Horticulture

Elderflowers are used in products as either fresh or, more commonly, as dry flowers and as either whole or homogenized flowers . Therefore, each of these parameters were evaluated resulting in the following types of samples: fresh whole flowers ; dry whole flowers ; fresh homogenized flowers ; dry homogenized flowers . Phenolic compounds were quantified using HPLC-DAD-FLD and information regarding the standard curves can be found in Table 1. Significantly more phenolic compounds were extracted from FHF compared with FWF, DWF and DHF indicating that phenolic compounds are released more readily from the vacuoles during homogenization while the flower is still fresh. There was no significant difference in the sum of all measured phenolic compounds between FWF, DWF, or DHF; however, levels of most phenolics were slightly higher in the DHF, suggesting that homogenization also increases the extraction efficiency in dry flowers. Furthermore, a statistically significant interaction was found between the fresh and dry flowers and homogenization of the sample for most phenolic compounds, due to the uniquely high levels present in the FHF and the absence of an equally high increase in DHF . This trend can also be seen in the totals of each phenolic class , vertical vegetable tower as the FHF were significantly higher than all other sample types, with the exception of total flavan-3-ols in DHF .

The levels of IR were significantly higher in FHF, with a maximum concentration of 78.73 ± 4.84 mg g-1 . This is a significant difference as compared with the European and American subspecies, in which rutin is the predominant phenolic compound in flowers and at much lower concentrations. Levels of IR in European elder flower levels range from about 0.200 to 0.900 mg g-1 fresh weight, though higher levels were found in elder flower tea, ranging from 4.260 to 13.500 mg g -1 . This key difference in the flowers of the blue elderberry provides an opportunity to create unique products for consumers looking for high levels of bioactive phenolic compounds, as studies have shown that IR can induce apoptosis in cancer cells. The other flavonol glycosides found in the flowers include rutin, kaempferol-3-Orutinoside, and isorhamnetin-3-O-glucoside. Rutin ranged from 3.20 ± 0.395 µg g-1 in FWF to 10.01 ± 0.97 mg g-1 in FHF . The concentrations of rutin vary greatly across studies of European elder flowers. For example, levels are reported as 1.65490 ± 0.10951 mg g-1 fresh weight,6 1.8877 ± 0.2691 mg g-1 fresh weight, between 15.70 and 23.90 mg g-1 in dried elder flower tea which varied with cultivar,and 4.14443 ± 0.11469 mg g-1 in a traditional beverage called sabesa which is made from fresh flowers. Herein, kaempferol-3-O-rutinoside ranged from 1.24 ± 0.03 to 3.010 ± 0.37 mg g-1 dry weight, and isorhamnetin-3-O-glucoside ranged from 0.77 ± 0.05 to 1.42 ± 0.40 mg g-1 dry weight . Quercetin was the only flavonol aglycone identified in the flower extracts and was low relative to the flavonol glycosides.

Though this compound may be due to the degradation of a quercetin glycoside, quercetin aglycone has been measured in other elder flower studies, and our results are similar to those reported by Viapiana et al. .The flavan-3-ols monomers found in the flowers include -catechin and -epicatechin, highest in the FHF at 1.110 ± 0.30 and 1.24 ± 0.19 mg g-1 , respectively . Epicatechin, but not -catechin, had an interaction between the fresh and dried and homogenization of the sample, as it was significantly higher in FHF. Proanthocyanin B type was also tentatively identified via HPLC-MS/MS analysis in the flowers, and was present in relatively low quantities in all samples . A procyanidin trimer was identified in elder flowers extracts and beverages by Mikulic-Petkovsek et al. .Chlorogenic acid was identified as the main phenolic acid in the flowers of the blue elderberry, like the flowers of the American elderberry, whereas the predominant phenolic acid in the flowers of the European elderberry is neochlorogenic acid .Neochlorogenic acid and other caffeoylquinic acid isomers were also present in the elder flowers of S. nigra ssp. cerulea . Two isomers of 5-caffeoylquinic acid in addition to 3- and 4-caffeoylquinic acid have been identified in elder flower products. Evaluation of the phenolic content of elder plants grown in different locations and altitudes indicate, in general, that plant material from shrubs at higher altitudes had higher levels of hydroxycinnamic acids and flavonols. The authors postulated that the stress of harsher climates at higher altitudes may have led to the increase in hydroxycinnamic acids and flavonols to cope with the increase in UV radiation. They also reasoned that the high amounts of sun and cool nights may increase the metabolism of phenolic compounds.

The flowers in the present study experience hot, dry summers with cool breezes from the Sacramento-San Joaquin Delta at night, and these conditions may contribute to the unique phenolic profile in this flower. The average day/night temperatures for Davis, California while the flowers were growing were 24/7 °C in April 2021, 28/12 °C in May 2021, and 31/13 °C in June 2021, with less than 3 inches of rain during that time span. A phenolic compound unique to the blue elder flower was identified as 5-hydroxypyrogallol hexoside . This compound has also been identified in the berries of this subspecies grown in the same locations. The concentrations of 5-HPG ranged from 1.26 ± 0.20 mg g-1 in DWF to 2.00 ± 0.72 mg g-1 in FHF . Because no commercial standards exist for this compound, the tentative identification of this compound was determined by high resolution QTOF-MS/MS data. The mass spectrum shows the molecular ion [M-H]- at m/z 303.0728 and fragment ion showing the loss of the sugar molecule [M-hexose-H]- at m/z 141.0199. Although the biological properties of this unique phenolic compound have not yet been investigated, 5-HPG hexoside can serve as a marker for S. nigra ssp. cerulea, especially since it is present in relatively high levels in the flower and berry and is not identified in other elderberry subspecies. Elderflower tea is one of the most traditional and simplest ways that the flowers are used in the preparation of beverages. To make elder flower tea, the flowers are infused in hot water to extract the flavor and biologically active phenolic compounds from the flowers. Recommended steeping times can vary widely, however there are no studies investigating the impact of steep time on the extraction of phenolic compounds in the elder flower tea. To address this, the impact of time on the extraction of phenolic compounds from teas made from dried flowers was evaluated. The profile of phenolic compounds extracted in hot water infusions was similar to the profile obtained in ethanol/water extracts, however the concentrations were lower in the hot water extracts . Phenolic compounds were quantified at 5, 10, 15, and 20 minutes of steep time. Over time, the concentrations of total measured phenolic compounds increased 47% from five minutes . These results suggest that longer infusion times are beneficial for extracting the highest level of compounds. The overall levels of phenolic compounds in elder flower of S. nigra ssp. cerulea are comparable to the levels in the European flowers. However, vertical farm tower variability between studies due to post-harvest conditions, extraction solvent, and analytical method make it challenging to make direct comparisons. For example, in this study, FWF, DWF, and DHF all had about 50-60mg g -1 dry weight, while the FHF had ~120 mg g -1 dry weight. The unique composition of phenolic compounds in these flowers provides an opportunity to make different products for the market, especially nutraceutical or functional food products that take advantage of the high levels of IR. Based on our results, it would be best to use fresh flowers and blend or homogenize the flowers to extract the highest levels of phenolic compounds. Before analyzing samples, method parameters were evaluated to find the optimal equilibrium time, temperature, and the extraction time for headspace VOCs. The optimal parameters for flower samples were determined to be 40 °C, 20 min equilibration , and 30 min extraction with a SPME fiber. The optimized conditions for the tea samples were 40 °C, 30 min equilibration, and 30 min extraction. The profiles of volatile compounds were evaluated in fresh whole, dry ground, and teas made from whole fresh and dry flowers by HS-SPME/GC-MS, and relative levels of identified volatile compounds were calculated.

