Horticulture

In California, they are larger than on Cleopatra mandarin, but smaller than those grown on Rough lemon, sour orange, or Troyer citrange. Seedlings of sweet orange are resistant to verrucosis according to Klotz . Again, this is not a problem in California, but in nurseries or grown trees it can be a problem in areas with more humid climates. Sweet orange is also resistant to mal secco , but this disease is not known to exist in the United States. It is a severe problem in some Mediterranean countries. Psorosis, or scaly bark, can be more serious on sweet orange than the resistant sour orange , since if both the scion and the stock are affected, the rate and degree of decline of the trees may be greater. Lemon trees on sweet orange do not express shell bark with the severity that they do on sour orange or Rough lemon. The sweet orange does not appear to be affected by exocortis, cachexia, xyloporosis, or woody gall . Trees on sweet orange root stock in Florida are not threatened by blight according to Lawrence and Bridges . The reaction of sweet orange root stocks to tristeza inoculations has generally been negative . However, in my Baldwin Park experiments in California,blueberry container there was some light stunting as a result of the inoculations. A seedling cultivar of ‘Bessie’ sweet reacted severely enough to be classed as susceptible.

The mother tree source did not. Trees on ‘Koethen’ sweet were also somewhat stunted and borderline in their reaction. In Brazil, trees on ‘Caipira’ sweet orange are also adversely affected. Sweet orange is susceptible to the citrus nematode . In fact, it is one of the most susceptible root stocks. Certain selections of sweet orange like the ‘Ridge Pineapple’ and the ‘Sanguine Grosse Ronde’ have shown some resistance to the burrowing nematode . Not only are sweet orange trees very susceptible to nematodes, they are very susceptible to gummosis . This is true even though the trees are budded high, planted high, and the best cultural practices are used. This is probably the greatest disadvantage that sweet orange has. It is remarkable, considering the susceptibility of sweet orange, that so many seedling orchards survived for a half a century or more, in the early history of California and Florida plantings. Surviving affected trees often bear scars of the infection by this disease. Sweet orange trees are as susceptible as trees on Rough lemon, often worse, but not nearly as tolerant as sour orange, Alemow, or Troyer citrange. It is interesting that at the Citrus Experiment Station, Riverside, and in some of my other root stock plots, that although the sweet oranges in the original plantings were remarkably free of gummosis, that in replant situations, even though the soil was fumigated and good cultural practices used, it is almost impossible to successfully re-establish trees on sweet orange root . It should be pointed out, however, that the soil fumigation is generally for eliminating the citrus nematode. The fumigant rate to kill nematodes is lower than the dosage to act as a fungicide. Because of roots the higher dosages are generally not used.

Sweet orange has very poor root generation capacity and once infected, usually succumbs to the attack. Klotz and Fawcett reported that the ‘Indian River’ variety, an ambiguous name, from Florida was somewhat more resistant to gummosis than other sweet orange varieties tested in the variety collection at Riverside, or in existing root stock trials also at the Citrus Experiment Station. However, the tests were not replicated and the gummosis differences were small. The difference was not enough to attract any grower interest and consequently, it was never used as a commercial root stock. The gummosis resistance of ‘Precoce de Valence’ reported by Olson, et al. in Texas would also warrant further investigation. When the author came to Riverside in the mid-l940’s, there were still many seedling orange orchards in Riverside and adjacent areas. The first sweet oranges were planted in Riverside in 1871. One of these, the ‘Koethen,’ was probably planted prior to 1880, and was selected by H. B. Frost in the early 1900’s, and later incorporated into the 1927 root stock trials by H. J. Webber and reported on elsewhere in this text. The ‘Koethen’ turned out to be one of the better performing sweet orange cultivars and is still grown commercially, especially in Southern California. Seedling orange trees were first planted in San Bernardino County in 1857. One of these, the old Cram tree, at East Highlands is shown on page 36 of Volume I of The Citrus Industry, with Dr. H. J. Webber standing beside it. It was widely used in the San Bernardino area. Nearby was the ‘Hinekby’ orchard at Bryn Mawr. It also was used commercially.

In Ventura County, the ‘Olivelands’ sweet from the Limoneira Company has also been widely used as a root stock in that area and its other seedling orange cultivars have also been used, and their performance has been good. The Limoneira Company has continued to use the ‘Olivelands’ sweet as its principal root stock for lemons. They state “we know sweet orange, its performance and limitations. We feel that with good cultural practices, inspection, and sanitary procedures we can control gummosis. There is nothing we can do about the phloem necrosis of the Eurekas on C. macrophylla, the sudden incompatibilities of Eureka on ‘Troyer’, on ‘Swingle’ citrumelo, or #1452 citrumelo, and the bud-union overgrowth and compression girdling on ‘Cleopatra’ mandarin. Much of the success of an orchard depends on its longevity and orchards which have to be replaced every decade or several decades cannot be considered successful.” There was always interest in a “good-performing” sweet orange. About 1950 the author introduced the ‘Kona’ from the Kona district of the island of Hawaii on the basis the trees were quite old and grew quite well in an area characterized by 250 cm of rainfall. Its performance in California districts was mediocre. In 1940, H. S. Fawcett introduced sweet orange seeds from the Santa Ana Mission in North Argentina. The author incorporated these in later root stock trials and the performance has been good. The author visited many sweet orange seedling orchards in Southern California,maceta cuadrada which persisted well into the 1950’s. The very striking thing in most of these orchards was the freedom of the trees from gummosis which certainly was not a factor in their ultimate removal. In an effort to develop cold hardy edible citrus to extend its commercial growing range, Swingle hybridized the navel orange with trifoliate orange, and the ‘Troyer’ and ‘Carrizo’ citranges are part of the result. The ‘Washington’ navel, aside from producingno or few seeds, has nothing to recommend it as a root stock. More recently, Cameron and Soost hybridized the ‘Ruby’ blood orange with a trifoliate orange from which resulted the ‘C-32’ and ‘C-35’ citranges. However, the ‘Ruby’ blood has never been used as a root stock and has nothing to recommend it. Dr. Cameron’s defense was that they thought the ‘Ruby’ produced more hybrids than some of the other sweet oranges. However, that seems a poor excuse, when a few more pollinations or the use of developing tissue culture techniques may have increased the number of zygotic embryos obtained. One wonders how much better ‘Troyer’, ‘Carrizo’, ‘C-32’, and ‘C-35’ citranges might have been if the sweet orange female parent would have been a more desirable and proven root stock type. If new citranges are to be developed for root stocks, then certainly the plant breeders should consider using the ‘Argentina’ sweet, or the ‘Olivelands’ sweet, as the female parent. One of the disadvantages of some of the commercial citranges is their exceptional vigor, which doesn’t fit in with today’s interest in close spacing. However, the ‘Fuya Menuda’ cultivar is a genetic dwarf. Hybridized with a dwarfing strain of trifoliate orange, it is possible that more dwarfing hybrid citranges might be obtained.

Positive identification or verification of the ‘Fuya Menuda’ is essential. Unfortunately the specimen in the Citrus Variety Collection at Riverside is not true to type and hopefully has not been distributed in seed or budwood requests. None of the sweet oranges tested have ever shown any resistance to the citrus nematodes. However, the ‘Ridge Pineapple’ and the ‘Sanguine Grosse Ronde’ show resistance to the burrowing nematode . Perhaps they should be hybridized with select strains of trifoliate orange to produce hybrids possibly resistant to both the citrus and burrowing nematode. Also, the California strain of ‘Carrizo’ citrange which shows resistance to the burrowing nematode might be hybridized with the above sweet orange cultivar to provide improved resistance to this destructive soil organism. The sour orange, C. aurantium, was once the world’s most widely used citrus root stock. It was intensively used in the United Sates , all of the Mediterranean area, including the Near East, all of South America, Cuba, Central America, and to a limited extent in Australia and New Zealand. It was never successful in the Orient, Java and South Africa due to a virus disease called tristeza . Because of the rapid spread of this disease around the world, the acreage on this stock has continued to dwindle. It is still an important root stock in the United States in Arizona, Texas and Florida. It is still used in the Mediterranean area, but the occurrence of tristeza in Spain and Israel threatens adjacent countries as well as the Near East, and other root stocks are rapidly being employed. The stock is still used in Cuba, the western half of South America , in Central America and a few other scattered areas. As a root stock the sour orange possesses many fine qualities and it has been a major disaster to the citrus areas which have been decimated by the tristeza virus. A sour orange root stock fully tolerant to the tristeza virus would still be a tremendous asset to the growers and the plant breeders. All of the standard sour orange varieties are very seedy, producing large plump seeds, which are moderately to highly nucellar, not being as high as sweet orange, Rough lemon, ‘Troyer’ citrange, ‘Cleopatra’ mandarin, etc. This is not always true of the socalled aberrant types like ‘Chinotto’ or putative hybrids like the ‘Nansho daidai’ which will be discussed later. A minimum of rogueing is therefore required in the citrus seed bed or nursery and container growing. The seeds are also more resistant to drying out than seeds of many other citrus species like Rough lemon and trifoliate orange. The seedlings are fairly vigorous growers, not as much so as Rough lemon, Alemow, or ‘Troyer’ citrange, but more so than sweet orange or grapefruit. The seedlings develop mainly a strong single trunk which requires less shaping in the nursery than sweet or grapefruit, but more than ‘Troyer’ citrange and Rough lemon . This growth habit has facilitated easy budding, and the seedlings bud well to all common scion varieties. The sour orange may be grown quite easily from cuttings, but they are more difficult to root than cuttings of Rough lemon and sweet orange. Due to its seediness and the uniformity of its seedlings, there is no need for the cuttings. Seedlings are perhaps more resistant than other root stocks commonly used with the exception of the trifoliate orange and some of its hybrids like ‘Troyer’, ‘Carrizo’, ‘C-32’, ‘C-35’, ‘Swingle’ citrumelo, etc. When frozen back, the seedlings readily sprout from the base of the trunk and a sprout can easily be trained to a new leader. Sour orange seedlings are very susceptible to citrus scab , which sometimes causes serious injury to nursery seedlings in humid climates . The bud unions of the common sour orange variety are nearly normal with scions of sweet orange, mandarin, and grapefruit varieties, but rather commonly show a strong scion overgrowth with most varieties of lemons , limes, and citrons. This lack of congeniality was called “miriñaque” with lemon in Spain . With most scion varieties there is a slight bulge at the union, with the stock tending to be smaller than the scion. Grapefruit scions tend to overgrow the stock to a greater degree, but this is slight compared to the reaction with lemons and citron . Certain strains of Lisbon lemons such as the ‘Keen,’ ‘Monroe,’ ‘Bradbury,’ etc. on sour orange show a more congenial reaction.

The cultural practices within an orchard can materially affect root systems. In Ceylon , Gandhi cites a difference in citrus root distribution between irrigated areas and rainfall areas. However, when he grew grapefruit trees on Rough lemon with various degrees of cover crop the citrus roots would not compete with the cover crop. They either turned back into open areas or penetrated deeper. If the roots extended to the cover crop they grew under it, but not through it. Those trees without cover crop extended roots laterally 2 to 3 times the spread of the branches. Where cover crops were used the roots only extended to the periphery of the branches. A nine-year-old grapefruit tree with a cover crop of Indigofera and Ecaphylla [could not identify this plant] also had lateral roots extending a little beyond the periphery of the crown. They did not appear to appreciably mingle with roots of other trees only 4.3 M away. His observations clearly indicate citrus roots are not able to maintain the same growth rate in association with permanent cover as without it, and that for free development of their roots they require the soil beyond the drip of the tree crown to be free of competition for water by other roots. Figure 64 by Wallace and Nauriyal [figure could not be included due to copyright restrictions] shows citrus rootlet distribution as influenced by cultivation. The tilled orchard had few roots at the 0-15 cm level and most were in the 60-150 cm.

By contrast, under non-tillage there were 7 times as many roots in the 0-15 cm and the highest concentration was at the 15-30 cm zone. Deep soil was essential for making furrows, basins,how to set up a vertical farm or block-and-ridge systems for water. Such tillage severed most of the surface laterals and caused severe damage. Figure 65 shows such a root system with the laterals cut at several locations, and Figure 66 [Image could not be located] shows such a severed root which then regenerates a multitude of short stubby roots, with no single root gaining dominance. Water and nutrients are most available in the upper soil zones, which is rendered unavailable by tillage. Diseased trees have reduced root systems. Ford reports that declining grapefruit trees had 43% less roots than healthy trees. Oranges on Rough lemon at 7 years of age replanted on spreading decline soil had 18 per cent more feeder roots in the upper 25 cm of soil than good trees the same age. However, the decline trees had no roots below 75 cm. Ford says that mature citrus trees affected by the spreading decline nematodes had 40 per cent fewer feeder roots than healthy trees. Decline trees had more feeder roots in the upper 25 cm of soil but almost none below 75-105 cm. Roots examined in affected groves showed significant disintegration at the 50-64 cm level with a rapid increase in deterioration with increase in depth. Practically all roots of decline trees at 50-105 cm were dark and discolored. Trees replanted in spreading decline affected soil had no lateral roots or feeder roots below 75 cm. Montenegro found poor root systems under trees affected with exocortis or tristeza.

