Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Influence of limiting and regulating factors on populations of Asian citrus psyllid and the risk of insect and disease outbreaks

Diaphorina citri, known as the Asian citrus psyllid (ACP), is the insect vector of a devastating citrus disease (huanglongbing; HLB), which has caused billions of dollars in damage in Florida since its detection in 2005. Data from long‐term monitoring programs in two Florida citrus groves were used to assess ACP demography and population ecology, which is needed to implement more effective management strategies for HLB. Seasonal patterns and correlations between ACP density estimates and a suite of environmental and community indicators, previously shown to influence ACP demography, were described and interpreted. Data were evaluated for evidence for spatial clustering and density‐dependent recruitment of ACP in a major outbreak event using Taylor power law analysis and by fitting a stochastic Beverton–Holt recruitment model using state‐space approach. Strong evidence for density‐dependent recruitment and spatial clustering was found and should be considered for future modelling work as it can greatly influence ACP populations by affecting their growth rates, dispersal behaviour and morphology, as well as HLB transmission. Observations of ACP density and dispersion in space and time, along with the estimated parameters from the Beverton–Holt model, suggested that intraspecific competition for resources may initiate both local, then widespread dispersal process, thus affecting grove‐wide and area‐wide HLB transmission. When these results were synthesised with those of parallel studies, the removal of several of these regulating factors in a single year could lead to widespread disease of the entire crop in a grove, and likely surrounding groves as well. We provide the first field evidence of the consequences of back‐to‐back ACP colonisation on the rapid spread of the HLB in a grove (a mechanism previously demonstrated in laboratory settings). We stress the importance of proper integrated pest management and area‐wide management to prevent such outbreaks.     

Ecological effects of multi‐species,ant–hemipteran mutualisms in citrus

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Automated Needle‐free Injection Method for Delivery of Bacterial Suspensions into Citrus Leaf Tissues

A prototype needle‐free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand‐held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand‐held injection. Results from inoculations with the automated device are in accord with the results with the hand‐held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ‘Hamlin’ sweet orange are susceptible.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck)

Two dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to study the somatic embryogenesis (SE) in Valencia sweet orange (Citrus sinensis Osbeck). Twenty-four differentially expressed proteins were identified at five time points of citrus SE (0, 1, 2, 3, 4 weeks after embryo initiation) covering globular, heart/torpedo and cotyledon-shaped embryo stages. The general expression patterns for these proteins were consistent with those appeared at 4 weeks of citrus SE. The most striking feature of our study was that five proteins were predicted to be involved in glutathione (GSH) metabolism and anti-oxidative stress, and they exhibited different expression patterns during SE. Based on that oxidative stress has been validated to enhance SE, the preferential representation for anti-oxidative proteins suggests that they could have a developmental role in citrus SE. Some proteins involved in cell division, photosynthesis and detoxification were also identified, and their possible roles in citrus SE were discussed.     

Targeted cybridization in citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness

CMS (cytoplasmic male sterility) can be controlled by the mitochondrion genome in higher plants, including Satsuma mandarin. Somatic fusion experiments in citrus combining embryogenic callus protoplasts of one parent with leaf protoplasts of a second parent often produce cybrid plants of the leaf parent, a phenomenon occurring most often with interspecific fusion combinations. In an attempt to practically exploit this cybridization phenomenon, we conducted somatic fusion experiments combining embryogenic suspension-derived protoplasts of Satsuma mandarin, Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a male-sterile cultivar, with leaf protoplasts of other seedy types—Hirado Buntan Pink pummelo (HBP) [Citrus grandis (L.) Osbeck], Sunburst mandarin (C. reticulata Blanco), Orie Lee hybrid (C. reticulata cv. Clementine × Murcott tangor), and Murcott tangor [C. reticulata × C. sinensis (L.) Osbeck], respectively—in an attempt to generate seedless cybrids by the targeted transfer of CMS. The genetic identities of regenerated plants from all four parental combinations were determined by flow cytometry, SSR, CAPS (or PCR-RFLP), RFLP, and chloroplast-SSR analyses. Regenerated plants from the first three parental combinations were diploids, and the cybrid nature of G1 + HBP with the mitochondrion genome from G1 and the chloroplast genome from HBP was confirmed, whereas the cybrid nature of the remaining two combinations was difficult to confirm because of the close phylogenetic relatedness of both fusion parents, as expected. Plants from G1 + Murcott were confirmed as tetraploid somatic hybrids. This is the first report of targeted citrus cybrid production by symmetric fusion with male-sterile Satsuma as the callus parent and other seedy cultivars as the leaf parents.Abbreviations CAPS: Cleaved amplified polymorphic sequence - CMS: Cytoplasmic male sterility - cp-SSR: Chloroplast simple sequence repeat - PEG: Polyethylene glycol - SSR: Simple sequence repeat - RFLP: Restriction fragment length polymorphism Communicated by G.C. Phillips     