The fresh flowers were evaluated as whole flowers because homogenizing them can cause oxidation and create artifacts in the volatile headspace profiles. This led to higher variability in the relative peak areas of compounds, but it is believed to be truer to the real headspace VOC profile as compared to a homogenized fresh flower sample. Overall, 25 compounds in the fresh whole flowers, 44 compounds in the dry ground flowers, and 18 compounds in the tea preparations were identified. Table 1S contains the m/z ion of the base peak and the average match factor for each of the compounds identified in the headspace of samples. In the headspace of fresh flower, the most concentrated compounds were pentadecane > methyl eugenol > cis-3-hexenyl acetate > α-farnesene > and cis-3-hexenyl-α- methylbutyrate . The contribution of each compound’s odor to the overall aroma of these elder flowers cannot be determined from the concentration alone, as each compound has its own odor activity and threshold. However, it can be useful to know the characteristic odors of these compounds as a way to understand what comprised the general aroma. These compounds are described to have odors such as waxy; clove, spice; fresh, green, sweet, fruity, apple, pear, melon;wood, sweet; ; fruity, sweet, minty, fresh, and green apple, respectively. In addition to pentadecane, several straight chain hydrocarbons were also present, which may be released from the cuticle of the petal or peduncle of the flower. These include 1-pentadecene, heptadecane, 8- heptadecene and 6,9-heptadcadiene. Flowers also contain 4.6% methyl salicylate a compound with a sweet, minty odor80 that is frequently used as an analgesic in liniments to relieve pain. Methyl salicylate has been identified in several other studies on the volatile profile of elder flowers. The profile of headspace VOCs in elder flowers of S. nigra ssp. cerulea differ significantly from the European elder flowers as linalool oxides and other derivatives predominate in the European flowers, and are absent in the present study. Furthermore, the present study indicates a unique headspace VOC profile in the blue elder flowers because pentadecane and methyl eugenol have been identified as major contributors to the headspace VOC profile. Pentadecane has been identified at trace levels in some European elder flower extracts, however methyl eugenol has not been identified in European elder flowers. Methyl eugenol, which has a clove-like aroma19 , appears to be unique to the S. nigra ssp. cerulea elder flower, and could be a unique volatile marker for this subspecies. Tea made with fresh whole elder flowers presented a slightly different headspace VOC profile as compared to fresh flowers. Although methyl eugenol was still a prominent compound in the headspace , there were also two ketones in relatively high concentrations: 2,2,6-trimethyl-4H-1,3-dioxin-4-one and 4-methyl-2-heptanone . Aroma descriptors were not found for these compounds. In addition, two aldehydes including heptanal and nonanal were also present in the headspace of the tea but not the fresh flowers. Heptanal odor is described to be fresh, fatty, green, and herbal, whereas nonanal is described to have waxy, rose, orange peel or fatty notes. Methyl salicylate comprised only 1.27 ± 0.29% of the headspace volatiles in the tea made from fresh flowers. In the headspace of dry elder flowers, the most concentrated compounds were -3-hexen-1-ol > 1-penten-3-ol > 3-methyl-butanal > heptanal, > isocyanato-methane . In general, the dry flowers contain a wider range of volatiles than the fresh flowers, including more aldehydes, alcohols, alkanes, and other hydrocarbons. Other notable volatiles identified include methyl salicylate , dihydroedulan , which is a driver of typical elderberry aroma, and linalyl acetate which is the only linalool derivate identified any preparations of the elder flowers of S. nigra ssp. cerulea, unlike European elder flowers which typically have high concentrations of linalool derivatives. In tea prepared from dried flowers, the headspace aroma less complex than the dried flowers, but many of the aldehydes were still identified, including nonanal , heptanal , and hexanal .

The most significant impacts of the vineyard floor treatments were of the cover-crop treatments on soil parameters in the middles. Soil organic matter in cover-cropped middles was higher than in bare middles each year . Cover crops affected key soil nutrients in the middles; for instance, cover crops greatly reduced nitrate-nitrogen , and to a lesser extent, extractable phosphorus , which may be beneficial in reducing loss of these nutrients in runoff during winter storms, but which also may have reduced the phosphorus content in the vines. In addition, cover crops in the middles also significantly reduced soil boron , extractable sodium and pH , and increased chloride and zinc when compared to bare soil.Soil microbial biomass. Microbial biomass varied as a result of both the cover-crop and weed control treatments. In both the middles and vine rows, microbial biomass was higher in rye cover-crop plots compared to bare plots . These results confirm earlier observations by Ingels et al. that microbial biomass carbon was higher in cover-cropped middles compared to bare middles. In the vine rows, microbial biomass was greater in plots adjacent to rye cover-cropped plots compared to bare plots. The effect of cover crops grown in the middles on soil in the vine rows may be due to cover-crop roots or tops extending into the vine rows and their subsequent decomposition, providing a food source for soil microbes.

