Category: hydroponic agriculture

Eight of the nine markers showed highly significant association with die back resistance, consistent with the Tvr1 gene being located in this region. Although the threshold for declaring association significant was set at p < 0.001, most of the associations were significant at p ≤ 0.00001. The only exception was marker Cntg4252, where the most significant association reached only p = 0.0042. The low association between SNPs at this marker and die back resistance was somewhat unexpected, since Cntg4252 co-segregated with the resistance allele in the × Salinas mapping population. While unexpected, it is not uncommon that markers closely linked with a trait in a mapping population do not show association when tested on a set of diverse accessions. This problem is well documented in potato, where markers linked to the Gro1 and H1 resistance genes in the mapping population were tested on 136 unrelated cultivars. The Gro1-specific marker was not correlated with the resistance phenotype, while H1-specific marker was indicative of resistance in only four cultivars. A similar example can be shown for lettuce, where markers most tightly linked to the cor resistance gene were the least useful for diagnostic when tested in a large collection of cultivars. There are several other examples of markers tightly linked to resistance genes, but whose use present problems in material different from the original in which they were identified.Therefore, an important requirement for any molecular marker used in MAS is not just its applicability in a specific cross, but its association in a wide gene pool. From SNPs that were significantly associated with die back resistance, the best fit was observed for those located in marker Cntg10192. This is the second of two markers, the other being Cntg4252, that co-segregated with the resistance allele in the mapping population. It is intriguing that one of the two markers co-segregating with the Tvr1 allele in the mapping population showed no significant association in a set of diverse accessions,30 litre plant pots bulk while the other showed a perfect match. Although these two markers were not separated in the intraspecific population, the linkage map developed from the Salinas × UC96US23 cross indicates that they are 1 cM apart.

Therefore it is possible that testing more RILs from the intraspecific population would separate the two markers and Tvr1. Association of SNPs from marker Cntg10192 with the resistance allele was validated in a larger set of 132 diverse accessions from several horticultural types. The marker-trait association was observed not only in L. sativa, but also in two L. serriola accessions included in the study. However, while the susceptible haplotype is identical in both species , the resistant haplotypes are different . To investigate the relationship between Tvr1 and the resistance observed in L. serriola, we screened 119 F8 RILs from the Salinas × UC96US23 population for resistance to die back. If Tvr1 and the resistance locus from UC96US23 were distinct and unlinked, approximately 25% susceptible offspring would be observed. However, since all RILs were resistant to the disease , we concluded that the resistance locus in UC96US23 is either allelic or linked to Tvr1. The same conclusion was reached for the resistance locus in the primitive romaine-type accession PI491224. The three resistance loci are associated with three distinct haplotypes; resistance in cv. Salinas with R1, in PI491224 with R2, and in UC96US23 with R3. Even though all 200 L. sativa accessions from the two testing sets showed the same haplotype-resistance association, it is unlikely that the EST from which this marker was derived is directly involved in die back resistance. A search for protein similarity in the NCBI database indicates that Cntg10192 is similar to the copper ion binding protein from castorbean and the plastocyanin-like domain-containing protein from Arabidopsis . The annotated functions of these two proteins do not imply an obvious role in plant-pathogen interactions.Moreover, the two substitutions at marker Cntg10192 that are the most significantly associated with die back resistance are synonymous, coding the same amino acid. Assuming that marker Cntg10192 is not directly involved in the resistance, it is probable that a recombinant genotype will eventually be identified. On the other hand, marker-trait associations can be very strong between some tightly linked alleles. For example, Rick and Forbes [51] documented linkage between allozyme Aps1 and tomato resistance gene Mi that did not break in as many as 30 backcross generations. Chromosomal linkage group 2 contains a large cluster of resistance genes that confer resistance to downy mildew and lettuce root aphid.

However, the Cntg10192 marker is well separated from thiscluster on the Salinas × UC96US12 map. Moreover, Tvr1 is one of the few resistance genes that was not at a genetic position coincident with any type of candidate resistance gene so far mapped in lettuce. Thus, it is possible that Tvr1 is different from the common types of pathogen recognition genes.We used HRM to directly detect sequence variations in PCR amplicons. High-resolution melting curves were recorded by the slow and steady heating of PCR products in a Light Scanner instrument.The method worked well for most of the analyzed markers, however, in a few cases, alleles could not be distinguished. When this occurred, we applied two alternative approaches to increase sensitivity through heteroduplex formation. In one approach, the heteroduplex formation was facilitated through mixing of samples prior to PCR. For example, if one sample contained DNA from cv. Salinas only, the other one would contain a mix of DNA from both cv. Salinas and Valmaine. The second alternative used an unlabeled probe 20 bp to 35 bp long that was designed for the region carrying the SNP. The probe was included in the PCR mix prior to cycling but was not consumed during amplification due to 3′ block. Genotyping was accomplished by monitoring the melting of probe-target duplexes post-PCR as described in Light Scanner manual. Both of the above alternatives improved allele detection; however, the probe-target duplex approach appeared to be more sensitive.Emerging plant diseases represent a continuous threat to economically important crop plants. Pandemics that affect supplies of grains and other annual subsistence crops are of particular concern . Diseases of perennial fruit trees are often very important for producers, due to the high costs of establishing intensively cultivated orchards and the extended amount of time needed to recover from outbreaks of diseases for which no effective chemical controls are available. For annual crops, a sensitive genotype can be replaced with disease-resistant varieties within a relatively short period of time. Diseases affecting fruit trees and vines may cause considerable losses through the gradual accumulation of newly affected trees over a number of years. This review describes how an important disease emerged, probably not from ingress of a new pathogen, but more likely from changes in cultural practices. It brings together information from a range of literary sources on the historical background of the emergence of xyloporosis and the linkage of this epidemic event with the history of citrus cultivation and with the natural history of Hop stunt viroid and other citrus viroids endemic to the Near East and the Mediterranean region.

Reichert and Perlberger reported on a “new” disease, xyloporosis, which appeared in 1928 among many of the newly planted citrus groves of Shamouti sweet orange trees that had been grafted onto Palestinian sweet lime , the most commonly used root stock in the British mandate-ruled area of Palestine. These authors described 3 essential phases of xyloporosis. First,wholesale plant containers small depressions appear on the stem bark of the root stock with small conoid pits with interfacing brownish pegs in the inner part of the bark. These symptoms often appear within 1 year of grafting and are most noticeable close to the bud union. In the second stage these symptoms intensify: The wood becomes discolored and, typically, the young tree becomes bent over and its leaves show symptoms typical of trees with root rot. In the third stage, there is blackish discoloration on the bark, the bark splits and leaves are small and yellow. Eventually, the branches wilt and die. Following intensive observations of a large number of infected trees of different ages in different planting areas, Reichert and Perlberger concluded that xyloporosis was present in all parts of the country and was not associated with any specific horticultural practice. Furthermore, although some differences were noticed among trees subject to different edaphic conditions, the disease could not be associated with any specific soil problem. The disease symptoms intensified as the trees aged. Seed source did not affect disease incidence. Pathological tests indicated that the disease was not associated with culturable fungal or bacterial pathogens. These observations led the authors to suggest 2 possible etiological causes of the disease: an unprecedented physiological disorder or an unknown viral pathogen. A major contribution to xyloporosis research was the observation of transmission by grafting of the cachexia disease affecting mandarins to a number of hosts, including a few that showed xyloporosis-like symptoms . These results suggested that the names of the 2 diseases, xyloporosis and cachexia, are synonymous and, based on the convention of prioritizing scientific names, the name cachexia was proposed for both diseases. Cachexia was found throughout most or all citrus-growing areas, particularly among Mediterranean varieties in Florida. Attempts by Norman and Childs to spread the cachexia disease via 5 different insect species failed and Olson showed that cachexia is not transmitted through seed. Later studies, reviewed by Bar-Joseph , showed that not only xyloporosis, but also exocortis and 3 other citrus viroids, were not seed transmitted to citron seedlings. The finding by Calavan and Christiansen that ‘Parsons Special’ mandarin showed more distinct symptoms than PSL supported the notion that the disease agents that cause xyloporosis and cachexia on different hosts are closely similar or identical. Recently, Langgut et al. reported the finding of typical citron pollen grains among the extracts from one layer of plaster, deposited to prevent leakage from an ancient irrigation pool belonging to a royal palace garden at present-day Kibbutz Ramat Rachel, near Jerusalem. Archeological evidence dated the construction of the facility to the Persian period and provides the first physical evidence for the earliest cultivation of citrus in the province of Judea. Initially, local growers refrained from grafting citron trees and were not familiar with the grafting technique .

The second edition of this book reported that some growers had started practicing grafting of citron on the PSL root stock, instead of propagation as seedling plants. Grafting was necessary because of the emergence of the destructive phytophthora gummosis disease, which rapidly decimated non-grafted trees and entire citrus industries throughout the Mediterranean basin . The dependence on grafting to save trees from diseases coincided with the selection of the Shamouti orange, which was obtained from a bud mutation of the local orange . Unlike the fruit of its seedy parent, the Shamouti fruit was almost seedless and these trees were propagated by grafting onto root stocks of an easily rooting citrus species, the PSL.Citrus cultivation expanded beyond the coastal plain , where the Shamouti orange on PSL root stocks was performing excellently, to new production areas just 10 km east of Jaffa, where all of the trees rapidly succumbed to the phytophthora gummosis disease. Phytophthora damage on PSL was initially blamed on the heavier soils of the new planting areas. However, when local citriculture expanded to sandy soils the root rot problems continued despite the improved edaphic conditions. It took almost 50 years for researchers and growers to realize that PSL root stocks made from cuttings were different from juvenile PSL seedlings, which were more sensitive to phytophthora root rot, and that the source of this difference was the absence in PSL seedlings of the viroid load commonly present in the root stocks that were made from cuttings. The positive effect of citrus viroid infection, the acquired resistance of phytophthora-sensitive root stocks induced by viroid infection, was first noticed by Rossetti et al. in Brazil. They noted that trees grafted onto Rangpur lime seedlings were succumbing to gummosis while those infected by the citrus exocortis viroid remained unaffected. Later studies using viroid-free and viroid infested buds of Shamouti grafted on PSL and also of other citrus stionic combinations confirmed this observation. Solel et al. showed that viroid infection also provides citron and Rangpur lime with tolerance to another serious fungal disease, mal secco . In retrospect, the change in susceptibility to phytophthora root rot could now be associated with the shift from the traditional practice of raising PSL root stocks from cuttings to producing root stocks from seed.

Only the smaller GFP variants moved beyond this zone . To add further complexity to protein trafficking and regulation, phosphorylation, and glycosylation are required for pumpkin CmPP16 to interact and form a stable complex with the mobility-endowing protein, Nt-NCAPP1, prior its phloem trafficking . Discrepancies in observed mobility from one study to another could be attributed to phosphorylation and glycosylation since earlier studies did not take these post-translational, covalent modifications into consideration. Two groups have demonstrated that non-endogenous proteins are retained in the root stock. The Gastrodia antifungal protein expressed by transgenic plum root stocks under the control of the constitutive CaMV35S promoter was identified in roots by immunoblot, but not in the soft shoot or leaf tissues of grafted, WT scions. This suggested that GAFP-1 was not moving into the WT-scion tissues of transgrafted plum trees . In the other example, transgenic watermelon root stocks over-expressing a cucumber mottle mosaic virus coat protein gene were transgrafted with WT watermelon. Protein expression and mRNA levels were detected in the transgenic root stock but not in the non-transgenic scion . Detection limits of the techniques utilized were not reported in either of these studies. A poke weed antiviral protein was expressed in transgenic Nicotiana tabacum root stocks and provided resistance to potato virus X in NN and nn grafted non-transgenic scions. However, the antiviral protein was detected only in the root stocks and not in the grafted scion tissues . The basis for resistance expression in this situation is not clear. Protein translocation from a transgenic root stock to a WTscion will likely depend on the species and/or type of protein in the transgene construct. Should proteins encoded by transgenes manage to migrate to the scion,plants in pots ideas their longevity is a consideration. For example, NPTII and GUS proteins have estimated half-lives of 6–7 min and 36 h, respectively, in planta .

