Response of carbon fixation, allocation, and growth to source-sink manipulation by defoliation in vegetative citrus trees.

Source-sink balance in plants determines carbon distribution, and altering it can impact carbon fixation, transport, and allocation. We aimed to investigate the effect of altered source-sink ratios on carbon fixation, transport, and distribution in 'Valencia' sweet orange (Citrus x sinensis) by various defoliation treatments (0%, 33%, 66%, and 83% leaf removal). Gas exchange parameters were measured on 0 and 10 days after defoliation using A/Ci response curves, and leaf export was measured two days after defoliation using radioisotope tracer techniques. Greater defoliation increased the maximum rate of carboxylation (Vcmax), electron transport rate (J1200), and triose-phosphate utilization rate (TPU). Leaf export was unaffected by defoliation but increased in leaves closer to the shoot apex. Basipetal translocation velocity in the trunk remained unaltered, indicating that more photosynthates remained in the shoot rather than being transported directly to the root sink. Defoliated plants initiated more new flush shoots but accumulated less shoot biomass per plant after 8 weeks. Carbon allocation to fine roots was smaller in defoliated plants, suggesting defoliation led to retention of carbohydrates in aboveground organs such as the trunk and other shoots from previous growing cycles. In conclusion, the low source-sink ratio increased carbon fixation without impacting individual leaf export in citrus. The results suggest that intermediate sinks such as the aboveground perennial organs play a role in mediating the translocation velocity. Further research is necessary to better understand the dynamics of source-sink regulation in citrus trees.

Genome Wide Identification of Respiratory Burst Oxidase Homolog (Rboh) Genes in Citrus sinensis and Functional Analysis of CsRbohD in Cold Tolerance.

Respiratory burst oxidase homologs (Rbohs) are critical enzymes involved in the generation of reactive oxygen species (ROS) that play an important role in plant growth and development as well as various biotic and abiotic stresses in plants. Thus far, there have been few reports on the characterization of the Rboh gene family in Citrus. In this study, seven Rboh genes (CsRbohA~CsRbohG) were identified in the Citrus sinensis genome. The CsRboh proteins were predicted to localize to the cell membrane. Most CsRbohs contained four conserved domains, an EF-hand domain, and a transmembrane region. Phylogenetic analysis demonstrated that the CsRbohs were divided into five groups, suggesting potential distinct functions and evolution. The expression profiles revealed that these seven CsRboh genes displayed tissue-specific expression patterns, and five CsRboh genes were responsive to cold stress. Fourteen putative cis-acting elements related to stress response, hormone response, and development regulation were present within the promoters of CsRboh genes. The in-silico microRNA target transcript analyses indicated that CsRbohE might be targeted by csi-miR164. Further functional and physiological analyses showed that the knockdown of CsRbohD in trifoliate orange impaired resistance to cold stress. As a whole, our results provide valuable information for further functional studies of the CsRboh genes in response to cold stress.

Genome-Wide Characterization and Expression Analysis of the SBP-Box Gene Family in Sweet Orange (Citrus sinensis).

SBP-box is an important plant-specific transcription factor family and is involved in diverse biological processes. Here, we identified a total of 15 SBP-BOX genes in the important fruit crop sweet orange (Citrus sinensis) and characterized their gene structures, conserved domain and motif, chromosomal location, and cis-acting regulatory elements. SBP genes were classified into four subfamilies based on the amino acid sequence homology, and the classification is equally strongly supported by the gene and protein structures. Our analysis revealed that segmental duplication events were the main driving force in the evolution of CsSBP genes, and gene pairs might undergo extensive purifying selection. Further synteny analysis of the SBP members among sweet orange and other plant species provides valuable information for clarifying the CsSBP family evolutionary relationship. According to publicly available RNA-seq data and qRT-PCR analysis from various sweet orange tissues, CsSBP genes may be expressed in different tissues and developmental stages. Gene expression analysis showed variable expression profiles of CsSBP genes under various abiotic stresses, such as high and low-temperature, salt, and wound treatments, demonstrating the potential role of SBP members in sweet orange response to abiotic stress. Noticeably, all CsSBP genes were also downregulated in sweet orange upon the infection of an important fungal pathogen Diaporthe citri. Our results provide valuable information for exploring the role of SBP-Box in sweet orange.