Microbial biomass varied between the weed treatments in the vine rows but not middles . In the vine rows, stackable planters microbial biomass was significantly higher in the cultivation plots compared to the pre-emergence weed control plots . The most likely explanation is the incorporation of greater amounts of weed-derived carbon into the surface soil of the cultivated plots. Mycorrhizae. AMF can benefit grapevines by improving the nutritional 35 30 25 20 15 10 5 0 Cultivation Post-emergence Pre-emergence herbicide herbicide Bare ground Merced rye Trios 102 status of the plant and producing a highly branched root system. We quantified AMF reproductive structures in grapevine roots to determine if the weed control treatments in the rows and/or cover-crop treatments in the middles had significant effects on mycorrhizal colonization from 2003 through 2005. Based on ANOVA, the effects of weed control on colonization were not consistent among covercrop treatments . Grapevines adjacent to ‘Merced’ rye had higher colonization compared to those adjacent to ‘Trios 102’ triticale or bare ground, in both the cultivation and pre-emergence treatments . In contrast, grapevines in the post emergence treatment had the lowest colonization when adjacent to ‘Merced’ rye. These findings were consistent in each study year, based on the absence of significant main or interactive effects of time . It is possible that low colonization of grapevines in the post-emergence-by-‘Merced’ rye treatment is associated with this treatment’s weed community. Indeed, weed species vary in their ability to host AMF , so their presence or absence may affect mycorrhizal colonization of grapevines. Indeed, reports on the influence of plant community composition on AMF suggest that plant diversity has a strong effect on AMF diversity , and this may affect the colonization of individual plant species. All yield, fruit quality and vine growth parameters varied by year, and this was the only significant effect for these parameters, with the exception of berry weight and titratable acidity . No differences in crop yield or fruit composition were observed from 2001 to 2005 due to weed control treatments . Cover-crop treatments also had no significant effect on yield or fruit composition, although in 2001 and 2004, there was a reduction in berry size in the ‘Trios 102’ triticale treatment. Weed control treatments also had no effect on vine growth , based on shoot counts and pruning weights taken at dormancy. Cover-crop treatments had no significant effect on vine growth when averaged over 5 years, although in 2001 and 2005 the ‘Trios 102’ triticale treatment significantly reduced pruning weights. The trend for lower pruning weights may be related to the greater decline in soil moisture in the middles where this cover crop was used. It appears that vine growth, yield and grape quality are more significantly affected by annual precipitation than by vineyard floor management practices. In low rainfall areas the choice of cover crop is critical because of its effect on available soil moisture. We observed that late-maturing ‘Trios 102’ used more soil moisture during the vine growing season; if irrigation water does not compensate for water used by the cover crop, reduced vine growth and yield losses may result. The clear benefits of cover crops were increased organic matter in the middles and reduced sediment loss. Microbial biomass was increased in cover-cropped middles and there were indications that this effect extended to under the vines. Although there were no negative impacts of weed control treatments on vine productivity, we observed increased compaction over time from the use of cultivation. This study indicated that the choice of weed control strategy and cover crop must be carefully considered to maximize the benefits and minimize negative impacts of the practices. The benefits of cover crops are concentrated in the middles,and future research should focus on evaluating practices that improve the quality of soil under the vines.Elderberry is a part of the Viburnaceae family and grows all over the world, including Europe, North America, and Asia. Due to the vast geographic and morphological variety within Sambucus, there have historically been many species within the genus. However, a reorganization by Bolli reclassified some of the most common species under Sambucus into subspecies of S. nigra. More recently, elderberry was moved out of the Adoxoaceae family, which had already been changed before when elderberry was taken out of the Caprifoliaceae family. These changes have impacted the three subspecies most of interest in this work: the European elderberry S. nigra ssp. nigra; the American elderberry S. nigra ssp. canadensis; and the blue elderberry S. nigra ssp. cerulea . However, due to wide acceptance of this naming scheme for the subspecies, it will be used through this work to align with the current naming, but previous works cited may use the former species names. Furthermore, some sources refer to the entire plant as an “elder”, while others refer to the plant as “elderberry”, which is also used to denote the fruit of the plant. In this work, “elderberry” is used to discuss the plant as well as the fruit. “Elderflower” is used to refer to the blossoms of the plant. European elderberry is the most well-studied and widely used subspecies of elderberry in the market. This subspecies grows throughout the European continent, including countries such as Slovenia, Portugal, and Austria. The fruit and flower have been studied for decades for their composition and bio-activity, and while elderberry and elder flower are not new ingredients to the market, they have garnered more attention in the last several years as consumers look for more natural remedies and supplements to support their health. This has been especially true during the COVID-19 pandemic, in which elderberry became a popular ingredient in immunity-supporting supplements. Thus, investigating other elderberry subspecies like the blue elderberry, the focus of future chapters, allows for farmers in the United States to capitalize on this demand, but more information is needed on this particular plant if it is going to be used in consumer products.There is a long, rich history of the use of different parts of the elderberry plant by many cultures. For example, the wood has been used for kindling and musical instruments. Indeed, stackable flower pots the name of the plant is derived from various ancient words related to instruments. The flowers and berries have been used in a variety of beverages, foods, and other herbal supplements. Folklore has many stories about the healing power of the elderberry and elder flower. The plant itself has been revered by many cultures, with a story about the “Elder Mother” living within the plant would protect those near the plant. It was even expected to ask the Elder Mother for the berries or flowers before taking them; without permission, she may seek revenge. The leaves, branches, flowers, and berries were believed to have protective powers for a home and the leaves were also used during burial rituals by some Celtic people. The personification and deep reverence for the elderberry show the importance of the plant through generations. Hippocrates and Pliny the Elder both wrote about elderberry and its medicinal properties. Tribes indigenous to North America used flowers and fruit for medicinal and beverages. Berries were also used as a natural dye for baskets and branches were used to make musical instruments. Elderberry is a perennial, deciduous plant native to many regions of the northern hemisphere. Elderberry plants are neither tree nor bush, as the plant sends new canes up each season, which without pruning, can lead to a large, shrub-like plant that can be several meters tall and wide. They prefer to grow in sunny, riparian climates with moist, well-drained soil, though subspecies in North America can be drought-tolerant. While pruning even down to the ground level of the elderberry can improve yield and accessibility for harvesting, there is a limitation on pruning of the blue elderberry in the Central Valley of California. Due to the threatened status of the Valley Elderberry Longhorn Beetle , which lives only in the elderberry, branches larger than one inch in protected areas should not be pruned or removed from a growing site. Elderberry shrubs typically produce small white flowers with five petals in the spring, though the elder flowers of the blue elderberry are a creamy yellow color. Small, dark blue-purple berries ripen in the summer in large clusters called umbels or cymes, though there are examples of subspecies that have some variation to these morphologies, such as the blue elderberry that has a white bloom on the berries, causing the berries to look blue, and S. racemosa, which are red. Variation can also occur within a subspecies due to growing conditions, such as soil type, precipitation, and temperatures, as well as a key differentiation tool: cultivars or genotypes. There are established cultivars or genotypes of the European subspecies , such as Sambu or Haschberg, as well as of the American subspecies , like Bob Gordon or Wylewood. Cultivars can have more consistent growing patterns, such as blooming or ripening all at once, and desired chemical compositions, such as increased anthocyanins, thus are more desirable to use in large scale growing of elderberry for commercial use. Blue elderberry does not have any established genotypes to date. If commercial interest in this subspecies continues to expand, effort should be made to develop cultivars with consistent quality and improved harvest ability, which is hampered right now due to flowers and berries ripening throughout a season, instead of a smaller window of time like the American and European subspecies. Indeed, starting this work can help increase the commercial interest viability of the blue elderberry. A primary driver in interest in the composition of elderberry and elder flower is for their potential health benefits. Several reviews have recently been published on this topic; thus, it will not be explored in depth here. European elderberry has been studied for its antioxidant, antimicrobial, anti-inflammatory, anticancer, immunomodulatory, and antidiabetic properties, as well as neuroprotection and cardiovascular protection in vitro and in vivo. These activities have been mainly attributed to the phenolic compounds like cyanidin 3-glucoside and cyanidin 3- sambubioside, but some other compounds have been shown to be bioactive as well, including terpenes, lectins, pectin, peptides, and malic acid. Using data from randomized, controlled clinical trials, a recent review found that elderberry could reduce symptoms from upper respiratory viral infections, providing support for the use of elderberry supplements by consumers to combat colds and flus without the use of antibiotic medicine. In a more unique application, elderberry and elder flower extracts have both shown to be effect in combatting gingival inflammation using a topical herbal patch and elder flower tea, respectively. A study of the mechanism of cyanidin 3-glucoside to treat against the influenza virus showed that elderberry extract had some inhibitory effect during the early stages of virus cycle with stronger impacts during post-infection.

Because of their limited protein-coding capacities, geminiviruses rely heavily on host cellular machinery and interact with an assortment of plant proteins and pathways to promote infection . Although three of these genes have been assigned putative functions in viral DNA replication and coat formation , their specific functions in pathogenesis are yet to be elucidated. Our results indicated that the developmental stage of the berries may not influence the expression of GRBaV viral genes and that host factors could play a more critical role in the establishment of successful infections. Red blotch symptoms in grape berries include abnormal chemical composition and asynchronous ripening in the clusters , both reflecting alterations in host metabolic homeostasis and developmental processes. Interestingly, we determined that GRBaV infections induced some processes associated with early fruit development in berries at late stages of ripening, strawberry gutter system while repressing pathways involved in fruit ripening . Abiotic stress responses to hypoxia and temperature were also suppressed by red blotch in grape berries.

These observations may imply that the virus, as a biotrophic pathogen, could redirect host metabolic processes to sustain higher energy demands due to viral replication and at the same time suppressing ripening related events and responses to stress , potentially counterproductive to viral infections. However, further evidence using infected berries from different grape cultivars and environmental conditions will be necessary to understand how both grapevine genotype and environment may influence the disease outcome. It is also important to consider that the effects of GRBaV infections on berries may not be comparable with those occurring in vegetative tissues; for instance, photosynthetic pathways are generally inhibited during viral infections of grape leaves . GRBaV infections restricted the biosynthesis and accumulation of phenylpropanoids and derivatives, which preferentially accumulate after véraison in red-skinned berries. These secondary metabolites function as antioxidants and phytoalexins to protect the berries against a variety of stresses, and are important contributors of berry quality parameters, such as color, flavor, and aroma . The inhibition of phenylpropanoid metabolism appears to be a hallmark of viral infections in red-skinned berries, as has previously been reported in leaf roll-affected berries . In particular, the anthocyanin biosynthetic pathway was greatly impaired as a result of leafroll and red blotch, correlating with the reduced coloration observed in GRBaVinfected berries from several red-skinned cultivars .

