Search

Chitinase plays a vital role in the virulence of entomopathogenic fungi (EPF) when it infects host insects. We used gene recombination technology to express chitinase of three strains of Lecanicillium lecanii: Vl6063, V3450, and Vp28. The ORF of ChitVl6063, ChitV3450 and ChitVp28 were inserted into the fungal expression vector pBARGPE-1, which contained strong promoter and terminator, respectively, to construct a chitinase overpressing plasmid, then transformed the wild-type strain with blastospore transformation method. The virulence of the three recombinant strains against Toxoptera aurantii was improved by overproduction of ChitVl6063, ChitV3450, and ChitVp28, as demonstrated by significantly lower 3.43 %, 1.72 %, and 1.23 % fatal doses, respectively, according to an insect bioassay. Similarly, lethal times of recombinants (ChitVl6063, ChitV3450 and ChitVp28) were also decreased up to 29.51 %, 30.46 % and 33.90 %, respectively, compared to the wild-type strains. Improving the expression of chitinase is considered as an effective method for the enhancement of the EPF value. The efficacy could be enhanced using recombinant technology, which provides a prospecting view for future insecticidal applications., Competing Interests: Declaration of competing interest All authors have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Mycorrhizal plants show enhanced resistance to biotic stresses, but few studies have addressed mycorrhiza-induced resistance (MIR) against biotic challenges in woody plants, particularly citrus. Here we present a comparative study of two citrus species, Citrus aurantium, which is resistant to Tetranychus urticae, and Citrus reshni, which is highly susceptible to T. urticae. Although both mycorrhizal species are protected in locally infested leaves, they show very distinct responses to MIR., Results: Previous studies have indicated that C. aurantium is insensitive to MIR in systemic tissues and MIR-triggered antixenosis. Conversely, C. reshni is highly responsive to MIR which triggers local, systemic and indirect defense, and antixenosis against the pest. Transcriptional, hormonal and inhibition assays in C. reshni indicated the regulation of jasmonic acid (JA)- and abscisic acid-dependent responses in MIR. The phytohormone jasmonic acid isoleucine (JA-Ile) and the JA biosynthesis gene LOX2 are primed at early timepoints. Evidence indicates a metabolic flux from phenylpropanoids to specific flavones that are primed at 24 h post infestation (hpi). MIR also triggers the priming of naringenin in mycorrhizal C. reshni, which shows a strong correlation with several flavones and JA-Ile that over-accumulate in mycorrhizal plants. Treatment with an inhibitor of phenylpropanoid biosynthesis C4H enzyme impaired resistance and reduced the symbiosis, demonstrating that phenylpropanoids and derivatives mediate MIR in C. reshni., Conclusion: MIR's effectiveness is inversely correlated to basal immunity in different citrus species, and provides multifaceted protection against T. urticae in susceptible C. reshni, activating rapid local and systemic defenses that are mainly regulated by the accumulation of specific flavones and priming of JA-dependent responses. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: Among the Citrus species, lemon (Citrus limon Burm f.) is one of the most affected by the two-spotted spider mite (Tetranychus urticae Koch). Moreover, chemical control is hampered by the mite's ability to develop genetic resistance against acaricides. In this context, the identification of the genetic basis of the host resistance could represent a sustainable strategy for spider mite control. In the present study, a marker-trait association analysis was performed on a lemon population employing an association mapping approach. An inter-specific full-sib population composed of 109 accessions was phenotyped through a detached-leaf assays performed in modified Huffaker cells. Those individuals, complemented with two inter-specific segregating populations, were genotyped using a target-sequencing approach called SPET (Single Primer Enrichment Technology), the resulting SNPs were employed for the generation of an integrated genetic map., Results: The percentage of damaged area in the full-sib population showed a quantitative distribution with values ranging from 0.36 to 9.67%. A total of 47,298 SNPs were selected for an association mapping study and a significant marker linked with resistance to spider mite was detected on linkage group 5. In silico gene annotation of the QTL interval enabled the detection of 13 genes involved in immune response to biotic and abiotic stress. Gene expression analysis showed an over expression of the gene encoding for the ethylene-responsive transcription factor ERF098-like, already characterized in Arabidopsis and in rice for its involvement in defense response., Conclusion: The identification of a molecular marker linked to the resistance to spider mite attack can pave the way for the development of marker-assisted breeding plan for the development of novel selection coupling favorable agronomical traits (e.g. fruit quality, yield) with a higher resistance toward the mite., (© 2024. The Author(s).)

