Effect of host‐plant and infection with ‘Candidatus Liberibacter asiaticus’ on honeydew chemical composition of the Asian citrus psyllid,Diaphorina citri

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the citrus greening pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) by feeding on citrus phloem sap. Because phloem sap is rich in sugars but low in amino acids, ACP sucks large quantities and excretes most of it as honeydew. We studied the chemical composition of ACP honeydew on various host plants. Honeydew samples were analyzed with gas chromatography–mass spectrometry. Fourteen sugars, 13 amino acids, and six organic acids were detected in the honeydew of ACP. Sugars composed about 95% of the total compounds. Sucrose and trehalose were the predominant sugars, composing about 58 and 23% of the total sugars, respectively. Proline, asparagine, aspartic acid, and glutamic acid were the most abundant amino acids in ACP honeydew. The host plant and its infection with CLas had some effect on the honeydew composition. Glucose, chiro‐inositol, myo‐inositol, inositol, maltose, and turanose were lower in honeydew collected from CLas‐infected citrus compared to that collected from non‐infected trees. In CLas‐infected citrus (pineapple sweet orange, Citrus sinensis L. Osbeck) and Bergera koenigii (L.) Spreng. [curry leaf tree (both Rutaceae)] honeydews, valine, alanine, serine, glutamine, glycine, and the organic acids were lower than in honeydew from healthy citrus. Mannose, galactose, inositol, mannitol, an unknown disaccharide, and proline were higher in the honeydew collected from B. koenigii than in honeydew collected from healthy citrus (pineapple sweet orange), whereas fructose, chiro‐inositol, myo‐inositol, trehalose, and lactic acid were lower. The findings of this study help us understand the metabolism and the nutrient needs of ACP that transmits CLas, the pathogen of huanglongbing in citrus.     

Behavioral,Ultrastructural and Chemical Studies on the Honeydew and Waxy Secretions by Nymphs and Adults of the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Psyllidae)

The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) is the primary vector of the bacterium causing citrus huanglongbing (citrus greening), the most serious disease of citrus worldwide. Psyllids and other hemipterans produce large amounts of honeydew, which has been used previously as an indicator of phloem sap composition and insect feeding or metabolism. Behavioral, ultrastructural and chemical studies on ACP, its honeydew and waxy secretions showed important differences between nymphs, males and females, and suggested some mechanisms by which the psyllids, especially nymphs and adult females, can minimize their contamination with honeydew excretions. The anal opening in ACP, near the posterior end of the abdomen, is on the ventral side in nymphs and on the dorsal side in adult males and females. Video recordings showed that adult males produce clear sticky droplets of honeydew gently deposited behind their body on the leaf surface, whereas adult females produce whitish honeydew pellets powerfully propelled away from the female body, probably to get their excretions away from eggs and newly hatched nymphs. ACP nymphs produce long ribbons or tubes of honeydew that frequently stay attached to the exuviae after molting, or drop when feeding on the lower side of citrus leaves. Furthermore, honeydew excretions of both nymphs and adult females are covered with a thin layer of whitish waxy material ultrastructurally composed of a convoluted network of long fine filaments or ribbons. This material is extruded from intricate arrays of wax pores in the circumanal ring (around the anus) that is found in nymphs and females but not in males of ACP or other psyllid species. Infrared microscopy and mass spectroscopy revealed that, in addition to various sugars, honeydew excretions of ACP nymphs and females are covered with a thin layer of wax similar in profile to ester waxes.     

Caterpillar‐induced plant volatiles attract conspecific herbivores and a generalist predator