Genome‐wide characterization and expression analysis of genetic variants in sweet orange

Heterozyosity is an important feature of many plant genomes, and is related to heterosis. Sweet orange, a highly heterozygous species, is thought to have originated from an inter‐species hybrid between pummelo and mandarin. To investigate the heterozygosity of the sweet orange genome and examine how this heterozygosity affects gene expression, we characterized the genome of Valencia orange for single nucleotide variations (SNVs), small insertions and deletions (InDels) and structural variations (SVs), and determined their functional effects on protein‐coding genes and non‐coding sequences. Almost half of the genes containing large‐effect SNVs and InDels were expressed in a tissue‐specific manner. We identified 3542 large SVs (>50 bp), including deletions, insertions and inversions. Most of the 296 genes located in large‐deletion regions showed low expression levels. RNA‐Seq reads and DNA sequencing reads revealed that the alleles of 1062 genes were differentially expressed. In addition, we detected approximately 42 Mb of contigs that were not found in the reference genome of a haploid sweet orange by de novo assembly of unmapped reads, and annotated 134 protein‐coding genes within these contigs. We discuss how this heterozygosity affects the quality of genome assembly. This study advances our understanding of the genome architecture of sweet orange, and provides a global view of gene expression at heterozygous loci.     

Effects of timing of defoliation on nitrogen assimilation and associated changes in ethylene biosynthesis in mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

Mustard (Brassica juncea L.) is characterized by oblong-shaped leaves on the lower layers of axis. These leaves are poorly illuminated, remain below light photosynthetic compensation point and abscise at maturity. Earlier research has shown that the removal of these shaded leaves improves photosynthetic potential of the rest of the leaves, the overall growth and yield of the crop. Now we show that 50% removal of these leaves at pre-flowering stage, i.e. 40 days after sowing (DAS) enhances nitrogen assimilation in the leaves more substantially than defoliation at post-flowering stage, i.e. 60 DAS. Further, the changes in N assimilation were concurrent with the ethylene evolution. It is suggested that strategies that lead to the abscission of lower leaves at early stage of growth may be adopted for efficient N utilization, and ethylene may be considered as an important physiological tool.     

Incidence and transmission patterns of tuberculosis among indigenous populations in Brazil

Approximately 10% of the Brazilian indigenous population lives in the state of Mato Grosso do Sul (MS), where a large number of new cases of tuberculosis (TB) are reported. This study was conducted to assess TB occurrence, transmission and the utility of TB diagnosis based on the Ogawa-Kudoh (O-K) culture method in this remote population. The incidence of TB was estimated by a retrospective review of the surveillance data maintained by the Notifiable Diseases Surveillance System for the study region. The TB transmission pattern among indigenous people was assessed by genotyping Mycobacterium tuberculosis isolates using the IS 6110 restriction fragment length polymorphism (RFLP) technique. Of the 3,093 cases identified from 1999-2001, 610 (~20%) were indigenous patients (average incidence: 377/100,000/year). The use of the O-K culture method increased the number of diagnosed cases by 34.1%. Of the genotyped isolates from 52 indigenous patients, 33 (63.5%) belonged to cluster RFLP patterns, indicating recently transmitted TB. These results demonstrate high, on-going TB transmission rates among the indigenous people of MS and indicate that new efforts are needed to disrupt these current transmissions.     

Progress, spread and natural transmission of Bahia bark scaling of citrus in Brazil

Progress, spread and natural transmission of Bahia bark scaling of citrus were evaluated in a trial where 240 screenhouse‐nursed nucellar grapefruit plants –‘Clason’, ‘Little River Seedless’, ‘Red Blush’, ‘Reed’ and ‘Howell Seedless’ cvs – were planted alongside and 5 m apart from a 10‐year‐old symptomatic ‘Marsh Seedless’ grapefruit orchard. Plants were distributed in 16 rows of 15 trees, with three plants of each cultivar per row. Eight trial plants were kept in screen cages. Incidence of symptomatic plants was assessed at 3‐months intervals, for 5 years, and for further 2 years at irregular intervals. Cumulative maps of disease incidence were produced for each assessment date and used in all analyses. Temporal progress was analysed by nonlinear fitting of three disease progress models. Spread was characterised in three levels of spatial hierarchy by the following analyses: ordinary runs, binomial dispersion index, binary power law fitting, isopath mapping and nonlinear fitting of disease gradient models. The first symptomatic plant was detected 2 years after planting. In the last disease assessment, 5 years after the first, 98% of the unprotected plants were symptomatic. None of the screen‐caged trees showed any symptoms. Bahia bark scaling progress was polyetic and best described by the logistic model. Ordinary runs analysis showed little if any evidence of transmission between adjacent trees. Diseased plants showed a very aggregated pattern inside quadrats (D > 5 and b > 1.53). Isopath mapping showed that main spread was only because of the primary inoculum source. Secondary foci were also observed, but they were never dissociated from main initial disease focus. Disease gradient followed wind direction, starting near the original inoculum source and was best described by exponential model. These results support a hypothesis of Bahia bark scaling transmission by air‐borne vectors with limited dispersion ability.     