Root-knot nematodes of the genus Meloidogyne parasitize the roots of thousands of plants and can cause severe damage and yield losses. Here, we report a new root-knot nematode, Meloidogyne limonae n. sp., parasitizing "lemon" ( Citrus limon ) in Hainan Province, South China. Lemon trees infected by the root-knot nematode showed poor-quality lemons, chlorosis of foliage, weak growth, and numerous root galls with white females and egg masses protruding outside. Phylogenetic trees of sequences within the ribosomal and mitochondrial DNA demonstrated that this species differs clearly from other previously described root-knot nematodes. Morphologically, the new species is characterized by an oval-shaped perineal pattern and the lateral field marked by a ridge of cuticle on one or both sides; the dorsal arch is low, with fine to coarse, smooth cuticle striae; the vulva slit is centrally located at the unstriated area; the spicules of males are arcuate and curved ventrally; the gubernaculum is distinct and curved; the labial disc of second-stage juveniles is prominent and dumbbell shaped; stylet knobs are oval and sloping backwardly; pharyngeal glands are not filling the body cavity and overlapping the intestine ventrally; and the conical tail is gradually tapering. Phylogenetic trees based on the ITS1-5.8S-ITS2, D2-D3 of the 28S rDNA, and COI and COII-16S rRNA genes of the mtDNA showed that M. limonae n. sp. belongs to an undescribed root-knot nematode lineage that is separated from other species with the resemblance in morphology, such as M. floridensis , M. hispanica , M. acronea , and M. paranaensis ., Competing Interests: The author(s) declare no conflict of interest.

" Candidatus Liberibacter asiaticus" (CLas) is the causal agent of citrus Huanglongbing (HLB, also called citrus greening disease), a highly destructive disease threatening citrus production worldwide. A novel Microviridae phage (named CLasMV1) has been found to infect CLas, providing a potential therapeutic strategy for CLas/HLB control. However, little is known about the CLasMV1 biology. In this study, we analyzed the population dynamics of CLasMV1 between the insect vector of CLas, the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) and the holoparasitic dodder plant ( Cuscuta campestris Yunck.); both acquired CLasMV1-infected CLas from an HLB citrus. All CLas-positive dodder samples were CLasMV1-positive, whereas only 32% of CLas-positive ACP samples were identified as CLasMV1-positive. Quantitative analyses showed a similar distribution pattern of CLasMV1 phage and CLas among eight citrus cultivars by presenting at highest abundance in the fruit pith and/or the center axis of the fruit. Transcriptome analyses revealed the possible lytic activity of CLasMV1 on CLas in fruit pith as evidenced by high-level expressions of CLasMV1 genes, and CLas genes related to cell wall biogenesis and remodeling to maintain the CLas cell envelope integrity. The up-regulation of CLas genes were involved in restriction-modification system that could involve possible phage resistance for CLas during CLasMV1 infection. In addition, the regulation of CLas genes involved in cell surface components and Sec pathway by CLasMV1 phage could be beneficial for phage infection. This study expanded our knowledge of CLasMV1 phage that will benefit further CLas phage research and HLB control.

The citrus red mite (Panonychus citri) is a challenge to manage in citrus orchards due to resistance against several pesticides. There is a necessity therefore to find new pesticides for effective control of P. citri. This study was designed to evaluate the lethal and sublethal effects of emamectin benzoate against P. citri. The results showed that the LC 50 of emamectin benzoate to adults of P. citri was 0.35 (0.26-0.43) mg a.i. L -1 and the LC 90 was 1.44 (1.16-1.96) mg a.i. L -1 . The sublethal concentration exposures (LC 10 and LC 30 ) had a significant negative impact on the larval, protonymph, and deutonymph developmental periods. Male longevity was much lower in LC 30 treatments than in the controls. Although female longevity was unaffected, the fecundity (eggs per female) was decreased in the sublethal concentration treatments. Results revealed that the adult pre-oviposition period (APOP) and total pre-oviposition period (TPOP) were increased. Other growth parameters r, λ, and R 0 decreased, whereas mean generation time (T) increased due to pesticide exposure. The survival rate (S xj ), age-specific fecundity and net maternity, life expectancy (E xj ), and reproduction (V xj ) was reduced by LC 10 and LC 30 exposure. An increase in malondialdehyde (MDA) contents with increasing emamectin benzoate concentration demonstrates that emamectin benzoate induces oxidative stress in P. citri. The activity of antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) was decreased due to LC 30 and LC 10 treatments compared to the control. Detoxification enzyme activity (cytochrome P450, glutathione-S-transferases, GST and acetylcholinesterase, AChE) was increased in treated mites compared to the control. This study demonstrates that emamectin benzoate has both a lethal effect on citrus red mite and sublethal effects on its biology and physiology. It is, therefore, potentially an effective pesticide for management of P. citri., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Panonychus citri, a major citrus pest. In pest management, bifenazate is a novel acaricide with high biological activity against red mites, such as Tetranychus urticae Koch. However, in the field, pests are frequently exposed to sublethal or lethal concentrations of pesticides. At present, its sublethal effects on P. citri have not been reported. Therefore, in order to investigate sublethal effect of bifenazate on biological traits and enzymatic properties of P. citri. The newly emerged females were treated with two concentrations of bifenazate: LC 10 and LC 30 , the development and fecundity were observed. The results showed that female adult duration, fecundity, oviposition days, longevity were decrease compared with control, but pre-oviposition period was longer, net reproductive rate (R 0 ), mean generation (T) were decreased, intrinsic rate of increase (rm), finite rate (λ) were decreased in LC 30 , however, doubling time was increased. Enzymatic tests showed that CAT, POD, CarE activities were higher in treatments than control. The SOD and GST activities were lower in LC 30 than control and LC 10 , the CYP450 activity was decreased with the increasing concentrations. This study demonstrated that low lethal concentrations of bifenazate adversely affected life table parameters, enzymatic properties in P. citri. Therefore, bifenazate has the potential to control this pest., (© 2021. The Author(s).)

