Author: naturehydro

Rhizoboxes offer the advantage of localized sampling in soil using sorption media such as paper and membrane filters, compound specific ion exchange binding resin or micro-suction cups placed closed to root zones of inTherest to collect exudates . Moreover, in a rhizobox fitted at the bottom with a porous rootimpenetrable membrane, a root mat is allowed to be formed which is then further transferred onto a collection compartment . The collection compartment containing soil could then be cut into thin slices parallel to the membrane to represent differing distances from the rhizosphere . While this approach can be used to investigate exudate release and sorption under soil conditions, the root mat growth generalizes exudate production in terms of the whole root system and occludes spatial exudation patterns. In a hybrid set up by Oburger et al. , the rhizobox is transplanted to a second specialized rhizobox for continued vertical root growth. This specialized rhizobox consists of a nylon membrane close to the transparent side to restrict root growth into the soil except for root hairs . This creates a vertical flat root mat onto which localized exudate samples can be collected. A comparison of this novel set up to conventional collection methods showed that amino acid exudation rates were most varied among the different methods , further highlighting the need for specialized chambers. Nonetheless, successful implementation of these chambers is still limited to fast-growing plants which can form active root mats. The high density of root mats could also lead to unnatural root exudate levels and an overestimation of rhizosphere effects. In addition, care has to be given to the choice of membrane as selective sorption of certain root exudates onto the membrane may also occur . Free-living nematodes are ubiquitous in the soil. They are beneficial to the plants by playing a role in nutrient cycling and in defense against insects and microbial infections through signaling interactions with the roots .

Conversely, infections by parasitic nematodes in the roots increase the plant’s susceptibility to stress and other pathogenic bacteria, fungi,growing hydroponically and viruses creating major losses in crop productivity . With an impending rise in nematode infections due to climate change, understanding nematode behavior and interactions in the rhizosphere becomes important to develop appropriate biocontrol methods to ensure long term food security . Traditional nematode studies are performed in petri dishes with agar or culture media . However, these substrates do not accurately emulate the physical textures and heterogeneity of soil and create homogenous solute and temperature gradients which could impact nematode behavior and interactions with the roots . Indeed, nematode motility speed and dispersal decreased in substrates more closely mimicking sand . On the other hand, studying nematode behavior in the soil is a difficult endeavor as its near-transparent body and small size makes it almost indistinguishable from soil particles. Cross-sectioning and staining infected roots make it possible for nematode visualization but they are destructive and provide only static snapshots of cellular changes or nematode behavior during infections . On the other hand, microscopy rhizosphere chambers provide non-invasive detection and observation of nematode activity in the rhizosphere . The roots in these chambers grow between a glass slide and a nylon membrane . The membrane restricts movement of roots except root hairs into the soil while the transparent glass enables microscopy of the roots at high resolution . Coupled with fluorescently stained nematodes, microscopy rhizosphere chambers allowed for non-destructive in situ observations of nematode infection in its host species over the entire life of the parasite . Nonetheless, staining nematodes is an additional challenge as nematode cuticles are impermeable to stains . This can, however, be alleviated by using advanced imaging technologies which eliminates the need for staining.

A recent study demonstrated live screening of nematode-root interactions in a transparent soil-like substrate through the use of label-free light sheet imaging termed Biospeckle Selective Plane Illumination Microscopy coupled with Confocal Laser Scanning Microscopy . Using this set up, researchers were able to monitor roots for nematode activity at high resolution and suggest its possible use in rapid testing of chemical control agents against parasitic nematodes in soil-like conditions . Fungal communities in the rhizosphere are involved in the degradation of organic matter in the soil and subsequent nutrient turnover affecting plant health as well as the microbial community . Fungal biomass often reaches a third of total microbial biomass carbon and almost all terrestrial plants are able to form symbiotic associations with mycorrhizal fungi . The majority of these associations are with arbuscular mycorrhiza fungi  which penetrate into root cortex cells to form highly branched structures . The investment of photosynthetic carbon by plants to AMF is rewarded with increased nutrient availability made possible by the extended hyphal network in the soil. For instance, up to 90% of phosphorus uptake in plants can be contributed by symbiosis with AMF . AMF networks in the soil also influence water retention and soil aggregation further impacting plant growth . Moreover, next-generation sequencing technologies and advances in imaging techniques have greatly improved our knowledge on the taxonomical and functional properties of fungal communities in the rhizosphere . However, these methods are optimized for fine scale analysis and are not capable of assessing the foraging capabilities of hyphal networks which can span across centimeter to meter scales. Toward this end, several researchers have used compartment setups with physical barriers created by 20–37 µm nylon membranes which restrict movement of roots but not mycorrhizal fungi. This separation creates root-free and plant free soil compartments connected only by mycorrhizal fungi to examine the transport of various compounds across these compartments. Using this set up, the importance of mycorrhizal fungi in the flow of different elements such as carbon , nitrogen and phosphorus between plants, soil and microbes over centimeter distances have been validated.