If NPTII were translocated to scions it would be lost rapidly, but the GUS protein would not be reduced to 1% of the initial level accumulated in scions for 10 days. Research on the production of proteins encoded by transgenes in root stocks for delivery to scions arguably is more advanced than analogous work with the use of nucleic acids. For example, researchers at the University of Florida have engineered grape root stocks that deliver hybrid lytic peptides to control bacterial and fungal diseases .Work in our lab has shown that delivery of a protein that inhibits microbial maceration of plant cell walls is possible . While advances to date have focused on delivery of single gene products with specific functions to scions, future advances may target transport of transcription factors that influence expression of multiple genes,which could coordinate concerted scion responses to complex challenges such as pathogens, pests, or abiotic stresses.Proteins that are delivered to and function in the apoplast can provide protection against pathogens, particularly those pathogens that target the cell wall. In many plant–microbe or plant– pathogen interactions, the plant cell walls are a major obstacle to colonization or expansion within plant tissues. To overcome this barrier, most fungal pathogens produce a variety of enzymes, which degrade the host cell wall. Polygalacturonases  are often the first enzymes secreted during the infections . PGs cleave α- linkages between d-galacturosyl residues in pectic homogalacturonan, causing cell separation and tissue maceration. Botrytis cinerea expresses six PGs during infection and growth on plant hosts and the PG-inhibiting protein produced in pear fruit , inhibits some but not all of these PGs . Given the importance of PGs in pest and pathogen interactions with plants, it is not surprising that PGIPs are components of the defenses against invasion by pathogens and pests . Tomato foliar and ripe fruit resistance to the fungal pathogen, B. cinerea, is improved about 40% by the constitutive over-expression of pPGIP in tomatoes . The Miridae insect, Lygus hesperus, produces PGs that cause damage to alfalfa and cotton flflorets and PGIPs can inhibit these PGs and may, therefore, reduce the damage to plant tissues . The nematode, Meloidogyne incognitacausing root knot disease expresses PGs , but it is not known if they can be inhibited by PGIPs. PGIPs expressed in root stocks, therefore, are potential anti-pathogen proteins thatcould be delivered from the root stock to the scion in transgrafted plants. Our work has shown that pPGIP expression reduces the effects of Pierce’s Disease in grapevines, caused by the bacterium, Xylella fastidiosabecause it inhibits the X. fastidiosa virulence factor, PG .

As with other vascular pathogens, the X. fastidiosa PG contributes to disease development by digesting the polysaccharides in the pit membranes of the xylem network. When intact, these so-called “membranes” help to prevent the pathogen’s vessel-to-vessel spread from the initial sites of infection of grapevines . Because pPGIP inhibits the X. fastidiosa PG and because pPGIP can enter the xylem, PGIPs in the xylem of both the root stock and the scion could provide protection against other PG-utilizing pathogens in the water transport system. We have observed that when pPGIP-expressing transgenic plants are used as root stocks onto which non-expressing scions are grafted, the pPGIP protein, but not the pPGIP-encoding nucleic acids, are exported to the scion, crossing the graft union via the xylem system . In grafted tomato plants expressing pPGIP in the root stock, pPGIP protein has been detected in scion leaves . Similarly, in grafted grapevines, we have observed the pPGIP protein in the wild-type scion tissue grafted onto pPGIP-expressing root stocks . Furthermore, we have observed that expression of pPGIP in root stocks reduces pathogen damage in scion tissues . Thus, defense factors in roots can be made available to scions via grafting, improving the vigor, quality, and pathogen/pest resistance of the food-producing scion and its crop.DNA barcoding is an effective tool to identify many plant species rapidly and accurately. However, there is no single universal barcode that can be successfully used to identify all plants to the species level. Consequently, two alternative strategies have been proposed to distinguish among plant species: the first one is the use of complete chloroplast genomes, named ‘super-barcoding’, and the second one is an approach that involves searching for mutational hotspots, or using comparative plastid analyses to find loci with suitable species-level divergence. Analyses of entire chloroplast genome sequences provide an effective way to develop both of these strategies. In most angiosperms, the chloroplast genomes are inherited maternally and have a consistent structure, including two inverted repeats , one large and one small single copy region. Te chloroplast genome always contains 110–130 genes that exhibit a range of levels of polymorphism. Thus, chloroplast genome sequence data are extremely valuable for studies of plant population genetics, phylogeny reconstruction, species identification, and genome evolution.

The Ranunculaceae is a large family, which includes approximately 59 genera and 2500 species. Many plants of Ranunculaceae are pharmaceutically important. The genus Pulsatilla Adans. consists of about 40 species which are distributed in temperate subarctic and mountainous areas of the Northern Hemisphere. There are always long, soft hairs covering plants of Pulsatilla species. Most of the fowers of Pulsatilla are large and showy, and therefore the genus has horticultural importance. The fowers are solitary and bisexual. In one flower, there are always six tepals, numerous stamens and carpels, with the outermost stamens resembling degenerated petals, excluding P. kostyczewii. In China, there are eleven species of Pulsatilla. Some species of Pulsatilla have been used in traditional Chinese medicine for many years, such as for “detoxifcation” or “blood-cooling”, because Pulsatilla species contain numerous secondary metabolites, including phytosterols, triterpenoid saponins and anthocyanins. At the same time, all members of Pulsatilla produced the lactone protoanemonin. In Europe, some species of Pulsatilla are rare, endangered and endemic. Those taxa are protected due to their small populations and disappearing localities,container size for blueberries and those species have been placed on the Red Lists of Endangered Species. Taxonomically, Pulsatilla is an especially complex and challenging group. In all treatments published before, three subgenera have been recognized: subgenus Kostyczewianae , subgenus Preonanthus, and the largest subgenus Pulsatilla. However, the intragenic morphological variability of Pulsatilla was especially complicated. Te recognition and identifcation of wild Pulsatilla species is particularly difcult based on traditional approaches. Molecular markers are significant to explore the phylogenetic relationships of the genus Pulsatilla. Phylogenetic relationships between Pulsatilla and closely related genera have been dedicated during the past years. Previous studies have attempted to identify these species among Pulsatilla with universal molecular markers, but the species resolution was relatively low. In this study, we present seven complete cp genomes from two subgenera of Pulsatilla obtained through next-generation sequencing and genomic comparative analyses with four previously published cp genome sequences of Pulsatilla from NCBI, with Anemoclema glaucifolium as the out group. We identify microsatellites , larger repeat sequences, and highly variable regions, with the aim of developing DNA barcodes and testing the feasibility of phylogenetic analyses of Pulsatilla using the chloroplast genome.In most angiosperms, the IR regions of cp genomes of angiosperms are highly conserved, but the expansion and contraction of IR region boundaries are ever present. At the same time, several lineages of land plant chloroplast genomes show great structural rearrangement, even loss of IR regions or some gene families. The expansion and contraction in IRs are significant evolutionary events, because they can change gene content and chloroplast genome size. Expansion of the IRs has been reported in Araceae. Sometimes, the size of LSC increases and that of SSC decreases, becoming only 7000 bp in Pothos. At the same time, a linear chloroplast genome was also reported in some groups, e.g. maize. Expansion and contraction of the IR regions can also lead to duplication of certain genes or conversion of duplicate genes to single copy, respectively. Changes in the size of the IRs can also cause rearrangement of the genes in the SSC as recently observed in Zantedeschia. The Pulsatilla chloroplast genomes were compared to previously published data and showed typical Anemoneae genome structure. As reported for Anemoclema, Anemone, Clematis and Hepatica, the IR regions of genus Pulsatilla are roughly 4.4 kb longer than those of other genera of the family Ranunculaceae, such as Aconitum, Coptis, Talictrum, Megaleranthis, Ranunculus, and Trollius. The gene orders located within the IR-SSC and IR-LSC boundaries are similar among tribe Anemoneae but diferent from those of other genera of Ranunculaceae . We compared the IR/SC boundary regions of Pulsatilla, and the junction positions are very similar and conserved within genus Pulsatilla. In the four boundary regions of seven Pulsatilla cp genomes, the LSC/IRa and IRb/LSC border was in the intergenic region, and the adjacent genes is rps36, rps8 and rps4, respectively. The genes ycf1 andψycf1 have crossed the SSC/IRb and IRa/SSC boundary, respectively, which was also found in Monsteroideae. The pseudogene ycf1 has been found in other groups. The IR regions were highly conserved, with nucleotide diversity values in those regions less than 2%.Chloroplast genome markers, especially several universal chloroplast regions, have been widely used in plant systematics and identifcation at multiple taxonomic levels. Highly suitable polymorphic chloroplast loci have been identifed and designed as unique markers in diferent groups. However, relationships within the genus Pulsatilla have not been well resolved because of the lowpolymorphism of these universal markers. In order to facilitate identification of closely related species of Pulsatilla, we sought to identify highly variable regions of the chloroplast genome, as previously described. As a result, we identified nine divergent hot spot regions, including six intergenic spacer regions and four protein-coding regions. Most commonly employed loci, e.g. trnL-trnF, trnH-psbA were not selected in our finding. The nine highly variable regions included 684 variable sites, including 181 indels. However, these indels are not suitable for the phylogenetic inference because Maximum likelihood model used only substitutions not indels. Their nucleotide diversity values ranged from 0.00802 to 0.02212. The region of ccsA-ndhF showed the highest variability, the next most variable regions were rps4-rps16, ndhC-trnV, and psbE-petL. Te diversity level of two protein-coding regions was the lowest. Among the nine divergent hotspot regions, the ndhI is difcult to align. There are large numbers of indels in ndhI and the intergenic spacer between ndhI and ndhG, these regions were not considered suitable for the phylogenetic inference of the Pulsatilla. Thus, we selected eight regions, four in the LSC and four in the SSC, with relatively high variability as potential molecular markers for the study of species identification and phylogeny in Pulsatilla.

In 73 of the 519 nights included in analysis , nocturnal bouts of activity were detected by actimetry that were sufficient to divide nocturnal sleep into two bouts . In these cases, Actiware sofware chose sleep onset and wake times from the longer of the two sleep bouts to represent the sleep period for that night. If the following criteria were met, then the two sleep bouts were manually joined to allow reported sleep onset and wake time to represent one sleep period across the entire nocturnal period . Combining the two sleep periods did not change total nocturnal sleep duration, as any periods of awakening during the night were not included as sleep. Te combining procedure served to consolidate the sleep bouts into one sleep period with reduced sleep efficiency. Criteria for combining sleep periods were, the period of nocturnal activity must have occurred during a time when the subject was “usually” asleep/inactive , the subject must have been asleep for at least 2 h prior to the period of awakening, or the period of nocturnal awakening had to be shorter than the shortest period of sleep. For example, if a subject slept for 30minutes, awoke for 2h and went back to sleep for 6h, this would not qualify for fragmentation removal, and the 6h sleep period would be considered their nocturnal sleep period. However, if a subject slept for4h, awoke for 2h, and then slept for 4h, this would qualify for fragmentation removal .Dependent variables were calculated as follows. Actiware 6.0.9 sofware was used to score bedtime, sleep onset, wake time, rise time, nocturnal sleep duration , sleep efficiency , and nap duration. Nocturnal sleep duration and nap duration were summed to yield 24 h total sleep time . Activity data were imported into Clocklab 6.0 in 1min bins for calculation of non-parametric circadian variables, including L5 , M10 , relative amplitude , intradaily variability and interdaily stability . JMP 14 and Prism 7.0 were used for inferential statistics and to produce figures. Te primary aim was to assess the relationships between sets of independent variables and dependent variables using separate analyses of variance for each dependent variable.

Prior to conducting these ANOVAs, we explored the effects of potential covariates, such as age, number of co-sleepers,blueberry container size and body fat percentage by observing whether the potential covariates significantly correlated with the various dependent variables. Te only significant correlation observed was between age and sleep efficiency. Accordingly, to assess the relationships among community type and adult type with sleep timing variables and sleep duration variables, separate 2×3 ANOVAs were conducted for each dependent variable. To account for the significant relationship between age and sleep efficiency, a ANCOVA was used to emulate the ANOVAs, but with age entered into the model as a covariate. Tukey’s post hoc tests were used to further explore significant main effects of adult type, or significant interactions. In cases where parametric tests were not appropriate , Mann-Whitney U non-parametric independent samples tests were performed between community types, foregoing analysis by adult type. Statistical significance was defined at p<0.05 . Figures including means are plotted±standard error of the mean .Te Actiware 6.0.9 sleep scoring algorithm divided nocturnal sleep into multiple bouts on at least one night in 56% of the electric community sample and 47% of the non-electric sample, for a total of 73 out of 519 nights . Among individuals exhibiting sleep fragmentation, the average percentage of nights with fragmentation was 30% in the electric and 20% in the non-electric communities. Prevalence was highest in breastfeeding females in the electric communities. Fragmentation contributed to the nocturnal sleep efficiency score, which was 3.0% lower in the electric communities compared to the non-electric . An analysis of covariance model was used to test the effects of community type , adult type , and the covariate age on sleep efficiency. Te analysis showed a statistically significant interaction between the covariate age and community type =8.32, p=0.005). Sleep efficiency was related to age only in the non-electriccommunity. For this reason, separate tests were conducted for both electric and non-electric communities. In the electric community, there was a main effect of adult type =4.44, p=0.019), with lower sleep efficiency in males and breastfeeding females compared to females . In the non-electric community, there was no main effect of adult type =0.47 p=0.629).