The sour orange, because of its tendency to develop deep tap roots, was used extensively in the West Indies as a root stock for limes. Such trees were reported by growers to withstand hurricane injury better than lime on its own roots. Fennah in a study of the root systems of budded trees at St. Lucia Island in the Caribbean found that sour orange seedlings in the nursery all had tap roots. However, out of 24 orchard trees examined at three years of age only three had tap roots and that of 110 trees at five years of age only three had tap roots, and the root system was horizontal. Fennah explained this circumstance as due mainly to damage by the citrus weevil and to injury at transplanting. Kaufmann, Boswell, and Lewis , in a spacing trial of Washington navels on Troyer citrange in California, studied root distribution at different spacings. They found that the distribution of the roots varied with the distance from the tree and the availability of soil water . The trees at a 2.7 x 4.7 M spacing at eight years of age had utilized the full rooting area and this was leading to a reduced tree performance. In Florida, Ford observed that the root system of sour orange was influenced more by freeze injury of the scion than the root system of Rough lemon, and that the root system of sour orange recovered more slowly. In California, Biely, Wallace, and Kimball report pruning citrus trees affected root distribution. In a period of five months following skeletonization there was a great reduction in feeder roots at all depths.

Essentially all the feeder roots had disappeared. One year later the amount of rootlets under skeletonized trees was greater in number than before the trees were severely pruned. In a Florida root stock experiment, Castle and Krezdorn report that the depth of rooting was correlated with tree height; the tallest trees had the deepest root systems. Feeder root weight and tree height were not correlated. The tallest scion trees were on Rough lemon, Palestine sweet lime and Cleopatra mandarin. The shortest scion trees were on Rusk citrange and various trifoliate orange selections. Scion trees on Carrizo and Troyer citranges, sour orange and sweet orange were intermediate. The depth of rooting varied from 465 cm for Rough lemon to 377 cm on trifoliate orange and 366 cm on Rough lemon to 206 cm on Rusk citrange. Rootstocks had a pronounced effect on total feeder root weight. Deep-rooted trees on Rough lemon and sweet lime had more than 50 per cent of their feeder roots below 75 cm. Intermediate-sized trees were about the same as the tall trees above and below the 75 cm level. Exceptions were Cleopatra, Rusk citrange, and Rubidoux trifoliate which had over 60% of their roots above 75 cm. Rough lemon was the only root stock with roots below 457 cm. Reports on drought resistance are somewhat variable. Webber says sour orange is tolerant. Hume confirms this and states Rough lemon is more susceptible. Evans states Rough lemon is tolerant and grapefruit and sour orange are more susceptible. Brown indicates the Egyptian lime is tolerant. Ford reports sweet orange to require more frequent irrigations than Rough lemon. Bhattacharya and Dutta indicate the Soh-myndong to have drought resistance. Various visitors to the Citrus Research Center have indicated that the Palestine Sweet Lime and the Rangpur lime have drought resistance. All of this may vary with soil type, temperatures, etc. Hilgeman et al. compared Rough lemon, Rangpur lime, Ocklawaha sour orange, Sacaton citrumelo, Troyer citrange, Koethen sweet orange,plastic pots for planting arid Wilking mandarin as root stocks for Lisbon lemon in a sandy soil near Yuma, Arizona, and a sandy loam near Phoenix. They found that young trees on sour orange and Troyer citrange developed greater moisture stress between irrigations than trees on Rough lemon and Rangpur lime. The growth of the fruit was restricted at the differential irrigation levels. They suggested the root growth on the Rough lemon was more rapid than on sour orange stock and hence a larger soil volume was available to provide water to the trees on Rough lemon than those on sour orange. Gardner and Horanic in Florida’s humid climate compared the transpiration of young trees on four root stocks by weighing container-grown trees at frequent intervals. The rate of transpiration of Hamlin orange tops was lowest on Rough lemon, highest on sweet orange and intermediate on Cleopatra mandarin and sour orange, although the actual amount of water transpired was essentially the same for all four root stocks. The transpiration rates were inversely proportional to the leaf areas of the scion variety tops developed by these root stocks, indicating the tops carried the greatest leaf area on Rough lemon and had the least resistance to transpiration. The effect of the root stocks on transpiration rates apparently resulted from differences in leaf resistance. Horanic and Gardner , following a prolonged dry period in Florida during September 1958, found a marked difference in wilting of Parson Brown and Valencia oranges in a root stock experiment on a Lakeland fine sand.

Marked differences between root stocks were evident. Sour orange, Rough lemon, and Cleopatra mandarin were outstanding in their survival resistance to drought as compared to trees on grapefruit, sweet orange, and Rusk citrange. Unbudded Parson Brown seedlings in the planting also showed severe wilting. They felt the differences in apparent drought resistance were attributable to the root stock effect rather than to a greater or less soil moisture depletion by trees of different size. Ongun and Wallace , using the water weight loss in plants grown in containers in a greenhouse, studied the effect of both root stocks and root temperature on the transpiration rate of Washington Navel orange scions grafted on rooted cuttings of a number of root stock varieties. They reported that of the root stocks used, the transpiration rate was highest on Rough lemon and lowest on sour orange and that while root temperatures influenced transpiration, not all the root stocks were affected to the same extent. It descending order the combinations were rated best on Rough lemon, then Poncirus trifoliata, Troyer citrange, grapefruit, sweet orange, Cleopatra mandarin and sour orange. Mendel in Israel agrees that trees on Rough lemon transpire at a faster rate and, although a large tree, shows considerable drought resistance. He remarks of the remarkable drought resistance of trees on Rough lemon in spite of the high transpiration rate. Trees on sour orange had a strong tendency toward lower transpiration losses than trees budded on sweet lime. O’Byrne lists Rough lemon as most drought resistant followed in descending order by sweet orange, sour orange and grapefruit. Crocker, Bell, and Bartholic using a modified Scholander pressure bomb found it was sensitive enough to detect significant differences in relative leaf water stress on Orlando tangelo trees on several root stocks.In descending order the trees ranked Rough lemon, Palestine sweet lime, sour orange, Carrizo citrange, and trifoliate orange. Perhaps the largest contingent of trees observed for drought resistance was made in Brazil by Moreira et al. on nucellar Bara and Valencia Late orange, Dancy tangerine and Eureka lemon budded on 77 tristeza-tolerant root stocks which were grown under nonirrigated conditions in Brazil. They categorized the drought resistance into three groups: low, fair and good. If one ignores the early reports from Florida on the drought resistance of sour orange which seemed to be a special circumstance, it would appear that those root stocks which have the greatest amounts of fibrous roots appear to be the most drought tolerant irrespective of the tap root system, and that conversely those root stocks lacking quantities of fibrous roots regardless of the depth of penetration are more susceptible to drought.Undoubtedly, the good drought resistance of Rough lemon root stock in Florida is the deep root penetration into the warm sandy subsoil of the “ridge” area. These soils are very deep and [text incomplete] drained in the poorly drained soils in eastern Florida the drought resistance of Rough lemon is not so good. In California during the summer months, there are periods of high temperatures and low humidity accompanied by hot, dry, desiccating winds. Under these extreme conditions leaves of trees may suffer severely from mesophyll collapse. After one such occasion in Coachella Valley, several of the author’s root stock plantings were rated in 1960 for severity of mesophyll collapse . Leaf damage was very severe on Brazilian sour orange, Bessie sweet orange, Batangas mandarin, Cleopatra mandarin, Suen Kat mandarin, #653 tangor, Carrizo citrange, Savage citrange, Troyer citrange, Uvalde citrange, Calamondin, and Shekwasha. It was much less on stocks of #343 grapefruit, Ponkan mandarin, Sunshine tangelo, Citrus macrophylla, C. pennivesiculata, Rangpur lime and Rough lemon. This correlates very well with observations on the extent of fibrous roots for the various stocks. One of the most striking effects on root system development is that exerted by the scion variety.

Notably, although recruited from across the United States, nearly all participants were sheltering in place at the time of study enrollment due to the COVID-19 pandemic, which may have affected substance use patterns and mood as well as interest in a digital health intervention. Notably, however, alcohol sales in the United States increased during the COVID-19 pandemic. The primary outcomes of substance use, cravings, confidence, mood, and program acceptability were standard measures with demonstrated validity and reliability. The limitations were that all were self-reported, and acceptability measures were not open-ended or qualitative. Few participants were misusing opioids, likely due to study exclusion designed to mitigate risk, namely, the requirement of engagement with medication-assisted treatment and no history of opioid overdose requiring Narcan . Notably, nearly 1400 people with interest in a program for those with substance use concerns were excluded due to low severity on the CAGE-AID screener. Worth testing is the utility of digital health programs for early intervention on substance misuse that is sub-syndromal. Building upon the findings of this study, future research will evaluate W-SUDs in a randomized controlled trial with a more racially or ethnically diverse sample,vertical plant growing balanced on sex and primary problematic substance of use; will employ greater strategies for study retention ; and will be conducted during a period with less restrictions on social contacts and physical mobility.

Randomized controlled evaluations of conversational agent interventions relative to other treatment modalities are required. Self-cutting can be understood clinically as a symptomatic behavior, on the one hand, and as a bodily practice embedded in a cultural imaginary and identity on the other. It is present in a variety of ways including the 1993 memoir of Susanna Kaysen “Girl, Interrupted” , the 1995 acknowledgment by Princess Diana that she identified herself as a “cutter,” and the 2011 video “F**kin’ Perfect” by the pop music performer Pink. The Internet has become a massively popular resource for cutters to share information , and one study identified more than 400 message boards about cutting generated via five search engines . Youths may identify with “Emo” or “Goth” culture which lionize depression and cultivate self-cutting as a cultural practice . Popular concern about perceived dangers of self-cutting has at times been heightened to the point that one cultural historian suggested that “Cutting has become a new moral panic about the dangers confronting today’s youth” . Anthropology has not been disposed toward addressing cutting as a problematic cultural or clinical phenomenon given the disciplinary propensity to understand body mutilation and modification in terms of rituals and cultural practices. This is perhaps because ritual meaning is not so dependent on distinguishing whether harm is inflicted by others or by oneself or on differentiating cultural practice from psychopathology.

These distinctions are clear in one of the rare instances in which an anthropologist has addressed the issue of self-injury, here in the context of commenting on an article relating it to borderline personality disorder.One other anthropological observation has been provided by Lester, who notes that current explanations of self-harm can be grouped into four categories: communicating emotional pain, emotional or physiological self-regulation, interpersonal strategy, and cultural trend. She notes that these categories share the idea that self-harm manifests individual pathology or dysfunction, with the cultural assumption of the individual as a rational actor. In contrast, an anthropological perspective emphasizes the “cultural actor who embodies and responds to cultural systems of meaning to internal psychological or physiological states” . Emphasizing the powerful symbolic significance and long cross-cultural record of self-harm and blood shedding as ritual and even therapeutic practices, she suggests that contemporary cutting may be seen as privatized and decontextualized social rituals affecting transformation parallel to collective initiation rituals that operate in a cycle of self-harm and repair, especially in the case of adolescent girls struggling with the aftermath of sexual abuse and/or with contradictory gender messages . Sociocultural characteristics of a typical “self-cutter” emerged in the 1960s as Euro-American, attractive, intelligent, and possibly sexually adventurous teenage girls, that Brickman claimed was partially taken up in medical discourse in a manner that “pathologizes the female body, relying on the notion of ‘femininity as a disease’” . Gilman took exception to assumptions of pathology with the provocative claim that “self-cutting is a reasonable response to an irrational world” .

From a clinical vantage point, self-cutting is often viewed as a type of injury or harm to the self. The historical backdrop to this development can be traced to Menninger’s attention to self-mutilation as distinguished from suicidality. The distinction between “delicate” and “coarse” self-cutting was made by Pao , with Weissman focusing on wrist-cutting syndrome and Pattison and Kahan proposing the existence of a deliberate self-harm syndrome. Favazza provided cases of extreme and highly unusual forms of self-mutilation in excruciating detail, with an attempt to classify types based on severity. With the provisional emergence of non-suicidal self-injury disorder criteria in the fifth version of the Statistical and Diagnostic Manual of Mental Disorders DSM-V ,1 the distinction between self-harm as within a normative or pathological range remains equivocal. This is illustrative of the manner in which conceptualizations of self-cutting continue to be embedded in a complex cultural history of changes in the incidence, popular awareness, and social conditions in which such phenomena occur.While it is possible to find clinical, psychometric, survey, and historical approaches to the phenomenon of self-cutting, we lack an ethnographic account with a substantive locus in the interactions of individuals, grounded in the specificity of bodily experience and the immediacy of struggle in the face of existential precarity . In this article, we take a step toward such an account with a discussion situated at the intersection of two anthropological concerns. First is the ethnographic understanding of experiential specificity through anthropological adaptation of phenomenological method . Drawing on this approach, we understand experience as meaningful sensory perception in temporal context and within particular cultural, social, and interpersonal settings and subjectivity as the more or less enduring structure of experience. With respect to mental illness, this approach invites anthropological recognition of struggle as a fundamental human process that comes to light in the context of lived experience.

