is not well characterized, we might predict that non-native plants escape their natural enemies in urban areas and experience increased herbivory rates when they move into natural areas where they encounter a higher diversity of herbivores.Responses to global change, such as those in space and time, discussed above, encompass plasticity in behaviour or physiology and distributional shifts, which may be rapid. However, there is growing evidence that evolutionary responses might also be rapid, assuming there is sufficient standing genetic variation for selection to act upon. While fluctuating selection can maintain this standing variation, providing the raw material for future adaptive responses, strong directional selection, such as that imposed by anthropogenic climate warming, plastic gardening pots can erode genetic variation and potentially impede evolutionary adaptation, elevating population extinction risk.

Insect herbivores and the plants they feed upon are locked in an evolutionary arms race, and insect herbivory drives contemporary plant evolution, changing plant allele frequencies within a few generations. It is likely that climate-induced shifts in herbivory will impose additional selection on both plants and insect herbivores already under pressure from direct effects of climate change. Natural history collections that span multiple generations can provide a record of evolutionary changes and constraints. It can be difficult, however, to disentangle plastic and evolutionary responses. 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. Herbaria provide a record of this genetic diversity even when it is no longer present in the wild. In addition, 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. In a recent study, we quantified historical insect damage on a crop species, the low bush 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, blueberry pot size 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. This loss of material can reduce the use of insect specimens for morphological and genetic analyses. 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, 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.The volume before you is the first systematic, comparative attempt to use the concepts and models of behavioral ecology to address the evolutionary transition from societies relying predominantly on hunting and gathering to those dependent on food production through plant cultivation, animal husbandry, and the use of domesticated species embedded in systems of agriculture. Human behavioral ecology is not new to prehistoric analysis; there is a two-decade tradition of applying models and concepts from HBE to research on prehistoric hunter-gatherer societies . Behavioral ecology models also have been applied in the study of adaptation among agricultural and pastoral populations. We review below a small literature on the use of these models to think generally about the transition from foraging to farming, while the papers collected here expand on these efforts by taking up the theory in the context of ethnographic or archaeological case studies from eleven sites around the globe.There are older transformations of comparable magnitude in hominid history; bipedalism, encephalization, early stone tool manufacture, and the origins of language come to mind . The evolution of food production is on a par with these, and somewhat more accessible because it occurred in near prehistory, the last eight thousand to thirteen thousand years; agriculture also is inescapable for its immense impact on the human and non-human worlds . Most problems of population and environmental degradation are rooted in agricultural origins. The future of humankind depends on making the agricultural “revolution” sustainable by preserving cultigen diversity and mitigating the environmental impacts of farming. Simple population densities tell much of the story. Huntergatherers live at roughly 0.1/km2 ; rice agriculturists in Java at 1,000/km2 , a ten-thousand-fold difference. There were an estimated ten million humans in the world on the eve of food production ; now oversix billion people live on this planet, an increase of 600% in only ten millennia. Agriculture is the precursor, arguably the necessary precursor, for the development of widespread social stratification, state-level societies, market economies, and industrial production . Social theory maintains that present-day notions of property, equality and inequality, human relationships to nature, etc., are shaped, at least in part, by the social organization, technology, or food surpluses entailed in our dependence on agriculture.

Random effects were modeled with plant identity nested within site to account for the block design of the experiment and to control for variation between our sites. To model count data and to correct for over dispersion, we used a Poisson-lognormal model with a log link function by including a per-observation random effect as described above .We modeled CBB removal by ants using a GLMM. We included treatment , coffee plant distance to nest tree, ant activity on coffee plants after string placement, the interaction between treatment and distance, and the interaction between treatment and ant activity on plants as fixed effects . Random effects were modeled with plant identity nested within site to account for the block design of the experiment and to control for variation between our sites. To model count data in our response variable we used a Poisson distribution with a log link function.We constrained model selection to include biologically pertinent terms for inference and to aid in model interpretation. A full model of these terms was tested, along with subsequent models of different covariate combinations and a null intercept-only model of random effects . The best fit model was determined via backwards model selection compared to the full model, blackberries in containers where the model that resulted in the lowest AIC score with ∆AIC > +2 was selected. Overall significance in models was assessed using Wald type II Chisquared tests. Statistical differences among treatments were compared by Wald Z tests .