Plants release volatiles in response to caterpillar feeding that attracts natural enemies of the herbivores, a tritrophic interaction which has been considered to be an indirect plant defence against herbivores. On the other hand, the caterpillar‐induced plant volatiles have been reported to either repel or attract conspecific adult herbivores. This work was undertaken to investigate the response of both herbivores and natural enemies to caterpillar‐induced plant volatiles in apple orchards. We sampled volatile compounds emitted from uninfested apple trees, and apple trees infested with generalist herbivore the pandemis leafroller moth, Pandemis pyrusana (Lepidoptera, Tortricidae) larvae using headspace collection and analysed by gas chromatography/mass spectrometry. Infested apple trees uniquely release six compounds (benzyl alcohol, phenylacetonitrile, phenylacetaldehyde, 2‐phenylethanol, indole and (E)‐nerolidol). These compounds were tested on two species of herbivores and one predator in apple orchards. Binary blends of phenylacetonitrile + acetic acid or 2‐phenylethanol + acetic acid attracted a large number of conspecific male and female adult herbivores. The response of pandemis leafroller to herbivore‐induced plant volatiles (HIPVs) was so pronounced that over one thousand and seven hundred conspecific male and female adult herbivores were caught in traps baited with HIPVs in three‐day trapping period. In addition, significantly higher number of male and female obliquebanded leafroller, Choristoneura rosaceana (Lepidoptera, Tortricidae), was caught in traps baited a binary blend of 2‐phenylethanol + acetic acid, or a ternary blend contains 2‐phenylethanol and phenylacetonitrile + acetic acid. This result challenges the current paradigm hypothesized that HIPVs repel herbivores and question the indirect defensive function proposed for these compounds. On the other hand, a ternary blend of phenylacetonitrile and 2‐phenylethanol + acetic acid attracted the largest numbers of the general predator, the common green lacewing, Chrysoperla plorabunda. To our knowledge, this is the first record of the direct attraction of conspecific adult herbivores as well as a predator to the caterpillar‐induced plant volatiles in the field.     

Behavioral Response of <Emphasis Type="Italic">Tamarixia radiata</Emphasis> (Waterston) (Hymenoptera: Eulophidae) to Volatiles Emanating from <Emphasis Type="Italic">Diaphorina citri</Emphasis> Kuwayama (Hemiptera: Psyllidae) and Citrus

Tamarixia radiata Waterston (Hymenoptera: Eulophidae) is an effective idiobiont ectoparasitoid of the psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae), vector of the huanglongbing (HLB). We examined the olfactory responses of T. radiata to volatiles emanating from D. citri or plant volatiles using a custom designed T-maze olfactometer and open arena bioassays. We also examined the behavioral response of male and female T. radiata to conspecifics of the opposite sex to determine whether olfactory signals mediate mate location. T. radiata adults exhibited a sexually dimorphic response to volatiles emanating from D. citri and citrus. Female T. radiata responded positively to the odors emanating from D. citri nymphs in both olfactometer and open arena bioassays. However, female wasps showed no response to odors emanating from D. citri adults, D. citri honey dew secretions, intact citrus or orange jasmine leaves. Odors emanating from D. citri damaged citrus were not attractive to T. radiata females but stimulated attraction of wasps to D. citri on damaged plants. T. radiata females were not attracted to D. citri immatures when they were presented as visual cues. Male T. radiata did not show attraction to D. citri nymphs or other putative odors that were attractive to female T. radiata. In olfactometer bioassays, more male T. radiata responded to the odor of female conspecifics than blank controls in the absence of associated citrus host plant volatiles. Odors emanating from female T. radiata were not attractive to male T. radiata. Male or female T. radiata were not attracted to the odors emanating from same sex conspecifics. Both male and female T. radiata adults showed positive phototactic behavior. Collectively, our results provide behavioral evidence that: 1) female T. radiata use volatiles emanating from D. citri nymphs to locate its host and: 2) female T. radiata release a volatile pheromone that attracts male conspecifics.     

Drought stress affects response of phytopathogen vectors and their parasitoids to infection‐ and damage‐induced plant volatile cues

1. The response of a phytopathogen vector to pathogen‐induced plant volatiles was investigated, as well as the response of the phytopathogen vector's parasitoid to herbivore‐induced plant volatiles released from plants with and without drought stress. 2. These experiments were performed with Asian citrus psyllid (Diaphorina citri), vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas) and its parasitoid Tamarixia radiata as models. Candidatus Liberibacter asiaticus is the presumed causal pathogen of huanglongbing (HLB), also called citrus greening disease. 3. Diaphorina citri vectors were attracted to headspace volatiles of CLas‐infected citrus plants at 95% of their water‐holding capacity (WHC); such attraction to infected plants was much lower under drought stress. Attraction of the vector to infected and non‐stressed plants was correlated with greater release of methyl salicylate (MeSA) as compared with uninfected and non‐stressed control citrus plants. Drought stress decreased MeSA release from CLas‐infected plants as compared with non‐stressed and infected plants. 4. Similarly, T. radiata was attracted to headspace volatiles released from D. citri‐infested citrus plants at 95% of their WHC. However, wasps did not show preference between headspace volatiles of psyllid‐infested and uninfested plants when they were at 35% WHC, suggesting that herbivore‐induced defences did not activate to recruit this natural enemy under drought stress. 5. Our results demonstrate that herbivore‐ and pathogen‐induced responses are environmentally dependent and do not occur systematically following damage. Drought stress affected both pathogen‐ and herbivore‐induced plant volatile release, resulting in concomitant decreases in behavioural response of both the pathogen's vector and the vector's primary parasitoid.     