The Asian citrus psyllid (ACP) survival in the presence of contact insecticides may be through physiological adaptations or by behaviorally avoiding. Curiously, although the first alternative is the object of frequent attention, the second was often neglected, but both may lead to insecticide resistance. In this paper, we characterize the growth dynamics of ACP population using a novel impulsive differential equation model to account for the effect of physiological and behavioral resistance, and investigate the threshold conditions for the extinction of ACP population. Furthermore, we discuss the optimal switching methods for insecticides based on two different criteria. Our numerical result suggests that ignoring both resistances or behavioral resistance would underestimate the transmission risk of Huanglongbing, whereas only considering behavioral resistance leads to an overestimation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Predatory mites in the family Phytoseiidae (Acari: Mesostigmata) are of great importance as biological control agents of pest mites and other arthropods. Correct identification of species is crucial to implement effective biological control of target pests. Here, we provide re-descriptions of seven phytoseiid mite species collected from citrus orchards in Florida. The several important morphological features including dorsal setae lengths, dorsal solenostomes, shape of calyx of spermatheca, chelicera dentition, measurements, and shape of macrosetae on legs currently used to discriminate phytoseiid species were missing in the original descriptions and re-descriptions of these species. Additionally, we observed the presence of a previously unnoted taxonomically important character on Proprioseius meridionalis Chant. Therefore, the re-description was essential for further diagnosis of this species. Accordingly, the validity of the presence/absence of this structure as a diagnostic character to separate species groups in the genus Proprioseius should be re-considered. Furthermore, Typhlodromalus peregrinus, a species for which a series of morphological variations are reported in previous descriptions, is re-described and illustrated from Clermont, Florida, a location very close (10 km) to its type location (Minneola), and the leaves of type host citrus. The macrosetae StIV was knobbed apically in all our specimens of T. peregrinus indicating invalidity of sharp-pointed or knobbed StIV to separate this species from a closely related species, T. aripo De Leon. These re-descriptions and species are important to utilizing authentic and promising candidates for biological control., Competing Interests: The authors have declared that no competing interests exist.

Food resource access can mediate establishment success in invasive species, and generalist herbivorous insects are thought to rely on mechanisms of transcriptional plasticity to respond to dietary variation. While asexually reproducing invasives typically have low genetic variation, the twofold reproductive capacity of asexual organisms is a marked advantage for colonization. We studied host-related transcriptional acclimation in parthenogenetic, invasive, and polyphagous weevils: Naupactus cervinus and N. leucoloma. We analyzed patterns of gene expression in three gene categories that can mediate weevil-host plant interactions through identification of suitable host plants, short-term acclimation to host plant defenses, and long-term adaptation to host plant defenses and their pathogens. This approach employed comparative transcriptomic methods to investigate differentially expressed host detection, detoxification, immune defense genes, and pathway-level gene set enrichment. Our results show that weevil gene expression responses can be host plant-specific, and that elements of that response can be maintained in the offspring. Some host plant groups, such as legumes, appear to be more taxing as they elicit a complex gene expression response which is both strong in intensity and specific in identity. However, the weevil response to taxing host plants shares many differentially expressed genes with other stressful situations, such as host plant cultivation conditions and transition to novel host, suggesting that there is an evolutionarily favorable shared gene expression regime for responding to different types of stressful situations. Modulating gene expression in the absence of other avenues for phenotypic adaptation may be an important mechanism of successful colonization for these introduced insects., Competing Interests: The authors have declared that no competing interests exist.