Relationships between age and sleep efficiency by adult type were weak for both communities , and significant only for breastfeeding females in non-electric villages, with sleep efficiency decreasing with age =10.5, p=0.002). Subjective reports from interviews indicate that 90.7% of individuals in the electric and 92.3% in the non-electric communities, report waking up during the night. Te cause of sleep interruptions in both community types was most frequently attributed to infant care , and dogs barking . Despite this, the majority of individuals in each community reported feeling that they slept “enough”.Sleep timing, duration, and efficiency can be affected by environmental stimuli, including light, temperature, humidity, and co-sleepers. Residents in both communities went to bed approximately 3–3.5h afer sunset, which occurred between 17:17h−17:32h during the study, and awoke very close to sunrise, which occurred between 05:52h−06:00h . Wake times were on average closer to sunrise than to transitions in ambient temperature and relative humidity, which, at the time of recording, occurred after sunrise, as measured by iButtons in sleeping huts. A role for evening light exposure in the relationship between sleep onset time and sleep duration is suggested by a significant negative correlation between evening light and nocturnal sleep duration in the electric community . Evening light was not significantly correlated with sleep duration in the non-electric community , and morning light exposure was not significantly correlated with sleep duration in either group. In neither community was sleep efficiency or sleep duration significantly related to average nighttime temperature or humidity . Te absence of a relationship may be due to relatively low variability of average temperature and humidity across the nights of this study. A few of the sleeping huts in villages with electricity were constructed with tin and cement, and these had lower humidity at night compared to grass huts, but the small number of these huts precluded analyses by hut types.All participants in this study shared sleeping quarters with multiple children or adults. Higher numbers of co-sleepers might be expected to increase the number of nocturnal awakenings, and thereby reduce sleep efficiency and potentially sleep duration, as has been previously reported. In the present study, the average number of co-sleepers was slightly greater in the non-electric communities compared to the electric communities , yet the non-electric communities had both longer nocturnal sleep and higher sleep efficiency.

This is the first actigraphy study of sleep timing and duration in indigenous Melanesians living small scale, traditional horticultural lifestyles in the south pacific island nation of Vanuatu. We found that habitual sleep duration among the Ni-Vanuatu of Tanna Island is long compared to several small-scale hunter-gatherer, agrarian and pastoralist societies in Africa and Bolivia,raspberries in containers and compared to most samples from industrialized western populations studied by actimetry using Actiwatches . We also found that nocturnal sleep onset was delayed by 23minutes and duration was shorter by 28 minutes in participants living in coastal villages with on-demand access to electric light at night. A significant interaction with adult type suggests that the difference in sleep duration is driven primarily by breastfeeding females in the communities with electricity. Reduced nocturnal sleep in this group may have been causally related to increased light exposure during nighttime infant care, compared to breastfeeding females in the non-electric communities who had the same nocturnal responsibilities without on-demand availability of electric light. We interpret these results as supporting the view that sleep timing and duration in humans is shaped in part by lifestyle adaptations to the opportunities and challenges of particular ecologies. Hunter-gatherer and pastoralist societies, living at virtually the same latitude as Tanna Island and also studied using wrist-worn actigraphy averaged markedly less daily sleep than the Ni-Vanuatu on Tanna Island. Life on Tanna Island is characterized by reliable food access, a mild subtropical climate with relatively low daily and seasonal variability in temperature and day length, absence of predators, and minimal social conflict. Under these conditions, there may be no special fitness advantage of short sleep. Conceivably, there may be a fitness advantage favoring a short sleep genotype in hunter-gatherer and pastoralist societies that is not present in the horticulture-based lifestyle on Tanna Island. Alternatively, short sleep durations in some groups may reffect less favorable sleeping conditions, which could imply that these groups are chronically in sleep deficit. Te similar latitude, and thus daylength, sunrise, and sunset times, rule these out as explanations for differences between the Ni-Vanuatu and hunter-gatherer and pastoralist societies studied to date . We also interpret these results within the context of the developing economy sleep degradation hypothesis and the postindustrial sleep degradation hypothesis. Although sleep duration on Tanna Island was long by comparison with most actigraphy studies of industrialized western populations , sleep efficiency was low. This may reffect environmental disturbances, such as having multiple co-sleepers, and housing that offers little protection from surroundings . Compared to Western homes, the walls of dwellings in Vanuatu are thin and uninsulated and allow greater exposure to outdoor temperatures, and greater sound transmission when wild dogs bark or neighbouring babies cry. Reported sleep disruptions seem to reffect differences in location. For instance, the electric communities are closer to developed roads and reported more automobile related noise disruptions, which would be expected to increase as industrialization progresses. Noise is proposed as a large component of the ‘developing economy sleep degradation hypothesis’ since increasing population density paired with traditional housing offers little bufer. In addition, participants living in villages with electric lighting exhibited delayed and shorter nocturnal sleep. This was associated with increased exposure to evening light, and was particularly prevalent in breastfeeding females, who would be expected to experience more nocturnal waking and evening light exposure. Thus, sleep on Tanna Island exhibits characteristics of developing economy sleep degradation and post-industrial sleep degradation . While modern standards of living may improve sleep, access to lighting around the clock and other factors may counteract some of these improvements.Te difference in average nocturnal sleep duration between the electric and non-electric villages in our study sample was 28minutes. A reduction of this magnitude in industrialized populations is thought to be physiologically and behaviorally significant, especially if accumulating over days of the work week or longer. It is possible that the 7.88h average nocturnal sleep duration in Tanna Island villages without electricity represents a surfeit, and that a roughly half-hour reduction in villages with electricity is of no functional consequence. If 28min less sleep at night does represent a deficit, then we might expect to see an effort to compensate by increased daytime napping. While both study groups exhibited some daytime sleep, naps were significantly more prevalent in villages with electricity. When these naps were combined with nocturnal sleep to yield total daily sleep time, the difference in sleep duration between villages with and without electricity was no longer statistically significant. This suggests that daytime naps are at least in part compensatory and that the shorter nocturnal sleep duration in electric communities represents a deficit. Presumably, the magnitude of differences in sleep timing and duration between communities with and without electric lighting on Tanna Island is limited by continuous exposure to natural light throughout the day in both groups. Morning light opposes the phase delaying effect of evening light, and increased daytime light decreases sensitivity to artifcial evening light. Given the similarity of daytime light exposure patterns in the coastal and inland villages, evidence for a significant effect of on-demand electric light in coastal villages is notable. Another factor that may limit differences between groups is the use of solar torches after sunset in both communities. However, torches provide only low intensity light that is typically directed toward objects and away from the eyes. Despite the use of torches in the non-electric communities, those living in the electric communities showed more light exposure during the first hours of the night , and this was associated with delayed sleep onset times and less nocturnal sleep.

The traditional root stock breeding programs have produced the interspecific hybrid ‘GF-6770 , GN series, ‘Root-Pac 400 , ‘Vlach’, ‘RX10 , ‘VX2110 , ‘UCB10 , ‘Newberg’, and ‘Apache’ root stocks in different nut trees. However, for tree nut crops, which have long extended juvenility, long productive lives and high heterozygosity, the traditional breeding approaches employed in annual crops are too slow, and costly. Understanding how root stocks and scion interact can provide modern breeders new techniques to improve tree nut crops productivity. Incorporating the newly emerging technologies including high-throughput phenotyping and genotyping as well as genome-wide transcriptome analysis into investigations of the genetic and domestication processes of nut trees root stock species will address pertinent questions for root stock biology and breeding. Among these questions are how the root stock/scion interactions affect graft compatibility, vigor, water and nutrient uptake and efficiency, biotic and abiotic stresses, yield, and quality. Of particular value in root stock breeding programs is germplasm collection and construction of grafting experiments to identify the genes associated with phenotypic variation in both the root stock and the scion. The collection of genomic data for nut trees is accelerating as the cost of next generation sequencing decreases. The almond, hazelnut, walnut, pistachio, and pecan genomes have been fully sequenced and are available. In the near future reliable phenotypic data will be the rate limiting step in root stock improvement. As tree nut crops are highly heterozygous with long juvenility periods and productive lives, genomic based approaches, such as marker-assisted selection , genome-wide association study , genomic selection ,blueberry pot size and genetic transformation offer promise for root stock breeding. Comprehensive germplasm collections, coupled with genomic approaches, has the potential to yield significant advances in grafted tree nut crops.

Predicting the flowering time of angiosperm taxa under projected climate conditions or in locations at which flowering has not been observed is essential to the prediction of a wide array of ecological processes, including risk of frost damage to floral tissues , nectar and pollen availability to pollinators , and the intensity of competition for pollinators among co-flowering taxa . Phenological prediction can also be important to local tourism, and for determining the optimum time for herbicide or pesticide treatment. For example, accurate predictions of flowering time can prevent the planned application of pesticides during flowering, when beneficial insects and birds are visiting flowers. Similarly, the planned use of herbicides to suppress invasive plant species should occur before or during flowering, so as to minimize seed production. Consequently, the ability to predict the flowering times of angiosperm species is relevant not only to ecologists and other researchers, but also to land managers and other professionals across a wide array of disciplines. In recent years, some tools have emerged to predict phenological timing under various climate conditions, such as the phenological forecast maps produced by Phenology Forecasts or univariate phenological models produced by the USA National Phenology Network . To date, however, species-specific phenological models have been developed for only a small number of species, and such models have often required daily growing degree-day or chilling degree-day information, which until recently have not been readily available across the vast majority of locations, and have required significant technical expertise to utilize effectively. Furthermore, the output of such models is rarely bundled in such a way as to facilitate phenological predictions in the absence of extensive calculations or data manipulations on the part of the user.In this paper, we present PhenoForecaster, a software package that allows users to predict quickly and easily the mean flowering date for each of 2320 angiosperm species.

PhenoForecaster uses readily accessible climate data in combination with species-specific phenological models that were generated by the authors using a simplified version of a method previously used to evaluate phenological responses to climate using digital herbarium records . Specifically, PhenoForecaster uses estimates of five climate parameters to predict the day of year on which the selected angiosperm species will reach its mean flowering date at a location experiencing those conditions. These parameters represent the climate cues to which MFD was found to be most sensitive across the majority of these species using similar data and modeling techniques to those used by PhenoForecaster . In order to facilitate PhenoForecaster’s use, all of the phenoclimate models that it uses were limited to these climate parameters, which were sufficient to retain the majority of the predictive power produced by more complicated models . This package allows both manual entry of climate parameters as well as bulk entry of data in cases where phenological predictions are required across multiple locations or climate scenarios. PhenoForecaster has been designed to accept climate input in a comma-separated value format that is compatible with climate data generated by ClimateNA , a freely available software package that produces spatially explicit estimates of historical climate conditions throughout North America, and which utilizes a user-friendly graphical interface and requires only that the user provide the latitude and longitude of all points of interest. Thus, while predictions of phenological timing for a given plant species previously required extensive observation, modeling, and calculation, PhenoForecaster represents a simple-to-use tool through which the phenology of many angiosperm species can be readily predicted under any observed or theoretical climate.To install the package, the user simply needs to download and run the installer. The executable has been successfully tested on Windows 7, 8, and 10.

PhenoForecaster has an intuitive graphical user interface that allows users with minimal prior experience with phenological prediction or with PhenoForecaster to predict the phenological timing of any targeted species by implementing the following steps. First, the user must select the subset of species-specific models from which they wish to choose, based on the minimum model reliability they desire. By default, only the 490 species-specific models for which expected mean absolute error ≤15 days were considered to be “good” model fits, and are therefore displayed for selection. Depending on user preference, however, this list of species may be expanded to include species-specific models that exhibit higher MAE, or contracted to only display those species for which more accurate phenological models are available . Having filtered the species by the minimum MAE desired, the user must then use the species selection drop down menu to select the species for which phenological predictions are to be generated . Second,grow blueberries in pots the specific climatic conditions for which phenological predictions are desired may then be entered manually or uploaded as a CSV data file . For the latter, the first line of the input file is a header line with column descriptions. The first two columns of the file, labeled ‘ID1’ and ‘ID2’, represent any string data the user desires to include for the purpose of identifying each row of data in a unique fashion. The remaining columns may be in any order, but must include the following: ‘NFFD_wt’, ‘NFFD_sp’, ‘PAS_wt’, ‘PAS_sp’, and ‘BFFP’. Data in the column ‘NFFD_wt’ should consist of a count of the number of frost-free days from January 1 to March 31 in the year for which flowering time is to be estimated. Data in the column ‘NFFD_sp’ should consist of a count of the number of frost free days from April 1 to June 30 in the year for which flowering time is to be estimated. Data in the column ‘PAS_wt’ should consist of the total precipitation that fell as snow from January 1 to March 31 in the year for which flowering time is to be estimated. Data in the column ‘PAS_sp’ should consist of the total precipitation that fell as snow from April 1 to June 30 in the year for which flowering time is to be estimated. Data in the column ‘BFFP’ should consist of the DOY on which the annual frost-free period began. PhenoForecaster allows any number of additional data columns to be placed into the input file. In cases where the user desires that data from such additional columns be preserved in the output file created by PhenoForecaster, they may select the ‘retain all input data’ option in the lower left of the user interface. If this option is selected, PhenoForecaster will preserve all columns from the input data, appending a new column with the header ‘DOY_Predicted’ that consists of the predicted MFD for a given row of data, and output all data as a CSV file. Otherwise, PhenoForecaster will generate output in the form of a CSV file, with the headers ‘ID1’, ‘ID2’, and ‘DOY_Predicted.’ PhenoForecaster utilizes phenoclimate models that were constructed for each species from herbarium-based phenological data using a total of 556,322 digital records of herbarium specimens collected in flower across 72 herbaria throughout North America , collected between 1901 and 2015 and structured in Darwin Core format. Specimens that did not include either the decimal latitude and longitude from which the sample was collected or the precise date of collection were eliminated. Specimens that were not explicitly recorded as being in flower within either the Darwin Core fields ‘reproductive condition’ or ‘life stage’ were eliminated. Specimens that were only listed as ‘in bud’ or ‘fruiting’ were not considered to be in flower for purposes of this analysis. Duplicate specimens were also excluded from analysis. Each remaining specimen therefore represented a single phenological observation. Phenological models derived using herbarium-based observations of flowering phenology have been found to accurately predict shifts in phenological events that were observed in situ in response to climate changes . Species-specific models of MFD for each species were conducted using elastic net regularization, which has previously been demonstrated to be an effective method for predicting the flowering times of angiosperm taxa using herbarium specimens .