House Fly (Musca domestica L.) Attraction to Insect Honeydew

House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.     

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one. Received: 5 October 1999 / Accepted: 3 November 1999     

Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease‐susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9‐targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^? allele. The promoter of both alleles contains the effector binding element (EBE_PthA4), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%–64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2‐6 and S5‐13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.     

Suppression of citrus canker disease mediated by flagellin perception

Citrus cancer, caused by strains of Xanthomonas citri (Xc) and Xanthomonas aurantifolii (Xa), is one of the most economically important citrus diseases. Although our understanding of the molecular mechanisms underlying citrus canker development has advanced remarkably in recent years, exactly how citrus plants fight against these pathogens remains largely unclear. Using a Xa pathotype C strain that infects Mexican lime only and sweet oranges as a pathosystem to study the immune response triggered by this bacterium in these hosts, we herein report that the Xa flagellin C protein (XaFliC) acts as a potent defence elicitor in sweet oranges. Just as Xa blocked canker formation when coinfiltrated with Xc in sweet orange leaves, two polymorphic XaFliC peptides designated flgIII-20 and flgIII-27, not related to flg22 or flgII-28 but found in many Xanthomonas species, were sufficient to protect sweet orange plants from Xc infection. Accordingly, ectopic expression of XaFliC in a Xc FliC-defective mutant completely abolished the ability of this mutant to grow and cause canker in sweet orange but not Mexican lime plants. Because XaFliC and flgIII-27 also specifically induced the expression of several defence-related genes, our data suggest that XaFliC acts as a main immune response determinant in sweet orange plants.     

Host plant volatiles responsible for the invasion of Stenotus rubrovittatus (Heteroptera: Miridae) into paddy fields

We investigated the attractiveness of synthetic volatile blends or individual volatiles of flowering rice panicles or flowering Scirpus juncoides spikelets to the sorghum plant bug Stenotus rubrovittatus (Matsumura). None of the individual chemicals tested attracted either sex of the bug. Synthetic volatile blends of flowering rice panicles composed of geranyl acetone, β‐caryophyllene, n‐decanal, methyl salicylate, β‐elemene and n‐tridecene attracted females. The synthetic blend of volatiles was just as attractive as natural flowering rice panicles to females. Other synthetic blends did not attract the bug. We sampled headspace volatiles from flowering S. juncoides spikelets with solid‐phase microextraction and analysed them using gas chromatography–mass spectrometry. The main volatile emitted from S. juncoides was β‐caryophyllene, one of the major volatile components of flowering rice panicles. β‐Elemene was a common volatile found in flowering rice panicles and flowering S. juncoides spikelets. Therefore, we investigated the attractiveness of synthetic blends of flowering rice panicles and S. juncoides spikelets composed of β‐caryophyllene and β‐elemene. The synthetic blend of flowering S. juncoides spikelets significantly attracted males but not females. The synthetic blend of flowering rice panicles composed of β‐caryophyllene and β‐elemene did not attract either sex. These results suggest that β‐caryophyllene and β‐elemene are common active compounds responsible for attractiveness of flowering rice panicles and S. juncoides spikelets although some of the other volatile components act synergistically with these two compounds in natural plant odours.     