An intracellular bacterium, strain IAS T , was observed to infect several species of the plant-parasitic nematode genus Xiphinema ( Xiphinema astaregiense , Xiphinema incertum , Xiphinema madeirense , Xiphinema pachtaicum , Xiphinema parapachydermum and Xiphinema vallense ). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAS T was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EB T (92.9 % sequence identity) and ' Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes ( atpD , lepA and recA ) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAS T as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAS T is therefore proposed as representing ' Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Jellyfish are rich in resources and widely distributed along coastal areas. As a potential approach to respond to jellyfish blooms, the use of jellyfish-derived products is increasing. The citrus spider mite ( Panonychus citri ) is one of the key citrus pests, negatively impacting the quality and quantity of oranges. Due to the resistance and residue of chemical acaricides, it is important to seek natural substitutes that are environmentally friendly. The field efficacy of the venom from the jellyfish Nemopilema nomurai against P. citri was assayed in a citrus garden. The frozen N. nomurai tentacles were sonicated in different buffers to isolate the venom. The venom isolated by PBS buffer (10 mM, pH 6.0) had the strongest acaricidal activity of the four samples, and the corrected field efficacy 7 days after treatment was up to 95.21%. This study demonstrated that jellyfish has potential use in agriculture.

Climate change has not only exacerbated abiotic stress, but has also rendered external conditions more feasible for pests to spread and infest citrus fruit. Citrus leafminer (Phyllocnistis citrella) is a potential pest that directly feeds the newly sprouted leaves and twigs of all three spring, summer and autumn flushes. Increasing temperatures in spring and autumn, leafminer accrued more heat units or developmental degree days to accelerate the biological stages of its life-cycle, thereby increasing the pressure of infestation. Present work was conducted at three different environmental conditions in Sargodha, Toba Tek Singh (TTS) and Vehari districts of the Punjab province, Pakistan; all three experimental sites were located in different agro-ecological zones. More infestation was recorded in all three flushes at TTS and Vehari than in Sargodha. Overall, more damage was observed due to higher temperatures in TTS and Vehari than in Sargodha. After May-June heat stress, spontaneous vegetative growth continued from July to November, produced newly spouted tender leaves for feeding the leafminer larvae, and was seen more in TTS and Vehari. Leafminer larva prefers to enter young and tender leaves with a maximum entrance in leaves up to 1 cm 2 in size while observing no entrance above 3 cm 2 of leaf size. Physiological response of leaves primarily attributed to chlorophyll and carotenoid contents, both of which were recorded lower in the mined leaves, thereby reducing leaf photosynthetic activity. Similarly, lower levels of polyphenols and antioxidant activity were also recorded in the mined leaves. The on-tree age of mined leaves of three vegetative flushes of Kinnow plant was also less counted than non-mined leaves. Climate change has affected vegetative phenology and become feasible for pests due to extemporaneous leaf growth, particularly leafminer, and eventually causes economic loss by supplying low carbohydrates either to hanging fruits or next-season crops.

The Asian citrus psyllid (ACP), Diaphorina citri, is a harmful pest of citrus trees that transmits Candidatus Liberibacter spp. which causes Huanglongbing (HLB) (citrus greening disease); this is considered to be the most serious bacterial disease of citrus plants. Here we detail an anatomical study of the external and internal anatomy (excluding the reproductive system) using micro-computed tomography (micro-CT). This is the first complete 3D micro-CT reconstruction of the anatomy of a psylloid insect and includes a 3D reconstruction of an adult feeding on a citrus leaf that can be used on mobile devices. Detailed rendered images and videos support first descriptions of coxal and scapus antennal glands and sexual differences in the internal anatomy (hindgut rectum, mesothoracic ganglion and brain). This represents a significant advance in our knowledge of ACP anatomy, and of psyllids in general. Together the images, videos and 3D model constitute a unique anatomical atlas and are useful tools for future research and as teaching aids.

The Chinese citrus fly, Bactrocera minax, is a notorious univoltine pest that causes damage to citrus. B. minax enters obligatory pupal diapause in each generation to resist harsh environmental conditions in winter. Despite the enormous efforts that have been made in the past decade, the understanding of pupal diapause of B. minax is currently still fragmentary. In this study, the 20-hydroxyecdysone solution and ethanol solvent was injected into newly-formed pupae to obtain non-diapause- (ND) and diapause-destined (D) pupae, respectively, and a comparative proteomics analysis between ND and D pupae was performed 1 and 15 d after injection. A total of 3,255 proteins were identified, of which 190 and 463 were found to be differentially abundant proteins (DAPs) in ND1 vs D1 and ND15 vs D15 comparisons, respectively. The reliability and accuracy of LFQ method was validated by qRT-PCR. Functional analyses of DAPs, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction, were conducted. The results revealed that the diapause program of B. minax is closely associated with several physiological activities, such as phosphorylation, chitin biosynthesis, autophagy, signaling pathways, endocytosis, skeletal muscle formation, protein metabolism, and core metabolic pathways of carbohydrate, amino acid, and lipid conversion. The findings of this study provide insights into diapause program of B. minax and lay a basis for further investigation into its underlying molecular mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Asian citrus psyllid (ACP) causes direct and indirect damage to the citrus industry. Extractive electrospray ionization mass spectrometry (EESI-MS) and high performance liquid chromatography (HPLC) were used to detect the metabolites of C. limon leaves at 0, 12, 24, and 72 h after ACP treatment. The EESI-MS results showed that ACP infestation significantly affected metabolites within a short feeding duration with 8 metabolites identified. The metabolites in leaves of these four groups could be distinguished, with 55 peaks showing significant differences including methyl N-methylanthranilate, caffeic acid, and syringic acid. The quantification of 15 phenolic compounds with HPLC-UV method in C. limon leaves after ACP infestation showed that the total content of them reached a peak of 3504.69 μg g -1 at 12 h, with 9 phenolic compounds changing significantly (P < 0.05). A total of 21 metabolites identified in this study were involved in the biosynthesis pathways of flavonoid, flavone and flavonol, isoflavonoid and phenylpropanoid, and the degradation of aminobenzoate. Contents of epicatechin and caffeic acid increased with the feeding time of ACP as detected by both EESI-MS and HPLC. This may be related to plant defense. This study provides novel insights into the biochemical relationship of ACP and its host plants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium., Competing Interests: The authors have declared that no competing interests exist.