Repeated disruption of the hyphal connections also led to a decreased resistance in plants to drought stress . The membranes can also be placed horizontally to create different depth gradients to investigate hyphal contributions to water uptake . In some studies, an additional 1.5–3 mm air gap is created between two membranes with a wire net to restrict solute movement between two chambers . A common feature of these set ups is the size-exclusion membranes which proved to be critical in distinguishing fungal hyphae processes in the rhizosphere soil. In addition to AMF interactions, a split root set up, which separates the roots of one plant into halves, can be introduced to investigate the systemic response of plants . In essence, the split-root system directs the growth of the roots to generally two different growth conditions and enables the investigation of whether a local stimuli have a local or global response which can be observed at the root or shoot level . Split-root systems are widely studied and have been adapted to rhizoboxes as well as to pots and tubes . In the rhizosphere, plants host a wide diversity of bacteria on the surface of the root as well as within roots in the vascular tissues . Due to its abundance and importance,ebb flow tray the bacterial community in the rhizosphere is perhaps the most widely studied among other microbial members in the rhizosphere ecosystem. While the study of endophytic bacteria requires inevitable destructive sampling due to its localization, several non-destructive approaches have been developed to study microbes inhabiting the rhizoplane. One of the most widely studied plant-microbe interactions in the rhizosphere is that of the symbiotic relationship between legumes and rhizobia . Once a potential nodule forming bacteria is isolated, it is often required to authenticate its nodule forming phenotype by inoculating on host plants. However, conventional methods such as the use of soil pouches do not allow long term incubation, while “Leonard jars,” consisting of two stacked glass jars forming the top soil layer and the bottom nutrient solution layer, can be expensive and time consuming . A recent study challenges this by describing the use of clear plastic CD cases as minirhizotrons with potential for use in phenotyping root traits such as legume formation, and demonstrated innovation that democratizes research opportunities in rhizosphere research . Other microbial interactions in the rhizosphere, however, may not result in visible changes to the root system and often rely on next-generation omics technologies. As such, physical separation of the rhizosphere from the bulk soil becomes paramount in elucidating changes to microbial community and interactions. One approach to this end is the use of nylon bags with differing pore sizes . The nylon bag restricts the movement of roots and the soil inside the bag is then regarded as the rhizosphere soil to compare against the surrounding root free bulk soil . Developing further on this concept, Wei et al. designed a specialized rhizobox that allowed repeated non-destructive sampling by adding individual nylon bags of root-free soil surrounding the root compartment which are then used as a proxy for the rhizosphere . These methods allowed easy distinction of the rhizosphere and the bulk soil but, we now know that the rhizosphere community is not only distinct from the bulk soil but also varies with type, part and age of the root, largely as a consequence of varying root exudation patterns . Studying this phenomenon in situ in the soil requires separation of desired roots from others without disturbance to plant growth or soil. To address this, researchers have used a modified rhizobox design with a side compartment to regulate root growth and quarantine specific roots from the main plant chamber . This additionally creates easy distinction between old and new roots and allows testing on specific quarantined roots despite plant age. A study using this set up showed specific microbial chemotaxis toward different exudates on an individual root whereas another showed spatial and temporal regulation of niche differentiation in microbial rhizosphere guilds .