Use of herbivore‐induced plant volatiles as search cues by Tiphia vernalis and Tiphia popilliavora to locate their below‐ground scarabaeid hosts

Japanese beetle, Popillia japonica Newman, and oriental beetle, Anomala orientalis (Waterhouse) (both Coleoptera: Scarabaeidae) are considered invasive species and have been reported as key pests of urban landscapes in the Northeastern USA. Tiphia vernalis Rohwer and Tiphia popilliavora Rohwer (Hymenoptera: Tiphiidae) were introduced as biocontrol agents against these beetles. These parasitic wasps burrow into the soil and search for grubs. When a host is found, the wasp attaches an egg in a location that is specific for the wasp species. It is unknown if these wasps can detect patches of concealed hosts from a distance above ground and what role, if any, herbivore‐induced plant volatiles play in their host location. This study evaluated the responses of female T. vernalis and T. popilliavora to grub‐infested and healthy plants in Y‐tube olfactometer bioassays. Also the effect of root herbivory on the composition of turfgrass (Poaceae) volatile profiles was investigated by collecting volatiles from healthy and grub‐infested grasses. Tiphia wasps were highly attracted to volatiles emitted by grub‐infested tall fescue (Festuca arundinacea Schreb.) and Kentucky bluegrass (Poa pratensis L.) over healthy grasses. In contrast, wasps did not exhibit a significant preference for grub‐infested perennial ryegrass (Lolium perenne L.) as compared with the control plants. The terpene levels emitted by grub‐infested Kentucky bluegrass and tall fescue were greater than that of control plants. Low levels of terpenes were observed for both test and control perennial ryegrass. The elevated levels of terpenes emitted by grub‐infested Kentucky bluegrass and tall fescue coincided with the attractiveness to the tiphiid wasps. Here, we provide evidence that plant exposure to root‐feeding insects P. japonica and A. orientalis resulted in an increase in terpenoid levels in turfgrasses, which strongly attracts their above‐ground parasitoids.     

Incidence of “Candidatus Liberibacter asiaticus” in a Florida population of Asian citrus psyllid

The incidence of a bacterium “Candidatus Liberibacter asiaticus” was assessed in a Florida population of Asian citrus psyllid, Diaphorina citri. The bacterium is the presumed causal agent of Asiatic huanglongbing, a serious citrus disease. Adult D. citri were periodically collected between May 2010 and September 2012 in a block of diseased trees located in east‐central Florida. The psyllids were individually subjected to molecular analyses (quantitative polymerase chain reaction assays using HLBaspr primers) to determine whether the bacterium was present and, if so, the population level of the pathogen based on qPCR cycle threshold (CT) values. Significantly greater percentages of females tested positive for the pathogen than males, but there were no significant differences between females and males with respect to population levels of the pathogen within the psyllids. No significant differences were found among the three D. citri colour morphs with respect to percentages of adults testing positive for the pathogen. Among 47 sample dates, a mean of 17.5% of adults per sample date tested positive (CT < 36) for the pathogen with a mean CT value of 31.1. The incidence of the pathogen was generally higher during late fall or early winter and often lower during mid‐ to late summer. There was a significant negative correlation between percentages of D. citri testing positive and air temperature. Increases in the incidence of the pathogen may not necessarily correspond to increases in transmission, as a number of factors both internal and external to D. citri can influence transmission. Transmission rates may be highest during periods when D. citri infestation levels are large, a high percentage of adults carry a high population of the pathogen in their salivary glands, and citrus flush is abundant.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

The response of citrus plants to the broad mite Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae)

1. Polyphagotarsonemus latus (Banks) (Acari: Tarsonemidae) is a common polyphagous mite in tropical and subtropical areas and is considered as an important citrus pest.
2. To understand the response of citrus to P. latus infestation, we have characterized the volatile profile and the molecular defence mechanisms of two citrus genotypes, namely sour orange (Citrus aurantium) and Cleopatra mandarin (Citrus reshni), to P. latus infestation. These two species are important rootstocks for the citrus industry and display differential resistance to Tetranychus urticae Koch (Acari: Tetranychidae), with sour orange showing elevated levels of constitutive and induced resistance associated with the jasmonic acid (JA) pathway compared with Cleopatra mandarin.
3. P. latus infestation activated both the JA- and the salicylic acid-dependent pathways in sour orange but not in Cleopatra mandarin. However, this differential activation resulted in the production of similar volatile blends (a mixture of green leaf volatiles and aromatic compounds).
4. Contrary to T. urticae infestation, sour orange supported larger densities of P. latus than Cleopatra mandarin with similar injury levels.
5. Therefore, sour orange may be more tolerant to P. latus than Cleopatra mandarin.