The Asian citrus psyllid (Diaphorina citri) transmits the bacterium 'Candidatus Liberibacter asiaticus' (CLas), which causes huanglongbing (citrus greening) disease, in a circulative-propagative manner. We compared CLas inoculation efficiency of D. citri nymphs and adults into healthy (uninfected) citron leaves when both vector stages were reared from eggs on infected plants. The proportion of CLas-positive leaves was 2.5% for nymphs and 36.3% for adults. CLas acquisition by early instar nymphs followed by dissections of adults and 4th instar nymphs revealed that CLas bacterium had moved into the head-thorax section (containing the salivary glands) in 26.7-30.0% of nymphs and 37-45% of adults. Mean Ct values in these sections were 31.6-32.9 and 26.8-27.0 for nymphs and adults, respectively. Therefore, CLas incidence and titer were higher in the head-thorax of adults than in nymphs. Our results suggest that following acquisition of CLas by early instar D. citri nymphs, emerging adults inoculate the bacteria into citrus more efficiently than nymphs because adults are afforded a longer latent period necessary for multiplication and/or translocation of CLas into the salivary glands of the vector. We propose that CLas uses D. citri nymphs mainly for pathogen acquisition and multiplication, and their adults mainly for pathogen inoculation and spread.

The citrus borer, Diploschema rotundicolle, is a Neotropical longhorn beetle that has become a serious citrus pest in southern South America. Management strategies for this insect rely on trimming off damaged shoots, which is expensive and inefficient. We studied the chemical communication system in D. rotundicolle in search of attractants for monitoring or control. GC-MS and enantioselective GC analyses of volatile extracts from field-collected adults showed that males produce (R)-3-hydroxy-2-hexanone, irregularly accompanied by minor amounts of 2,3-hexanediol (all four stereoisomers) and 2,3-hexanedione. Males emit the compounds only at night, when the adults are active. GC-EAD analyses of natural and synthetic compounds showed that both male and female antennae respond to the natural enantiomer (R)-3-hydroxy-2-hexanone, suggesting that it may function as an aggregation-sex pheromone as seen in many cerambycines. The non-natural (S) enantiomer as well as the minor component 2,3-hexanediol did not trigger antennal responses. Field tests with the racemic 3-hydroxy-2-hexanone, enantiomerically pure (R)-3-hydroxy-2-hexanone, as well as a mixture of racemic 3-hydroxy-2-hexanone and 2,3-hexanediol, showed in all cases low capture levels of D. rotundicolle. However, increasing the elevation of the trap and the emission rate of dispensers enhanced field captures in traps baited with racemic hydroxyketone. Incidental catches of another native cerambycine, Retrachydes thoracicus, in traps baited with 3-hydroxy-2-hexanone are also reported. This is the first report of pheromone chemistry in the genus Diploschema and in the tribe Torneutini, reaffirming the pheromone parsimony well established for the Cerambycinae. Potential factors explaining the weak attraction of D. rotundicolle in the field are discussed.