Second is the ongoing anthropological concern with adolescence as a stage in the life course at which identity is consolidated and people approach full cultural membership but which is also fraught with challenges to well-being that anthropology can contribute to understanding in a way relevant to mental health policy and practice . The contemporary anthropological approach to childhood is strongly influenced by child standpoint theory that aims at an account of society from where children are socially positioned and in which they are not passive social “others” but agentive participants in social life,growing strawberries vertically hence co-constructors of social knowledge and by extension of knowledge generated by research2 . In particular, anthropologists have taken up the idea that “children have agency and manifest social competency” . Guided by these concerns, we will focus specifically on self-cutting among a group of adolescents who have been psychiatric inpatients; by attending to experience and subjectivity articulated in the youth’s own voices, we will come to understand self-cutting as a crisis of agency enacted on the terrain of their own bodies. There is scant literature on how young people conceive and understand mental health , let alone experiential accounts of adolescent mental illness from the standpoint of the child . In addressing the experience of cutting among a clinically defined and diagnosed group of youth, our stance is not to fall prey to accepting a false dichotomy between ethnographic and clinical sensibilities; that a young person is following a regimen of psychotropic medication is as much an ethnographic as a clinical fact, and that a young person lives in a fragmented family environment may have clinical as well as ethnographic implications. Self-cutting can be understood as a troubling symptomatic behavior or as a creative struggle for agency and may exhibit elements of both pathological obsession and ritual transformation, but in either case it is an enactment of a vexed relation between body and world.This discussion is based on SWYEPT, our study of youth in New Mexico who were inpatients in the state’s flag ship Children’s Psychiatric Hospital at the University of New Mexico .

New Mexico is a state whose total population according to the 2010 United States Census was 2,059,179. In 2010, according to the US Census Bureau’s categories, by race the largest population proportions were designated “white” and American Indian/Alaska Native , with 23 federally recognized Indian tribes in the state comprising various groups of Pueblos, Navajos, and Apaches; other racial categories were minimally represented. By ethnicity, Hispanics or Latinos accounted for the largest single block , while among non-Latinos the largest blocks identified themselves as generically white or American Indian . New Mexico is one of the poorest states in the nation. According to the US Statistical Abstract, as of 2008 the median household income was $43, 508 or 44th among the 50 states, and the proportion of persons living below the poverty level was 17.1% or 5th in rank among the states. New Mexico ranks as one of two states within the United States hardest hit by child poverty, with the rate of 30% in New Mexico . Relatedly, home foreclosures have also been inordinately high. Along with poverty comes a serious drug problem, with parts of the state severely afflicted by heroin and methamphetamine use, and the presence of violent gangs, with one anti-gang website listing 178 in the Albuquerque area. The SWYEPT study examines cultural meaning, social interaction, and individual experience among adolescent patients and their families, with the long-term goal of producing knowledge of broad use to those concerned with the treatment of adolescents suffering from mental illness in the context of significant cultural differences. The aspects of this knowledge include: types of problem, illness, or psychiatric disorder experienced by afflicted adolescents; trajectories of adolescent patients from the community into treatment and back into the community; patient experience of therapeutic process and family response to that process; alternative and complementary resources brought into play by families on behalf of patients; difference between the experience of afflicted adolescents and that of counterparts who have not been diagnosed or treated for emotional disturbance. Notably for present purposes, ours was not explicitly a study of self-cutting or self-harm, but cutting emerged within the ethnographic interviews as a theme deserving of the particular attention we devote to it here. We recruited participants for the study with the assistance of three clinicians at Children’s Psychiatric Hospital who referred to us patients aged 12–18 they judged as not so severely cognitively disabled or developmentally impaired as to be unable to participate in interviews and not so emotionally fragile or clinically vulnerable that their participation would be unduly stressful. We obtained informed consent from youth and their parent or primary guardian based on these referrals, recognizing the ethical responsibility of respecting the vulnerabilities of individual patients and the need for continued rapport in the relationship between therapists and families, as well as the importance of our respect as researchers for the clinical expertise of the referring therapists. All participants entered the project as inpatients at CPH. Assisted by a team of graduate student ethnographers and clinically trained diagnostic interviewers, we conducted ethnographic interviews covering life history and experience with illness and treatment with the young people and their primary parent/guardian three times at approximately six months’ intervals.During this period, we also conducted the child version of the Structured Clinical Interview for DSMIV, a clinician-administered research diagnostic interview , the Adolescent Health Survey , and the Youth Self Report and Child Behavior Check List for children and their parent/guardian respectively. Although initial interviews occasionally took place in the hospital, it was rare for a participant still to be there at the time of the second and third ethnographic interviews. Yet it was not always the case that they were at home, since it was not uncommon for them to be placed instead at another treatment facility of in-treatment foster care. This often led us far afield from the hospital in Albuquerque, such that our ethnography ranged across the entire state of New Mexico and occasionally beyond. In this respect, our work was not strictly speaking a clinical ethnography in the sense of ethnography primarily situated in a clinical context that focuses on the institutional cultural milieu and interactions among patients and staf .

Several trees of Webb Red Blush grapefruit on Tavares limequat also showed problems at the bud union.Olson postulated that since seedlings of calamondin, lemonquat, red grapefruit, and sweet orange grew normally, whereas certain budded combinations showed bud union crease, it would seem possible that the disorder was related to interaction between specific root stock and scion varieties. It seemed desirable, therefore, to determine whether the disorder was of virus origin and other scions were susceptible. He found that old-line grapefruit on sour orange was compatible, but observed bud union crease with the same scion on calamondin and lemonquat. Olson showed that nucellar Red Blush grapefruit, old-line Red Blush grapefruit, nucellar Valencia and old-line Valencia all displayed bud union crease on calamondin root stock within two to three years. However, nucellar Eustis limequat, old-line Eustis, nucellar Lakeland limequat, and Meyer lemon showed no symptoms on calamondin. He also found that calamondin, Cleopatra mandarin, Columbian sweet lime, Sunki mandarin, and Rangpur lime on Eustis limequat showed no bud union problems in two years time, but on sour orange and Sunshine tangelo there were problems. He concluded the problem was not a virus, but rather a localized incompatibility,vertical tower for strawberries and probably could be prevented by the insertions of a proper inter stock. Olson and Frolich reviewed the calamondin bud union problem.

They point out that this disorder was also reported by Salibe in Brazil and also by Salibe in Florida as well. It was also emphasized that while the work of Weathers and Calavan showed that, while six different root stock varieties which produced bud union crease when propagated with old budline calamondin tops grew normally when budded with nucellar calamondin buds, when nucellar calamondin buds were inoculated with old budline calamondin tissue the trees also remained free of bud union crease. However, when Weathers top worked old-line calamondin onto the nucellar calamondin on the same six root stocks so that the old budline calamondin formed the tops and the nucellar calamondin formed the inter stock, the trees developed bud union crease at the lower union between the nucellar calamondin and the root stock. Evidently, something was translocated from the old budline calamondin across the nucellar calamondin inter stock, because nucellar calamondin on the same root stocks failed to show bud union crease when they were eight years old. Weathers suggested that his old budline and nucellar calamondin were different, even though the nucellar line was a seedling from the old budline . Olson and Frolich also report that at Weslaco, Texas, eight trees of old budline calamondin propagated on C. taiwanica had only one tree of the eight show bud union crease. They emphasize that off type seedlings of C. taiwanica are common and may explain this occurrence. At Indio, California , nucellar calamondin trees on Carrizo and Troyer citranges grew vigorously for seven years free of bud union crease. However, nucellar and old budline kumquats on the same root stocks died a few years after budding, and they emphasized that compatibility reactions of kumquat and calamondin are not necessarily identical.

Olson and Frolich budded a large number of varieties on calamondin root stock. Table 13 shows the varieties which were compatible and showed no bud union crease after 2-1/2 years. Table 14 shows a list of those varieties which Olson found to be incompatible on calamondin stock and showed bud union crease at the end of 2-1/2 years. When old budline calamondin was grown on sweet orange root stock in California, or a nucellar grapefruit top was grown on calamondin root stock in Texas, bud union crease developed. However, when Cleopatra mandarin on Rough lemon was used as an inter stock between incompatible components, bud union crease failed to develop within a three-year period, see Table 15. Although Swingle considered calamondin to be a mandarin-kumquat hybrid, the incompatibility reactions of the kumquats were not identical with those of the calamondin. In California, old budline kumquat trees grow well on calamondin and on sweet orange root stocks, and make excellent unions. However, old budline calamondin tops with kumquat inter stock on sweet orange root stock developed mild bud union crease between the sweet orange and kumquat. Also, calamondin grows well on Rough lemon root stock whereas kumquat does not. Old budline kumquat trees on Rough lemon root stock show a swelling like a collar at the bud union. In Florida, this disorder termed “Podagra” was neither infectious nor transmissible according to Knorr . Olson concludes, as do others like Weathers and Calavan , that bud union crease of certain varieties of citrus on calamondin root stock appears to result from tissue incompatibility. Salibe in Brazil budded old-line and nucellar calamondin on Brazilian Rough lemon and Florida Rough lemon. Three years later there was severe creasing and gumming at the bud union of all trees on the Brazilian Rough lemon, but they were normal on Florida Rough lemon except there was stem pitting and wood discoloration similar to xyloporosis. Salibe also found Eureka lemon and old-line Selecta de Itabuna orange incompatible on calamondin, and nucellar line calamondin on Natsumikan, Caipira sweet orange, Coachella Eremocitrus, and Brazilian Rough lemon also showed bud union crease. In the 1927-1930 root stock plantings at the Citrus Experiment Station, described and discussed by Webber and Batchelor and Rounds , various scions budded on the calamondin generally did very poorly.

The poorest results were obtained with the Eureka and Lisbon lemon scions, and those trees were removed from the orchard very early. The Washington navel, Valencia orange and grapefruit scions did slightly better, but such trees were all discarded before the author arrived at Riverside, 20 years later. All of these combinations showed bud union crease. However, as a root stock for Satsuma, results were quite good, the bud unions were normal and there was no bud union crease. Webber shows a picture of the bud union and the root system; however, yields were only average. In the 1948 tristeza plantings at Baldwin Park , the Valencia oranges on calamondin did show bud union crease , but did not react to tristeza. The trees showed little distress up to the time when the experiments were discontinued 13 years later. In the same plantings, the bigaraldin reacted severely to tristeza, but showed no indication of any bud union problem. The calamondin was also included in root stock trials by the author in the desert area of California in 1960. Scions included the marsh grapefruit, Valencia orange, and the Dancy tangerine. The grapefruit and Valencia trees showed a bud union crease problem within a few years, but managed to survive with some distress for at least 20 years. The Dancy tangerine trees did not develop any bud union crease, and were vigorous, healthy trees up to the time of their removal. The Glen citrangedin is incompatible on Carrizo citrange . At South Coast Field Station in California, Valencia oranges on Faustrimedin were incompatible,vertical growing but Eureka and Lisbon lemons on this same stock were still healthy at ten years of age with no sign of bud union problems . Some discrepancy in calamondin performance may be the result of incorrect identification. Even Hodgson on page 531 of volume I of The Citrus Industry, describes the calamondin as “sweet and juicy.” It is juicy, but it definitely is not sweet, possessing an acidity that ranks with commercial lemons and limes. In fact, in the Philippines where commercial lemons and limes are not grown, the calamondin is widely grown and used as a substitute for those varieties. While Swingle suggests that the calamondin “is very probably and orangequat that arose in China by insect cross-pollination of a sour, loose-skinned mandarin orange and a kumquat,” the cultivar in the first place should not have been designated as an “orangequat.” While it has some morphological similarities with the kumquat and some of its physical responses in scion-root stock combinations also resemble certain kumquat reactions, recent unpublished isozyme studies at the Citrus Research Center, Riverside, would seem to confirm that there is no genetic relationship between these two cultivars. As early at 1905, Coit in Florida referring to rebudding frozen trees, stated with reference to the kumquat that one does not bud directly onto the Rough lemon since it is incompatible, but rather buds onto the sprouts of the oranges and grapefruit. This may have been the first report of incompatibility with kumquat and Rough lemon. Hodgson on page 580 of volume I of The Citrus Industry shows a 30-year-old Nagami kumquat on sweet orange root stock. In the old citrus variety collection at Riverside, Nagami did very well on trifoliate orange and sweet orange as root stocks. Later plantings of Nagami on Troyer citrange root stock also did well at Riverside. W. Reuther presented the author with a picture of a 13-year-old Nagami on Swingle in Venezuela. The bud union looks fine . Nagami can also be successfully propagated on calamondin. The problem seems to be more acute with the Meiwa variety. In Japan, the Meiwa is grown exclusively on trifoliate orange root stock. In my many visits to Japan, the author is unaware of any incompatibility problems. The original source of Meiwa at Riverside was on trifoliate orange and was healthy up to the time of the orchard’s removal nearly 30 years later.