     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits ‘Candidatus Liberibacter asiaticus’ (Clas), the pathogen responsible to cause HLB. Understanding of vector, pathogen, and host plant interactions is important for the management of this vector‐disease complex. We used the direct‐current electrical penetration graph (DC‐EPG) system to evaluate feeding behavior of Clas‐infected D. citri adults, and their potential to transmit the pathogen to healthy citrus, Citrus reticulata Blanco cv. Sunki (Rutaceae), following a 24‐h inoculation access period. Plants were tested for the presence of Clas by qPCR 6 months after inoculation. Findings suggest that inoculation was associated with salivation into the phloem sieve elements (waveform E1). The minimum feeding time for successful transmission by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min. Regression analysis indicated a significant relationship between E1 duration and transmission efficiency. The adults successful in transmitting Clas to healthy citrus were able to penetrate and feed in the phloem much earlier than those which did not transmit. The minimum duration of E1 for a female was shorter than that of a male, but transmission was higher. However, durations of other EPG parameters were not significantly different between male and female. Feeding by single Clas‐infected D. citri adults on 6‐month‐old plants (Sunki) resulted in 23% HLB‐positive plants 6 months after inoculation. Multiple nymphs or adults could transmit the pathogen more efficiently than individual adults in the field, and further enhance the severity of the disease. Effective tactics are warranted to control D. citri and disrupt transmission of Clas.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

Effects of quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested maize on the attraction of the larval parasitoid Cotesia kariyai

Cotesia kariyai Watanabe (Hymenoptera: Braconidae) is a specialist larval parasitoid of Mythimna separata Walker (Lepidoptera: Noctuidae). Cotesia kariyai wasps use herbivore‐induced plant volatiles (HIPVs) to locate hosts. However, complex natural habitats are full of volatiles released by both herbivorous host‐ and non‐host‐infested plants at various levels of intensity. Therefore, the presence of non‐hosts may affect parasitoid decisions while foraging. Here, the host‐finding efficiency of naive C. kariyai from HIPVs influenced by host‐ and non‐host‐infested maize [Zea mays L. (Poaceae)] plants was investigated with a four‐arm olfactometer. Ostrinia furnacalis Guenée (Lepidoptera: Crambidae) was selected as a non‐host species. One unit (1 U) of host‐ or non‐host‐infested plant was prepared by infesting a potted plant with five host or seven non‐host larvae. In two‐choice bioassays, host‐infested plants fed upon by different numbers of larvae, and various units of host‐ and non‐host‐infested plants (infestation units; 1 U, 2 U, and 3 U) were arranged to examine the effects of differences in volatile quantity and quality on the olfactory responses of C. kariyai with the assumption that volatile quantity and quality changes with differences in numbers of insects and plants. Cotesia kariyai was found to perceive quantitative differences in volatiles from host‐infested plants, preferring larger quantities of volatiles from larger numbers of larvae or plants. Also, the parasitoids discriminated between healthy plants, host‐infested plants, and non‐host‐infested plants by recognising volatiles released from those plants. Cotesia kariyai showed a reduced preference for host‐induced volatiles, when larger numbers of non‐host‐infested plants were present. Therefore, quantitative and qualitative differences in volatiles from host‐ and non‐host‐infested plants appear to affect the decision of C. kariyai during host‐habitat searching in multiple tritrophic systems.     

The Role of Chemical Cues in Host and Mate Location in the Pear Psylla Cacopsylla bidens (Homoptera: Psyllidae)

Chemical communication was shown to play a role in the pear psylla, Cacopsylla bidens. Electrophysiological (EAG) and behavioral responses were investigated in males and females pear psylla . Males were found to be attracted to females, and especially to those on host plants, but not to males, uninfested host plants, or plants infested with conspecific larvae. On the other hand, females were not attracted to males or females but displayed some attraction to host plants. Furthermore, females showed a preference for uninfested pear versus plants infested with conspecific larvae. The antennae of males gave highest electroantenographic response to volatiles from pears infested with females but not males, while females, responded also toward the volatiles of pear alone. These results indicate that females of C. bidens emit sex pheromones that are attractive to the males and suggest that, host volatiles may play a role in host selection by pear psylla females.     

Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.