Variations in phenolic acids and antioxidant activity of navel orange at different growth stages.

Ripe navel orange has abundant amounts of phenolic compounds. Few studies monitored changes in these compounds during ripening. In this study, the effects of navel orange maturation on dynamic changes in antioxidant activity, total phenolic content (TPC), total flavonoid content (TFC) and phenolic acids were investigated. Five growth stages of navel orange were studied, and nine phenolic acids were detected via high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS). Results showed that antioxidant activity, TFC and TPC decreased gradually with fruit ripening. The concentrations of most phenolic acids also declined during fruit maturation, except for free fractions of sinapic acid and bound fractions of ferulic and caffeic acids. Ferulic acid was the most dominant of all phenolic acids at all growth stages. Partial least-squares showed significant differences among fruits of different maturities. A significant correlation between antioxidant capacity, TPC, TFC and some phenolic acids was found.

Anthocyanin Biosynthesis and DNA Methylation Dynamics in Sweet Orange Fruit [Citrus sinensis L. (Osbeck)] under Cold Stress.

The blood red color of pigmented orange fruit varieties [Citrus sinensis L. (Osbeck)] is due to the presence of anthocyanin pigments that largely contribute to determine the high organoleptic qualities and the nutritional properties of the fruits. The content of pigments in sweet orange depends primarily on genetic factors and on environmental conditions. In particular, it has been extensively shown that cold temperature induces an increase of anthocyanin content that is achieved by the induction of the related gene expression. The purpose of our work is to understand the mechanism underlying the color variegation occurring inside the blood oranges during the cold induction of anthocyanin biosynthesis, despite the fact that the entire fruit is genotypically programmed to produce pigments. Therefore, the amount of anthocyanin and the expression of both structural and regulatory genes have been monitored in either high-pigmented (HP) or not/low pigmented (NP) segments of the same fruit during the storage at 4 °C for a total experimental period of 25 days. Our results clearly indicate that the anthocyanin content is directly correlated with the levels of gene transcription, with higher pigmented areas showing higher enhancement of gene expression. Furthermore, we analyzed the reshaping of the DNA methylation status at the promoter regions of genes related to anthocyanin biosynthetic pathway, such as DFR and Ruby. Our results unequivocally demonstrate that in the promoter regions of both DFR and Ruby, the amount of cytosine methylation strongly decreases along the cold storage in the HP areas, whereas it increases in the NP areas of the same fruit, probably causing a partial block of the gene transcription. Finally, by measuring the changes in the expression levels of the Citrus DNA demethylases, we found that DML1 might play a crucial role in determining the observed demethylation of DFR and Ruby promoters, with its expression induced by cold in the HP areas of the fruits. This is the first report in which different levels of gene expression implicated in anthocyanin production in blood orange fruit is correlated with an epigenetic control mechanism such as promoter methylation.

Optimization of sampling and monitoring of vegetative flushing in citrus orchards.

Citrus trees produce flushes throughout the year, but there are no criteria established for a precise shoot monitoring in orchards under tropical climate. Methods for quantification of flush dynamics would be useful for horticultural and pest management studies because different insect vectors feed and reproduce on flushes. We estimated the minimum number and distribution of trees for sampling and determined the flushing pattern over time in 'Valencia Late' orange trees grafted onto 'Swingle' citrumelo rootstock. Shoots within a square frame (0.25 m2) on two sides of the canopy were counted and classified by their phenological stage. The minimum number of samples was estimated using the mean number of shoots and area under the flush shoot dynamics (AUFSD). The temporal and spatial distribution analysis was performed by Taylor's power law and by multiple correspondence analysis (MCA). Additionally, a shoot maturity index (SMI) based on visual qualitative assessment of flushes is proposed. Considering the mean number of shoots, it was necessary to sample two sides of 16 trees to reach a relative sampling error (Er) of 25%, whereas by the AUFSD, only five trees were necessary to reach an Er of 10%. Flushes were predominantly randomly distributed over time and space. Testing eight transects, sampled trees should be distributed throughout the block, avoiding sampling concentration in a certain area. MCA showed that the west side and the upper sampling positions of trees were more likely to be associated with younger shoots. AUFSD and the evaluation of both sides of the canopy yielded a smaller number of trees to be assessed. The SMI was a reliable metric to estimate the shoot phenology of orange trees, and correlated well (R2 > 70%) with the mean number of shoots within the square frame. Therefore, SMI has the potential to make shoot monitoring in the field more practical.