The author also has a Meiwa on trifoliate orange in his back yard, which is now 21 years old and showing no bud union problem. In California, Meiwa kumquat is incompatible on Troyer citrange . It is also incompatible on the calamondin and on Citrus shunkokan . Also, in the tristeza trials at South Coast Field Station , Valencia trees on Meiwa, and F. hindsii are incompatible . Eureka and Lisbon lemons on these same stocks are also incompatible . In the Baldwin Park tristeza tests , Valencia oranges on Thomasville citrangequat showed no tristeza problems and no bud union problems after 13 years. Thomasville and Sinton citrangequat at Riverside have also not shown any bud union problems when grafted on several miscellaneous root stocks. In Italy, Russo reports that all trees of seedling Meiwa kumquat budded on sour orange showed decline symptoms within 2-3 years. He also reports observing the same situation in Spain. McClean and Engelbrecht report that Nagami kumquat budded on seedlings of Nagami were incompatible. This is not surprising since Nagami produces only hybrid seedlings. There are many problems of incompatibility of various scions on Rough lemon root stock. Nour-Eldin noted bud union crease on more than 90 per cent of the trees of Baladi sweet orange budded on Rough lemon root stocks. He also observed bud union crease in about 80 per cent of the blood oranges budded on Rough lemon, but doesn’t state specifically the varieties of bloods. Salibe , and Knorr report problems of kumquats budded on Rough lemon. Fernández Valiela also found problems of blood oranges budded on Rough lemon. Salibe found Shamouti and Pera oranges incompatible on Florida Rough lemon, but when he inoculated with Pera buds into nucellar Bahianinha on Florida and Mazoe Rough lemon, the trees were normal; however, buds from the same Pera budded into two other cultivars of Rough lemon showed bud union crease on both. The list of scion root stock combinations exhibiting bud union crease included many sweet orange varieties budded on Rough lemon . Rodriguez in Argentina indicated all Pera sweet oranges budded on Rough lemon showed bud union crease. Bhutani, Bakhshi, and Knorr pointed out that Mosambi sweet orange and Blood red orange showed decline on Rough lemon root stock. In South Africa, McClean found no problem with Marsh seedless and Ruby Red grapefruit on Rough lemon. However, Triumph grapefruit, probably a hybrid, had bud union crease. The same applied to Jackson grapefruit, a seedling sport of the Triumph, and also to a seedling of Jackson. Three Ruby grapefruit seedlings scions on Rough lemon also showed mild symptoms at the union following inoculation with buds from trees of sweet orange on Rough lemon with a faulty union. McClean states that the abnormal union that some sweet oranges make on Rough lemon varies in degree. The most common orange varieties on Rough lemon affected are Valencia and Washington navel. He only found problems in one orchard of Valencia old-line, but most navels were affected.

Although knowledge of the art of budding, or grafting, goes back several millennia , it was only in the late 1800’s that budding of citrus became a general practice. The change was partly due to the introduction of superior varieties like the Washington navel orange, the Valencia orange and other named cultivars in which it was a disadvantage to grow seedlings or marcots, and partly because of the susceptibility to soil pests and diseases . The height of budding in most citrus areas has been somewhat standard and probably varies from a height of 5 to 15 cm, although it is now changing. However, the height of budding in some instances has varied from the ridiculous to the sublime. Nowhere are trees budded higher than they are in Italy, where sour orange seedlings may be budded at a height of 90 to 150 cm in attempts to escape gummosis, which so severely devastated their seedling orchards in the 1800’s . On the other hand, the author years ago observed a nursery near Fairhope, Alabama, in which the nurseryman hoed the soil away from the base of trifoliate orange seedlings and fall budded them to Satsuma mandarin just above the crown roots.In California, height of budding has generally tended to be directly related to the inherent vigor of the kind of root stock seedlings grown. Vigorous seedlings like Rough lemon tended to be budded somewhat high; low vigor seedlings like trifoliate orange, somewhat low. Seedlings of sweet orange, grapefruit and Cleopatra mandarin were budded at more of an intermediate height.

When trees were in great demand, even the vigorous seedlings might be budded low,vertical growing systems and occasionally nurserymen have selectively budded the largest seedlings in the nursery row and left the slower growing seedlings for a later budding. In addition, those root stock cultivars which tend to branch and sucker excessively and thereby develop a bushy character were often budded lower than cultivars with a strong central leader merely because the nurseryman didn’t want to go to the expense of continually removing the lower suckers and branches. Figure 4 [Image could not be located] is a composite picture of six typical root stock cultivars used in California citrus nurseries in the early 1950’s. The sweet orange, grapefruit, and Sampson tangelo seedlings were characteristic of the slow growing category which tended to branch and sucker excessively, requiring a great deal of pruning. They were not conducive to high budding, although the trunk caliper was fairly good. The Cleopatra mandarin seedlings grew more upright, but tended, in the nurseryman’s terms, to be “leggy”, of slender caliper, and often times in need of staking for support. Cleopatra does grow more rapidly and obtain better caliper under tropical conditions. The Rough lemon and the Troyer citrange tend to send out a strong central leader and a minimum of lateral branches. Troyer seedlings, of course, do not attain the caliper of Rough lemon seedlings, but can easily be budded to desired heights. Within the trifoliate orange, there is also considerable variation in seedling types comprised of the small-flowered, large-flowered, and of course the flying dragon. Figure 5 shows a nursery comparison of Rubidoux trifoliate on the left, compared to Pomeroy trifoliate on the right. The Rubidoux seedlings do not have the strong central leader and are quite prone to lateral branching. Whether all small-flowered trifoliate strains have the same nursery characteristics as the Rubidoux, or all large-flowered strains have the same nursery characteristics as the Pomeroy, has not been verified by adequate observation, but it would seem to be a likely possibility. The flying dragon trifoliate is something else.

It is more spindly than the other trifoliates, with a twisting, corkscrew type of trunk growth, making bud insertion difficult at some points on the trunk. The retrorse, or strongly recurved, spines hinder the budding procedure. The first significant experiment on height of budding was reported by Murray in Trinidad. Included in a series of root stock trials was the height of bud insertion of grapefruit and Jaffa sweet orange scions on sour orange root stock. Six heights of bud insertion were used: 5, 13, 25, 38, 51, and 64 cm. There were eight trees of each combination and the trees were completely randomized. There was some incidence of gummosis in these experiments, and while the incidence was low, it was obvious that to avoid gummosis the trees should have been budded at 38 cm or higher. The effect of bud insertion on average number of fruits per tree, average total weight of fruit per year over a ten year period, as well as final stock and scion girth measurements in 1945 are presented in Tables 3 and 4. Several things deserve to be pointed out. The largest trees were those budded at the 5 cm height, and the size of the tree became successively smaller with each increment increase of height. The sour orange stock was noticeably larger than the scion at the 5 and 12 cm insertion, the stock and scion girth was about equal at the 25 cm insertion, but above the 25 cm insertion, the girth of the scion was larger than the sour orange root stock. The greatest number of fruits per tree per year was at the 5 cm level, but there does not appear to be a significant difference in number of fruits at the different heights of budding. The average yearly weight of fruit seemed to be less at the 5 and 13 cm levels and was greater at 25 cm and above. Somewhat of a parallel situation existed with the Jaffa sweet orange . While there was no reversion in bud union character, the largest trees were those budded at 5 and 13 cm, with a break in size at 25 cm, and the smallest trees were at the 64 cm level as in grapefruit. Highest yields were at the 5 cm level of insertion, and a rather marked reduction in yield at the 64 cm level. Fruit weight gradually increased from the 5cm level up to the 51cm level, with a sharp drop at the 64 cm level.

Since these plantings were replicated and the experiment ran for 13 years, Murray concluded that the combinations gave the overall best performance budded at a height of 38 cm. Blondel , in Algeria, budded Clementine mandarin on trifoliate orange stock at essentially three height groups. There were 24 trees budded 0-10 cm; 102 trees at 10-30 cm; and 65 trees budded over 30 cm. Observations were made on the presence or absence of exocortis, character of the bud union,vertical greenhouse volume of the trees, and yield over a four-year period. He found that at the 0-10 cm budding only 13 per cent of the trees showed exocortis; at 10-30 cm, 58 per cent of the trees showed exocortis; and those budded over 30 cm, 86 per cent of the trees showed exocortis. This variation is a little difficult to explain merely on the height of budding and also the knowledge now available on mechanical transmission of the disease by contaminated budding knives . Blondel also found a marked variation in the size of the trees at the different budding heights, which he divided into three classes; large, medium, and small. At the 0- 10 cm level, 70 per cent of the trees were large, 13 per cent were medium, and 17 per cent were small. At the 10-30 cm level, 31 per cent of the trees were large, 16 per cent were medium, and 53 per cent were small. However, at the 30 cm level and above, only 10 per cent of the trees were large, 1.5 per cent of the trees were medium and 89.5 per cent were small. Obviously, the trees became smaller with increased height of budding and this might be due in this experiment to the increased incidence of exocortis. The character of the trifoliate stock also changed with the height of budding. At the 0-10 cm level, 91 per cent of the stocks were ribbed and 49 per cent were smooth. However, at the 30 cm level or higher, only 15 per cent of the trifoliate stocks were ribbed and 85 per cent were smooth. The author has also noticed that the bud unions of trifoliate stock and trifoliate hybrids become smoother with increased height of budding . Yields declined with height of budding. For the four-year period 1948-51, yields on the 0-10 cm height averaged 25 kg, at the 10-30 cm height it declined to 21 kg, and at the 30 cm level plus, it averaged only 13 kg. While it is unfortunate that Blondel’s experiment was confounded by the presence of exocortis, the results still agree pretty well with what Murray found in Trinidad, and with subsequent observations the author has made in California. Batchelor and Webber set out very extensive root stock plantings with all the major scion varieties at the Citrus Experiment Station, Riverside, and also with grower co-operators, during 1927, 1928, and 1929. The trees were budded at from 15-30 cm, somewhat higher than commercial practices at that time. The reason for this was to provide full bud union expression, and permit the girth of the trunk to be measured 10 cm above the union and 10 cm below the union as an index of scion or root stock volume, and avoid any measurements near the union where there might be over growths or under growths or flaring of the crown roots.

A comparison of these old experimental root stock trees and trees in commercial orchards , suggested that the greatest single difference between the two groups of trees was the height above ground at which the trees were budded . Further observations indicated that while bud union malformations are greatest with lemons , it might also occur with other scions to a lesser degree, and also differ with root stocks. This fact is consistent and obvious. The higher the trees are budded once a critical height is reached, the smoother the bud union. The lower the trees are budded, usually the more malformed the bud union. The incidence of gummosis was lower on the higher budded trees. With lemons affected by shellbark , shellbark is slower in expression on the high-budded trees and the severity of the symptoms is much less . Lemon shell bark would not be a problem if the trees were budded around 90 cm. The author felt that around 30 cm was a fairly critical height and, pending more observations and research, recommended to nurserymen in California that this height be considered as a bare minimum, especially for lemons. Reasons for the variation in tree response and bud union reaction with the height of budding are unknown. Some researchers say that it changes the top-root ratio, but it is difficult to understand how a few cm of trunk more or less can have such an effect on bud union and scion reaction. Neither can it be explained by the root-stem transition area. While in some plants the transition area is completed at leaves above the cotyledonary nodes, In the subgenus Citrus, the seeds are hypogynous in germination and the cotyledons remain below ground. Hayward and Long found that in Citrus, the transition area was completed at the cotyledonary node. In 1937, L. D. Batchelor planted out a small experiment with different heights of budding. The experiment consisted of two trees each of Eureka and Lisbon lemon budded on Rough lemon root stock at heights of 30, 45, 60, 90, 120 and 150 cm. Unfortunately, they served as guards for another experiment, were next to a road adjacent to a eucalyptus windbreak, and suffered some pilferage. Consequently, the trees were somewhat neglected and observations were few and sporadic. However, 20 years’ yield data is available. It must be pointed out that lemon trees in California are usually pruned annually. The trees were finally pulled in 1960 when the trees were 23 years old. Some final measurements of height and width and resultant top volume was done at that time. The data are presented in Table 5.

To achieve this one needs a healthy, productive, long-lived tree. It is increasingly evident that one of the most basic factors in achieving this end is the use of the best available root stock. The initial cost of a good tree is no more than a bad tree; but it sure makes a difference over the life of the orchard. differ, it is not surprising that there is, and has been, a wide range of diversity in citrus root stocks used throughout the world. Fawcett pointed out the resistance of the sour orange to gummosis or foot rot after the disease had decimated trees on sweet orange root stocks in the Azores about 1834. Also see Klotz , and Klotz and Calavan , or the section on disease later on in this monograph. This discovery led to sour orange root stock being the most widely used in most of the Mediterranean areas of Europe and Africa, in South America and the United States, and to a lesser extent in Australia, Central America, Mexico, the Caribbean, Iran, Iraq, Jordan, etc. The occurrence and spread of tristeza , a virus disease which is insect transmitted and affects principally various scions on sour orange root stock, precluded its general establishment in South Africa, and later rapidly eliminated plantings in Brazil, Argentina and other countries of South America, in California and Florida, and more recently in Spain and Israel.