In this study, we demonstrated that GRBaV infections compromised the regulation of ripening by: suppressing specific ripening events; altering the expression patterns of transcription factors that control the transition from the growth to ripening phases and the activation of ripening pathways ; and causing hormonal imbalances. Most of the alterations in the ripening regulatory networks occurred in infected berries at véraison, the same developmental time when red blotch symptoms initiated, suggesting a link between mechanisms involved in the control of grapevine responses to viruses and berry development. Similar observations were made in leaf roll-affected berries, which presented lower expression levels of MYB transcription factors at late stages of ripening . Plant hormones modulate ripening processes and stress responses in grape berries. Red blotch had a profound impact on ABA, ethylene, and auxin pathways. ABA is considered the triggering signal of berry ripening, since its accumulation coincides with véraison and ABA responsive transcription factors have been implicated in the activation of ripening processes . Particularly, the induction of anthocyanin biosynthesis in a variety of grape cultivars has been linked to ABA-mediated signaling pathways . Our results indicate that alterations of ripening events in the GRBaV-infected berries, including the reduced anthocyanin content at late stages of ripening, could be a direct result of lower ABA levels at véraison. ABA has been shown to stimulate ethylene-mediated pathways in berries, and together both hormones appear to regulate the progression of ripening . GRBaV infections also affected genes involved in ethylene biosynthesis and responses , which could further account for the antagonistic effect of red blotch on ripening events and immune responses. In contrast, red blotch promoted auxin-mediated pathways, known to suppress berry ripening. Auxins play crucial roles in the early development of grape berries and are known to inhibit ripening processes by delaying ABA-triggered processes .

The mechanisms by which grapevine viruses cause disease in ripening berries need to be investigated. GRBaV may actively interfere with the regulation of berry ripening by hijacking the plant post-transcriptional control. We identified elements of the host post-transcriptional machinery that were misregulated in GRBaV-infected berries. Post-transcriptional regulation intersects plant immune responses, developmental transitions, and hormone signaling . Plant viruses, in particular geminiviruses, encode multiple silencing suppressors that interfere with host siRNA production and alter plant DNA methylation and miRNA pathways, causing developmental defects . A previous study on leaf roll indicated that the viral infection caused up-regulation of Dicer-like genes in ripening berries . Therefore, the relationship between the viral-induced repression of host RNA silencing processes and the post-transcriptional regulation of ripening should be addressed from the perspective of compatible grapevine– virus interactions.Shriveled berries on ripening clusters are not uncommon in California vineyards. They usually occur in only a small proportion of a vineyard’s fruit , but in particular vineyards and years, shriveling can affect more than half of the crop . Most shrivel disorders make the fruit less desirable for winemaking, with subsequent yield and production losses. Before taking steps to reduce the incidence of fruit shriveling in vineyards, it is necessary to differentiate between shrivel disorders. We describe four common causes of fruit shriveling and detail compositional differences between normally developing fruit and that affected by sugar accumulation disorder .Fruit exposed to direct sunlight for all or part of the day, especially in the heat of the afternoon, can be damaged by sunburn, which may be caused by high temperature, ultraviolet radiation or a combination of the two . The physical appearance of sunburned fruit depends on the grape variety and stage of development — white grapes and red grapes exposed before pigment accumulation begins develop brown discoloration, which varies depending on severity.Veraison and early postveraison red varieties with sunburn often exhibit poor color development, and may remain pink for the remainder of the season. Post-veraison sunburn leads to fruit with less color and a shiny appearance. Sunburned berries often crack, presumably due to damaged epidermal tissues. Extreme sunburn leads to complete berry desiccation and the formation of raisins in both red and white varieties. Sunburn only affects berries that are directly exposed to sunlight. The non-exposed side of a sunburned cluster often develops normally. If a cluster is fully exposed to the sun on both sides, hydroponic nft gully or if the rach is itself is damaged, then the cluster may be completely affected. Obvious signs of sunburn may only occur on the exposed portions of individual berries. Sunburn can be avoided by reducing the fruit’s exposure to direct sunlight, especially in the afternoon. While leaves are removed in the fruit zone in many growing regions to increase cluster exposure to indirect light, in north-south row orientations leaves are removed on the east side of the canopy to reduce direct exposure in the afternoon and the probability of sunburn. This practice does not completely eliminate the risk of sunburn, however, because morning sun can also cause damage.Natural dehydration is another type of shrivel that may affect berries late in ripening but prior to commercial harvest. These berries appear similar to fruit with bunchstem necrosis, but the rachis look green and healthy. For this type of shrivel, which is especially pronounced in Syrah , berries lose weight due to water loss, and sugars are concentrated . Both increased transpiration and decreased phloem infl ux have been suggested as causes for late-season dehydration, but recent studies provide evidence that several varieties of grape berries remain hydraulically connected to the parent vine and therefore may lose water back to the parent plant late in ripening as well as to dry, ambient air .Clusters affected by bunch stem necrosis are identifi ed by necrotic rachis tissue, with shriveled berries distal to the necrotic tissue. The visible symptoms of bunch stem necrosis begin as small black spots on pedicels , and progress to the lateral stem structure and rachis .

Usually, necrosis symptoms are not noted until the rachis is affected. Bunchstem necrosis may affect an entire cluster as well as the wings and tips of otherwise healthy clusters . It can occur in many varieties, but is especially prevalent in Cabernet Sauvignon on California’s North Coast. It has been described in the literature in many different countries, with descriptive terms that include waterberry , bunchstem dieback , shanking , stiellähme , palo negro , desséchement de la rafl e and dessichimiento della rachide . No specifi c cause of bunchstem necrosis has been identified, despite many years of research. In some cases, varietal differences in susceptibility have been correlated to xylem structure, specifically a reduction on the area of xylem distal to branch points in the peduncle . The incidence of bunchstem necrosis has also been correlated to various concentrations or ratios of mineral nutrients, including magnesium, calcium, potassium and nitrogen . Work in Chile and Australia has shown that the amino acid metabolite putrescine is associated with bunchstem necrosis. More light in the canopy can also reduce bunchstem necrosis . Bunchstem necrosis can appear very early in fruit development or after veraison. The terms “inflorescence necrosis” and “early bunchstem necrosis” have been used to describe bunchstem necrosis around bloom . The composition of such fruit varies depending on when during fruit development the rachis becomes necrotic. Presumably, the necrosis prevents both sugar and water transport to the berry. Hence, if the rachis becomes necrotic early in the ripening period before the berry has accumulated much sugar, fruit will have low Brix . On the other hand, if the rachis becomes necrotic after the berries have accumulated appreciable sugar, subsequent shriveling can concentrate the sugars. Bunchstem necrosis in Cabernet Sauvignon on the North Coast is usually the latter type. Fruit with bunchstem necrosis can have a Brix as high as 42 .Another disorder with symptoms that occur during the ripening period has been called “berry shrivel”; we recently proposed that it be called “sugar accumulation disorder” . This disorder was first described in Emperor table grapes from California’s San Joaquin Valley and is characterized by poor coloration and low sugar accumulation. Sugar accumulation disorder has been found in a number of varieties and is present in many areas of California. In general, it affects only a small proportion of clusters in a vineyard, though in certain years and vineyards up to 50% of the fruit can be affected. Regardless of the variety or location, fruit affected by sugar accumulation disorder has lower pH, berry weight and Brix compared with normally developing fruit . When multiple rachises and fruit with sugar accumulation disorder were tested for minerals, the only consistent difference from normally developing fruit or rachises was increased calcium in the rachis tissue . To test the hypothesis that fruit exhibiting sugar accumulation disorder may have altered nitrogen metabolism, we measured the amounts of nitrogenous compounds at harvest in fruit with the disorder compared to normally developing fruit. The vines were located at the UC Oakville Experimental Vineyard in the Napa Valley. Samples were taken at harvest on Oct. 21, 2005. Berries with sugar accumulation disorder came from clusters on six vines that historically exhibited the disorder and showed symptoms in 2005 . Normally developing berries came from clusters on three nearby vines that had no history of sugar accumulation disorder and did not display symptoms at harvest. Two berries were sampled from each cluster and eight to 10 berries were pooled to ensure enough material for analysis. Berries were peeled, their seeds removed and flesh homogenized. One milliliter of the homogenate was used for the analysis of nitrogenous compounds. Individual amino acids in three samples of berries with sugar accumulation disorder and normally developing berries were measured at the UC Davis Molecular Structure Facility . Briefly, juice samples were acidified with sulfosalicylic acid to precipitate any intact protein before analysis. Free amino acids were separated using a Li-citrate buffer system with ion exchange chromatography on a Hitachi L-8900 amino acid analyzer. Amino acids were quantified by a postcolumn ninhydrin-reaction detection system. Amino acid concentrations were quantified from peak areas using standard curves. Data was analyzed by ANOVA .