Genuine Carotenoid Profiles in Sweet Orange [Citrus sinensis (L.) Osbeck cv. Navel] Peel and Pulp at Different Maturity Stages.

The carotenogenesis in the endocarp and flavedo of Navel oranges over four consecutive maturity stages was assessed by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-multistage mass spectrometry. After optimization of the extraction method, 77 carotenoids, including 26 monoesters and 33 diesters of violaxanthin, ß-citraurin, and antheraxanthin, were characterized. Whereas chloroplast-specific pigments, such as (all-E)-lutein and (all-E)-ß-carotene, predominated in the flavedo of green-ripe fruit, a highly complex pattern of xanthophyll esters was found in the mature oranges. Total carotenoid contents of flavedo were approximately 9-fold higher [12 605 µg/100 g of fresh weight (FW)] than those in the endocarp (1354 µg/100 g of FW) at the fully mature stage. The mature endocarp abundantly contained violaxanthin mono- and diesters, in addition to diverse antheraxanthin esters, which were exclusively detected in this fruit fraction. Likewise, ß-citraurin esters were found to be unique flavedo constituents of mature fruit. Therefore, they may support the detection of fraudulent use of peel fractions during orange juice production.

Evaluation of California-Grown Blood and Cara Cara Oranges Through Consumer Testing, Descriptive Analysis, and Targeted Chemical Profiling.

The acceptability of Moro, Tarocco, Cara Cara, Shahani, Bream Tarocco, Boukhobza, and Sanguinelli oranges from both commercial and research orchards was tested with adult (n = 152) and child (n = 72) consumers. Qualitative focus groups were also conducted to understand consumer familiarity and thoughts about the fruit. Sensory descriptive and chemical analyses were carried out to identify drivers of liking. Overall, consumers preferred the lighter colored varieties consisting of Tarocco, Cara Cara, and Boukhobza. One cluster of adults (n = 80) showed preferences towards sweet and fruity flavors and away from sourness and citric acid. The second adult cluster (n = 72) was tolerant of the sour fruit but did not like fruit high in bitterness and flavonoid content. The largest child cluster (n = 42) showed preferences for samples higher in orange and tropical flavors (Cara Cara, Tarocco, and Boukhobza varieties). The appearance of the Cara Cara was strongly liked by the consumer population in both quantitative and qualitative settings. Hunter scale a color values strongly correlated to the higher berry/dried fruit flavors, and concentrations of naringenin. Focus group participants noted that they were relatively unfamiliar with blood oranges. Growers and producers may want to invest in the lighter colored varieties, such as Cara Cara, Tarocco, Boukhobza and Shahani, as these were liked by a majority of consumers and were low in less desirable sensory characteristics, such as bitterness and sourness. PRACTICAL APPLICATION: Through consumer tests, sensory evaluation, and chemical analyses, this research uncovered which sensory properties may drive consumer acceptance of blood and Cara Cara oranges, and informed potential production and marketing strategies for increasing their consumption. This information should benefit the citrus industry as a whole and may enhance the use of specialty oranges by the food, beverage, and food service industries.

Multiyear life energy and life cycle assessment of orange production in Iran.

Increasing agricultural production requires the application of more chemical inputs during the growth of different crops. This study aims at assessing the energy indicators and environmental impacts during the initial 7-year growth of orange orchards. Through the life cycle assessment (LCA) technique, the hotspots in 11 environmental indicators of the orange orchard growth were determined for different years. The system boundaries were considered to be the orange orchard gates, and the functional units were 1 kg of orange and 1-h orange orchard. The energy input of nitrogen fertilizer, diesel, and human labor has the biggest share in the total energy inputs. The total average energy input and energy output (in 7 years) were calculated as 62,917.027 MJ/ha and 47,618.17 MJ/ha, respectively. The results showed that the energy indices (energy efficiency and net energy) were increasing from year first to seventh and also the share of renewable energy increased. According to the results, with respect to the highest share for production of 1 kg orange in different criteria, nitrogen fertilizer was the main contributor to abiotic depletion and human toxicity, orchard field emissions and nitrogen fertilizer had the highest shares in global warming and photochemical oxidation, fossil fuels and nitrogen fertilizer were the highest contributors to ozone layer depletion, whereas ecotoxicity was mainly affected by chemical fertilizers and orchard surface emissions. Finally, the main contributor for acidification and eutrophication was surface emissions. In the study of environmental impacts from the first to the seventh year, it should be noted that if the functional unit is considered mass based (1 kg orange), due to the unproductive of the trees in the first to third years (low fruit production), the environmental effects are high and then it decreases after the third year due to increasing the yield of the product. Generally, with consideration of the tree growth period, the useful data of energy and environmental impacts for production horticultural products can be provided so that we can avoid multiple interpretations of results associated to reporting annual energy and environmental impact variations.

Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis.

BACKGROUND: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. RESULTS: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. CONCLUSIONS: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.

Fruit fly identification, population dynamics and fruit damage during fruiting seasons of sweet oranges in Rusitu Valley, Zimbabwe.

In 2003, the pest species Bactrocera dorsalis (Hendel) was reported for the first time in Kenya, Africa, and subsequently on many other African countries. In this work, 20 locations along the Rusitu Valley (Zimbabwe) were sampled in 2014 during the sweet oranges fruiting seasons, to verify the fruit fly taxonomy, invasion source, population dynamics, and fruit damage. The trapped fruit flies were identified using morphological traits and molecular techniques, as B. dorsalis. The haplotype network analysis revealed that Zimbabwe COI sequences were identical to other African B. dorsalis sequences. Fruit fly trappings per day varied during the year, although it remained always high. The same applies to fruit damage, most likely due to the permanent availability of cultivated and wild fruit varieties during the year. Rusitu Valley was invaded by B. dorsalis, most likely from neighbouring countries. Ten years after the first report in Kenya, the complete or near complete invasion of Africa has been achieved by B. dorsalis. In northern Africa the distribution is clearly limited by the Sahara desert. The large population size, the polyphagous nature of the species, and the continuous availability of suitable host fruit species during the year complicates the eradication of this species.

Spatiotemporal association between the mite Brevipalpus yothersi and Citrus leprosis virus C in orange orchards.

Citrus leprosis virus C (CiLV-C) is an economically important pathogen and the main causative agent of leprosis disease in citrus orchards. The main vector of this disease, the mite Brevipalpus yothersi, is widely distributed in Mexican orchards on a wide range of citrus species. Despite the importance of both the virus and the mite, field studies recording their occurrence and co-occurrence are practically non-existent. We systematically sampled orange orchards for both CiLV-C and B. yothersi throughout the year. The distribution of the CiLV-C and B. yothersi was evaluated on each sampling occasion and their spatiotemporal associations were determined. Specifically, 100-112 orange trees, distributed in 18 rows (five or six trees per row), were sampled monthly between March 2017 and February 2018 (11 sampling dates). Twenty leaves per tree were sampled on each occasion. The number of mites per tree and the percentage of leaves per tree with disease symptoms were recorded. On each sampling occasion, spatiotemporal associations between mites and disease were determined using the Spatial Analysis by Distance Indices (SADIE) method. CiLV-C and B. yothersi were identified using molecular methods. Throughout the study, the distribution of CiLV-C was aggregated and the distribution of B. yothersi was random. No association was found between the virus and the mite on any of the sampling dates. In total, 173 mites were collected, but only 43 mites were found to be carrying CiLV-C. The reason for this lack of association between the virus and the mite, as well as the impact of our findings on the epidemiology of the disease in orange orchards, are discussed.

Excess copper effects on growth, uptake of water and nutrients, carbohydrates, and PSII photochemistry revealed by OJIP transients in Citrus seedlings.