For the models used by PhenoForecaster, winter and spring climate conditions at the location and DOY from which each specimen was collected were first estimated using the software package ClimateNA . Each species specific phenoclimate model was then constructed using elastic net regularization, a multivariate regression method that, rather than selecting or removing parameters in a binary fashion as with forward or backward selection, enforces parsimony by penalizing model complexity using two penalty terms: the sum of the absolute value of all parameter coefficients , and the sum of all parameter coefficients squared .This method has substantial advantages over stepwise forward selection or backward elimination regression techniques, particularly when handling data sets in which multiple explanatory factors are likely to exhibit some degree of collinearity, such as is common in climatic data . Elastic net regression has been found to generate models that remain highly stable in cases where multiple explanatory factors exhibit collinearity , while avoiding the variance inflation that often occurs when using stepwise regression techniques . For each angiosperm species that was represented by 100 or more specimens in our herbarium-based data set, phenological models were constructed to predict the MFD of that species from local climate conditions using the elasticCV class contained within Scikit-Learn 0.814-4 in Python, which conducts an internally cross-validated version of elastic net regularization that selects the optimal values for the weighting terms ρ and α in order to minimize both model complexity and standard error . The models used for each species in this study were constructed through iterative fitting along a regularization path, using 100 values of α and 22 values of ρ . The optimal model coefficients were then selected using 25-fold cross-validation. The MAE for each model represents the mean MAE of the 25 iterations in which it was trained and tested using separate data sets; this value therefore represents the expected degree of error that may be expected for phenological predictions of a given species under novel conditions . Additionally, the accuracy of these species-specific models was tested for three species using observations of mean flowering time derived from in situ phenological observations provided by the USA National Phenology Network database. The models used by PhenoForecaster predicted the timing of both in situ and herbarium-based observations of mean flowering with similar accuracy . Species for which phenoclimate models produced MAE values of <15 days were considered to exhibit “good” model fits by default. However, PhenoForecaster allows users to alter the MAE threshold that they consider to represent “good” model performance to accommodate cases where higher or lower predictive accuracy is required.

In good agreement with other studies, Solibacteres, mainly Candidatus Solibacter, previously suggested to be adapted to nutrient-limited environments, was associated with the conventional farming system. Interestingly, taxa capable of degrading xenobiotic compounds were also enriched. The nitrification process was enhanced, as ammonia oxidizing bacteria and archaea wereparticularly induced after applying the conventional fertilization, and that response was consistent over the 3 months of the experiment, similar to longer-term studies. Thaumarchaeota archaea’s enrichment has been previously observed after a long-term application of organic fertilizers and in several long-term fertilization experiments with inorganic N treatment. In addition, several denitrifying bacteria responded to the chemical fertilization, such as Gemmatimonas , Pseudomonas , Achromobacter , Nocardia, and Rhodococcus . In this study, functional profiles were more resistant to intervention than community composition. This agrees with the conclusions of Pan et al., who proposed that the coexistence of organisms with overlapping ecological functions confers functional stability. Fierer et al. found that, under high concentrations of inorganic nitrogen, the relative abundance of the DNA/RNA replication, electron transport, and protein metabolism functions increase. Likewise, Carbonetto et al. evidenced that the relative abundances of intracellular trafficking, secretion and vesicular transport, energy production and conversion, and amino acid transport and metabolism were greater in soils under conventional farming system than in uncultivated soils, consistent with a copiotrophic strategy. Ding et al. reported changes in functional groups associated with nitrogen cycling when conducting a metagenomic analysis, observing the greatest effect for functional groups associated with aerobic ammonia oxidation, nitrite reduction, anaerobic ammonia oxidation, and nitrate reduction. Chen et al., besides, found no significant differences in functional genes, predicted from 16S RNA using PICRUSt, involved in denitrification , nitrification ,blueberries in containers growing and N-fixation when analyzing soils managed over 18 years that included organic and conventional farming.

Similarly, in the present study, when evaluating PICRUSt predicted functions, the Kruskal–Wallis test did not detect any differentially abundant functions between the conventional and organic soil samples, which included those N transformation functions. However, according to LDA, organic soils had greater predicted abundances of glutathione metabolism, which plays an important role in the defense of microorganisms and plants against environmental stresses. In addition, it is also involved in the regulation of sulfur nutrition and plays a key role in the nitrogen-fixing symbiotic interaction. However, the functional results reported here are based on predictions obtained from the 16S rRNA gene, which resulted in NSTI values that were moderately high, as expected for phylogenetically diverse samples such as soil, suggesting that those predictions must be interpreted with caution. In addition, functional differences might have been hidden, as de novo OTUs were eliminated for the analysis to conduct PICRUSt predictions. Nut trees are among of the most important horticultural tree crops. Both production and consumption are increasing dramatically due to strong economic returns and the nutritional value of their products. The world’s tree nut production has increased by 48% over the last 10 years . The world-wide export value of tree nut crops amounted to approximately 34.5 billion dollars in 2019, an increase of ~107% compared to the prior 10-year period. Technical knowledge regarding nut tree production has also rapidly increased as a result of the demand for higher production and quality, multiple destinations of nuts fruit in current consumption and food industry, but also of the growing importance accorded to the nuts in a balanced and healthy diet and in the prevention of various diseases. Among the areas of interest and progress has been the use of root stocks to adapt to climate and edaphic factors including soil borne diseases and abiotic stresses, control scion vigor, increase yield, and improve fruit quality. the selection of the scion cultivar is the grower’s top consideration for long-term productivity and profitability, root stock selection is becoming more important. Now, the root stock scion and interaction per se is considered when planting an orchard. The advantages of selected root stocks have been recognized and utilized in the nut trees’ production, but they do not have a long history of use in many species.

Although nut trees are grown around the world, root stock studies are limited to only a few tree nut species. Initially, most root stocks were open-pollinate seedling, or seed stock. Seed stocks are not as genetically uniform as clonal root stocks, but they have advantages such as deep root system and tolerance to edaphic abiotic stresses. However, seed stocks have high heterozygosity in terms of different traits. Hence, the type of seed and location in which it is grown is important for choosing seed stocks. Seed stocks should be uniform, vigorous, disease resistant, and readily available. Therefore, several studies have been performed to study the growth vigor of seed stocks and improve seed germination in nut trees. In addition to seed stocks, a wide range of clonal root stocks are now being developed. Numerous root stock breeding programs have begun to introduce clonal root stocks to meet important challenges, including excess vigor, low yield, poor nut quality, poor soil, climate change, drought and salt stress, suckering, diseases, and graft incompatibility. Common tree nut root stocks, especially clonal root stocks, and their main characteristics are listed in Table 1. Advances in the development of temperate nut trees root stocks until 2003 were last reviewed by Grauke and Thompson. Given the recent advances in root stock breeding for tree nut crops, this review will focus on the physiological and molecular effects of root stocks on scions under different edaphic and climatic conditions. The main purpose of this review paper is to present studies on various aspects of breeding and physiology of nut trees root stock, as well as, draw a comprehensive vision to accelerate future research in this field using combination of traditional and modern methods. To this end, we first provide overall information on vigor, root stock-scion com-patibility, suckering, and rooting ability which can be useful for tree nut crops researchers and growers. Next, we review water and nutrient uptake on nut trees. In the following, we review phenology and yield related traits which are important in industry and marketing. Then, we comprehensively review abiotic and biotic stresses studies on tree nut crops. Finally, we briefly review root stock-scion transfer of macromolecules and small interfering RNAs in nut trees. Since nut tree crops have a long juvenile period, development of a new variety or root stock may take more than 20 years via classical breeding. Therefore, in the conclusion and perspectives section, we note the future prospects of molecular breeding in nut tree crops using novel technologies for rapid generation advancement.The nut trees growth is strongly controlled by the distribution of organic and inorganic constituents within the tree trunk, canopy, and the root system. The vascular system plays a role in this long-distance signaling.

Hypothetically, root stocks impact scion vigor by controlling water and nutrient transfer and hormones signaling and RNAs which move up through the graft union. Numerous studies have been conducted regarding the effect of root stocks on the growth of nut trees. Pistachio growers and breeders are seeking vigorous root stocks. Kallsen and Parfitt reported ‘Kerman’,planting blueberries in containers the previously primary female pistachio cultivar in California, has a rapid growth habit that produces trunk circumferences larger than that of the root stocks. Matching the scion and root stock growth rates produces stronger graft unions. Highly vigorous root stocks produce more uniform graft unions and reduce bark damage from trunk shaking harvesters by uneven graft unions. They report that UCB1 is a better root stock for ‘Kerman’ as it produces a smoother trunk compared to Pistacia integerrima root stocks. Caruso et al. evaluated one seedling and eight clonal pistachio root stocks and reported that root stock had a significant effect on growth rate of the scion and nut yield. Clones of P. integerrima and P. atlantica are highly to intermediately vigorous root stocks. The pistachio cultivar ‘Bianca’ onto P. integerrima seedling root stock had significantly better growth than on P. terebinthus or P. atlantica clonal root stocks. Scions grown on P. terebinthus root stocks had the least vigor. When ‘Bianca’ scions were budded onto eight in vitro propagated clonal root stocks and observed for 4 years, trunk cross-sectional areas on P. integerrima were three times higher than on P. terebinthus root stocks. Ak and Turker reported the cultivars, ‘Kirmizi’ and ‘Siirt’, grafted onto P. vera, P. khinjuk, and P. atlantica demonstrated different budbreak, flowering time and vegetative growth. P. vera flowered earlier and P. atlantica and P. khinjuk had greater stem diameters. Rahemi and Tavallali studied the effect of ‘Badami’ , ‘Sarakhs’ , and ‘Beneh’ seedling root stocks on growth, yield, and nut quality of the Iranian cultivars, ‘Ohadi’, ‘Kalleh- Ghouchi’, and ‘Ahmad-Aghaei’. ‘Sarakhs’ seedlings had the least vigor, while ‘Badami’ root stocks produced the highest yields and best nut quality. Ghazvini et al. evaluated the ecophysiological characteristics of four seedling root stocks, ‘Badami’, ‘Sarakhs’, P. mutica, and P. atlantica. Photosynthesis, stomatal conductance, and transpiration was highest in trees on the ‘Sarakhs’ root stock and lowest on the P. mutica root stock. P. integerrima is the most vigorous root stock now commonly used in pistachio cultivation but is also the least cold tolerant. It is rapidly being replaced by the more coldand salinity-tolerant hybrids, available as both a seedling and a clone, and P. integerrima × P. atlantica, now available as a clone . In contrast to pistachio, there is no a specific walnut breeding program to select high vigorous root stock. Nevertheless, the major walnut clonal root stocks introduced in the last few years are vigorous. Among the clones of ‘Paradox’ which was introduced by the University of California-Davis, ‘VX2110is highly vigorous and nematodes-tolerant root stock. Furthermore, ‘Grizzly’ clonal walnut root stock has been recently introduced as a highly vigorous root stock. The mother tree of ‘Grizzly’ is a Tulare variety grafted on a seedling Paradox root stock. This root stock showsgood performance in poor soil structure with low nutrition and heavy populations of lesion nematodes. In addition, high vigorous trees are very important for the wood industry. Numerous interspecific hybrids were carried out in Juglans genus between J. regia with J. cinerea, J. nigra, and J. major. Compared to the parent, most of them such as ‘NG230 , ‘NG380 , and ‘MJ2090show high vigor, disease resistance, greater winter-hardiness, and high wood quality. Walnuts are highly vigorous trees with an extended juvenility phase. Dwarf walnut trees could potentially decrease labor costs and increase yields per hectare by allowing increased plant density. Although dwarfing has not generally been the most important objective of walnut root stock breeding programs, identifying sources of this trait is of great interest in countries with high genetic diversity such as Iran, China, Turkey, and Central Asian countries. In these countries, traditional orchards of giant walnut trees are difficult to harvest mechanically. Harvest injuries and death of laborers during manual harvesting have precipitated interest in dwarfing root stocks. Reportedly dwarf walnut trees have a short life span. Therefore, in some countries, breeders are attempting to combine slow-growing scions with vigorous root stocks. Juvenile and mature walnut tree vigor is highly heritable. Wang et al. evaluated Persian walnuts in China and selected six dwarf walnut root stocks; ‘Xinwen 6090 , ‘Xinwen 7240 , ‘Xinwen 9080 , ‘Xinwen 9150 , ‘Xin 9160 , and ‘Xinwen 9170 as potential root stocks for breeding. Analysis of growth traits of ‘semi-cultivated’ local genotypes of Juglans regia on their own roots, in the sands area of south-west Romania, showed that climatic and edaphic factors significantly influenced the annual growth ring width of the trees, but also their adaptability to environmental factors. Precocious and dwarf walnut trees have been evaluated in Iran. These genotypes induce dwarfing and precocity in scions in preliminary experiments, apparently due to a slower growth rate. They have fewer nodes, shorter internodes, and smaller shoot length, smaller root system, and lower sap flow and hydraulic conductivity which are the typic traits of dwarf root stocks in other fruit trees. They also have a better rooting ability and higher grafting success. Dwarfing is a desirable trait for other tree nuts. In China, dwarfing chestnut root stocks are being evaluated. In the USA, Anagnostakis et al. attempted to breed dwarfing chestnut root stocks and suggested that hybrids with Castanea seguinii could be a source of dwarfing. Researchers at the University of Missouri identified various chestnut cultivars as potential sources of dwarfing. Studies of graft compatibility, vegetative growth, and productivity of these trees are continuing to determine if dwarf chestnut root stocks are feasible.