Sour orange may still be the world’s leading root stock because of existing plantings as yet unaffected by tristeza, especially in certain of the Mediterranean countries,vertical farming supplies western and northern South America, Central America, Mexico, and the Caribbean. However, the use of sour orange as a root stock is rapidly decreasing because of the imminent threat of tristeza to all citrus areas. The discovery of mal secco disease in Sicily about 1923 , to which the sour orange root stock is particularly susceptible, has discouraged somewhat its use for lemon plantings in that area. The disease is caused by a fungus and root stock resistance would be desirable. The future of sour orange in California, where mal secco does not occur, appears to be limited to certain types of Lisbon lemon, because of phloem necrosis with Eureka types and sour orange necrosis . The sour orange never became established as a root stock in Japan, China, and Taiwan, which grow predominantly mandarin types of citrus, because growers for decades were aware the two were not congenial—perhaps, again, due to the presence of tristeza disease. In the United States during the early citrus developments in both Florida and California, sweet orange seedlings were commonly used. In the period after 1870, the expansion of commercial orange growing in Florida included the top working of scattered areas of sour orange seedlings growing wild in that state. The success of these top-worked groves in the often imperfectly drained but rich “hammock” lands of eastern Florida focused attention to the success of sour orange as a root stock for sweet orange varieties. When gummosis became prevalent in sweet orange seedling orchards, the sour orange was generally adopted as a root stock.

Later experience in Florida indicated that the sour orange stock was not satisfactory on the deep, well drained, very sandy soils of the “ridge area” in the central part of the state, nor in the shallow oolitic limestone soils of the southeastern coastal section. Thus, it came to be that the Rough lemon became the dominant stock until the 1970’s. At that time it became clear that a disease known as “blight,” or “young tree decline ” was particularly devastating on Rough lemon stock . In California, budded trees were first employed instead of seedling trees after the introduction of the Washington navel in 1873 and the Chapman Valencia in 1876. Sweet orange was commonly used as the root stock because at that time there were thousands of sweet orange seedlings in the Riverside area and probably very few sour oranges. After 1900, the sour orange and the sweet orange were used almost equally, with the predominant use of the sour orange in the heavier soils and the more saline and calcareous soils of the desert and adobe soils elsewhere. After 1920 the tendency was somewhat away from the sour orange, especially for lemons and oranges, but not for grapefruit. Thus, more sweet orange root stock was probably used in California than elsewhere. There was very little use in Florida and in South Africa. The identification of the virus nature of tristeza by Fawcett and Wallace almost completely eliminated the further use of sour orange in California. The threat of tristeza around the world to trees on sour orange root stock will result in complete replacement of this root stock in the very near future. About 1952, the trifoliate orange and a hybrid, the Troyer citrange, were introduced to the California growers , and the use of sweet orange has since been on a marked decline. In California, trees on sweet orange stock would not generally be grown by the nurserymen today, except on special order. There may still be some interest in South Africa, but it cannot be used in areas where gummosis is a serious hazard. In spite of the fact that there is no problem with tristeza, sweet orange is now almost completely replaced by other root stocks.

Until recently, Rough lemon was probably the world’s second leading root stock behind sour orange. Rough lemon has long been used in India, where it is commonly known as “jambhiri” and other names. In South Africa it was in use before 1900 due to the failure of sour orange stock because of tristeza. It is frequently referred to there and in Australia as the “citronelle.” Rough lemon had been extensively used in Florida for many years in the sandy “ridge” section and along the southeast coast. However, in recent years the incidence of “young tree decline” or “blight” on this stock has almost eliminated its use in Florida . It was never widely used in California except in the sandy soils of Coachella Valley,mobile vertical grow racks and most plantings since about 1950 in the sandy soils around Yuma, Arizona are predominantly on this stock. Recent decline of trees on Rough lemon stock in the Yuma area are a matter of great concern. The decline appears to be principally from the severe incidence of gummosis, which was probably aggravated by their cultural practices and irrigation methods. Citrus volkameriana, Pasq. used in Italy and Sicily since the mid-l900’s as a mal secco resistant stock for lemons, is one of the many Rough lemon cultigens. It would best be called the “Volkamer” lemon and not “volkameriana.” This cultivar is not recognized as a valid species by Swingle , Tanaka , and Hodgson , or anyone else. Tests show that it has exactly the same isozyme pattern as the type specimen of Rough lemon, which suggests that it is not a hybrid. Taxonomically the variety should be referred to as C. limon under the Swingle system and C. jambhiri under the Tanaka system. The grapefruit , Citrus paradisi, has not been used extensively as a root stock. Although tried to some extent in Florida, it did not gain favor, and this is also true in California. In California perhaps its best performance was with lemon tops, but its low and variable yields, its erratic performance, and its sensitivity to tristeza with orange scions have all but eliminated its use as a root stock anywhere. To some extent it may still have some limited use in some of the citrus growing areas of the Caribbean. The pummelo, or shaddock has never been used commercially as a root stock, although it may have been tried experimentally in a few areas—the Philippines, Indonesia and parts of the Malayan archipelago. The fact that the species is monoembryonic and produces only hybrid seedlings indicates variability would have been the most serious problem.

As a root stock the species is extremely susceptible to tristeza, which would further remove any consideration. The Cuban shaddock is an obvious hybrid. It differs from the regular shaddocks in that it is highly polyembryonic and that as a root stock it is tolerant to tristeza. In the early 1900’s it saw limited use as a root stock in Cuba, and hence the name. It was incorporated into the 1927 root stock trials at the Citrus Experiment Station, Riverside, but its performance was only mediocre and did not result in any commercial plantings. An ornamental citrus nurseryman in California does use it as a dwarfing stock for some of the Citrus varieties he grows. The trifoliate orange, Poncirus trifoliata, has been used for decades in Japan as a root stock for Satsuma mandarin and other mandarin and pummelo types. Japan grows over 400,000 acres of citrus on this stock, making it the principal user of trifoliate orange. The People’s Republic of China also has extensive plantings, principally mandarin, but some oranges and miscellaneous. In Taiwan it is also very popular. These countries predominantly use the “small-leaved” strains of the trifoliate orange as root stocks. This root stock has been used sparingly in Australia, Argentina, South Africa, Spain and Turkey, but is now seeing extended usage. This root stock’s cold hardiness, precociousness, and disease resistance have been its biggest assets. When interest was high in the United States about 1900 regarding establishing a Satsuma mandarin industry along the coast of the Gulf states, the trifoliate orange was also introduced and tried as a root stock. The introduction of citrus canker and the freeze of 1916-1917 essentially eliminated all orchards and the use of the stock, except for a limited area south of New Orleans, Louisiana. In California, old orchards on this stock were a curiosity and most were seriously affected by exocortis. Large commercial plantings did not start until around 1950 after the seriousness of tristeza was recognized, accompanied by the release of nucellar strains of the major varieties which were free of exocortis. In some areas of Japan, for some reason, it has been a relatively short-lived stock, necessitating inarching with Yuzu at an early age. Since about 1980 in California there has been interest in the Flying Dragon strain as a true dwarfing root stock and high density plantings. Also in Australia, considerable interest involves the use of selected trifoliate orange strains inoculated with a graft transmissible agent to attain controlled dwarfing or “viroidal dwarfing” as it may be called. All these aspects of the trifoliate orange will be discussed in detail at more appropriate sections of the text. The mandarins have not been widely used as root stocks. In the People’s Republic of China, the Ponki and Sunki have been principally used, although the Ponki is not used today. Taiwan also uses the Sunki . The Cleopatra mandarin has been used to a slight extent in Florida, particularly on the heavier soils, but it did not gain favor in Texas or Arizona. In California it was used to a limited extent after 1950, but that use has declined, although a few plantings may still be made on saline or alkaline soils. The Empress mandarin was used sparingly as a root stock in South Africa. Ponkan types have been occasionally used to a very limited degree elsewhere. Interest has persisted in the Philippines in the use of mandarin stocks, and current interest for nearly the past 50 years is centered on the Calamandarin. The taxonomic status of the variety is not known. The heen-naran and the nas-naran have occasionally been used in India. The limes have been frequently tried as root stocks, but few have endured. The East Indian lime has been used to some extent in Iran and Egypt and in a few other areas, but mostly with little success or commercial importance. In Israel, the Palestine sweet lime was the most commonly used stock for over a half century. Because of xyloporosis, it was shortlived and was inarched with sour orange at an early age. Both it and the sour orange are susceptible to tristeza and the recent occurrence of this disease has forced Israel in pursuit of new root stocks.

Heterozygous recombinant BC6S1 individuals were allowed to self-pollinate and progeny were marker-selected to obtain homozygous BC6S2 sub-NILs. These plants underwent self-pollination to obtain ample BC6S3 seed for replicated field experiments. We evaluated 62 BC6S3 sub-NILs in the 2009 field experiments. In the 2010 field experiments, a subset of 42 of the 62 subNILs was evaluated to allow increased replication per location while reducing genetic redundancy, as explained previously by Johnson et al. . Graphical marker genotypes for the 62 selected BC6S3 sub-NILs used in field experiments for the present study are presented in Supporting Information, Table S1.The chromosome 11 BC6S3 sub-NILs and the parental NIL from which they were derived were evaluated in replicated experiments at field locations in Salinas, California and in Davis, California over 2 yr. Summer and early fall in Salinas are generally cool and humid, which is conducive to late blight disease development, whereas Davis summers are warm and dry, with no rain, as is typical of California’s Central Valley tomato production areas. Additional information about field sites has been described by Haggard et al..Sixty-five genotypes and 45 genotypes were included in the 2009 and 2010 experiments, respectively.

Experiments were arranged in randomized complete block design . For both years,vertical hydroponic garden one plot per genotype per block was included, except for controls, for which there were two plots per block. In 2009, three blocks per location were used. In 2010, use of a reduced number of 42 sub-NILs enabled replication to be increased to five blocks in locations 1 and 2 and to four blocks in locations 3 and 4. At each of the four locations, each plot consisted of five plants spaced 0.30 m apart in rows separated by 1.02 m in locations 1 and 2, and by 1.52 m in locations 3 and 4. Border rows and plots with the cultivar E6203 surrounded each experiment at each location to reduce edge effects on the experimental plots. Standard horticultural field practices for processing tomato were used at all locations. Locations 1 and 2 were sprinkler irrigated, whereas locations 3 and 4 were furrow-irrigated, as needed.All traits were evaluated on a per-plot basis, as described by Haggard et al. . Vegetative horticultural traits were evaluated in all four locations. Late blight disease was only evaluated in Salinas because, as expected, this disease did not occur in Davis due to typical warm, dry summer conditions. Reproductive traits were only evaluated at Davis due to logistics of timely sampling of ripe fruit. Vegetative horticultural traits measured were plant height and width in cm, canopy density , and plant habit . H, W, CD, and HAB were obtained at both locations at 71 and 46 days after planting in 2009 and 2010, respectively. At locations 3 and 4, these traits were evaluated at 80 DAP in 2009 and at 68 and 73 DAP in 2010. From plant height and width, two secondary traits were derived, plant size and plant shape .

The reproductive horticultural traits measured or scored were as follows. DAP to maturity was evaluated at two stages of maturity: when each plant in the plot had its first ripe fruit and when 50% of fruit in a plot were ripe . Weight of 30 ripe fruit was evaluated when 50% of fruit in a plot were ripe . Yield in kg was evaluated when 95% of the fruit in a plot were ripe. Ripe fruit were used to obtain the weight of 100 seeds , which was measured only in 2009 due to labor limitations. The ripe fruit quality traits pH and Brix were measured using a pureed sample of 10 whole fruit obtained from plots with 50% ripe fruit using a pH Testr2 and a Reichert AR200 digital refractometer , respectively. Size traits obtained on ripe fruit were perimeter , width , and height . These traits were measured on flatbed scanner images of eight longitudinally sliced fruit per plot using Tomato Analyzer software , which refers to fruit length as height and fruit longitudinal circumference as perimeter. From FH and FW, the secondary variable fruit shape was obtained. Trait names, abbreviations, and brief descriptions are provided in Table 1. On 15 September 2009, Salinas locations 1 and 2 were inoculated with a local P. infestans isolate as described in Johnson et al. . In 2010 in mid-September, a natural P. infestans infection occurred in both locations, precluding the need for inoculation. As detailed in Johnson et al. , phenotypic scoring of late blight disease symptoms was performed visually and symptom data were used to calculate area under the disease progress curve for foliar and stem disease symptom progression . Lower AUDPC values indicate less disease symptom progress and therefore are indicative of increased disease resistance.A linkage map for the chromosome 11 introgressed region was constructed using DNA marker genotype data across 21 loci for 852 BC6S1 progeny .