The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites per se, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility. The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, hydroponic channel plant health and fruit production, as well as for those concerned with C storage.

The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.Grapes are the most valuable fruit crop in the United States, valued at over $6.5 billion annually , but climate change is projected to reduce grape production and quality . Climate affects grape quality by impacting the concentration of sugars, organic acids, and secondary compounds . The climatic conditions producing the highest quality wine cause the berries to reach optimal ratios between sugar and acid concentrations and maximum concentrations of pigment, aroma, and flavor compounds simultaneously . Hot temperatures accelerate sugar accumulation, forcing growers to harvest earlier, before berries reach optimal flavor development, to avoid the high alcohol content and insipid wine flavor from excessive sugar to acid ratios . Harvest dates have shifted earlier historically, and climate models predict further acceleration of ripening .

Growers can partly compensate through management practices, such as trimming canopies or using shade clothes to reduce the ratio of sugar supply to demand , though these practices are costly and increasingly ineffective in the face of climate change . Planting existing cultivars or developing new cultivars with slower sugar accumulation are promising alternative strategies to mitigate these climate change impacts, but these efforts have been hindered by uncertainty around the plant traits controlling sugar accumulation . Grape cultivars vary in berry maturation and sugar accumulation rates, and in their response to abiotic stress, but the main anatomical and physiological mechanisms driving these differences remain unknown . Multiple physiological processes influence berry sugar accumulation and its responses to climate, including photosynthesis, long-distance sugar transport, and local transport and metabolism in the berries . However, the relative importance of these factors in regulating sugar concentrations and fruit growth is debated . Photosynthetic responses to heat and water stress could impact cultivar differences in accumulation rates by affecting the sugar supply for ripening . Further, sugar is transported from the photosynthesizing leaves to the berries through the sugar-conducting vascular tissue – the phloem. At the onset of ripening , the berries significantly accelerate sugar accumulation by initiating active sugar unloading from the phloem, making the phloem the primary pathway for water and resource influx into the berries . The importance of phloem transport to ripening suggests that phloem traits could be important drivers of cultivar differences in sugar accumulation, and that modifying phloem traits to slow sugar accumulation under hot conditions could help mitigate the impacts of climate change on wine quality.

However, the main traits controlling sugar accumulation in grape remain unclear . The rate of phloem transport is determined by both the hydraulic resistance to the flow of sugar sap, and the activity and kinetics of water and sugar transporters in the sources, sinks, and along the transport pathway . Modeling studies suggest that increasing the hydraulic resistance of the phloem reduces sugar export to the sinks . Therefore, selecting grape cultivars with lower total phloem conductance could decelerate sugar accumulation and improve the synchronization of sugar accumulation with flavor development under hotter conditions. However, a higher hydraulic resistance can make the phloem more susceptible to declines or even complete failures in transport under severe water stress . Thus, we expect cultivars that produce high-quality wine in hot, dry conditions to exhibit phloem hydraulic resistances that slow berry sugar accumulation while avoiding phloem failure. The phloem transport pathway is composed of individual sugar-conducting cells with porous end walls stacked to form conduits . The anatomy of the transport pathway, including the total cross-sectional area of sieve tubes in plant organs, lumen area of individual sieve tubes, and porosity of the sieve plates, significantly impacts pathway resistance . Plants with a greater cross-sectional area dedicated to phloem , sieve tubes with wider lumen areas , and larger and more abundant pores in the sieve plates are expected to have a lower hydraulic resistance . Total phloem cross-sectional area in the shoots has been found to vary between several grape cultivars , and a greater cross-sectional phloem area has been linked to faster sugar accumulation in the fruit in other crop species . However, the variation of phloem structural traits across cultivars adapted to a diverse range of climatic conditions and the relationship of these traits to sugar accumulation is largely unknown for grapevines. Establishing these anatomical links could allow breeders to modify sugar accumulation by selecting for phloem traits, instead of management practices that can negatively impact the fruit zone environment or yield . In this study, hydroponic dutch buckets we used a common garden experiment to evaluate the links between phloem anatomy and sugar accumulation across 18 winegrape cultivars typically grown in climatically diverse grapegrowing regions. We assessed phloem and xylem vascular anatomy in leaf petioles and midveins and berry pedicels, to capture hydraulic resistance along the long-distance transport pathway. We also measured maximum berry sugar accumulation rates in the post-veraison ripening period to capture the greatest capacity for sugar transport . We predicted that traits that reduce hydraulic resistance, including larger total cross-sectional phloem areas, larger mean lumen areas for individual sieve tubes, and more porous sieve plates would increase maximum sugar accumulation rates. We also predicted that cultivars typically grown in hotter wine regions would havetraits that increase hydraulic resistance, as an adaptation to increase wine quality by reducing the rate of sugar accumulation. In addition, we measured photosynthesis and vine water stress to compare the impacts of phloem anatomy, vine carbon supply, and vine water status on sugar accumulation rates. Overall, our goals were to determine the most influential traits for sugar accumulation in grape berries and evaluate the role of phloem anatomy in adapting grape cultivars to a wide range of different climates.Leaves and berries were sampled to measure petiole, midvein, and pedicel anatomy in the morning on three days at the end of the growing season . Two berries and leaves per vine were excised with a razor blade.