Similar physical perturbations to regulate root growth in response to microbial stimuli have also been applied in the microscale and are explored in the next section. Our assessment of the major growth chambers showed that most of the systems applied share similarities in basic structural components such as in the use of two parallel sheets in rhizobox based devices. While these growth chambers brought many of the rhizosphere processes to light, limitations do exist. One limitation is with the scale of applicability. Most of these growth systems are mesoscale and can easily reproduce potscale studies but may not be easily translatable to interactions occurring at the microscale nor recapitulate processes occurring at field-relevant scale. The next section describes advances in technology resulting in a new wave of unique devices making use of microfluidic processes and fabricated ecosystems which are specifically made to investigate specific rhizosphere processes.A complex web of biochemical processes and interactions occur in microscale dimensions in the rhizosphere. Having the ability to interrogate and manipulate these microscale processes and environmental conditions with high spatiotemporal resolution will elucidate mechanistic understanding of the processes. Microfluidics has proven to be a powerful approach to minimize reagent usage and to automate the often-repetitive steps. The microscale of the channels also allows precise control of reproducible conditions utilizing the laminar flow and automated fluidic operations . In addition, the microfluidic devices are well integrated with conventional imaging techniques by using a glass slide or coverslip as a substrate bonded with polydimethylsiloxane . These characteristics, as well as the ability to rapidly prototype and reproducibly manufacture using soft lithography technique, have enabled new ways of interrogating and studying the rhizosphere environment in a reproducible manner.They have an opening port, sometimes with pipette tips inserted into the PDMS body where the seed of the seedling rests and a microchannel where the primary root grows into. The dimension of the channel depends on the type and age of the plant. For example, an Arabidopsis thaliana’s seedling is typically grown in a microfluidic device up to 10 days, with chamber dimension around 150 to 200 µm in height, whereas the Brachypodium distachyon seedling chamber is 1 mm in height due to its thicker roots . Media and/or inoculation of the microbiome is achieved through additional channels to the main chamber. The PDMS body with the channels is typically bonded on a 50 mm by 75 mm microscope slide, and is made to accommodate multiple plants to increase throughput.

The International Seed Testing Association has as one of its important objectives to develop and test methods used to quantify seed germination. Existing methods to test seed viability include X-ray analysis, tetrazolium staining, a cut test, in some cases the careful extraction and sterile culture of zygotic embryos under aseptic in vitro conditions, but germination tests are probably the most widely used. Each method has advantages and disadvantages and consequently no single method provides a definitive means of accurately quantifying seed viability. For example, while relatively new and non destructive the X-ray assessment of seeds only indicates whether seeds are structurally intact , not whether the seeds are actually alive so while the seeds can be placed back into storage following assessment the use of this test on its own does not provide any certainty as to whether any structurally sound seeds are specifically alive. Although the germination test is probably one of the most accurate ways to assess seed lots, it is labor intensive, time consuming , and it may dependent upon knowledge concerning the optimal germination conditions and dormancy breaking requirements of the species in question. In addition, a significant concern from a seed bank management perspective is the fact that germination tests are ‘‘irreversible’’, so if seeds are viable and therefore germinate, the genetic information that they represent will be lost if not grown to maturity which may not possible in large seed banks. This is a particular concern with critically endangered species, as seed stocks may be exceptionally rare and in some cases impossible to source again. With currently available seed viability tests being largely irreversible, generally destructive, and sensitive to subjective data interpretation,hydroponic grow systems there is a pressing need for development of non-destructive and quantitative methods to assess viability and germination of precious seed stocks.