The map was constructed with JoinMap 3.0 using the Kosambi mapping function and a 3-LOD significance threshold. We used a comparison of marker locations between our map and to the S. lycopersicum genome sequence  to estimate the physical size of the S. lycopersicum region replaced by the S. habrochaites introgression. QTL mapping for each trait was performed with the composite interval mapping module in WinQTLCartographer 2.5 using sub-NIL means obtained from ANOVA for each trait. QTL mapping was performed using CIM Model 6 and the forward and backward regression method with a walk speed of 1 cM and a window size of 2 cM. Trait-specific permuted LOD thresholds were empirically established for each trait using 1000 permutations in WinQTLCartographer. A QTL for a trait was considered significant at P # 0.05 if the peak LOD value exceeded the trait-specific permuted threshold. Multiple QTL were declared for a single trait when the LOD values between significant peaks within the introgressed region decreased below the significance LOD threshold for at least two contiguous markers. Each significant QTL was denoted by trait name, location, and year. For example, DAP1st34_2009 is a QTL detected in the analysis of DAP1st data from locations 3 and 4 in 2009. A linkage map figure showing locations of significant QTL was constructed using MapChart2.1 . QTL locations were indicated as 1-LOD bars and 2-LOD whiskers . For easy reference and purposes of discussion, QTL were assigned to QTL trait groups based on coincidence of their 1-LOD support intervals. Although a few of the QTL had 1-LOD support intervals that extended beyond the boundary of their assigned group, their peak locations supported their placement in these groups. Comparisons were made among QTL for disease resistance traits and horticultural traits for QTL coincidence by visual inspection of their chromosomal locations on the linkage map. A statistical test based on the hypergeometric probability distribution was used to calculate QTL correspondence,vertical home farming the probability of obtaining the observed number of matching QTL by chance. A QTL match was declared when the one-LOD support intervals overlapped. The number of comparison intervals was six, based on the average size of our QTL and the overall map distance of the introgression . Our QTL locations were also compared with those previously reported for both disease resistance and horticultural traits on chromosome 11 in tomato and in potato, based on common markers as well as genomic sequence data for both crop species. Sources used for QTL location comparisons included the following: tomato ; potato ; and genomic sequences . When common markers were not available, the Tomato-Expen 2000 map available on the Sol Genomics Network was used to facilitate map alignment.Truncation selection was applied sequentially for LEAF, YLD, FP, and 30Wt to identify breeding lines potentially useful for development of tomato varieties with improved resistance to P. infestans. Out of42 sub-NILs, the first round of truncation removed 9 lines with leaf resistance scores below that of E6203 in 2 years or locations. The second round removed 2 lines with YLD ,66% of E6203 in 2 years or locations, whereas the third round removed 7 lines with FP ,92% of E6203 in 2 years or locations. The final round removed 2 lines with 30Wt ,80% of E6203 in 2 years or locations. Maturity was also considered; however, two lines with significantly later maturity than E6203 in at least 1 year or location were selected due to their relatively high levels of foliar resistance to P. infestans . At the end of the process, 11 lines were chosen.There were significant differences among genotype means for all traits, except for fruit pH in 2009 . In general, subNILs with S. habrochaites introgressions at the marker loci At5g04590 and TG400 were significantly later maturing than control cultivar E6203 in at least one trait and year or location combination; however, some lines were exceptions to this trend.

Most sub-NILs with S. habrochaites introgressions at the marker loci At5g04590 and TG400 also had significantly reduced FH compared with E6203. Relative to E6203, NIL11 exhibited significantly greater foliar resistance to P. infestans , but only in 2010. NIL11 also had later maturity , reduced FH , FP, 30Wt, SW, and W, increased H and pH , taller, narrower SH, and more prostrate habit than E6203. Sub-NILs 08GH3723, 08GH3999, 08GH4018, 08GH4106, and 08GH8032 displayed significantly greater foliar resistance than E6203 in 2010; however, only 08GH8032 performed significantly better than E6203 in 2009. 08GH4228 had significantly better stem resistance than E6203, but only in one location in 2010. None of the lines with significantly greater resistance showed any significant decrease in YLD or BRIX; however, several of them had significantly delayed maturity , reduced fruit size and 30Wt, and more upright HAB.None of the QTL in Hort 11-1 was detected in all years and locations. The fruit size QTL in this group were identified at both Davis locations, but only in 2010. Similarly, the Brix QTL was mapped only in 2009. The maturity QTL in this group were only detected in a single year and location, suggesting an environmental influence on QTL expression. Similarly, none of the QTL in Hort11-2 was identified in all years and locations. The two QTL for SW were detected in both locations; however, data were only collected for this trait in 2009, so their stability over the years is unknown. Other QTL, such as those affecting Brix, pH, and FP were mapped in both locations, but only in a single year. QTL for plant size were detected over both years, but only at the two Salinas locations in 2009 and only at a single location in 2010. Their effect may have been accentuated by the narrow row spacing, relative to that at the Davis locations. QTL for SH were detected in this group with opposite effect depending on location, another manifestation of QTL · E. In 2010, the S. habrochaites allele conferred a shorter, wider phenotype at the Davis locations, but a taller, narrower phenotype at the Salinas locations. This may also be due to the differences in row spacing between the two pairs of experiments. The difference in LOD peak location and only minor overlap between these two QTL may also indicate that these are separate, linked loci of opposite effects that are most pronounced in contrasting environments. In the Hort11-3 QTL group, the DAP50, FH, and FP QTL appear to be stable, being identified in each year and location. However, the LOD peaks for the FH and FP QTL over the 2 yr are sufficiently far apart to suggest that there may be multiple linked loci contributing to the effects for each trait. QTL for H were mapped at both Davis locations in both years but only at one of the Salinas locations, and only in 2010. QTL for CD were also relatively stable, being detected in three of the four year/location combinations; however, an additional CD QTL was found at location 4 in 2009, distal to those three QTL. QTL for FW, FS, and pH were identified in both locations in 2009, but not in 2010. The HAB QTL was mapped only in the Davis locations in 2010 and was not detected in 2009.

Further investigation found that transcription factor NAC4 acted downstream of AFB3 to regulate lateral root growth in response to nitrate, but did not affect primary root growth, indicating that AFB3 is likely to be involved in two dependent pathways to modulate root system architecture. Furthermore, AFB3 and NAC4 gene expression in response to nitrate treatment depends on the nitrate transport function of NRT1.1. Moreover, peptide-mediated signaling has been found in N control of root growth. The Arabidopsis CLE gene was found to be induced by N-deficiency, and over expression of CLE inhibits lateral root elongation but not initiation. The peptide sequence of CLE is homologous to CLV3, which binds to CLV1 and the clv1 mutant showed increased lateral root length under low N conditions. The transcript levels of CLE were increased in the clv1 mutant, suggesting a feedback regulation of CLE by CLV1. Transgenic lines with increased CLE levels in clv1 did not inhibit lateral root growth, indicating that the inhibition of CLE3 on lateral root development requires CLV1. Altogether, the N-responsive CLE-CLV1 peptide-receptor signaling module restricts expansion of the lateral root system in N-deficient environments. Although nitrate is a crucial nutrient and signaling molecule, its distribution in soils is heterogeneous. To adapt the prevailing nitrate conditions, plants have evolved a systemic response mechanism. NRT2.1 was the first molecular target identified in long-distance signaling reflecting root responses to environmental nitrate conditions.

Plants were grown using a 1 mM NO3 − solution, then the root was split into two parts,vertical aeroponic tower garden one subjected to N-free treatment and the other one treated with 1 mM NO3 −. Both 15NO3 − influx and the transcript level of NRT2.1 were increased in the NO3 −-fed root. Recent findings revealed that the C-terminally encoded peptide originated from N-starved roots; located in xylem vessels, it sends root-derived ascending signals to the shoot before being recognized by a leucine-rich repeat receptor kinase, CEPReceptor 1 , and then inducing the expression of CEPD polypeptides. CEPD sent long-distance mobile signals translocated to each root and upregulated the expression of NRT2.1. The activity and expression of NRT2.1 in plants were inhibited when supplied with high N. Lepetit’s lab configured a forward genetic approach using a transgenic line expressing the pNRT2.1::LUC construct as a reporter gene. The mutant hni9, showing increased expression of NRT2.1 under high N supply, was isolated and the mutation was found in IWS1, a component of the RNAPII complexes. Further investigation revealed that the levels of the H3K27me3 on NRT2.1 chromatin decreased, resulting in the upregulated expression of NRT2.1 in response to high N supply in the iws1 mutants. Thus IWS1 is likely to be involved in the transduction of N systemic signals through controlling the expression of NRT2.1 in plants. Another important player participating in root foraging, TCP20, was identified by Crawford’s lab using the yeast one-hybrid system to screen the transcription factors that can bind to the fragment of nitrate enhance DNA. TCP20 was found to be able to bind to the promoters of NIA1, NRT1.1, and NRT2.1.

The tcp20 mutants exhibited deficiencies in preferential lateral root growth on heterogeneous media in split-root experiments, indicating that TCP20 can regulate the preferential growth of lateral roots in high nitrate zones, thus playing an important role in the systemic signaling pathway. Recently, using an electrophoretic mobility shift assay , the DNA-binding sites of TCP20 in a 109 bp NIA1 enhancer fragment were found to be in close proximity to NLP7 and NLP6 binding sites. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that NLP7 and NLP6 can interact with TCP20 and both the PB1 domains of NLP6&7 and the glutamine-rich domain of TCP20 are necessary for protein–protein interaction. Further work will be needed to elucidate the underlying molecular mechanism explaining the involvement of TCP20 in systemic signaling.Root microbiota associate with every land plant and show community compositions and dynamics that are distinct from the surrounding soil microbial community . Both rhizosphere and root endosphere microbiomes affect plant health and soil health via processes such as mineral and nutrient turnover and pathogen suppression . Attribution of specific processes to distinct microbial players or populations is challenging because soil ecosystems are among the most complex environments on Earth . Soils are made up of a multitude of heterogeneous abiotic and biotic components that interact in a dynamic fashion over a range of spatial and temporal scales.Soil type, together with climatic characteristics, allows for the development and activity of biological constituents that are specific to a given soil in a particular location and can vary dramatically among soils and locations .

Those biological constituents can include plants, insects, bacteria, archaea, and fungi, which all contribute to and feed off of the bio-geochemical cycles in a given soil. The resulting complex network of interactions is extremely challenging to disentangle due to technological limitations and insufficient information in biological and chemical reference databases . Furthermore, soils contain a vast diversity of microorganisms, which are heterogeneously distributed and engage in frequent horizontal gene transfer. Despite this, most root microbiome studies present data from single time points or single locations and primarily conduct amplicon sequencing combined with limited information on plant or environment. Although the average values provided by such studies may suggest some interactions or mechanisms, few studies follow up with the comprehensive sampling necessary to definitively understand these mechanisms and interactions. In addition, single-point studies are difficult to compare or extrapolate to other environments or plants because measured values can vary dramatically over time . Soil and other environmental characteristics can be important indicators of bio-geochemical processes that have occurred in the past or are ongoing. Generally, few root and soil microbiome studies take advantage of the relatively inexpensive techniques to measure soil characteristics. Data on parameters such as pH, volumetric water content, temperature, and salt concentration could allow researchers to draw correlations between microbial activity, plant productivity, and environmental parameters and facilitate opportunities to cross reference studies conducted under comparable conditions. In the last decade, the root microbiome research community has made tremendous progress in understanding the complexity of soil ecosystems through improvements in experimental methods at both laboratory and field scales. These exciting technological and scientific advancements pave the way forward in root microbiome research. This review summarizes recent technological advancements and the resulting research opportunities categorized by ecosystem component and scale ,vertical gardening in greenhouse and ends with an outlook and potential applications for phytobiome research.Microbial colonization of the root and rhizosphere can significantly affect root phenology and metabolism. Roots demonstrate enormous phenotypic plasticity with respect to anatomy, shape, cell type, cellular structure, metabolism, and biochemical composition, and these characteristics contribute tremendously to root exudation variation and, as a result, to microbial community differentiation . These reciprocal interactions between roots and microbes are not well understood but their direct link showcases the fact that, for understanding root microbiomes, a foundational understanding of root biology is required. Although hyperspectral imaging of leaves has been broadly applied to monitor plant health, even simple imaging of intact roots has lagged behind due to the challenges presented by the opaqueness of soil . Ideally, imaging of root architecture, microbes, and chemical composition as well as visualization of fluxes such as carbon flow through plant compartments and into the soil would be conducted at multiple temporal and spatial scales. Most current methods for analyzing root growth either require artificial growing conditions , are severely restricted in the fraction of roots detectable , or are destructive . For example, many root phenotypic datasets have employed coring or “shovelomics”, subsequent root picking and washing, and imaging using light imagers such as the RhizoVision Crown platform . This method provides valuable information about root architecture; however, it is extremely laborious, it is often not feasible to excavate deep roots, it can remain unknown how much of the root system was recovered and scanned, and root excavation often times terminates the experiment for the selected plants. All of these methods are severely limited because they are destructive, low throughput, or artificial. The later point is particularly important because root architecture can be significantly affected by plant genetics, environmental conditions, soil type, and rootcolonizing bacteria and fungi . Magnetic resonance imaging presents a noninvasive modality that addresses some of the limitations of other root measurement techniques.