Spatial dispersion and sampling of citrus whitefly,Dialeurodes citri,for control decisions in a citrus orchard

1 Monitoring the citrus whitefly Dialeurodes citri (Ashmead) in citrus orchards in Israel is necessary for control decisions in IPM in the period before the establishment of natural enemies. The main damage caused by D. citri is due to sooty mould, which colonizes the sweet secretions of this pest. 2 Using Taylor’s Power Law to describe the variance–mean relationship, the dispersion of the citrus whitefly was found to be aggregated at all developmental stages. The action threshold was estimated using regression of sooty mould level on egg or larval counts. 3 Two-stage sampling plans were constructed in different pest generations, for eggs and larval stages, on the basis of the variance between leaves on a tree and the variance between trees in a plot. 4 Fixed sample size plans were prepared, with possible numbers of trees and the numbers of leaves per tree to be sampled, in order to attain a given precision level. 5 Sequential sampling plans were also drawn up, on the basis of four leaves per tree, where trees are sampled until a decision can be reached. The maximum number of trees to be sampled was set according to the corresponding fixed sample size plan. 6 Simulation of the sequential sampling plans showed them to be highly reliable in terms of the decision reached with economy in sample size for high and low infestation levels.     

Species‐specific regulation of herbivory‐induced defoliation tolerance is associated with jasmonate inducibility

Induced changes in root carbohydrate pools are commonly assumed to determine plant defoliation tolerance to herbivores. However, the regulation and species specificity of these two traits are not well understood. We determined herbivory‐induced changes in root carbohydrates and defoliation tolerance in seven different solanaceous plant species and correlated the induced changes in root carbohydrates and defoliation tolerance with jasmonate inducibility. Across species, we observed strong species‐specific variation for all measured traits. Closer inspection revealed that the different species fell into two distinct groups: Species with a strong induced jasmonic acid (JA) burst suffered from a reduction in root carbohydrate pools and reduced defoliation tolerance, while species with a weak induced JA burst maintained root carbohydrate pools and tolerated defoliation. Induced JA levels predicted carbohydrate and regrowth responses better than jasmonoyl‐L‐isoleucine (JA‐Ile) levels. Our study shows that induced JA signaling, root carbohydrate responses, and defoliation tolerance are closely linked, but highly species specific, even among closely related species. We propose that defoliation tolerance may evolve rapidly via changes in the plant's defense signaling network.     

Genetic transformation of sweet orange with the coat protein gene of <Emphasis Type="Italic">Citrus psorosis virus</Emphasis> and evaluation of resistance against the virus

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.     

Delivery of dsRNA through topical feeding for RNA interference in the citrus sap piercing‐sucking hemipteran,Diaphorina citri

RNA interference (RNAi) is a powerful means to study functional genomics in insects. The delivery of dsRNA is a challenging step in the development of RNAi assay. Here, we describe a new delivery method to increase the effectiveness of RNAi in the Asian citrus psyllid Diaphorina citri. Bromophenol blue droplets were topically applied to fifth instar nymphs and adults on the ventral side of the thorax between the three pairs of legs. In addition to video recordings that showed sucking of the bromophenol blue by the stylets, dissected guts turned blue indicating that the uptake was through feeding. Thus, we called the method topical feeding. We targeted the abnormal wing disc gene (awd), also called nucleoside diphosphate kinase (NDPK), as a reporter gene to prove the uptake of dsRNA via this method of delivery. Our results showed that dsRNA‐awd caused reduction of awd expression and nymph mortality. Survival and lifespan of adults emerged from treated nymphs and treated adults were affected. Silencing awd caused wing malformation in the adults emerged from treated nymphs. Topical feeding as a delivery of dsRNA is highly efficient for both nymphs and adults. The described method could be used to increase the efficiency of RNAi in D. citri and other sap piercing‐sucking hemipterans.