Several studies have demonstrated the potential of reflectance based spectroscopy methods in studies of plant seeds, including detection of internal infestations by weevils in dry field peas, classification of near isogenic maize lines, ageing of cabbage seeds, classification of near isogenic maize lines, differentiation between black walnut shell and pulp, sorting of lettuce seeds , and viability of horticultural seeds. These spectroscopy studies are based on the fundamental assumption that reflectance data acquired from the seed coat provides indicative information about the quality/ germination of the given seed. The research objective is therefore to identify portions of the wavelength spectrum, in which seeds show a significant and measurable change in certain parts of the examined reflectance spectrum and associated that change in reflectance with certain traits, such as, germination . A wide range of classification methods have been used as part of using reflectance data to characterize seeds and food products; these classification methods include support vector machine, variogram analysis, partial least square analysis, and linear discriminant analysis. LDA is based on discriminant functions, which are linear combinations of features and with one function for each target class. For each observation, a discriminant score is calculated and the observation is assigned to the class for which the discriminant function generates the highest discriminant score. In this study, we used hyperspectral imaging data to determine the germination of seeds from three native Australian tree species [Acacia cowleana Tate , Banksia prionotes L.F. , and Corymbia calophylla K.D. Hill & L.A.S. Johnson ]. These species represent the Australian flora with many different species held in seed banks across Australia as well as internationally including a number of critically endangered taxa. Seeds were exposed to standardized rapid ageing conditions, and at each assessment point were subjected to germination testing and hyperspectral imaging. We hypothesized that there would be detectable difference in seed coat reflectance between germinating and nongerminating seeds, and that changes in reflectance profiles would be most pronounced in spectral bands near known pigment peaks involved in photosynthesis and/or near spectral bands used in published indices to predict chlorophyll or nitrogen content in leaves.

The potential benefits of developing accurate machine vision systems to automate non-destructive monitoring of seed germination are discussed in the context of management of seed banks, botanic gardens, and implementation of vegetation restoration programs.A push-broom hyperspectral camera was mounted 40 cm above the seeds, and hyperspectral images were acquired with the spatial resolution of 50 pixels per mm2 under artificial lighting . The main specifications of the hyperspectral camera were: Firewire interface , 12 bit digital output, 240 spectral bands from 392 to 889 nm by 640 pixels . The objective lens had a 35 mm focal length with a 7 field of view, and it was optimized for the near-infrared and visible near-infrared spectra. During hyperspectral image acquisition in the lab, RH was between 30% and 40% and temperature 19–22C. A piece of white Teflon was used for white calibration. reflectance value was referred to relative reflectance and compared to that obtained from white Teflon. Colored plastic cards were imaged at all hyperspectral imaging events, and average reflectance profiles from these cards were used to confirm high consistency of hyperspectral image acquisition conditions .Immediately after hyperspectral imaging at each sampling time, the sub-samples of seeds were transferred to Petri dishes with semi solidified water agar for germination testing. Each seed was sequentially placed onto the dish and individually labelled with a permanent marker . Petri dishes were sealed with plastic film, wrapped in aluminium foil and incubated in a 15C growth chamber and checked weekly for germination. For determination of the predicted time required for germination to decline to 50% probit analysis was performed in Genstat version 10.0 . Seeds were aged at 60  C rather than 45C as is more common in seed ageing experiments. P50 values were calculated using the original data then adjusted to 45C by multiplying 60C P50 values by 8.44 as described in previous studies.Pixels with R660 reflectance values outside the stipulated ranges were excluded before average reflectance profiles were generated for each seed. We obtained 200 average reflectance profiles from each species , and relationships between results from the germination tests and hyperspectral imaging data were tested using LDA classification.

To select an optimized subset of the 10 ‘‘best’’ spectral bands, we conducted a forward stepwise LDA based on all average reflectance profiles for each of the three seeds species, and only these 10 spectral bands were used. Although not presented in this study, we conducted additional classification analyses with both more or fewer spectral bands,rolling benches and negligible classification accuracy was gained by including more than 10 spectral bands, and significant classification accuracy was lost by reducing the number of spectral bands. Thus, using the ‘‘best’’ 10 spectral bands was considered an optimum for this particular application. classification accuracies of each LDA was based on independent validation, as the original 200 average reflectance profiles and germination test results from each seed species were randomly divided into 80% training data and 20% independent validation data, This random division of data into training and validation data was repeated five times, and we calculated the average classification accuracy from the five randomized divisions of each data set.The effect of experimental ageing showed that within 0– 10 days, germination of Acacia and Corymbia seeds was above 90%, but both species of seeds showed a considerable decrease in germination after 10–20 or 20–30 days of experimental ageing, respectively . From the onset of ageing, there was an exponential decline in germination of Banksia seeds, and none of the seeds germinated after 30 days of experimental ageing. Germination for all three species was below 10% after 30 days of ageing, with B. prionotes seeds showing the steepest decline in germination with a P50 of only 7.0 days . For comparison, the P50 values for A. cowleana and C. calophylla seeds were 19.3 and 22.9 days respectively. Adjusted P50 values for all three species were 163.3, 59.1 and 193.3 days for C. calophylla, A. cowleana and B. prionotes respectively. With 25 seeds and eight sampling events , we obtained the following numbers of germinating and non-germinating seeds: Acacia , Banksia , and Corymbia .Average reflectance profiles acquired after 5 , 20 , and 20–30 days ofstandardized rapid ageing were used to illustrate the difference in reflectance between germinating and non-germinating seeds on days with similar numbers of seeds in both categories. Average reflectance profiles from the seed coat of the three species showed similar pattern with relative reflectance values commencing around 0.10 in spectral bands near 400 nm and gradually increasing reflectance in spectral bands near 900 nm. Loss of germination caused a decrease in reflectance in Banksia and Corymbia seeds, while it caused an increase in reflectance in Acacia seeds. We wish to highlight that it was virtually impossible to distinguish germinating and non-germinating seeds on the basis of visual inspection.