ABA, IAA and ethylene accumulation are altered by polyamine levels, which are positively correlated with fruit susceptibility to B. cinerea during strawberry ripening . Other hormones, such as brassinosteroids and jasmonic acid are present at lower levels during strawberry ripening. BR positively regulates vitamin C levels, sugar and anthocyanin biosynthesis during ripening, while negatively regulating acidity and concentration of other phenolic compounds .Endogenous JA levels are modulated by methyl jasmonate and the JA carboxyl methyltransferase that lead to high levels in white fruit and a decline during ripening, antagonistically to ABA . In strawberry, JA appears to be involved in defence responses against B. cinerea. For example, strawberries treated with MeJA had a delayed and much slower progression of B. cinerea infections .As indicated previously, B. cinerea releases enzymes and metabolites that act as virulence factors but may also induce plant responses that are beneficial for fungal infection . A relevant example of the manipulation of physiological processes in the host by B. cinerea is the interference with specific developmental processes. In tomato plants, B. cinerea infections modified host gene expression to increase susceptibility, such as the induction of senescence in leaves . Moreover, infected unripe tomato fruit show premature expression of genes involved in ethylene synthesis during tomato ripening . These findings suggest that B. cinerea could initiate ethylene production and thereby stimulate early ripening. As strawberries are non-climacteric fruit, ethylene production of B. cinerea may not have substantial effects on strawberry ripening; however,large plastic pots the fungus was also shown to induce genes involved in the biosynthesis of other plant hormones such as ABA. Moreover, B. cinerea can synthesize and secrete ABA that functions as a virulence factor .

Besides hormones, increased oxidative reactions caused by the pathogen may influence ripening progression .Defence mechanisms can be divided into preformed and induced defences. In strawberries, preformed defence compounds are especially abundant in the unripe stage, as reviewed in the section on quiescence of B. cinerea. Even though plants accumulate defence compounds, B. cinerea has mechanisms to cope with these metabolites by efflux and detoxification of inhibitory substances. ATP-binding cassette transporters are used by B. cinerea to facilitate the efflux of antifungal compounds, such as stilbenes . B. cinerea is capable of detoxifying inhibitory substances, like epicatechin by secretion of laccases . Active B. cinerea infections can result in a reduction of specific secondary metabolites. It has been reported that levels of flavan-3-ol, benzoic acid and phenylpropanoids drop in B. cinerea-infected strawberries . Strawberries respond to B. cinerea infection by triggering defences. In some cases, preformed and induced defences can overlap such as in the case of PGIPs. An endogenous PGIP appears to be constitutively expressed in fruit from various strawberry cultivars . However, this PGIP and six additional ones show higher expression levels upon infection with B. cinerea . Overexpression of FaPGIP1a and FaPGIP2a in cisgenic plants conferred enhanced resistance to grey mould . Other enzymes induced by B. cinerea infections are chitinases. Expression of the chitinases FaChi2-1 and FaChi2-2 peaked 16 hpi in B. cinerea-infected strawberries . Furthermore, heterologous expression of Phaseolus vulgaris chitinase cH5B in strawberry resulted in higher resistance to infection . Another study demonstrated that application of heat-inactivated cells of the yeast Aureobasidium pullulans promoted tolerance to B. cinerea in strawberries . This primed resistance is probably due to the fruit’s perception of chitin from the yeast leading to induction of chitinases or other plant immune responses. Moreover, fruit defence responses may be primed using mechanical stimulation as it was reported for strawberry leaves . Induced defences include accumulation of secondary metabolites and ROS. For instance, strawberries accumulate proanthocyanins around infection zones possibly to restrict fungal growth . The surroundings of infection sites generally display higher ROS production . ROS can serve as an effective defence against pathogens but also can lead to cell death, which is considered beneficial for necrotrophic fungi . B. cinerea itself produces ROS to induce host cell death, deplete plant antioxidants and increase lipid peroxidation . It is therefore interesting that, in unripe tomato fruit ROS production leads to resistance against B. cinerea, whereas in ripe fruit it seems to promote susceptibility . Future research will likely shed more light on the role of ROS in induced defences of strawberry fruit.

Basal immunity is activated upon fungal infection. Degradation of fruit cell wall pectins can produce demethylated oligogalacturonides that trigger basal immune responses . Expression of the F. x ananassa pectin methylesterase 1 FaPE1 in Fragaria vesca resulted in reduced methyl-esterification of oligogalacturonides in fruit. This reduced esterification activated basal defences via the salicylic acid signalling pathway that led to a higher resistance to B. cinerea . Involvement of SA signalling in responses against B. cinerea was previously suggested when strawberry plants and fruit treated with SA showed decreased post harvest decay . B. cinerea can suppress the expression of plant defence responses by hijacking the host sRNA regulatory pathways . In strawberry fruits, B. cinerea infections can alter the expression of microRNAs involved in the regulation of defence genes, including the plant intracellular Ras group-related LRR protein 9-like gene . Interestingly, B. cinerea can also take up plant sRNAs during its interaction with the host. For instance, transgenic plants expressing sRNA that targets B. cinerea DCL1 and DCL2 show significantly reduced fungal growth in strawberries . The suppression of fungal growth via host sRNA is not well understood, and it is yet to be demonstrated that this mechanism of defence naturally occurs in plants.The diverse arsenal of infection mechanisms employed by B. cinerea explains its extremely wide-host range. It is therefore not surprising that entirely resistant strawberry genotypes do not exist . Several authors have analysed field resistance of strawberries to B. cinerea by quantifying disease development without artificial inoculation. A multi-year study of three strawberry cultivars found a significant effect of year, cultivar and cultivar by year interaction on the incidence of B. cinerea infections . Moreover, there was a positive correlation between row density and disease. Other studies investigated field resistance in annual winter production systems and found that variation of B. cinerea incidence between years was larger than genotype differences within years . Even though field resistance assessments investigate conditions similar to commercial production, considerable variability between environmental conditions and years can interfere with the detection of genotype differences.Due to the confounding effects of different non-genetic variables in field studies,squre planter pots assessment of post harvest resistance to B. cinerea infections has been pursued to determine genotype differences between strawberry cultivars or species. A large study of grey mould development during post harvest storage of non-inoculated fruit reported variation in disease incidence and speed of progression amongst cultivars, but no complete resistance was observed .

Another approach to reducing environmental effects in disease tests is to inoculate fruit with B. cinerea conidia suspensions. Bestfleisch et al. tested quantitative resistance in 107 accessions of wild and cultivated strawberry. In this study, two wild ecotypes of F. virginiana showed high resistance to B. cinerea infections and slow disease progression. Such high tolerance in wild species was also reported in B. cinerea-inoculated leaves and fruit of F. chiloensis accessions from Chile . In these wild accessions, B. cinerea grew much slower. Comparative studies of disease progression indicated that fruit from the cultivar Chandler developed lesions at 24 hpi, while fruit from an F. chiloensis ecotype developed symptoms at 72 hpi . Fruit were entirely covered with mould at 6 days post-infection for the cultivar Chandler and at 9 dpi for the F. chiloensis ecotype. Considering that some accessions, particularly wild ecotypes, show reduced grey mould incidence and progression, there might be genetic sources of resistance against B. cinerea that could be used to increase resistance in strawberry. However, information about resistance mechanisms is mostly based on assumptions or empirical data. Differences in ripening patterns have been suggested as a potential explanation for resistance. For instance, some strawberries ripen from inside to outside, leaving the skin, which is the entry point of infections, unripe and thus resistant for a longer time . Some more tolerant cultivars remain white or unripe around the calyx , which is where many B. cinerea infections tend to initiate. Another mechanism of resistance could be the presence of fungal inhibitors or the induction of PR proteins. FcPR5 and FcPR10 are highly induced in resistant F. chiloensis accessions when compared to commercial F. x ananassa cultivars . Based on sequence homology, FcPR5 probably possesses anti-fungal activity, and FcPR10 is likely a ribonuclease. These findings reflect that even though efforts have been made to explore resistance mechanisms of strawberry to B. cinerea, very little is known. Therefore, more research is necessary to better understand the biology of strawberry interactions with B. cinerea infections using diverse germplasm accessions.Many disease management strategies have been implemented for the control of B. cinerea in strawberry as further described below. However, even combined approaches are only capable of reducing disease incidence and severity but cannot completely prevent or eliminate grey mould in strawberries .Historically, B. cinerea infections in strawberry production have been managed by agronomic and horticultural practices, such as removal of senescent plant material to avoid inoculum buildup . Preventing contact of fruit with soil is another common practice to avoid B. cinerea infections, as most of the inoculum is present on the ground and soil moisture promotes conidia germination . Selecting the right irrigation system could help reduce grey mould incidence; mainly, the use of drip irrigation and micro-sprinklers results in limited inoculum spread and reduction of water films on the fruit . As canopy characteristics influence microclimates , nitrogen fertilization can lead to dense canopies and favour grey mould .

Similarly, shorter plant spacings promote higher incidence of B. cinerea in the field . Additionally, plastic tunnels can avoid airborne inoculum and B. cinerea incidence is lower in non-fungicide treated tunnels than in fungicide treated fields , but tunnels favour powdery mildew and complicate harvest. In summary, cultural practices are essential to limit preharvest B. cinerea infections of strawberries, especially in organic agriculture.In modern production, pesticide applications are the most common management practice for B. cinerea control . In the previous two decades, the main pesticides used in strawberry production against B. cinerea belonged to the Fungicide Resistance Action Committee Groups 1 and 2, as well as captan . However, due to increasing fungicide resistance and new legal restrictions, producers have been forced to diversify their fungicide regimen . The frequency and timing of fungicide applications are crucial for B. cinerea control. One application of fenhexamid at anthesis can be as efficient as multiple weekly applications . Additionally, alternation and combination of different fungicides with different modes of action are recommended . Resistance of B. cinerea to fungicides is a real challenge in horticulture and fungicide resistance profiles can shift considerably even within a single season . A screen of 13 B. cinerea isolates in Louisiana showed that all were partial to full resistance to FRAC 1 fungicides, and several of the isolates also had different levels of resistance to FRAC 2 fungicides . A larger survey of 1890 B. cinerea isolates revealed that 7 isolates from different locations were resistant to all single-action site FRAC fungicides groups that are registered for B. cinerea control . B. cinerea resistance to fungicides is usually associated with over expression of efflux transporters or with modification of fungicide targets. These resistance mechanisms are acquired via mutations and recombination that occur frequently in B. cinerea due to heterokaryosis, sexual reproduction and the presence of abundant transposable elements in its genome . Efflux of fungicides or accumulation of altered fungicide targets has also been shown to lead to multi-resistances . The presence of resistant isolates against the most common multi-action site fungicides reinforces the need for innovative management practices. A new generation of RNA-based fungicides has been proposed, which relies on the application of sRNA or dsRNAs that target B. cinerea virulence genes to reduce fungal infections in strawberries . However, these RNA-based fungicides remain far from commercialization, which is why fungicide resistance management such as mixture and rotation of different fungicides or testing local isolates for resistance is necessary .