Asian citrus psyllid Diaphorina citri Kuwayama is an important economic pest of citrus, as it transmits Candidatus Liberibacter asiaticus, the causative agent of huanglongbing. In this study, we used RNA-seq to identify novel genes and provide the first high-resolution view of the of D. citri transcriptome throughout development. The transcriptomes of D. citri during eight developmental stages, including the egg, five instars, and male and female adults were sequenced. In total, 115 million clean reads were obtained and assembled into 354,726 unigenes with an average length of 925.65 bp and an N50 length of 1733 bp. Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were conducted to functionally annotate the genes. Differential expression analysis highlighted developmental stage-specific expression patterns. Furthermore, two trehalase genes were characterized with lower expression in adults compared to that in the other stages. The RNA interference (RNAi)-mediated suppression of the two trehalase genes resulted in significantly high D. citri mortality. This study enriched the genomic information regarding D. citri . Importantly, these data represent the most comprehensive transcriptomic resource currently available for D. citri and will facilitate functional genomics studies of this notorious pest., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The Asian citrus psyllid (ACP; Diaphorina citri) is the vector of Candidatus Liberibacter asiaticus (CLas) that is associated with the devastating Huanglongbing (HLB; citrus greening disease). This pest of Asian origin has spread into the Americas and more recently into a few countries in East Africa. During recent surveys, suspect ACP adults and nymphs were recorded for the first time infesting citrus trees in southwest Nigeria. Morphological identification and DNA barcoding confirmed the samples to be D. citri. Analysis of the obtained sequences revealed that the ACP recorded in Nigeria clustered with other taxa in the previously identified B1 clade that consists of populations from different continents. The presence of the endosymbionts Ca. Carsonella ruddii and Ca. Profftella armatura in ACP from Nigeria was also confirmed by PCR and Sanger sequencing. The ACP individuals were assayed for the presence of CLaf, CLam and CLas by qPCR, but none of the insects tested positive for any of the Liberibacters. The prolific nature of ACP and the tropical climate prevailing in the citrus-producing areas of Nigeria and other West African countries may favor its rapid spread and population increase, thus posing a grave threat to the sustainability of citriculture in these countries.

Trioza erytreae is the main vector for 'Candidatus Liberibacter africanus', the causative agent of African Citrus Greening disease. The insect is widespread in Africa, and has recently disseminated to Southwestern Europe. This study aimed at generating reference mitogenome sequences for T. erytreae, as a background for future genetic diversity surveys. Complete mitochondrial sequences of three specimens collected in Ethiopia, Uganda and South Africa were recovered using Ion Torrent technology. The mitogenomes of T. erytreae from Uganda and Ethiopia were highly similar, and distinct from that found in South Africa. The phylogeographic structure of T. erytreae was assessed using genetic clustering and pairwise distances, based on a dataset of public COI sequences recorded as T. erytreae. The dataset revealed ten haplotypes with strong phylogeographic structure in Africa and Europe. Three haplotypes found in Kenya on Clausena anisata belonged to pairs separated by distances as high as 11.2%, and were basal to all other sequences. These results indicate that not all sequences identified as T. erytreae belong to the same species, and that some degree of specificity with different plant hosts is likely to exist. This study provides new baseline information on the diversity of T. erytreae, with potential implications for the epidemiology of African Citrus Greening disease.

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., Competing Interests: The authors have declared that no competing interests exist.

The oriental red spider mite, Eutetranychus orientalis (Klein) is a major pest of citrus in many countries including Saudi Arabia (SA). The morphological variations among the different populations of E. orientalis were reported. In the present study, phenotypic variations based on 40 different morphological characteristics were evaluated in 10 E. orientalis populations collected from different hosts and regions of SA. Further, ITS2-rDNA sequences were used to confirm the identity of these phenotypically varying populations. Phenotypic variations in all populations were found in the shape and length of dorsal setae, striation pattern between setae d1 and e1, and leg chaetotaxy. The rDNA sequence analysis of these morphologically varying populations resulted in 10 different ITS2 Saudi haplotypes. The phenotypic and genetic variations were more related to the host plants rather than their geographic distribution. The E. orientalis population collected from Phoenix dactylifera was phenotypically distinct and genetically divergent. The populations collected from citrus species were also more phenotypically and genetically related to each other than to populations collected from non-citrus host plants. The haplotypes recovered from Ziziphus sp., Morus sp., and Azadirecta indica from different regions were grouped in the same sub-clade. Further, the ITS2 haplotypes of Saudi E. orientalis recovered from Citrus reticulata from Riyadh and Al Ula were 100% identical to the ITS2 haplotypes recovered from Citrus sp. from Israel. It is concluded that phenotypic variations exist among different populations of E. orientalis inhabiting different host plants. This species should be identified carefully by considering phenotypic intraspecific variations., Competing Interests: The authors have declared that no competing interests exist.