Leaf position was standardized as the 6th leaf from the shoot apex, to capture the most photosynthetically active leaves. Two leaf and one berry sample per vine were then prepared for light microscopy, and the other berry sample was prepared for scanning electron microscopy. For light microscopy, a 1-cm segment of leaf petiole and lamina and the entire pedicel of the berry were immediately excised and placed into a vial of chilled Formalin-Acetic Acid . For scanning electron microscopy, pedicels were immediately flash-frozen and immersed in liquid nitrogen for 1-min and placed into a chilled micro-centrifuge tube of 100% ethanol, then the tube was immersed in liquid nitrogen until the ethanol congealed . Samples were then immediately placed on ice and stored in a -20°C freezer for at least 24 hours before further processing.After 7 days in FAA, the light microscopy samples were soaked in 50% ethanol for 5 mins and then stored in 70% ethanol in preparation for paraffin embedding. Samples were first infiltrated with paraffin by using an Autotechnicon Tissue Processor to treat samples with the following sequence of solutions: 70%, 85%, 95%, 100% ethanol, 1 ethanol:1 toluene, 100% toluene , and paraffin wax , each for 1 hour. The infiltrated samples were then embedded into paraffin blocks with a Leica Histo-Embedder , and allowed to cool. A rotary microtome was then used to make 7μm-thick cross-sections for leaf laminas, petioles, and berry pedicels. Pedicel cross-sections were sampled from the receptacle and petiole and midvein cross-sections were sampled near the interface of the lamina and petiole. After the cross-sections were imaged, pedicels for four cultivars were remelted from their wax molds, oriented longitudinally and sectioned again at 7μm to obtain sieve element lengths. Sections were stained using a 1% aniline blue and 1% safranin solution following a modified staining procedure . Sections were then viewed under bright field or florescence microscopy using a Leica DM4000B microscope and a DFC7000T digital camera . Each pedicel , midvein , and petiole section was then measured for total phloem and xylem cross-sectional area using ImageJ software, by manually selecting relevant tissue areas. Vascular tissue in longitudinal sections and cross sections was identified by cell size and/or stain color. Safranin stained the secondary cell walls of the xylem red and phloem cell walls were stained blue by aniline blue. The phloem area measurements included sieve tubes and phloem fibers and parenchyma , and xylem area measurements included xylem vessels, fibers, and parenchyma. Xylem and phloem rays greater than 4 cell layers thick were excluded. The pedicel electron microscopy samples were processed following Mullendore . Briefly, samples were thawed at room temperature, washed in DI water, and cut into 1 mm cross sections with a fresh double-sided razor blade. Sections were then transferred to 1.5 ml of 0.15% Proteinase K solution and mixed at 55°C and 300 RPM rotation for 14 days with an Eppendorf Thermomixer . Samples were then washed in DI water and placed into an 0.1% amylase solution for 24 hours at 50°C. Samples were then washed in DI water again, lyophilized overnight, mounted on aluminum stubs, and viewed under a Field Emission Scanning Electron Microscope . Sieve plates were viewed under low vacuum , 20- KV of accelerating voltage and a spot size of 2.5.Phloem anatomy was a stronger predictor of maximum sugar accumulation rates than vine carbon gain or water stress. Maximum sugar accumulation rates were not significantly correlated with photosynthesis or midday leaf water potentials . Including photosynthesis and midday leaf water potential as additional predictors also did not substantively improve the relationships between maximum sugar accumulation rates and petiole or pedicel cross-sectional phloem areas. Akaike Information Criterion corrected for small sample size values were higher for the larger models than the univariate models predicting maximum accumulation rates from petiole or pedicel phloem area alone, indicating that accounting for vine carbon gain and water stress did not improve predictive capacity for sugar accumulation . In addition, only one correlation was found between phloem petiole area and minimum mid-day water potential , while other average photosynthesis and water potential variables did not correlate with the phloem anatomical parameters. Finally, a previous dataset measuring leaf area for each cultivar did not find any significant correlations with °Brix accumulation, or other parameters measured .

The models all use community fixed effects and, for simplicity, have farm size entering linearly.The estimated coefficients from models 1 – 5 are largely consistent. They indicate a strong inverse relationship between farm size and frontier TFP and that the frontier is increasing over time, reflecting positive technical change. The coefficients on inputs are positive and stable across specifications, with family labor and purchased intermediate inputs being significant. The variance of the inefficiency term 2 is roughly double the size of the variance of the noise 2 in all models, and lambda – the ratio of the two variances – indicates that estimation of a stochastic frontier is appropriate with the MxFLS data. The models indicate an inverse relationship between farm size and productivity at the technological frontier of the same order of magnitude as the farm size-TFP relationship estimated in the preceding analysis of the average production function. The coefficients on survey year dummies in Table 2.12 are all positive and significant, u plangting gutter indicating that the frontier is increasing over time. Thus, in contrast to the results from the average production function analysis where evidence of declining average TFP over time was found, here we find evidence of positive technical change at the frontier.

The interaction between farm size and the survey year dummies in model 5 identifies a positive and significant relationship between farm size and technical change, suggesting that technical change has been biased towards larger farms and that the inverse relationship along the frontier became less steep over time.Models 1 through 4 show that, while the variance of the inefficiency distribution increased over time, there is no relationship between farm size and inefficiency. The inclusion of interactions between farm size and survey year dummy variables in model 5, however, reveals a more nuanced dynamic relationship between farm size and technical inefficiency. Larger farms were indeed more efficient than smaller farms in 2002 but inefficiency is increasing faster for larger farms. These differential changes in inefficiency across the farm size distribution have caused the farm size – inefficiency relationship to disappear in the latter waves of the MxFLS.20 Model 5 reveals that rising technical inefficiency has accompanied technological change, suggesting that the majority of farms have been unable to keep up with the TFP growth of the most productive farms. This is particularly true for larger farms, who have experienced faster growth in both frontier productivity and technical inefficiency. Having secondary or college education reduces the variance of the one-sided inefficiency term when education is included in the inefficiency equation. When education of the household head is included in the frontier specifications but not in the explanation of inefficiency , having secondary education or a college education is positively associated with higher levels of productivity among frontier producers.

When education is included in both the frontier and inefficiency equations , almost none of the education dummies are significant as the model appears to struggle to identify the separate relationships with education. In models not shown here, we estimate a stochastic frontier including the household controls from Table 2.8 as explanatory variables of the inefficiency term. In addition to educational attainment of the household head, technical inefficiency is lower among Procampo participants and higher among farms practicing monocropping. When interacted with farm size, none of the interaction terms are statistically significant, suggesting that they do not fundamentally change the relationships observed in Table 2.12. The analysis of Mexican data reveals an inverse and time-invariant relationship between farm size and TFP. Underlying this IR is a negative relationship between farm size and frontier productivity that has diminished over time and a positive relationship between farm size and technical efficiency that disappeared over the sample period. This evidence suggests that, in the wake of NAFTA era reforms, the IR is weakening for the most productive farms along the production frontier but that this change is not widespread. Although frontier productivity is increasing most rapidly for larger farms, the higher growth of inefficiency for large farms leaves the farm size – TFP relationship unchanged over the period.