In all cases, fixed effect parameters and the variance of random effects was estimated by maximum likelihood with Laplace approximation using the ‘glmer’ function in the ‘lme4’ package in R . To aid in data interpretation, we removed one coffee plant replicate from our analysis where measured ant activity was more than double that of any other plant measured and may have resulted from an unusually high buildup of scale insects which are tended by A. sericeasur on coffee. Additionally, one nest tree replicate was not included in the tree activity analysis because the data were not collected at that site. Finally, coffee plant height and distance to nest tree were centered and scaled to aid model interpretation. All analyses were performed in R .Our experiment demonstrates that the addition of string to connect shade trees and coffee plants in coffee agroecosystems facilitates movement for A. sericeasur and potentially increases ant recruitment rates. Studies in natural systems have reported increases in ant activity with arboreal connections across the arboreal stratum , possibly driven by the easy access these pathways provide to resources . Other ants, such as Pogonomyrmex spp. prefer linear arboreal substrates and switch to cleared routes as a mechanism to reduce the energetic costs of ant foraging , and in some cases to decrease the risk of encountering predators . The observed increase in ant activity on connected coffee plants after the placement of strings suggests that structural connectivity can increase ant recruitment rates to foraging areas in coffee and may enhance the efficiency of movement for A. sericeasur.

This may lead to increased foraging efficiency for ants and enhanced resource capture rates on coffee. However, this could also reflect other benefits associated with using linear arboreal substrates, such as avoiding predators, a behavior that is known to occur in A. sericeasur . Using more efficient foraging pathways and thereby avoiding the leaf litter as a primary foraging substrate may potentially protect A. sericeasur workers from the attack of the phorid fly parasitoid Pseudacteon spp. . While ant activity only significantly increased after string placement on connected coffee plants, we also observed lesser increases in ant activity on control coffee plants and nest trees . This unexpected result could mean that strings, a novel element in the environment, acted as a form of habitat modification or disturbance, which increased overall ant activity in the local area. However, if our manipulation were the cause, we would have expected the ants to attack the jute strings , a behavior that we did not observe during the experiment. Experiments in tropical forests have shown that the long-term removal of lianas can influence ant richness on trees , and therefore may possibly also affect overall ant abundance and activity when promoted. It is also possible that other factors could potentially explain this result in control plants, such as changes in local abiotic factors that we did not measure systematically in our experiment. Future research which expands on the temporal scope of this study may be useful in assessing the long-term effects of artificial connectivity in this system. Ant activity post string placement was negatively affected by distance to the nesting tree .

This result is consistent with previous studies suggesting that within 5 meters A. sericeasur dominance in the leaf litter decreases with distance to the nesting tree . However, in our study, the effect of distance after string placement was significant only on control plants, but not on connected plants. This suggests that connections could buffer the negative effects that larger distances from the nesting tree pose to ant activity and potentially increase ant-provided biological control services in these plants. Connected coffee plants also had significantly higher CBB removal than control plants . Overall, greater ant activity on coffee plants was associated with higher CBB removal rates , suggesting that ant activity directly influenced CBB removal rates. However, while this effect was significant on control coffee plants it was only marginally significant on connected plants. While we believe that these results support the hypothesis that connectivity enhances ant foraging and bio-control services on coffee, the use of dead CBB in this experiment as a proxy to measure bio-control may explain the only marginally significant effect of ant activity on CBB removal in connected plants. It is possible that dead prey exhibit more variable recruitment responses from ants than live prey. Despite this, it is likely that strings facilitated ant movement to coffee plants by providing a smooth, linear substrate and indirectly increased CBB removal . In other systems, the leaf-cutting ant Atta cephalotes uses fallen branches to rapidly move between areas and thereby quickly discover new food resources . Similarly, these resources allow scouts to return quickly to the colony, minimizing the time taken for information transfer and recruitment of other foraging workers . The role of trunk trails and fallen branches has received extensive attention in the leaf-cutting ant system, however, fewer studies have looked at the influence of connectivity resources on foraging behavior of predatory arboreal ants. Surprisingly, CBB removal did not follow the same trend as ant activity with distance to the nesting tree. While control plants tended to have lower CBB removal rates than connected plants as distance to the tree increased, blackberry containers we did not find a significant effect of distance on CBB removal in either control or connected plant groups. Collectively, these results suggest that connections in the arboreal stratum have the potential to increase ant activity and therefore enhance plant protection from CBB attack, particularly in connected plants. Further studies should assess the effect of distance on CBB removal using plants located at distances larger than 3.5 m from the tree. It is important to note the potential for negative impacts of enhanced ant activity on coffee plants resulting from hemipteran tending behavior. While ant activity can increase the density of green coffee scale insects, which may harm coffee plants and reduce coffee productivity, the scale are not a major pest in the region of study, and are not as economically significant as the coffee berry borer . A recent study evaluating the benefits associated with the indirect Azteca–Coffea mutualism, which emerges from the Azteca-scale mutualism, found that the protective benefit ants provide to coffee plants is positively associated with high densities of the scale . Therefore, it is possible that enhanced CBB control associated with scale-tending by ants outweighs the costs associated with scale damage. However, these interactions may be context-dependent, and still need to be fully evaluated in the field to provide a holistic understanding of the impact of connectivity on scale density and coffee yield. Other ant species could also benefit from the addition of connections between coffee plants and shade trees, such as Cephalotes basalis and Pseudomyrmex simplex, which were observed using these connections during our study.