Citrus greening, or Huanglongbing (HLB), is currently the most devasting disease of citrus, creating unprecedented crisis for the multibillion-dollar global citrus industry. To-date, there is no effective cure and disease management relies on early detection and removal of infected trees. Thus, it is imperative that accurate, timely, and robust disease detection and diagnosis technologies are available to minimize the spread of disease. This study reports a sensitive and selective label-free biosensor that combines the physical and chemical advantages of carbon nanomaterials like single-walled carbon nanotubes (SWNTs) in a field-effect transistor (FET)/chemiresistor architecture with selective antibodies against Sec-delivered effector 1 (SDE1), a secreted protein biomarker, for the detection of HLB. The biosensor detected SDE1 biomarkers for citrus greening in plant tissue extracts with the dynamic range over three orders of magnitude in the low nanomolar to micromolar concentration range and limit of detection of 5 nM. The study also demonstrated the use of the standard additions assay method with the biosensor to attain a 90-percent signal recovery in concentrated plant tissue extract, allowing for quantitative detection without an external calibration. Adopting the novel detection strategy targeting the secreted protein biomarker, SDE1, addresses some of the challenges faced by current methods of nucleic acid-based assays and symptom-based diagnosis, which have been found prone to false negatives and misdiagnoses, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Symbiotic bacteria play a critical role in insect's biology. They also offer great opportunities to improve on current pest management techniques. In order to exploit and integrate the roles played by the gut microbiota on pest management programs, a better understanding of the structural organization of the microbial community in the Chinese citrus fly Bactrocera minax is essential., Results: The results revealed a total of 162 OTUs at 97% similarity interval. The dominant bacteria phyla were Proteobacteria, Bacteroidetes, Antinobacteria and Firmicutes, with the Proteobacteria having the highest relative abundance (more than 80% in all life stages). There was also a shift in the dominant OTUs from the early developmental stages to the late developmental stages and adult stages in B. minax. These OTUs related to Klebsiella pneumoniae, Providencia rettgeri and Enterobacter aerogenes, respectively. Six bacteria OTU were shared by all the life stages. These belonged to the Enterobacteriaceae and the Enterococcaceae families., Conclusion: The common bacteria groups shared by all the life stages and other fruit flies could be important targets for further research. This should aim towards realizing how these bacteria affect the biology of the fly and how their relationship could be exploited in the development of sustainable management strategies against fruit flies.

Numerous postharvest diseases have been reported that cause substantial losses of citrus fruits worldwide. Penicillium digitatum is responsible for up to 90% of production losses, and represent a problem for worldwide economy. In order to control phytopathogens, chemical fungicides have been extensively used. Yet, the use of some artificial fungicides cause concerns about environmental risks and fungal resistance. Therefore, studies focusing on new approaches, such as the use of natural products, are getting attention. Co-culture strategy can be applied to discover new bioactive compounds and to understand microbial ecology. Mass Spectrometry Imaging (MSI) was used to screen for potential antifungal metabolites involved in the interaction between Penicillium digitatum and Penicillium citrinum. MSI revealed a chemical warfare between the fungi: two tetrapeptides, deoxycitrinadin A, citrinadin A, chrysogenamide A and tryptoquialanines are produced in the fungi confrontation zone. Antimicrobial assays confirmed the antifungal activity of the investigated metabolites. Also, tryptoquialanines inhibited sporulation of P. citrinum. The fungal metabolites reported here were never described as antimicrobials until this date, demonstrating that co-cultures involving phytopathogens that compete for the same host is a positive strategy to discover new antifungal agents. However, the use of these natural products on the environment, as a safer strategy, needs further investigation. This paper aimed to contribute to the protection of agriculture, considering health and ecological risks.

Background: Phytophthora species secrete cytoplasmic effectors from a family named Crinkler (CRN), which are characterised by the presence of conserved specific domains in the N- and C-terminal regions. P. parasitica causes disease in a wide range of host plants, however the role of CRN effectors in these interactions remains unclear. Here, we aimed to: (i) identify candidate CRN encoding genes in P. parasitica genomes; (ii) evaluate the transcriptional expression of PpCRN (Phytophthora parasitica Crinkler candidate) during the P. parasitica interaction with Citrus sunki (high susceptible) and Poncirus trifoliata (resistant); and (iii) functionally characterize two PpCRNs in the model plant Nicotiana benthamiana., Results: Our in silico analyses identified 80 putative PpCRN effectors in the genome of P. parasitica isolate 'IAC 01/95.1'. Transcriptional analysis revealed differential gene expression of 20 PpCRN candidates during the interaction with the susceptible Citrus sunki and the resistant Poncirus trifoliata. We have also found that P. parasitica is able to recognize different citrus hosts and accordingly modulates PpCRNs expression. Additionally, two PpCRN effectors, namely PpCRN7 and PpCRN20, were further characterized via transient gene expression in N. benthamiana leaves. The elicitin INF-1-induced Hypersensitivity Response (HR) was increased by an additive effect driven by PpCRN7 expression, whereas PpCRN20 expression suppressed HR response in N. benthamiana leaves. Despite contrasting functions related to HR, both effectors increased the susceptibility of plants to P. parasitica., Conclusions: PpCRN7 and PpCRN20 have the ability to increase P. parasitica pathogenicity and may play important roles at different stages of infection. These PpCRN-associated mechanisms are now targets of biotechnological studies aiming to break pathogen's virulence and to promote plant resistance.