When coupled with an analysis pipeline in an automated system, MRI can monitor root mass, length, diameter, tip number, growth angles , and spatial distribution in a high-throughput manner . Similarly, X-ray computed tomography scanning can provide a comprehensive picture of root systems as long as the roots have a diameter larger than the instruments’ resolution . Hence, small plants or young roots are not likely to be resolved well. Another limitation common to both MRI and CT technology is that plants must be grown in pots that fit into the imaging machines and the applicability of MRI and X-ray CT in three-dimensional imaging of root systems across various pot sizes was recently evaluated . Although both MRI and CT were able to resolve high-quality 3D images of root systems in vivo, the reconstructed length and image details differed significantly between the two methods. In small pots, CT outperformed MRI and provided more details thanks to higher resolution whereas, in large pots, MRI was able to display root systems more comprehensively than CT. Soil features such as minerals and burrows can be resolved with CT, while MRI can measure water content in roots and soil. Both CT and MRI, struggled with roots thinner than 400 mm .There are a number of imaging tools that can resolve root vasculature. Using Synchrotron X-ray microtomography, Milien et al. contrasted the 3D images of vascular systems of successful and unsuccessful graft interfaces in vine rootstocks. Others have applied synchrotron X-ray microtomography to visualize drought-induced embolism in various plant species , to correlate root hair with rhizosphere soil structure formation , and to quantify root-induced changes of rhizosphere physical properties . Although synchrotron X-ray micro-CT can render unprecedented detail into the microanatomy of plants and microorganisms, the focus window is relatively limited and biological samples tend to lose viability as a result of the intense X-ray radiation. There are various other imaging methods that have been recently developed or applied to phytobiome research, including super resolution confocal imaging, which can enhance 3D mapping of root and microbial or fungal cells and showcase green fluorescent proteins , and correlative confocal and focused ion beam tool with integrated scanning electron microscope, which allows for extremely fine-scaled 3D mapping . When applied individually or in combination, the above mentioned imaging methods will provide opportunities to visualize plant tissue and attached or internally residing bacteria, fungi, and viruses at unprecedented resolution, as well provide information about their physical and chemical context. Because root development is vital for plant health, expansion of root image databases and novel correlations between above- and below ground plant features will enhance our understanding of plant response to environmental and biological stimuli.An important goal of the plant-microbiome field is to discover beneficial or deleterious effects of microbes. This means that recording and understanding plant phenotypes and linking them to microbiome variation is key. Similarly, plant microbiomes are intimately tied to the background soil; hence, monitoring soil characteristics is important but can be challenging and labor intensive at appropriate temporal or spatial scales.Unmanned aerial vehicles equipped with RGB cameras, infrared cameras, multi-spectral and hyperspectral cameras, GPS, navigation systems, programmable controllers, and automated flight planning have emerged as powerful tools for nondestructive, high-throughput field phenotyping that can be performed throughout the growth season . This has removed a bottleneck in phenotyping but automated processing of this data still presents various challenges, which are discussed elsewhere . Monitoring of agricultural fields using drones has become popular among researchers to more accurately plan and manage their experimental operations. Drones can produce precise maps of soil characteristics and plant characteristics , as well as determine irrigation needs, nitrogen levels, and pest occurrence . RGB, IR, and hyper- as well as multi-spectral cameras attached to drones can collect images of the above ground portion in a range of wavelengths.

Similarly, lateral roots are associated with distinct microbial communities, differing between tips and bases, as well as between different types of lateral root . One trait influencing the differential microbial colonization of root tissues could be the differential exudation profiles of the distinct root parts. This is illustrated in the following example. Clusterroots are densely packed lateral roots formed by some plants growing on extremely nutrient-poor soils; these roots exude high amounts of organic acids and, in some cases, protons, to solubilize phosphate. The low pH and carboxylate-rich rhizosphere of cluster roots is associated with a specialized rhizobiome, dominated by Burkholderia species that metabolize citrate and oxalate. Besides organic acids, mature cluster roots also exude isoflavonoids and fungal cell wall degrading enzymes, leading to a decrease in bacterial abundance, as well as fungal sporulation. Taken together, cluster root exudates not only solubilize phosphate, but also regulate microbes in such a way that they do not interfere with phosphate uptake. Beyond this example, spatial patterns ofmetabolite exudation are largely unexplored.We hypothesizethat such patterns exist in all root systems forthe following reasons: spatially distinct organic acid exudation is atrait of all root systems ; spatially distinct exudation was similarly detected for strigolactones, amino acids, and sugars;and root nutrient uptake,hydroponic net pots which is sometimes coupled with proton transport, can also exhibit spatial patterns.

Overall, spatially defined metabolite exudation by distinct root parts is likely an important factor in structuring the rhizobiome. Future studies should aim at characterizing spatially distinct rhizobiomes and their functional traits, and at investigating spatially distinct root exudation.Roottips are not only associated with high numbers of bacteria , but also produce border cells and mucilage , crucial for plant–microbe interactions. Depending on the root meristem organization,border cells are released into the rhizosphere either assingle cells or as border-like cells.Single maize border cells stayed alive in soil for months, likely due to the presence of starch deposits, whereas arabidopsis border-like cells survived for only 2 weeks. Border cells have a transcriptional profile distinct from root tip wells, with overall lower primary and higher secondarymetabolism. ABC transporters constitute a large fraction of differentially expressed genes, which is consistent with transport of secondary metabolites. Secondary metabolites are likely central to the role of border cells in defense against pathogens. Pathogen attack can result not only in higher border cell production and release, but also in higher mucilage production by border cells and root tip cells. Mucilage contains proteins with antimicrobial functions , as well as extracellular DNA involved in defense against fungi and certain bacteria. Importantly, mucilage is also produced under nonpathogenic conditions, serving as a lubricant for the root environment and stabilizing soil particles. Interestingly, mucilage also provides distinct carbon sources for microbes, thus influencing rhizobiome composition. Border cells similarly interact with nonpathogenic microbes : they release flavonoids that attract rhizobia, uncharacterized compounds that induce branching of mycorrhizal hyphae, and arabinogalactans that trigger biofilm formation of specific beneficial bacteria . The full extent of how border cells and mucilage shape root–microbe interactions remains unclear.

It is tempting to speculate that the specialized metabolism of the border cells results in a distinct exudation profile of not only proteins and mucilage, but also low molecular-weight compounds that could serve as microbial nutrients or as signaling compounds. Further research should focus on the genetic and physiological differences between border cells and border-like cells, as well as on the transport proteins involved in exudation of low-molecular-weight compounds, DNA, and proteins.Plant–microbe interactions are not only defined by plant root morphology and plant-derived exudates, but also by microbe–microbe interactions . Thus, we focus further here on microbial communities. Specifically, we discuss: how plant exudates influence microbial diversity; how plant-responsive microbes are identified; how microbes interact and how mycorrhizal fungi influence root–bacteria interactions. The rhizosphere serves as carbon-rich niche for the establishment of microbial communities, in contrast to bulk soil, which is rapidly depleted in carbon and other nutrients by heterotrophic microbes. Given that the ability of microbes to metabolize plant-derived exometabolites might determine their success in the microbial community, several studies have investigated whether the diversity of plant exudates correlates with microbial diversity. Some studies found higher plant diversity was associated with higher microbial diversity, and that the addition of a diverse exudate mix to plant monocultures increased microbial diversity. Interestingly, isolates from soils with a diverse plant community consistently exhibited less-narrow niches and displayed less resource competition than did isolates from low plant diversity environments. Although on a global scale, environmental factors had a larger impact on microbial diversity than did plant diversity, we can conclude that, on a local scale, high plant diversity likely promotes a diverse microbial community.

The large diversity of microbial communities is a current challenge for plant–microbe research, because it is impractical to study questions such as how members of a community interact, and what specific traits a microbial community has. Therefore, many studies currently aim at identifying the subset of microbes responsive to plants. Strikingly, only 7% of bulk soil microbes increased in abundance in the rhizosphere compared with bulk soil, which reduces the number of taxa to investigate from thousands to hundreds. Other approaches to identifying plant-responsive microbes have focused on transcriptional profiling. Compared with soilabundant microbes, plant-associated microbes exhibited distinct transcriptional responses to plant exudates and, intriguingly, displayed distinct phylogenetic clustering. Network analyses further revealed that rhizosphere microbes displayed higher levels of interactions than did bulk soil microbes. These studies illustrate the potential for the identification of a distinct set of plant-responsive microbes. The above points highlight how plants influence microbial communities. However, the members of microbial communities also interact with each other. Compellingly, it is still unclear whether microbe–microbe interactions are predominantly positive or negative. Network analyses reported predominantly positive intrakingdom interactions. By contrast, laboratory growth assays identified competition as the major factor in shaping isolate communities, and cooperation could only be detected for 6–10% of the isolates. One major difference between the two experimental approaches is that the former investigates a natural system, whereas the latter is based on the ability to culture microbes. Isolation of microbes introduces a bias, since it can select against cooperators, precluding obligate syntrophs. Further evidence that at least some microbes avoid competition was provided by co-cultivation experiments. Environmental isolates: displayed high substrate specialization; did not necessarily take up the compound with the highest energy; and diverged in substrate use when cultivated for several generations. In addition, some metabolites exuded by microbes could be metabolized by others,blueberry grow pot suggesting potential cross-feeding between community members. The above findings suggest complex interactions of microbes. It remains to be resolved in which situation competition or cooperation dominates communities. However, it is evident that microbial interactions are based on altered gene expression. Microbes responded to competing bacteria or even close relatives by differentially regulating genes involved in metabolite exudation and transport processes, making the study of microbial transporters a compelling topic for future studies. Thus, metabolite uptake, release, and sensing are important factors in shaping microbial communities. Metabolite turnover in soil is influenced not only by plants, but also by functionally diverse bacteria, fungi, and animals. Plant–fungal and plant–animal interactions in the rhizosphere go beyond the scope of this review, and are discussed elsewhere. Here, we provide a few brief examples focusing on the impacts of mycorrhiza on rhizobiomes and exometabolite turnover. Endomycorrhizal fungi receive a significant fraction of the carbon fixed by plants . Interestingly, these fungi also exude sugars,shaping a distinct bacterial community. Likewise, Ectomycorrhiza receive carbon from plants, and form a dynamic bacterial community; they even participate in plant-to-plant carbon transport. The field of fungal microbiomes is nascent: if and how fungi control exudation, whether fungal microbiomes have beneficial functions, and how plant and fungal microbiomes influence each other are all unknowns. Although many questions remain, these recent findings already suggest that a holistic view of rhizosphere nutrient cycling and signaling exchange via exometabolites requires a whole-community approach including all domains of life.Plant exudates shape microbial communities. Overall, plants exude up to 20% of fixed carbon and 15% of nitrogen, which includes an array of simple molecules, such as sugars, organic acids, and secondary metabolites, as well as complex polymers, such as mucilage.