The evolving relationships between farm size and frontier productivity and technical efficiency cast doubt on the ability to exploit the existing inverse relationship between farm size and TFP to generate productivity gains. These results are complemented by previous work on the farm size – productivity relationship in Brazil. Whereas the Brazilian experience suggests a dynamic farm size –TFP relationship, with an inverse relationship in traditional agriculture becoming flat and potentially positive with modernization, we observe no such dynamics in the Mexican sample. The relationship observed in the MxFLS is time invariant and persistently negative, contrasting with the emerging U-shaped relationship observed in the modernizing regions of Brazil. It is quite similar, however, to the more traditional agricultural regions in Brazil that display a persistent inverse relationship between farm size and TFP. The lack of corporate-run commercial farms is one limitation of using the MxFLS data, inhibiting analysis of the farm size-productivity relationship across all sectors of Mexican agriculture. This is especially true in light of findings that, in Brazil, larger commercial farms exhibit distinct advantages in achieving productivity growth . The frontier analysis using MxFLS data finds that technical change has been biased towards larger farms, weakening the farm size – productivity relationship at the frontier. This indicates that if inefficiency had not increased, the average inverse relationship between farm size and productivity would have weakened with modernization of the agricultural sector. This analysis indicates the potential for larger farms to be the key drivers of future productivity growth in Mexico. Policies geared towards smaller family farms may not have large returns in terms of increasing overall agricultural productivity, but they are likely very important for poverty reduction. Even if small farms generate an increasingly smaller share of agricultural output, they are likely here to stay because of their roles in generating livelihoods for rural households. Increasing their productivity remains an important component of facilitating poverty reduction in rural areas. These findings are largely consistent with earlier empirical work by Kagin et al. , who estimate both an average production function and a stochastic production frontier using a different panel of Mexican family farms. They find that both technical change and technical inefficiency increased over time and, as with the current analysis, planting gutter their fixed effects estimates show inverse relationships between farm size and both TFP and frontier productivity. Similarly, they find that smaller farms are more efficient than larger farms. In addition to highlighting the non-linearity in the farm size – TFP relationship, we provide evidence of a more nuanced and dynamic relationship between farm size and technical inefficiency and between farm size and productivity at the frontier. Larger farms have both more rapidly growing frontier productivity and technical inefficiency than their smaller counterparts, and these considerations are important for effective policy. We find evidence of declining average TFP over the period of analysis for the MxFLS sample of family farms. This appears to be driven by increasing average technical inefficiency offsetting the positive technical change and expansion of the productivity frontier. The largest farms in the sample and their relatively rapidly growing technical inefficiency are an important factor here, indicating a growing advantage for some large farms in harnessing more modern agricultural practices that has not been widespread enough to translate into sector-wide average TFP growth. Policies enabling broader inclusion in the benefits from technical change would both increase average TFP and likely further diminish the IR. Whereas policies promoting technical change are more relevant for smaller farms, policies improving technical efficiency, such as extension services, are exceptionally important for larger farms.

The growing technical inefficiency observed in Mexico indicates the potential for policies designed to promote and support the adoption and efficient use of best practices to achieve gains in agricultural productivity. The finding of declining average TFP over time is a curious result, running counter to both the body of long-run country-level analyses and the micro-level analysis of Kagin et al. over similar time periods. One important caveat is the MxFLS sample does not include corporate run commercial farms as do national-level studies such as an agricultural census. To the extent that such farms have more effectively harnessed the gains from technological change, as with larger family farms on the frontier, the potentially heightened productivity of such large farms is not included in the current evaluation of the farm size – TFP relationship in Mexican agriculture or growth in average TFP over time. This has important policy implications for the development impacts of agriculture productivity gains – if these gains are experienced primarily by corporate-run commercial farms and not by family-run farms, the potential impacts on poverty and broader rural economic development will not be fully realized. Productivity gains for smaller family farms not only reduce poverty directly but are also likely to contribute more to local development because of how they interact with the local economy. To be most effective, policy directed at spurring development and poverty reduction through agricultural productivity gains should be inclusive of smaller family farms. The lack of commercial farms does not, however, reconcile this finding with that of Kagin et al. , who find rising average TFP over a similar period in a different sample of rural households. One difference is the MxFLS includes more larger family farms, and these farms are experiencing the most rapid increase in technical inefficiency. The inclusion of more large family farms may be the source of this result. One possible explanation of the finding of declining average TFP over the first decade of the 21st century is that the productivity of Mexican family farms has declined in the wake of the NAFTA era reforms. This interpretation is consistent with claims that NAFTA era reforms were insufficient for generating positive change in Mexico’s agricultural sector, and that these reforms may have been detrimental to some segments of Mexican agriculture. Participation in Procampo and increased education are found to be positively correlated with the agricultural productivity of Mexican family farms, whereas the practices of monocropping and operating as a subsistence farm are found to be negatively correlated with TFP. We are tentative in drawing stronger conclusions about the causal impact of these variables, as they are likely endogenous. However, the frontier analysis suggests how these controls relate to productivity. Education appears to increase the efficiency with which inputs are used on family farms, and monocropping is found to be an inefficient use of inputs. In this light, farmer education – particularly in methods such as intercropping – is expected to increase technical efficiency on family farms. Procampois primarily an income support program it is unclear how participation would affect agricultural productivity. On the one hand, participation may relax income constraints and allow for adopting more productive methods because payments are distributed prior to planting season. This would suggest an emphasis on improving access to credit to improve the efficiency of Mexico’s family farms. On the other hand, the historical production requirements of Procampo participation may mean that participants are simply more experienced producers. Policies to ensure that farms have the necessary documentation could potentially help provide farms with the opportunity to keep abreast of technical change, as documented property rights are an important condition for accessing credit and thus facilitating adoption. This is especially true for ejido farms transitioning into participation with private credit and land markets. Nevertheless, we find no correlation here between agricultural TFP and property right documentation, access to credit, or ejido status, as we would have expected. Working with a sample of family farms from the Mexican Family Life Survey , we document a persistent inverse relationship between farm size and land productivity over the period 2002 to 2009. Similarly, when estimating an average production function we find a time-invariant inverse relationship between farm size andTFP, driven by the relatively high productivity of the smallest farms relative to those in the middle, and relatively low productivity of the largest farms. This is complemented by a stochastic frontier analysis, allowing for estimation of the relationship between farm size and frontier productivity and between farm size and technical inefficiency.

Plant morphology is more than an attribute affecting plant organization, it is also dynamic. Developmentally, morphology reveals itself over the lifetime of a plant through varying rates of cell division, cell expansion, and anisotropic growth . Response to changes in environmental conditions further modulate the above mentioned parameters. Development is genetically programmed and driven by biochemical processes that are responsible for physical forces that change the observed patterning and growth of organs . In addition, external physical forces affect plant development, such as heterogeneous soil densities altering root growth or flows of air, water, or gravity modulating the bending of branches and leaves . Inherited modifications of development over generations results in the evolution of plant morphology . Development and evolution set the constraints for how the morphology of a plant arises, regardless of whether in a systematic, ecological, physiological, or genetic context . In 1790, Johann Wolfgang von Goethe pioneered a perspective that transformed the way mathematicians think about plant morphology: the idea that the essence of plant morphology is an underlying repetitive process of transformation .

The modern challenge that Goethe’s paradigm presents is to quantitatively describe transformations resulting from differences in the underlying genetic, developmental, dutch bucket hydroponic and environmental cues. From a mathematical perspective, the challenge is how to define shape descriptors to compare plant morphology with topological and geometrical techniques and how to integrate these shape descriptors into simulations of plant development.Several areas of mathematics can be used to extract quantitative measures of plant shape and morphology. One intuitive representation of the plant form relies on the use of skeletal descriptors that reduce the branching morphology of plants to a set of intersecting lines or curve segments, constituting a mathematical graph. These skeleton-based mathematical graphs can be derived from manual measurement or imaging data . Such skeletal descriptions can be used to derive quantitative measurements of lengths, diameters, and angles in tree crowns and roots, at a single time point or over time to capture growth dynamics . Having a skeletal description in place allows the definition of orders, in a biological and mathematical sense, to enable morphological analysis from a topological perspective .