The ant P. simplex has been previously reported as an important CBB bio-control agent, acting in conjunction with other species of ants to effectively suppress CBB at various life stages . Therefore, this technique could support Azteca ants as well as other ant species that play an important role in suppressing CBB populations. Our results support the general hypothesis that connectivity, one measure of habitat complexity, can sustain important ecological processes in natural and managed ecosystems. In aquatic systems, more complex habitats with macrophytes allow for greater food capture and maintain higher levels of diversity . In terrestrial systems, higher complexity can influence trophic dynamics . In coffee agroecosystems, ants are highly sensitive to habitat change and management intensification, generally expressed as the reduction of shade, elimination of epiphytes, and use of chemical inputs . Such intensification can have a negative effect not only on vegetation connectivity and ant foraging, but may also cascade to affect ecosystem services, such as biological control. Our study supports the idea that promoting complexity at a local scale, in this case providing structural resources for ants in agroecosystems, can significantly enhance connectivity within the arboreal strata, and potentially improve biological control of coffee pests. This idea has already been successfully implemented in other agricultural systems, placing “ant bridges” made of bamboo strips or strings connecting neighboring trees in , and could be incorporated as a management strategy in coffee systems. Future research should evaluate the practical feasibility of adding connections between vegetation strata to enhance bio-control. For example,studies in timber plantations have estimated that the presence of ants increases timber production by 40%, and that ants can be maintained at lower costs by providing intra-colony host tree connections using rope, poles or lianas . It is important that future studies in coffee also consider the costs of other CBB control methods, such as the application of the pesticide endosulfan, which can lead to the development of resistance, can negatively impact natural enemies, and can have harmful impacts on human health . Further investigation into promoting ant bio-control with artificial connections in coffee should: 1) assess economic tradeoffs, management applicability, and farmers’ perceptions of this method in large and small coffee plantations, 2) compare the cost between string placement and other management approaches , and 3) assess coffee yields on connected and not connected plants to provide management recommendations. More broadly, incorporating conservation bio-control strategies in combination with vegetation connectivity is consistent with criteria identified as key for the sustainability of biological control, such as increasing local habitat quality and enhancing species’ dispersal ability . Generally, the maintenance of shade trees and natural vegetation in agroforestry systems may increase vegetation complexity and natural connectivity between plants to promote ant foraging and subsequent biological pest control. The extent to which biotic and abiotic factors shape species interactions and their distributions has been a central focus for community ecologists . Included in the wide spectrum of factors shaping community dynamics are the diversity and availability of food resources and habitat complexity; all are important determinants of species diversity, distribution and interactions . Recent theoretical advances and empirical work in community ecology suggest that further research is needed to understand how resource availability and environmental factors shape species interactions. Specifically, one research gap is in our understanding of how variation in resources along habitat complexity gradients affects species interactions . However, empirically demonstrating how resources vary and how species respond is methodologically complicated, in the sense that the manipulation of multiple resources and consumers is not always straight forward, especially in complex systems. Consequently, studies have focused primarily on one single resource and– disproportionately– on competitive species interactions. Further, the environmental context in which resources matter is also not well understood and will gain more importance as natural and managed systems continue to experience habitat simplification .