Usage would best be confined to lemons and in areas where salinity and gummosis may be critical factors. Hybrids of pummelos with other promising root stock cultivars should be considered.Almonds are California’s top agricultural export — 80% of those consumed worldwide are grown here. As water resources become increasingly scarce due to population growth, environmental needs and periodic drought, it will become more difficult both monetarily and politically to obtain sufficient water for crop irrigation. Drought tolerance in almonds has been documented in previous studies, but substantial irrigation is still required to maintain current production levels. Over the last 14 years there has been a steady increase in both bearing acres and yields — about 70 pounds per acre in almond yield improvement annnually , indicating a steady improvement in cultural practices, among them, irrigation. There is a pressing need to reliably maintain current almond production with less water. Surface-water allotments for irrigation during drought are often significantly reduced because precedence is given to other uses . Water reserves in California were low following the droughts of 2007, 2008 and 2009. In fact, spring 2008 was the driest on record . The current basis for estimating the irrigation need of a crop is to combine the water lost from the soil with the water lost through leaves , into an overall loss, the crop evapotranspiration . ETc is calculated by multiplying a weather based reference crop ET , by a crop coefficient , to give the final estimate . Research in the late 1980s and 1990s estimated the average seasonal ETc for almonds at 40 to 42 inches ,square pots with estimated seasonal irrigation requirements of 36 to 38 inches under typical soil and rainfall conditions of the southern San Joaquin Valley . But later field research suggested that almond ETc may average from 48 to 54 inches .

Reasons for the higher recent estimates probably reflect the many changes that have occurred in almond culture over the past two decades. Almond orchards are now intensively managed with pressurized rather than surface irrigation systems, and crop water status can also be monitored directly using midday stem water potential . SWP is measured directly on leaves sampled in the orchard using a pressure chamber, and it indicates the level of physiological water stress that is being experienced by the trees at the time of sampling, much as blood pressure or temperature can be a measure of any physiological stress in humans . Furthermore, nitrogen fertility management is more intensive than it was when the earlier research was conducted, and pruning practices have changed to manage canopy light differently, both producing more foliage and potentially higher ETc. In fact, a higher ETc rate and higher yields may both be responses to more intensive almond management. The ETc method of irrigation scheduling aims to maintain the crop in a non-stressed condition by supplying enough water to satisfy ETc. Alternative methods have been proposed that attempt to reduce unnecessary vegetative growth in orchard and vine crops in order to make water use more efficient; they include deficit irrigation, partial root-zone drying and regulated deficit irrigation . The objective of regulated deficit irrigation is typically to irrigate so that trees experience mild-to-moderate levels of water stress, in order to achieve an optimal horticultural balance between vegetative growth, which is very sensitive to stress, and fruit production, which is less sensitive . Previous studies in almonds and other crops have shown the beneficial effects of regulated deficit irrigation, including control of excessive vegetative growth, reduced hull rot and improved hull split in almonds , increased fruit density in prunes and pears and reduced vegetative growth in peaches . Previous studies of regulated deficit irrigation have created stress by applying a fraction of ETc, but for this 5-year study we used a plant-based indicator of stress and set a target level of mild-to-moderate stress during the hull-split period. We undertook this study to determine whether meaningful reductions in consumptive water use could be achieved with minimal impacts on orchard productivity.

Our study took place in a micro-sprinkler-irrigated, 270-acre almond orchard near Orland in the northern Sacramento Valley, which was planted with ‘Nonpareil’ and ‘Carmel’ trees spaced at 12 feet by 24 feet . The orchard was divided into five approximately equal blocks; two were planted in 1993 and three in 1999. From the first year of the experiment , the canopy shaded area in midsummer at noon was greater than 50% in all blocks, so all blocks were considered to exhibit fully developed crop water requirements . The five blocks were each subdivided into two sections to match the existing irrigation system design, with control and regulated deficit irrigation treatments assigned to the sections on alternating sides. Two rows of ‘Nonpareil’ almond trees in the center of each section were designated as the experimental plots, with two trees from each block used as the monitoring trees for SWP measurements. The rows averaged approximately 69 trees per block, and monitoring trees were positioned approximately one-third and two-thirds of the way into each row . SWP values were initially taken on weekly field visits using a pressure chamber, and were collected biweekly during the hull-split period. Leaves, still on the tree, were covered with an aluminized Mylar bag for a minimum of 10 minutes prior to measurements . Meters were installed on a single lateral line in each irrigation section to measure water applications. In 2004 and 2005, block-specific recommendations for regulated deficit irrigation were communicated to the grower, who was responsible for dayto-day irrigation management. In 2005, the orchard exhibited defoliation due to Alternaria leaf spot, and the grower was reluctant to withhold water from the large regulated deficit irrigation plots. In 2006, a separate irrigation system that could be monitored and controlled via a satellite-linked Internet service was installed for the experimental ‘Nonpareil’ row and the two adjacent Midday SWP and water meter data were collected weekly from early April until the hull-split period. Visual surveys were made weekly starting in mid-June to anticipate the beginning of hull split. Irrigation was reduced once the onset of hull split was observed in blank nuts, generally about a week before the onset of hull split in normal nuts. Before and following the hull-split period,drainage plant pot the water amounts applied to the regulated deficit irrigation and grower control treatments were equivalent. During the hull-split period, SWP was measured twice weekly and irrigation was adjusted to achieve a target mild-tomoderate stress level of −14 to −18 bars in each block.

By the last year of the study , block-specific irrigation was not necessary because the target SWP could be achieved using about the same level of deficit irrigation in all the treatment blocks. The target levels of midday SWP employed in this field trial were set to achieve mild-to-moderate water stress during the regulated deficit irrigation period. For almonds, Shackel reported about a 50% reduction in midsummer stomatal conductance with SWP values of −14 to −18 bars compared with a non-stressed SWP above −10 bars . Irrigation was returned to normal once visual surveys indicated 90% hull split in each block. The grower commercially harvested entire rows, and a weighing trailer was used to determine gross harvest weight in the field. We collected a 4-pound sub-sample from each of the blocks and used them to convert harvest weights into nutmeat yields. In this field trial, regulated deficit irrigation was limited to the hull-split phase of almond growth and development. ETc is typically highest during midsummer, so the opportunity is greatest at this time to impose crop stress in order to achieve significant irrigation reductions. In addition, Teviotdale et al. reported that both hull split and nut harvestability are improved and hull rot is reduced when regulated deficit irrigation is imposed during the hull-split period. Other stages of almond growth and development have shown greater susceptibility to negative impacts on tree growth and nut production . Crop stress is also difficult to impose from leaf-out through midMay due to rainfall, lower ETc rates and generally sufficient soil moisture.Soil moisture. We installed neutronprobe access tubes to measure the change in stored soil moisture from early spring to late summer, in order to quantify the contribution of soil water to the crop’s water needs . We installed two grids of 16 tubes in a single block, each in the southwest quadrant of a single monitoring tree for both the regulated deficit irrigation and control treatments. The tubes were arranged in 4-by-4 grids with overall dimensions of 6 feet by 12 feet . The grid spacing was measured from the center of the tube, with 2-foot spacing in the north-south direction and 4-foot spacing in the east-west direction. We tried to install the tubes to an overall depth of 60 inches and measure volumetric soil water content at 1-foot intervals, at depths of 8, 18, 30, 42 and 54 inches . However, due to the widespread variability in soils — including areas with significant gravel content, soil stratification and a shallow, temporarily perched water table — we achieved a depth of 54 inches for only 22 of the 32 tubes.

The remaining tubes were installed to a depth of 42 inches . Soil moisture readings were taken two or three times per season, typically around full bloom, in late summer and post harvest. The shallow water table receded during the course of each growing season, especially during the drought years of 2007 and 2008; it did not appear to influence orchard water status significantly during our study. If capillary flow of water from the shallow water table had contributed significantly to crop consumptive use, midday SWP would not have responded to the withholding of irrigation water during hull split. In addition, the gravel content and hard pan appeared to be barriers to deeper root development, so the roots may not have reached the soil water. Soil type. Soil types were variable throughout the orchard, but the majority of acreage consisted of three types: Cortina very gravelly sandy loam, Hillgate loam and Redding gravelly loam . These soils are described by a USDA land capability rating of 3 or 4, which generally groups soil types based on restrictions for field crops. The Redding soil typically has a restrictive layer at 20 to 40 inches , and the other soils extend to below 80 inches . Based on a nominal 60-inch soil profile, all have low available water — approximately 3.5 inches for the Cortina and Redding soils and 8 inches for the Hillgate soil . The two grids of neutron-probe access tubes were positioned in either a Cortina or Redding soil type. Ground cover. Ground cover varied between mowed resident vegetation in spring and winter, and bare ground in summer. Vegetation around the neutron-probe access tubes, where a mower could not be used, was controlled with herbicides each spring to match the surrounding vegetation. Reductions in water use Water savings. An average water balance summary for 5 years of this study showed overall savings of 4.8 inches of applied water in the regulated deficit irrigation regime . The neutron-probe readings showed an average seasonal contribution of approximately 5.0 inches of stored water in the control and 4.5 inches in the regulated deficit irrigation treatment, amounting to about 11% of overall consumptive water use. All in season precipitation was assumed to be an effective contribution. When the savings in applied water were combined with the contribution from soil storage, the regulated deficit irrigation regime resulted in a total average annual consumptive-water-use savings of 5.3 inches over the 5-year period, and yearly savings ranged from 10% to 15%, or 5.2 to 6.1 inches . Yield increases. Yields increased in both treatments during the 5-year study, with no clear trend of any reduction due to regulated deficit irrigation . The orchard’s increasing yields can be attributed to its relatively young age and continuing canopy growth. Canopy growth is typically very sensitive to deficit irrigation, so it is noteworthy that plant-based regulated deficit irrigation did not have a negative impact on yields over time, presumably because the deficit period was after the main period of vegetative growth.

Wolf also studied characteristic differences observable in cross-sections of one year-old roots of the same three species . The trifoliate orange is easily distinguished from the sour orange and the Yuzu by the large vessels in the wood, these being much larger and more numerous than similar vessels in the other two species, and by the more numerous groups of bast fibers in the bark, which form three or four broken concentric rings. In the Yuzu, only a few scattered groups of bast fibers are present in the bark, whereas in the sour orange the groups of bast fibers are numerous and close together in the inner row, with only a few scattered groups farther out, a condition intermediate between that of Yuzu and the trifoliate orange, but clearly differentiating the sour. In the differentiation of trifoliate stock from the Yuzu, Wolf also found that the Yuzu roots, when bruised, emitted a strong penetrating odor, disagreeable to many, and that the odor of the trifoliate is fainter and milder. The author also noticed some differences in the color of roots and their morphological appearance. In Israel, Cossman also studied the anatomy of citrus roots, including the root structure of sweet lime, Rough lemon, sour lemon, citron, ‘Baladi’ sweet orange, ‘Shamouti’ sweet orange, sour orange, grapefruit and shaddock. Cossman felt that characters which might be of taxonomic importance were the mode of lignification of the pith, the configuration of the protoxylem strands, suberization in the endodermis, and the thickening of the walls of the epiblema . Later, Hayward and Long described in detail the anatomy of the seedling and roots of the Valencia orange. In Israel, Green, Vardi, and Galun studied the plastomes of various citrus species and several citrus relatives. They found a resemblance between the plastomes of cultivars of lemon, orange, sour orange, grapefruit and pummelo. The plastomes of other citrus species, such as mandarin and citron,drainage gutter differed from each other as well as the plastomes of the above citrus species.

Furthermore, within the citrus relatives examined, the plastomes of the trifoliate orange and Microcitrus spp. were distinct from each other as well as from the citrus cultivars tested. They felt the result of their study constituted a useful tool for the identification of plastomes in hybrid plants of Citrus developed from protoplast fusion, i.e., somatic hybridization. Some excellent work on the anatomy of Citrus has been published by Schneider . However, most of this is developmental anatomy. He has also published crucially important papers on the seasonal production of xylem and phloem in the sweet orange tree trunk and the ontogeny of lemon tree bark , the relationship of the phloem to certain destructive diseases such as the Buckskin disease of peach and cherry to tristeza , and the incompatibilities and decline of lemons . Schneider et al. were extremely helpful in the early detection and diagnosis of these pathological and physiological problems. However, for some reason, Schneider ignored the structure of the xylem as it might differ between citrus species and how it might aid in identification. Some excellent work has been done on the structure of wood as an aid to identification with forest trees, both conifers and deciduous. One might consult Jane and [text incomplete] . The importance of knowledge of wood structure cannot be emphasized more than the convincing and convicting evidence provided by a wood expert in the trial of the kidnapper of the Charles Lindbergh baby in the early 1930’s. The expert from the regional U.S. Forest Products Laboratory at Madison, Wisconsin, successfully established that the wood in the ladder used by the kidnapper came from the attic of the kidnapper’s home, from which several pieces of wood were missing.Wagnon, Dobbins, and Breece used foliar gland characters in the identification of peach and nectarine varieties. It is possible that the nature, size, number, and arrangement of oil glands in Citrus leaves may also be useful in identification. Hirano , and Gianotti reported on the numbers and variation in stomata in Citrus and some related genera. This technique also might be of some benefit.