This statement is confirmed by the fact that reflectance values across the visual part of the spectrum were very similar. Based on independent validation of LDA classifications, we found that germination of Acacia and Corymbia seeds could be classified with over 85% accuracy, while it was about 80% for Banksia seeds . Regarding Acacia and Banksia seeds, we obtained similar classification accuracies of germinating and non-germinating seeds, but for Corymbia the classification accuracy associated with non-germinating seeds was considerably higher than for germinating seeds. We examined the relationships between days of ageing and classification accuracies, and it was revealed that the classification accuracy of : Corymbia seeds was above 80% at all time points, the classification accuracy of Acacia seeds was above 90% in the beginning and end of the study period but was below 65% around the time point with 50% germination, and Banksia seeds was below 80% during the time period with a marked decline in germination but around 90% in the beginning and end of the study period. In other words, seeds sampled during the gradual decrease in germination, in the transition from germination to non-germination, were generally classified with lower accuracy.Despite growing inTherest in use of reflectance based methods to determine the germination of horticulture and agriculture seeds, we are unaware of any published studies involving the assessment of native seeds for conservation or vegetation restoration purposes. We demonstrated that the germination of seeds can be accurately classified on the basis of commonly used classification methods, such as, LDA. Thus, it may be possible to replace time consuming and destructive germination tests with non-destructive reflectance based technologies as part of improved management of seed banks. The germination of Acacia and Banksia seeds decreased from above 90% to below 20% in about 10 days of experimental ageing. The decline in germination of Corymbia seeds was less pronounced from over 90% to about 10% in 20 days. Numerous studies have demonstrated successful use of reflectance-based spectroscopy as part of studies into seed germination. Shetty et al. used near-infrared spectroscopy to classify viable/ non-viable cabbage and radish seeds of different sizes with classification accuracies exceeding 90%. Similarly, Ahn et al.used a combination of hyperspectral imaging and fluorescence lighting to discriminate between viable and non-viable Brassica seeds with over 90%accuracy. Ahn et al. used the Fourier transform near-infrared reflectance technology technique to classify viable/non-viable water melon [Citrullus lanatus ] seeds and obtained classification accuracies also exceeding 90%. Finally, Esteve et al. used near-infrared reflectance technology to detect heat and frost damage to corn and to differentiate viable and non-viable corn and soybean seeds. The authors concluded that only heat damage could be accurately predicted. The exact associations between seed coat reflectance and primary seed metabolism are not known, so it is not possible to provide much more than speculations about the importance of certain changes in seed coat reflectance. However, some important insight may be gained by using knowledge gathered from reflectance studies of plant leaves. For instance, it is known that important plant pigments have maximum peaks at particular wavelengths: chlorophyll a , and carotenoids . In addition, there is a wealth of simple two spectral band indices used to estimate chlorophyll content in leaves, including : R430/ R680, R672/R550, R710/R760, R750/R550. Finally, there is a large body of research into use of reflectance based methods to quantify nitrogen content in leaves, and these have recently been reviewed. An important study analyzed the correlation between reflectance in spectral bands between R447–R1752 nm and leaf N accumulation in rice and wheat. The authors found that leaf N accumulation was strongly correlated with reflectance at R660, R810, and R870 nm.

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.