Although every plant produces exudates, the amount and composition of root exudates varies. First, exudation is defined by the genotype of the host, as observed in the distinct exudation patterns of 19 arabidopsis accessions. Strikingly, the amount of variation between the accessions depended on the metabolite class investigated. Glucosinolates displayed most, flavonoids medium, and phenylpropanoids low variability. Second, exudation changes with plant developmental stage: with increasing age, arabidopsis sugar exudation decreased, and amino acid and phenolic exudation increased. Third, exudation is modulated by abiotic stresses: the amounts of exuded amino acids, sugars, and organic acids changed in maize grown in phosphate-, iron-, nitrogen-, or potassium-deficient conditions. In addition, phosphate-deficient arabidopsis plants increased coumarin and oligolignol exudation, heavy metal-treated poplar induced organic acid exudation, and zinc-deficient wheat increased phytosiderophore exudation. Differential exudation is a plausible mechanism by which plants could modulate their interaction with microbes, as exemplified by the correlation between exudation patterns and rhizobiome variation reported for eight arabidopsis accessions. Differential exudation modulated by transport proteins is discussed below.Plant-derived exometabolites need to cross at least one membrane to transit from the cytoplasm of root cells into the rhizosphere. There is considerable discussion as to what degree plants are able to regulate this transport. In general, different modes of transport could be envisioned. First, small, hydrophilic compounds could diffuse from the root into the rhizosphere, driven by the large concentration gradient. Second, channel proteins could facilitate such diffusion. Third, active or secondary active transporters could shuttle compounds across membranes against a concentration gradient. Diffusion of compounds can only be relevant in young root tissue, which is still devoid of Casparian strips or suberized endodermis that both block apoplasmic flow in adult tissues. Transport proteins involved in exudation are mostly elusive. From a conceptual point of view, plasma membrane-localized exporters likely have a direct, and vacuolar transporters an indirect effect on exudation. The vacuole is a major storage organelle for many metabolites detected in exudates, such as sugars, organic acids, and secondary metabolites. Alteration of vacuolar transporter levels impacts vacuolar and cytosolic concentrations and, thus, can influence metabolite exudation into the rhizosphere. The few characterized transporters involved in exudation are essential for the transport of specific compounds, and are presented in Table 1. Since only a few transporters involved in exudation have been characterized, we suggest additional families that might be involved in the process. To complete the picture of metabolite exchange between roots and soil, Table 1 additionally contains a few important plasma membrane-localized metabolite uptake systems. Below, we discuss the evidence for transport processes involved in the import and exudation of compounds detected in root exudates, such as sugars, organic acids, and secondary metabolites.Sugars constitute a significant fraction of exudates, and are a main carbon source for microbes. Interestingly, many more sugar uptake than release systems have been described. Sugar Transport Proteins utilize high extracellular proton levels to import sugars, and mutation of STPs leads to higher external sugar levels. Sugars Will Eventually Be Exported Transporters are sugar uniporters, and all root-expressed members localize to the vacuole. Due to an alteration of root sugar homeostasis, SWEET mutant plants exhibited higher sugar export from roots compared with wild-type plants, and were more susceptible to disease. Intriguingly, no transporters directly exporting sugars into the rhizosphere have been characterized so far, and it is debated whether sugar exudation is a transport-driven process at all. Potential evidence for passive sugar efflux was supported by the observation of higher sucrose concentrations around young, permeable root tissue than around older, less-permeable root tissue. However, because sugars are synthesized in leaves, they still need to be unloaded either from phloem or from root cells to be exuded into the rhizosphere, a process likely depending on transporters due to the hydrophilic nature of sugars. A further indication of the presence of elusive transporters is the differential sugar exudation in various environments, as shown, for example, for maize grown in potassium-, phosphate-, or iron-deficient conditions.

For many ENMs such as nanoceria,reactivity is highly size dependent. To accurately model material fates thus requires understanding how material surface properties affect integrity, how both change under varying environmental conditions such as pH, clay content,and organic matter content,and how surface properties and particle reactivity affect physicochemical processes that are parametrized in far-field models. This is especially true for ENMs used as pesticide delivery mechanisms, including carbon nanotube composites with specifically reactive surface monomers. Yet only recently has modeling attempted to address differing properties of a material’s structural variants .Evaluating computational model predictions is a challenge for ENMs, which presently are estimated to occur in the environment at low concentrations.Also, detection methods for ENMs in environmental media and distinguishing ENMs from natural chemical analogs are still under development,with more evaluation strategies needed including a framework for validating new ENM analytical detection methods.Fullerenes from incidental sources were quantified in river sediments collected from locations across the globe and quantified in the atmosphere over the Mediterranean Sea.Perhaps related to a viable exposure scenario,stacking pots fullerenes were quantified at relatively high concentrations in treated wastewater effluent and at ng/L to μg/L concentrations in river waters receiving effluent discharge.

While not necessarily nanoscale, similarly high concentrations of TiO2 were reported for sediments sampled near a WWTP outfall.The greatest uncertainty in ENM exposures is near-field , at the receptor where toxicant dose manifests as internal dose. Heteroaggregation is a dominant fate process for ENMs when they interact with natural colloids.Given sufficient residence time for ENMs in environmental matrices, heteroaggregation and to a lesser degree homoaggregation will affect localized compartmentalization, including stability in the water column and therefore, sedimentation.However, these processes do not preclude biological impacts under simulated environmental conditions, as has been shown for nanoceria in a complex aquatic mesocosm.Exposure can be confirmed by quantifying receptor body burdens, thereby allowing for quantitatively relating near-field exposure to biological effects.Thus, in the absence of detailed, biologically complex, near-field models for local exposures to environmental receptors, the ability to trace ENMs to biological receptors sampled directly from the environment becomes the best available approach to relate far-field exposures to biological impacts.Overall, material flow models and multimedia modeling of ENMs have advanced to inform ENM ecotoxicology. Available far-field modeling frameworks are adaptable to changing inputs despite uncertainties in production volumes. Major uncertainties remain at the nexus of ENM surface and core chemistries as related to nanomaterial transport, aggregation, and degradation characteristics. However, fundamental research is needed to discover and parametrize complex fate processes.

New approaches, such as assays that can be used to rapidly probe surface associations,demonstrate how to populate far-field models and how to determine near-field exposures associated with effects. Although existing models can simulate particle movement, deposition, and some transformations, the knowledge state regarding ENM environmental exposure conditions via measurements or modeling simulations cannot be assumed to accurately represent actual conditions at biological receptors.Many of the outstanding research issues and recommendations for evolving ENM ecotoxicology are echoed in the discourse for other chemicals of emerging concern .These include the need for systematically understanding ENM and decomposition product toxicity across various receptors within linked levels of biological organization,quantifying actual exposures and uptake into environmental receptors,gaining mechanistic insights into and biological markers for acute and chronic low level exposures,and understanding how environmental factors including cocontaminants affect ENM transformation and biological impacts. Still, how can the potential for exposure and impacts of ENMs be anticipated, prevented, managed, or mitigated? Further, what data and tools do decision makers need to inform their work? Innovation in nanotechnology hinges on having the science to evaluate ENM safety. While no formalized process for incorporating all exposure conditions and concepts of ENM transformation, dose, and body burden into risk assessments currently exists, a proposed framework approach to risk characterization over the life cycle of ENMs has been published and is available.This framework advocates an initial decision cutoff in regards to exposure; in the absence of exposure, the need for further assessment is diminished or negated.In this available framework, ENMs that are certain to rapidly dissolve into ionic components in a destined environmental compartment would be assessed for risk based on the released components rather than the original nanoparticles.

Persistent ENMs are expected to accumulate in matrices such as sediments.The consequences of ENMs to successive generations, biodiversity, and ecosystem services are not addressed by model organism-specific assays of discrete growth and mortality.Nonetheless, in this available framework, toxicity end points associated with standardized testing protocols for sediment, aquatic, and terrestrial standard population-level end points over short and long time frames are advocated for assessing hazards of simulated ENM concentrations in the environment.In this framework, sunlight is an environmental variable, bio-accumulation is measured, and ENM modifications during product and material life cycles that may change bio-availability are considered.While such a framework has broad organizational appeal, priority setting within the framework is required and thus could focus on tests that are relatively well aligned with likely exposure scenarios. Even with a risk assessment framework that considers ENMs across product life cycles and considers sediments, water, and soil in testing end points,major hurdles hinder regulatory agencies, and research scientists, in using concepts such as exposure conditions, ENM transformation, dose, and body burden in interpreting biological and computational findings for assessing risks. Toxicity tests developed for dissolved chemicals typically require significant modification for use with ENMs.Tests may not apply to ENMs if they are not appropriate for solids.Additional scientifically based hazard information from the peer-reviewed literature may or may not be available for consideration. ENMs used in ecotoxicity tests,strawberry gutter system which are sometimes laboratory-synthesized to overcome uncertainty regarding proprietary coating or other commercial formulations, may be insufficiently analogous to allow for extrapolating information or risk comparisons. Issues include the need to know test material characteristics and how they relate to testing results and the ENM life cycle. Even if an initial risk assessment considers ENM solubility,ENM dissolution is not instantaneous; therefore, at what stage of dissolution does the contaminant no longer pose a hazard as an ENM? Also, where biological impacts stem from ENM surface characteristics, how can mass concentration be used to judge hazards? Environmental ENM effects in bench top experiments can be indirect, stemming from physical nutrient depletion,or amplifying organism uptake of cocontaminants.Other indirect physical effects derive from ENMs adhering to the organism surface,light shading,or internal food displacement.Near-field exposures can result in biological hazards from specific ENMs based on their properties .By definition, ecological risk assessment is “the process for evaluating how likely the environment will be impacted as a result of exposure to one or more environmental stressors.” ERA involves predicting effects for individuals, populations, communities and ecosystems, and concerns itself with valuable ecosystem services such as nutrient cycling.Thus, conducting ERAs for ENMs could benefit from an ecological outlook. All levels of biological organization, and interactions between them, would be considered when assessing responses to ENM exposure . Release and exposure scenarios , use of functional assays for assessing environmental compartmentalization ,and combined life-cycle and multimedia modeling have important roles in focusing ENM ecotoxicology. Less recognized is that mechanistically based models of dynamic biological effects are informed by hazard assessment research. Different types of process-based, dynamic models allow for predicting effects from exposures stepwise, starting at sub-cellular levels, into individuals, through populations, and conceivably to communities and ecosystems. Developing process-based models requires researching key effects processes and ecological feed backs.Models are formalized to describe interactive processes culminating in toxicity such as reactive oxygen species generation and cellular damage. Process-based mathematical expressions evolve with empirically based discoveries or through model reconciliation with experimental data. Parameters are independent of toxicity testing protocols, although models could be informed by standard test results. Thus, ENM ecotoxicity research could support predictive toxicology by informing and populating process-based, dynamic ecological effects models.

A comprehensive fate and effects research agenda is needed for addressing ENM quantification in complex media.Such an agenda has allowed for assessing experimental compartmentalization,and sensitively assessing environmental persistence, toxicity, bio-accumulation, trophic transfer,and indirect effects from the uptake of ENMs coated in other hazardous materials.Such research could substantially inform ENM risk assessment for a relevant environmental exposure scenario. However, most ENMs have not been studied comprehensively along the entire exposure and effects continuum . Further, the approach is not sustainable. Rather, the need is to develop efficient approaches applicable within an overall approach to rapidly evaluate the large number of ENMs under commercialization . A research agenda that focuses on distilling key determinants of exposure and hazard for ENM-environment systems that can be measured experimentally would be most compelling. Thus, while the science of ENM ecotoxicology and exposure characterization has advanced, there are disconnects between how regulators review ENM-based products for environmental safety and the research that is conducted to evaluate hazards. Except for results published in open source outlets or directly reported, research may be unknown to government bodies. Ongoing synthesis of published research results is challenging due to high variability across study conditions and ENMs tested, and due to effort needed to regularly update such comparisons. Moreover, there is a systematic resistance to publishing “no effect” studies in the peer-reviewed literature.As a result, relying only on published research to inform regulatory decisions can present challenges. A life cycle-based framework facilitates exposure modeling and hazard testing to support risk assessment. However, extrapolation of effects to untested concentrations, study, or environmental conditions, and across biological levels of organization, requires understanding dynamic biological process-based effects, which current standard tests neither deliver nor sufficiently inform. Ultimately, exposure scenarios are useful for framing and focusing ENM ecotoxicology, and some version of a tiered intelligent testing and risk assessment strategy is needed. Such a conceptual tiered strategy considering the impact of the ENMs’ varying properties on ecological risks at different life cycle stages was proposed in the EU FP7MARINA project and is being further developed in the EU NANoREG program. This strategy considers several domains represented by specific tools ranging from relatively simple in the lower tiers to more complex and specific in the higher tiers. The framework aim is to structure information collection and generation for cost-efficient risk assessment, compliant with 3R animal-use testing principles , which should also be pursued by means of grouping ENMs. A strategy for grouping ENMs based on releases, uses, physicochemical properties, bio-accumulation, bio-availability, and effects for both human and ecological risk assessment is currently in development across a number of EU research projects such as MARINA, NANoREG, SUN, and GUIDEnano. These efforts have been challenged by the complexity of ENM identity and interactions, but this approach is necessary, as the costs for safety assessment on a case-by-case basis would be exorbitant.Therefore, a vision on ENM grouping is needed, which should apply in a regulatory context.Applying grouping in regulatory risk assessments should enable read-across, that is, filling a data gap by using information on one ENM, or a nonENM, for another substance in the same group.The above-mentioned tools should be fitted into a risk assessment strategy for ENMs. This strategy should be flexible enough to address different assessment goals depending on the user’s needs, considering all data already available as a starting point, contingent upon data quality evaluation and selecting the most appropriate tools to fill existing data gaps. Such a strategy should ideally be exposure driven, starting with identifying the most relevant exposure scenarios in the ENM life cycle, and evaluating completeness and quality of the available data from a risk assessment perspective. This facilitates careful prioritization of ENMs to optimize testing efforts and can inform more realistic ecotoxicological investigations. Doing so can allow one to screen-out irrelevant exposure routes, eliminate unnecessary testing, and support prioritization of exposure scenarios. Exposure assessment should begin with an analysis of plausible exposure scenarios; where none is expected, further testing may be precluded for the applicable use patterns and volumes.Researchers and regulators need to understand actual exposures at biological receptors. This exposure-driven approach can also provide important information on realistic environmental conditions to affect test designs for improved interpretation of laboratory toxicology studies. Such practices can ensue in the interim, while research continues to discover best hazard assessment practices. Experimental ENM toxicity assessments, using ecologically relevant receptors and across linked biological levels of organization, should inform developing and parametrizing dynamic process-based models. Such models should respond to future scenarios and predict impacts.