Seedlings of 'Shatian pummelo' (Citrus grandis) and 'Xuegan' (Citrus sinensis) were supplied daily with nutrient solution at a concentration of 0.5 (control), 100, 200, 300, 400, or 500 µM CuCl2 for 6 months. Thereafter, seedling growth; leaf, root, and stem levels of nutrients; leaf gas exchange; levels of pigments; chlorophyll a fluorescence (OJIP) transients and related parameters; leaf and root relative water content; levels of nonstructural carbohydrates; H2O2 production rate; and electrolyte leakage were comprehensively examined (a) to test the hypothesis that Cu directly damages root growth and function, thus impairing water and nutrient uptake and hence inhibiting shoot growth; (b) to establish whether the Cu-induced preferential accumulation of Cu in the roots is involved in Cu tolerance of Citrus; and (c) to elucidate the possible causes for the Cu-induced decrease in photosynthesis. Most of the growth and physiological parameters were greatly altered only at 300-500 µM (excess) Cu-treated seedlings. Cu supply increased the level of Cu in the roots, stems, and leaves, with a greater increase in the roots than that in the stems and leaves. Many of the fibrous roots became rotten and died under excess Cu. These findings support the hypothesis that Cu directly damages root growth and function, thus impairing water and nutrient uptake and hence inhibiting shoot growth, and the conclusion that the preferential accumulation of Cu in the roots under excess Cu is involved in the tolerance of Citrus to Cu toxicity. The lower CO2 assimilation in excess Cu-treated leaves was caused mainly by nonstomatal factors, including structural damage to thylakoids, feedback inhibition due to increased accumulation of nonstructural carbohydrates, decreased uptake of water and nutrients, increased production of reactive oxygen species, and impaired photosynthetic electron transport chain. Also, we discussed the possible causes for the excess Cu-induced decrease in leaf pigments and accumulation of nonstructural carbohydrates in the roots and leaves.

CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot 'Kurodagosun' resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot 'Deep purple', resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.

The effect of moisture on soil microbial properties and nitrogen cyclers in Mediterranean sweet orange orchards under organic and inorganic fertilization.

Water shortage and soil degradation are common environmental stressors encountered in the Mediterranean area. We evaluated how different soil moisture levels, dependent on distance from drip irrigation points, impact on the biological, chemical and physical properties of citrus soil under organic and inorganic fertilization. We measured soil physicochemical properties, basal soil respiration, soil microbial biomass carbon, soil microbial community structure (phospholipid fatty acid assay), bacterial load (16S rRNA gene abundance), enzymatic activities (urease, dehydrogenase, ß-glucosidase and acid phosphatase) and abundance of microbial nitrogen cyclers (quantitative PCR). A field experiment was established in an orange orchard (Citrus sinensis) in southeast Spain and eighteen soil samples were taken from each plot to compare the impacts of soil moisture: near (wet, w) or away (dry, d) from drip-irrigation points, in plots with inorganic fertilizers under intensive ploughing (PI) or organic fertilization (OA). The results showed that changes in microbial properties and soil microbial indexes were strongly associated with soil moisture content under both organic and inorganic fertilization, and with organic carbon content. Soil moisture influenced soil aggregation, basal soil respiration, phosphatase activity, bacterial and fungal load (PLFAs) and the abundances of bacterial N cycling genes, including nifH (nitrogen fixation) nirS/K and nosZ genes (denitrification) and amoA-B (bacterial nitrification). The potential for N fixation and denitrification, two microbial processes that are crucial for determining the amount of N in the soil, were improved by increased soil moisture in the proximity of the drip irrigation. Soil OC and total N, which are higher under organic fertilization than under inorganic fertilization, were also shown to be highly correlated with the abundance of the N cycling genes. By controlling irrigation doses and applying organic amendments, it may be possible to increase the microbial abundance and function in soil and support greater fertility of soils.

Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

Spatial Distribution and Sampling Plans With Fixed Level of Precision for Citrus Aphids (Hom., Aphididae) on Two Orange Species.

Aphis spiraecola Patch, Aphis gossypii Glover, and Toxoptera aurantii Boyer de Fonscolombe are three important aphid pests of citrus orchards. In this study, spatial distributions of the aphids on two orange species, Satsuma mandarin and Thomson navel, were evaluated using Taylor's power law and Iwao's patchiness. In addition, a fixed-precision sequential sampling plant was developed for each species on the host plant by Green's model at precision levels of 0.25 and 0.1. The results revealed that spatial distribution parameters and therefore the sampling plan were significantly different according to aphid and host plant species. Taylor's power law provides a better fit for the data than Iwao's patchiness regression. Except T. aurantii on Thomson navel orange, spatial distribution patterns of the aphids were aggregative on both citrus. T. aurantii had regular dispersion pattern on Thomson navel orange. Optimum sample size of the aphids varied from 30-2061 and 1-1622 shoots on Satsuma mandarin and Thomson navel orange based on aphid species and desired precision level. Calculated stop lines of the aphid species on Satsuma mandarin and Thomson navel orange ranged from 0.48 to 19 and 0.19 to 80.4 aphids per 24 shoots according to aphid species and desired precision level. The performance of the sampling plan was validated by resampling analysis using resampling for validation of sampling plans (RVSP) software. This sampling program is useful for IPM program of the aphids in citrus orchards.