Nothing has been done recently with anatomical structure as an aid to root stock identification or taxonomic relationships. Clearly this method will become more complicated as more hybrids involving bigeneric and even trigeneric crosses are made. Furthermore, a microscope, good laboratory, technique, and a thorough knowledge of plant anatomy are required. The first attempt to identify root stocks by colorimetric chemical reactions was apparently that made by Henricksen , who based his method on the presence of varying quantities and kinds of glucosides containing phenol in all citrus roots. He used extracts from root pieces and, with ferric chloride as an indicator, found that the different color reactions or precipitations obtained were more or less characteristic for the four species he worked with, namely sour orange, grapefruit, sweet orange, and Rough lemon. Color density was greatest on sour orange and lightest with Rough lemon. Some confusion existed between sweet orange and Rough lemon. One, of course, needs known standard samples for comparisons. Halma and Haas developed a similar but more extensive method of identifying citrus species by employing colorimetric chemical tests with samples of dried bark since most of the reactive agent seemed to be concentrated there. A number of tests were used in these experiments, but the one that gave most consistent results was the Almen test developed by Cohn for carbolic or salicylic acid, which is practically the same as Millon’s reagent for albumens and phenols. Their experiments also indicated that three other reagents in various forms, molybdic acid, titanium chloride, and ferric chloride, were of value when identification was doubtful. The results obtained by these investigators was sufficiently uniform within commonly accepted limits of the species to lead Halma and Haas to “suggest the possibility that these colorimetric differences may be useful in citrus classification.” In a later paper, Halma described the preparation and use of the Almen test as it had been modified since its first use by Halma and Haas . The tests were only carried out with lemon, Rough lemon, grapefruit, sweet orange, and sour orange. Marloth , in South Africa, made extensive studies and experiments on the use of the four colorimetric reagents in identifying Citrus species, working mainly by the methods suggested by Halma and Haas . Both groups were able to distinguish between the commercial lemon and Rough lemon, and Marloth was able to separate grapefruit and pummelo. As the inroads of tristeza in Brazil became more prevalent and the relationship of root stock-scion combinations became more evident, Bacchi also used these colorimetric tests to distinguish root stocks.

He attempted to identify 15 species, hybrids, and cultivars and found that the reactions obtained were somewhat different from those described by Halma and Haas and Marloth . The differences between sweet orange and sour orange were quite apparent, but the situation becomes more complex with other species and varieties. Bacchi therefore proposed the separation of root stock species and varieties into four groups: sour orange, sweet orange, lemon, and “all the others.” When tristeza began to threaten California orchards and a variety of root stocks appeared to be involved, Masters made a review of laboratory tests for the determination of Citrus root stock varieties. He refined the technique somewhat and was more specific in his color chart,macetas para fresas which is perhaps the best available; it is reproduced here for the benefit of those who wish to conduct such colorimetric tests . Masters was the first person to point out that there is a difference in color reaction between above-ground and underground samples, and for these reasons an addendum is attached. Masters also proposed the use of ultraviolet light and fluorescence as an additional aid. Some of the differences between above-ground and below-ground samples may be pointed out, such as: above-ground sour orange extracts are clear with the ferric chloride test, belowground samples may become cloudy; sweet orange extracts show poor fluorescence above-ground and good fluorescence below-ground. Some of the differences in bark sample location may account for the discrepancies between previous investigators. Certainly it makes a difference as to how much the bark sample is scraped or washed to remove soil particles. The presence of contaminants such as fertilizer, pest control residues, fungicides, and other chemical agents may also make a difference in the color reactions. Of course, the importance of having knowns to compare unknowns with is critical to the tests. Furr and Reece also used a modification of the root stock color tests for the identification of hybrid and nucellar citrus seedlings with a reasonable degree of success. Similar tests were also used by Nishiura, Matsushima and Okudai to identify species and also distinguish hybrids from nucellar seedlings. Nakamura and Nakayama and Krishnamurthy, Singh and Deo also used the tests for studying phylogenetic relationships of the citrus species. Although these chemical tests were somewhat primitive by today’s standards, remarkable results were obtained by someone with care and experience. As chemical techniques and procedures improved, so have the diagnostic aids. Selle proposed a method of clearly identifying sour orange root stock from other stocks by paper chromatography. Essentially the method consisted of taking a piece of root stock bark, placing it upon a sheet of filter paper and hitting it with a hammer. Or, he used a bark extract made with a solution of ethyl alcohol-normal butyl alcohol-acetic acid and water, and placed drops of the extract on filter paper. The spots were allowed to dry, sprayed with a dilute solution of ninhydrin and again allowed to dry. The spots were examined with a long wave ultraviolet lamp and a characteristic flame pattern was observed for fluorescence. The hammer technique gave the most striking results. Selle also developed a spot chromatographic method. Root sections were taken and the bark removed, cut into very small pieces, placed in a bottle, and treated with 2,2-dimethoxypropane.

After standing for 30 minutes, single drops were placed on filter paper and the spots observed as they dried. Complete identification of all the root stocks was not obtained by using dimethoxypropane alone, and he got better differentiation by adding anhydrous aluminum chloride to the solution. He was thus able to identify sour orange, sweet orange, grapefruit, tangelo, mandarin, Rough lemon, trifoliate orange and Troyer citrange. The memory of the past is not always too reliable, but the author is quite certain that in conversation with Selle, he indicated he could also tell with the root bark what the scion variety was budded on it. Unfortunately, with Selle’s sudden death, perhaps this information was lost. Pieringer, Edwards, and Wolford and Kesterson et al. studied the application of gas-liquid chromatography to the citrus leaf oils for the identification of kinds of Citrus. Kesterson and his coworkers included eleven kinds of citrus and their data demonstrated that the oil composition for the different species is quite variable. They list the most prevalent and distinguishing features for each type oil in order of importance for sour orange, grapefruit, tangelo, mandarin, sweet orange and Rough lemon. They state that, “The percent of composition within species is shown to be sufficiently different to distinguish one variety from another.” Limits of normal deviation, tree variability, and seasonal variations are all factors which may affect leaf oil properties and they feel additional work will establish these limits. Pieringer, Edwards and Wolford studied the leaf oils of eight different citrus varieties and two sources of sour orange as subjected to four methods of instrumental analyses. These were: infrared and ultraviolet spectrophotometry, gas chromatography, and measurement of refractive indices. Some methods were more effective than others in separating closely related cultivars. They felt gas chromatography more successfully differentiated the varieties, whereas infrared and ultraviolet spectrophotometry appeared to be limited to the identification of Citrus species. The value of the refractive index was not fully determined. Burger , on page 109 of this thesis, obtained chromatograms with high pressure liquid chromatography of the phenolic present in the root stocks he worked with. Using this method, he could distinguish between Troyer and Carrizo citranges, which most other researchers could not do.

The unique character of Rough lemon under these conditions is the relatively high concentration of feeder roots at the 76-152 cm depths. On the poorly drained soils of the east coast of Florida, the results would be different. Results would also be different on the cooler and heavier soils of California. Frequent concentration in this zone may be greater than the total amount of roots in the upper 25 cm of soil. With sour orange the feeder roots have been found 3-4 M deep at nine years of age, which compares favorably with Rough lemon. Mature trees on sour orange usually have fewer feeder roots below 76 M than Rough lemon and differ from Rough lemon in having more feeder roots concentrated in the 0-25 cm zone and less in the 76-152 cm zone. Sweet orange differs from Rough lemon and sour orange in that the sweet orange has the greatest concentration of feeder roots of any stock used commercially.The high concentration of roots in rather limited zones may account for why growers in sandy soils claim sweet orange must be irrigated more frequently than Rough lemon. Ford also reports that 30-year-old trees on grapefruit roots had a shallow root development.He also says that Cleopatra is deep rooted. Temple oranges on Cleopatra at nine years of age had roots to 5.2 M. In regard to Webber’s comments regarding citrus root systems, the author is taking the liberty of interspersing his own comments since Webber did not live to the termination and removal of many of his root stock trials in 1960. At the time of the conclusion of these experiments, the root system of each tree was carefully analyzed by Kirkpatrick and Bitters , in which the roots of every tree and combination were counted, sized, and measured. Also, during the tristeza era in Southern California, when thousands of trees were pushed and pulled, it provided an opportunity to observe numerous root systems under diverse soil and environmental conditions. At the time of Webber’s demise ,best vertical garden system sour orange was still the world’s most popular root stock and the most widely used root stock in California. Webber points out that the sour orange has commonly been considered to be best adapted to growth on low, moist, and fairly heavy soils.

It first became established in Florida on such soils. On the very light sandy soils of the so-called “ridge” section of central Florida, it has been a failure. In California, especially with lemons , it was equally successful on light sandy soils such as in the Upland area. In root stock experiments it gave better results on a fairly light sandy soil at Riverside than a much heavier loam soil at Fillmore. His explanation to these different reactions was the difference in climatic and soil conditions as they reacted on the deep rooted sour orange. In California, where alkali in sub-soils and high water table is sometimes a problem, such as in the Imperial Valley, a deep rooted tree like sour orange may be seriously injured. Aeration may be a problem in high water tables and root asphyxiation may occur, or damage from high salt content. As the lower roots die back, they may be attacked by soil organisms like Phytophthora and cause further damage. A shallower-rooted tree would sustain little or no damage. The sour orange develops one or more tap roots which can grow to considerable depths, but lateral root development is more limited than in Rough lemon, sweet orange, or grapefruit. The tap root of sour orange cut back at transplanting from the nursery commonly branches and forms a small group of tap roots. Halma found one mature Eureka lemon grove in which the average number of main roots per tree was fifteen for 64 trees, of which 65 per cent were typical tap roots, whereas sweet orange was devoid of tap roots. Deep tap root penetration on deep soils renders the sour orange, to some degree, resistant to the effects of drought. Hume states that both nursery and grove trees on sour orange suffer much less in periods of protracted drought under identical conditions than Rough lemon, but Evans states in Dade County, Florida, orchard trees on grapefruit and sour orange may be actually dying of drought when adjacent trees on Rough lemon are satisfactory. Hume’s statement would not apply, perhaps, to results on the deep, sandy “ridge” soils of Florida which were planted mostly after 1926.

Webber further states the sweet orange does not develop a well differential tap root and is usually moderately shallow rooted. It does, however, have an abundant system of lateral roots. The author’s observations in California would agree. Mills states the sweet orange is a surface growing stock which has few or no penetrating roots, which does not agree with the observations of Ford . Webber makes no comment on mandarin root systems. However, when the old root stock plantings at Riverside were pulled, the author found the most extensive and massive root systems of all stocks observed with Washington navel, Valencia, grapefruit, and Eureka and Lisbon lemons to be on Cleopatra mandarin. It penetrated nearly as deeply as sour orange, had extensive laterals, but moderate fibrous roots on the sandy loam soil at Riverside which is underlain by a caliche hard pan at 1 M or more in depth. Dancy, Clementine, and Oneco were similar in structure to Cleopatra, but to a slightly lesser extent. Even the tractor driver removing the trees commented that the trees on Cleo were the most difficult to remove. Webber also made no statement regarding the pummelos. With all scion varieties at Riverside the root system of the pummelos was similar to that of sour orange, with deep penetrating multiple tap roots, a moderate lateral root system extending somewhat obliquely as in sour orange, and a scarcity of fibrous roots. On Rough lemon root stock, he states it develops a very wide spreading abundant system of lateral roots and commonly exhibits no marked tap root. The trees in these experiments did have abundant spreading laterals, profuse fibrous roots, but no tap roots. The root systems were not nearly as extensive as sweet orange, Cleopatra mandarin and several other stocks, and may account for the smaller tree size on the Rough lemon stocks. Webber also made no comment regarding lime root stocks. In the root-stock experiments at Riverside, the root system of sweet lime was similar to, but inferior to, Rough lemon. There were no tap roots, the laterals did not extend as far, but there were abundant fibrous roots. In other California experiments, the West Indian Lime had a root system similar to that described by El Azzouni and Wali , no tap root, extensive surface laterals with limited extension, and profuse fibrous roots.

The Rangpur lime had a somewhat similar root system but did not duplicate the mandarin root stocks in character. The citron also had no tap root, no extensive surface laterals and only fair amounts of fibrous roots. Many of these trees were “leaners”, further indicating that they had a weak root system. Relative to trifoliate orange, Webber remarks that it develops a well-branched root system with very abundant fibrous roots, but that the roots did not extend laterally as far as Rough lemon or sweet orange. The author’s observations on many trees indicate the complete absence of deep laterals,growing strawberries very shallow penetration, a very sharp angle of diversion from the trunk area and a moderate amount of fibrous roots. Most of the laterals were flattened in proximity to the trunk area with no surface laterals and many fibrous roots. No references were made to any of the citranges, although Morton, Savage, Rush, Coleman and Cunningham were in the plantings and Troyer and Carrizo were in later plantings. Most of the citranges have a poorly developed tap root system which only penetrates to a depth of about 1 M. They have, however, many laterals which emerge at an oblique angle and descend downward and may penetrate deeper than the tap roots. Fibrous roots are less extensive than the sweet orange parent, and in the surface layers less than the trifoliate orange parent. The root systems of Troyer, Carrizo, Savage and Morton were more extensive than the others observed and agrees well with Savage, Cooper, and Piper . No reference was made to Sampson tangelo. As expected from its parentage, its root system was extensive, but not as extensive as either mandarin or grapefruit. The tap root system was not well developed but the surface and subsurface laterals were prolific, and fibrous roots were moderate. Yuzu was very deep rooted with good surface laterals, but lacking in fibrous roots. The author would agree with Webber’s description of Calamondin, which has very large penetrating tap roots, at least several extending straight down to 150-180 cm or more. There were few laterals, only near the surface, and a scarcity of fibrous roots. Of 25 root stocks Webber observed, he felt Calamondin exhibited the most marked tap root system, followed by C. webberii, and sour orange next. The author’s observations would agree with this except, the shaddocks would also have to be included.