Topological analyses can be used to compare shape characteristics independently of events that transform plant shape geometrically, providing a framework by which plant morphology can be modeled. The relationships between orders, such as degree of self similarity or self-nestedness are used to quantitatively summarize patterns of plant morphology. Persistent homology , an extension of Morse theory , transforms a given plant shape gradually to define self-similarity and morphological properties on the basis of topological event statistics. In the example in Figure 2B, topological events are represented by the geodesic distance at which branches are “born” and “die” along the length of the structure. In the 1980s, David Kendall defined an elegant statistical framework to compare shapes . His idea was to compare the outline of shapes in a transformation-invariant fashion. This concept infused rapidly as morphometrics into biology and is increasingly carried out using machine vision techniques . Kendall’s idea inspired the development of methods such as elliptical Fourier descriptors and new trends employing the Laplace Beltrami operator , both relying on the spectral decompositions of shapes . Beyond the organ level, such morphometric descriptors were used to analyze cellular expansion rates of rapidly deforming primordia into mature organ morphologies . From a geometric perspective, developmental processes construct surfaces in a three-dimensional space. Yet, the embedding of developing plant morphologies into a three dimensional space imposes constraints on plant forms. Awareness of such constraints has led to new interpretations of plant morphology that might provide avenues to explain symmetry and asymmetry in plant organs or the occurrence of plasticity as a morphological response to environmental changes .

Computer simulations use principles from graph theory, such as graph rewriting, to model plant morphology over developmental time by successively augmenting a graph with vertices and edges as plant development unfolds. These rules unravel the differences between observed plant morphologies across plant species and are capable of modeling fractal descriptions that reflect the repetitive and modular appearance of branching structures . Recent developments in functional-structural modeling abstract the genetic mechanisms driving the developmental program of tree crown morphology into a computational framework . Similarly, functional-structural modeling techniques are utilized in root biology to simulate the efficiency of nutrient and water uptake following developmental programs . Alan Turing, a pioneering figure in 20th-century science, had a longstanding interest in phyllotactic patterns. Turing’s approach to the problem was twofold: first, a detailed geometrical analysis of the patterns , and second, an application of his theory of morphogenesis through local activation and long range inhibition , which defined the first reaction diffusion system for morphological modeling. Combining physical experiments with computer simulations, Douady and Coudert subsequently modeled a diffusible chemical signal produced by a developing primordium that would inhibit the initiation of nearby primordia, successfully recapitulating known phyllotactic patterns in the shoot apical meristem , the number of floral organs , the regular spacing of root hairs , and the establishment of specific vascular patterns .A true synthesis of plant morphology, which comprehensively models observed biological phenomena and incorporates a mathematical perspective, remains elusive. In this section, we highlight current focuses in the study of plant morphology, including the technical limits of acquiring morphological data, phenotype prediction, responses of plants to the environment, models across biological scales, and the integration of complex phenomena, such as fluid dynamics, into plant morphological models. There are several technological limits to acquiring plant morphological data that must be overcome to move this field forward. One such limitation is the acquisition of quantitative plant images. Many acquisition systems do not provide morphological data with measurable units. Approaches that rely on the reflection of waves from the plant surface can provide quantitative measurements for morphological analyses. Time of flight scanners, dutch buckets system such as terrestrial laser scanning, overcome unitless measurement systems by recording the round-trip time of hundreds of thousands of laser beams sent at different angles from the scanner to the first plant surface within the line of sight . Leveraging the speed of light allows calculation of the distance between a point on the plant surface and the laser scanner. Laser scanning and the complementary, yet unitless, approach of stereovision both produce surface samples or point clouds as output. However, both approaches face algorithmic challenges encountered when plant parts occlude each other, since both rely on the reflection of waves from the plant surface . Radar provides another non-invasive technique to study individual tree and forest structures over wide areas. Radar pulses can either penetrate or reflect from foliage, depending on the selected wavelength . Although more compact and agile systems are being developed for precision forestry above- and below ground , their resolution is too low to acquire the detail in morphology needed to apply hierarchy or similarity oriented mathematical analysis strategies. Image acquisition that resolves occlusions by penetrating plant tissue is possible with X-ray and magnetic resonance imaging . While both technologies resolve occlusions and can even penetrate soil, their limitation is the requirement of a closed imaging volume.

Thus, although useful for a wide array of purposes, MRI and X-ray are potentially destructive if applied to mature plant organs such as roots in the field or tree crowns that are larger than the imaging volume . Interior plant anatomy can be imaged destructively using confocal microscopy and laser ablation or nano- or micro-CT tomography techniques, that are limited to small pot volumes, to investigate the first days of plant growth. One of the outstanding challenges in plant biology is to link the inheritance and activity of genes with observed phenotypes. This is particularly challenging for the study of plant morphology, as both the genetic landscape and morphospaces are complex: modeling each of these phenomena alone is difficult, let alone trying to model morphology as a result of genetic phenomena . Although classic examples exist in which plant morphology is radically altered by the effects of a few genes , many morphological traits have a polygenic basis . Quantitative trait locus analyses can identify the polygenic basis for morphological traits that span scales from the cellular to the whole organ level. At the cellular level, root cortex cell number , the cellular basis of carpel size , and epidermal cell area and number have been analyzed. The genetic basis of cellular morphology ultimately affects organ morphology, and quantitative genetic bases for fruit shape , root morphology , shoot apical meristem shape , leaf shape , and tree branching have been described. Natural variation in cell, tissue, or organ morphology ultimately impacts plant physiology, and vice versa. For example, formation of root cortical aerenchyma was linked to better plant growth under conditions of suboptimal availability of water and nutrients , possibly because aerenchyma reduces the metabolic costs of soil exploration. Maize genotypes with greater root cortical cell size or reduced root cortical cell file number reach greater depths to increase water capture under drought conditions, possibly because those cellular traits reduce metabolic costs of root growth and maintenance . The control of root angle that results in greater water capture in rice as water tables recede was linked to the control of auxin distribution . Similarly, in shoots, natural variation can be exploited to find genetic loci that control shoot morphology, e.g., leaf erectness . High-throughput phenotyping techniques are increasingly used to reveal the genetic basis of natural variation . In doing so, phenotyping techniques complement classic approaches of reverse genetics and often lead to novel insights, even in a well-studied species like Arabidopsis thaliana. Phenotyping techniques have revealed a genetic basis for dynamic processes such as root growth and traits that determine plant height . Similarly, high-resolution sampling of root gravitropism has led to an unprecedented understanding of the dynamics of the genetic basis of plasticity .Phenotypic plasticity is defined as the ability of one genotype to produce different phenotypes based on environmental differences and adds to the phenotypic complexity created by genetics and development. Trait variation in response to the environment has been analyzed classically using ‘reaction norms,’ where the phenotypic value of a certain trait is plotted for two different environments . If the trait is not plastic, the slope of the line connecting the points will be zero; if the reaction norm varies across the environment the trait is plastic and the slope of the reaction norm line will be a measure of the plasticity. As most of the responses of plants to their environment are nonlinear, more insight into phenotypic plasticity can be obtained by analyzing dose-response curves or dose-response surfaces . Seminal work by Clausen et al. demonstrated using several clonal species in a series of reciprocal transplants that, although heredity exerts the most measureable effects on plant morphology, environment is also a major source of phenotypic variability. Research continues to explore the range of phenotypic variation expressed by a given genotype in the context of different environments, which has important implications for many fields, including conservation, evolution, and agriculture . Many studies examine phenotypes across latitudinal or altitudinal gradients, or other environmental clines, to characterize the range of possible variation and its relationship to the process of local adaptation . Below-ground, plants encounter diverse sources of environmental variability, including water availability, soil chemistry, and physical properties like soil hardness and movement. These factors vary between individual plants and within an individual root system, where plants respond at spatio-temporal levels to very different granularity . Plasticity at a microenvironmental scale has been linked to developmental and molecular mechanisms . The scientific challenge here is to integrate these effects at a whole root system level and use different scales of information to understand the optimal acquisition in resource limited conditions . Since it is extremely difficult to examine complex interdependent processes occurring at multiple spatio-temporal scales, mathematical modeling can be used as a complementary tool with which to disentangle component processes and investigate how their coupling may lead to emergent patterns at a systems level .

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 .