Critical Fungicide Spray Period for Citrus Black Spot Control in São Paulo State, Brazil.

The period of citrus black spot (CBS) control used in South Africa (SA) and Australia, from October to January or February, has not been as effective in São Paulo (SP), Brazil. This study aimed to evaluate different periods of protection and determine the critical period for CBS control in SP. A field trial was carried out for two seasons in a mature Valencia sweet orange orchard located in Mogi Guaçu, SP. Spray programs with a total of 60, 100, 140, 180, and 220 days of fruit protection (DFP) were evaluated. CBS symptoms and fruit drop decreased exponentially as the length of the period of protection increased. The reductions in CBS intensity and crop loss with these programs ranged from 34 to 96 and 50 to 77%, respectively. The programs with 180 and 220 DFP, which protected the fruit from September to March and May, showed the highest cost benefit. The critical period needed for CBS control in SP is longer than that in SA and Australia. The results obtained with the present study are helpful for scheduling a more efficient and rational program for CBS control not only in SP but also in other tropical and subtropical regions with similar weather conditions.

Comparative transcriptome and proteome profiling of two Citrus sinensis cultivars during fruit development and ripening.

BACKGROUND: Transcriptome and proteome analyses on fruit pulp from the blood orange 'Zaohong' and the navel orange 'twenty-first century' were performed to study Citrus sinensis quality-related molecular changes during consecutive developmental periods, including young fruit, fruit-coloring onset and fruit delayed-harvest for two months, during which fruit remained on the trees. RESULTS: The time-course analysis for the fruit developmental periods indicated a complex, dynamic gene expression pattern, with the numbers of differentially expressed genes (DEGs) between the two cultivars being 119, 426 and 904 at the three continuous stages tested during fruit development and ripening. The continuous increase in total soluble solids over the course of fruit development was correlated with up-regulated sucrose phosphate synthase (SPS) transcription levels in both cultivars. Eleven differentially expressed genes between the two cultivars involved in the flavonoid pathway were significantly enriched at the onset of the fruit-coloring stage when anthocyanins were detected in blood orange alone. Among 5185 proteins, 65 up-regulated and 29 down-regulated proteins were co-expressed with their cognate mRNAs with significant transcription and protein expression levels when the fruits from the two cultivars were compared at the fruit delayed-harvest stage. Additionally, important genes participating in the γ-aminobutyric acid (GABA) shunt were activated in blood orange at two significant expression levels in the fruit delayed-harvest stage. Thus, organic acids in fruit continuously decreased during this stage. CONCLUSIONS: This research was the first to provide a more comprehensive understanding of the differentially expressed genes involved in anthocyanin, sucrose and citrate metabolism at the transcriptome and proteome levels in C. sinensis, especially during the fruit delayed-harvest stage.

Honeybee spillover reshuffles pollinator diets and affects plant reproductive success.

During the past decades, managed honeybee stocks have increased globally. Managed honeybees are particularly used within mass-flowering crops and often spill over to adjacent natural habitats after crop blooming. Here, we uniquely show the simultaneous impact that honeybee spillover has on wild plant and animal communities in flower-rich woodlands via changes in plant-pollinator network structure that translate into a direct negative effect on the reproductive success of a dominant wild plant. Honeybee spillover leads to a re-assembly of plant-pollinator interactions through increased competition with other pollinator species. Moreover, honeybee preference for the most abundant plant species reduces its seed set, driven by high honeybee visitation rates that prevent pollen tube growth. Our study therefore calls for an adequate understanding of the trade-offs between providing pollination services to crops and the effects that managed pollinators might have on wild plants and pollinators.