The root systems of C. macrophylla and C. pennivesiculata are very similar to the lemon-lime group, that is no pronounced tap roots. Extensive trees on Severinia buxifolia were the smallest of any observed and it was surprising that the roots could support andanchor the tree. There was no tap root per se, but extensive surface laterals, most of which were less that 5 cm in diameter and did not extend more than 150-180 cm from the tree trunk, and very few fibrous roots. The root systems of cuttings varied tremendously with the scion cultivar. Those of navel were the poorest, consisting of only 3-5 very large surface laterals with little penetration and few fibrous roots. Those of grapefruit cuttings were intermediate. The Valencia cuttings, however, were very similar to sweet orange root stocks except they didn’t penetrate as deeply. There were lots of surface laterals and lower tier laterals and extensive fibrous root development. Huberty states that the dominant factors in determining root distribution of plants appeared to be irrigation and soil types and sub-soils. However, in an irrigation experiment at the Citrus Experiment Station which provided for irrigation on a two, four, and six-weeks’ schedule, no noticeable difference was apparent in the amount, or the pattern, of the roots as affected by the various irrigation treatments. A marked difference was shown by the type of root stock. Smith, Kinnison, and Carns in Arizona report the effect of variable frequency of irrigation treatments on the root development of young Marsh grapefruit trees on sour orange root stock in the light sandy soils of the Yuma Mesa. In these experiments, irrigation intervals of one, two, three, four, five, and six to nine weeks were followed during the summer irrigation season for three years. The weekly irrigation schedule followed on Plot I kept the surface soil moist and at a lower range of temperature than in the other plots, and permitted an extensive root development the first year in the top 15 cm of soil. The root development in the top 15 cm of Plot II, irrigated every two weeks, was quite pronounced, but in the remaining plots it was appreciably less. This related condition prevailed for several years and then indicated a tendency toward relatively shallow root development irrespective of soil-moisture conditions. The effect of increasing the interval between irrigations seemed to limit the total root structure rather than to force development into the lower soil depths where favorable moisture conditions existed. Huberty also points out that part of the root system of a 25-year-old Washington Navel orange tree on sweet orange root was exposed by careful digging. This tree was planted on a contour row in a sandy loam soil exceeding 150 cm in depth. The longest root found was growing along the tree row in soil which was not cultivated and to which irrigation water was not directly applied.

The Bittersweet differed from sour orange in fewer laterals and very few fine fibrous roots. However, the tap roots penetrated slightly deeper than sour orange. With the sweet oranges, Pineapple and Parson Brown, the root systems were very similar. Both showed well developed central roots, usually two, penetrating about the same depth as sour orange, about 90-125 cm deep, with numerous small laterals the full length of the tap roots. The laterals in the upper 30 cm of soil were not as long as sour orange. There were abundant fibrous roots in the first 30 cm. Their data did not agree with what Mills observed in California’s cooler and loamier soils, but rather with Hume’s observations in Florida, that roots of sweet orange and sour orange are about equal in development, which Oppenheimer also reports in the sandy soils of Israel. Rough lemon had the most vigorous root system of all 15 stocks. It had exceptionally large lateral roots with a spread of 150 cm or more from the trunk. The central tap extended as deeply as sour orange, but the fibrous roots were not as abundant around the crown as sweet orange or sour orange. For Duncan and Bowen grapefruit they found an abundance of fibrous roots and many large vigorous laterals and two or more large penetrating tap roots extending 92 cm with a mass of fibrous roots the full extension. The abundance of fibrous roots agrees with observations by Mills and Oppenheimer . The trifoliate orange had deeply penetrating central roots and numerous laterals with abundant fibrous roots in the upper 31-46 cm of soil. The root system was similar to sour orange except for the smaller spread of the laterals. Cleopatra mandarin had a cone-shaped root system with well developed central roots penetrating 125 cm or more. There were long fine-textured lateral roots in the upper 61 cm of soil well supplied with fibrous roots. Its root system didn’t differ much from sour orange except the tap root was straight, slightly longer, and less divided. The root system of Suen Kat was similar to Cleopatra. The root system of Morton Citrange had the tap root dividing into several which penetrated vertically and then fanned out,vertical farming equipments descending obliquely to about 91 cm. The laterals were similar to sour orange, with a good fibrous system.

The Rusk citrange was similar in root structure to Morton except there were fewer fibrous roots.There were no tap roots. The root system resembled grapefruit in fibrous roots, but grapefruit had a pronounced tap root. The Cuban Shaddock had a root system almost identical to Rough lemon except it had more fibrous roots. The calamondin had a group of vigorous central roots which penetrated deeply, in fact, more deeply than any of the other stocks observed. Yuzu displayed the smallest root system of all 15 stocks observed. It penetrated deeply, and there was a scarcity of laterals. Sweet orange cuttings had a shallow root system with many vigorous laterals which did not penetrate more than 61-91 cm. Savage, Cooper, and Piper thus concluded that Rough lemon and Cuban Shaddock had the most extensive root system, the calamondin the deepest, and sweet lime and grapefruit had the greatest amount of fibrous roots. In [date lacking] [“Check” appear here in typescript in the margin of the manuscript], Ross reported that trifoliate orange produces a well-branched root system with an abundance of fibrous roots which are very sensitive to drying. Robinson grew ten scion varieties on Cleopatra mandarin and sour orange in Norfolk sand in Florida, and at nine years of age reported that Cleopatra produced a deep tap root with numerous fibrous roots superior to sour orange. In India, Burns and Kulkarni exposed roots of Santara and Mosambi on Jamburi from fiveyear-old trees growing poorly. With the Santara trees 320 cm high, the lateral roots extended 42 M and average 2.5 M. The root spread was greatest parallel to the tree rows. With the Mosambi trees having a height of 2.1 M the diameter spread of the roots was 1.8 M. A 14-year-old tree of Santara had roots with a radius of 3 M with secondary roots of 4.9 M. The laterals were about 23 cm deep. He concluded Jamburi was surface rooting with a fair spread of laterals, but few deep penetrating roots under the tropical conditions of India. In Ceylon , Gandhi examined the root systems of Mosambi trees planted 5.5 x 5.5 M. The spread of the crown roots was 3.7 M, or less than 1.8 M between trees. Roots had spread 2.7-4.9 M from the crown and at four years the roots were intermingled. Grapefruit trees on Rough lemon at five years had a lateral spread of 4.9 M. Fifteen year-old Santara trees planted 4.6 x 4.6 M had roots which completely intermingled and extended trunk to trunk. The Santara trees on Rough lemon at 14 years had roots extending a radius of 3 M, some 4.9 M, and a similar situation occurred with Mosambi. He only studied the lateral spread of surface roots. He did not feel it necessary to ascertain the distribution of fibrous roots since he felt only the exposure of laterals to a depth of 10-20 cm was enough to give a general idea of the nature of the fibrous roots.

Gandhi also stated that the depth of rooting, lateral spread of main and subsidiary roots and their branching, and quantity of fibrous roots are specific characters. These specific characters may vary with different species, but the location of the fibrous roots on the root system did not seem to be a specific character. He felt it appeared to be more a result of environment and cultural treatment. Montenegro in Brazil found that sour orange seedlings had more superficial and less horizontal extensive root systems than Caipira sweet orange, and poorer feeder root systems as compared to the well-developed system of the Cravo tangerine. The root system of Caipira sweet orange was deep and had a vigorous feeder root system. The most vigorous root systems were Florida Rough lemon, sweet orange and Cravo tangerine. The least vigorous root systems were Rangpur lime, sweet lime, and trifoliate orange. Hamlin sweet orange scions strongly stimulated feeder root development in root stocks. This effect was moderate with Pera and slight in Baianinha sweet orange. Root development was more vigorous in well-drained and aerated soil than in imperfectly drained,vertical farm tower shallow layered soil. Exocortis and tristeza checked root development. Extensive root development was not necessarily associated with large tree crowns or high fruit yields. As root stocks for the sweet orange varieties Hamlin, Pera, and Baianinha, Rangpur lime stock showed poorer feeder root development than Caipira, Pera, Cravo tangerine, or Florida Rough lemon. Vigor was medium on Sampson tangelo which is deep rooted like Florida Rough lemon. Trifoliate orange on clay soils produced fewer roots than Florida Rough lemon, Cravo tangerine, or Caipira orange. With Baianinha scions Caipira was more abundantly rooted than Rangpur lime, Brazilian Rough lemon, or sweet lime. Nucellar clones of Baianinha on Caipira had excellent root development superior to that of an old clone on Pera orange or Cleopatra mandarin. Seedlings of Caipira orange rooted more deeply than those of sour orange and Cravo tangerine, and in these instances the roots tended to be massed around the trunk base. Ten-year-old Hamlin oranges on either trifoliate orange or sweet lime had 90 per cent of the root system within 90 cm of the surface, but for all of the remaining ten-year-old combinations of scion and stocks the top 60 cm of soil contained 90 per cent of the roots. On light soils the roots of grafted and seedling trees of Baianinha extended down to 90 cm.

In Japan, Okuchi et al. excavated a single tree of Satsuma mandarin on trifoliate orange which was 22 years of age. Roots extended down to 120 cm so the distribution was similar to that observed by others. Most roots were within 1 M of the trunk. The horizontal and vertical distribution of the roots showed 93 per cent within 2 M of the trunk and 94 per cent within 60 cm. Most feeder roots were within 20 cm of the surface with only a few at deeper depths. In Egypt, El Azzouni and Wali excavated the whole root system of 15- year-old Washington navels on Rough lemon, lime and sour orange. Roots were graded according to diameter and dry weights taken. There was some variability among trees on the same species. Roots of all stocks extended laterally outside the 5 M zone allocated. Sour orange extended laterally the most, with lime the lowest, and Rough lemon intermediate. The roots outside the 5 M radius were considered minor compared to the total root distribution. There was a high percentage of fibrous roots on all stocks. Woody roots .75 cm in diameter and greater extended 2.4 M from the trunk and beyond the 5 M radius, so greater extension was needed. Sour orange roots extended vertically 1.5 M, although Rough lemon and lime extended to 1.2 M with Rough lemon deeper than lime. Much of the root system of all three stocks was located in the 15-30 cm depth. There were considerable roots at .6 M, with lime having more roots in this zone than either Rough lemon or sour orange. Lime had the shallowest roots, Rough lemon intermediate, and sour orange the deepest. There were no direct orientation effects. Sour orange had the highest weight of total fibrous roots, with Rough lemon intermediate and lime the lowest. In contrast, the net weights of lime showed the highest percent of fibrous roots followed by Rough lemon and sour orange. All these stocks had fibrous roots located in the first .3 M and second .6 M, and with lime most were in the first .3 M, especially at 15 cm, even though the orchard was cultivated. Sour orange had the greatest root weight, followed by Rough lemon and lime, in that order. There were no tap roots on any of the stocks. Ford found Rough lemon roots penetrated to a depth of 4.3-5.2 M at 15 years of age in the warm deep well-drained Lakeland fine sand of Florida’s central ridge. At 25 years, the total amount of feeder roots was greater at the 76-152 cm zone than the 0-25 cm zone. Fifty per cent of the feeder roots were below 76 cm, and 15 per cent below 2.7 M. At 18 years sour orange feeder roots only penetrated occasionally to a depth of 5.2 M in sandy soils. Trees on Rough lemon had more feeder roots below 76 cm than trees on sour orange. The feeder roots of grapefruit were confined to 2.1-2.4 M zones. Temple oranges on Cleopatra had feeder roots to 5.2 M at nine years of age. Sweet orange at 15 years had root penetration to 3.4 M and had the greatest total weight of feeder roots per unit volume of soil than any other root stock observed. He reports Rough lemon feeder roots 7.6 M from the trunk. However, Ford reported Rough lemon roots had extended laterally a distance of 16.8 M. At Riverside, the author also observed the greatest lateral extension of roots on Rough lemon. One root extended parallel to the irrigation furrow a distance of over 9.1 M and was over 2.5 cm in diameter at the point at which it was severed during its removal. Ford reports that citrus trees in central Florida are very deep rooted. The root system of Rough lemon may penetrate to depths of 3 M at 6-8 years of age. Although 40 per cent of the feeder root system of a young five-year-old tree may be in the upper 25 cm of soil, by the time the trees are 20-30 years of age more than 50 per cent of the feeder root system may be below 76 cm in the soil, and of this amount 19 percent may be found below 2.7 M with roots extending down to 4.3-5 M.