The African citrus triozid, Trioza erytreae Del Guercio (Hemiptera: Triozidae) is one of the primary vectors of the bacterium Candidatus Liberibacter spp. which causes citrus greening, a disease of global economic importance in citrus production. Despite its economic importance, little is known about its chemical ecology. Here, we used behavioral assays and chemical analysis to study the chemical basis of interaction between T. erytreae and one of its preferred host plants, Citrus jambhiri. In dual choice Y-tube olfactometer assays, lemon leaf odors attracted females but not males compared to plain air or solvent controls. However, in a petri dish arena assay, both sexes were arrested by lemon leaf odors. Coupled gas chromatography-mass spectrometry (GC/MS) analysis revealed quantitative differences in the odors of flushing and mature leaves, dominated by terpenes. Twenty-six terpenes were identified and quantified. In Petri dish arena assays, synthetic blends of the most abundant terpenes mimicking lemon flushing leaf odors elicited varying behavioral responses from both sexes of T. erytreae. A nine-component blend and a blend of the three most abundant terpenes; limonene, sabinene and β-ocimene arrested both sexes of T. erytreae. In contrast, a six-component blend lacking in these three components elicited an avoidance response in both sexes. Furthermore, both sexes of T. erytreae preferred the three-component synthetic blend to lemon crude volatile extract. These results suggest that lemon terpenes might be used in the management of T. erytreae.

The ATP-binding cassette (ABC) transporters exist in all living organisms and play major roles in various biological functions by transporting a wide variety of substrates across membranes. The functions of ABC transporters in drug resistance have been extensively studied in vertebrates; however, they are rarely characterized in agricultural pests. The Asian citrus psyllid, Diaphorina citri, is one of the most damaging pests of the Citrus genus because of its transmission of Huanglongbing, also known as Yellow Dragon disease. In this study, the next-generation sequencing technique was applied to research the ABC transporters of D. citri. Fifty-three ABC transporter genes were found in the RNA-Seq data, and among these ABC transporters, 4, 4, 5, 2, 1, 4, 18 and 15 ABC proteins belonged to the ABCA-ABCH subfamilies, respectively. Different expression profiles of 52 genes between imidacloprid-resistant and imidacloprid-susceptible strains were studied by qRT-PCR; 5 ABCGs and 4 ABCHs were significantly upregulated in the imidacloprid-resistant strain. In addition, five of the nine upregulated genes were widely expressed in adult tissues in spatial expression analysis. The results suggest that these genes may play key roles in this phenotype. In general, this study contributed to our current understanding of D. citri resistance to insecticides.

The Asian citrus psyllid (ACP) is a vector of a pathogen associated with greening and thus a major problem in citriculture worldwide. Lures are much needed for improving ACP trapping systems for monitoring populations and surveillance. Previously, we have identified acetic acid as a putative sex pheromone and measured formic acid- and propionic acid-elicited robust electroantennographic responses. We have now thoroughly examined in indoor behavioral assays (4-way olfactometer) and field tests the feasibility of these three semiochemicals as potential lures for trapping ACP. Formic acid, acetic acid, and propionic acid at appropriate doses are male-specific attractants and suitable lures for ACP traps, but they do not act synergistically. An acetic acid-based homemade lure, prepared by impregnating the attractant in a polymer, was active for a day. A newly developed slow-release formulation had equal performance but lasted longer, thus leading to an important improvement in ACP trap capture at low population densities.

Native to sub-Saharan Africa, Delottococcus aberiae De Lotto (Hemiptera: Pseudoccidae) is an invasive mealybug that has been recently reported in Europe, seriously damaging citrus production in eastern Spain. In this study, we isolated and determined the structure of the sex pheromone of D. aberiae , to provide a highly specific and effective lure for detecting, monitoring, and potentially controlling this pest. The volatile profile of D. aberiae virgin and mated females was studied by aeration and collection of effluvia in Porapak-Q. The resulting extracts were analyzed by gas chromatography coupled to mass spectrometry (GC-MS), revealing a candidate compound specific of virgin females. GC-MS and nuclear magnetic resonance spectroscopy data evidenced a new compound, (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate, with an unusual β-necrodol skeleton. This compound was synthesized and shown to be attractive to male D. aberiae in both laboratory and field experiments. A GC analysis using an enantioselective stationary phase and polarimetry analyses of the synthetic enantiomers showed the natural compound emitted by virgin females to be the - enantiomer.

High-throughput sequencing is widely used for virus discovery, and many RNA viruses have been discovered and identified. A new negative-sense single-stranded RNA virus was identified in the brown citrus aphid and named Aphis citricidus bunyavirus. The genome consists of large (7037 nt), medium (3462 nt), and small (1163 nt) segments. Phylogenetic analysis and amino acid sequences identities of this virus with other bunyaviruses suggest that it is a new species belonging to the family Phenuiviridae. The small interfering RNA pathway could be involved against the infection of this virus in brown citrus aphid as supported by the viral derived small RNAs. The discovery of this virus illustrates the diversity of RNA viruses and contributes to the classification of bunyaviruses.

The larvae of Ceraeochrysa cubana and Ceraeochrysa valida, green lacewing species widely spread in Mexico, have been described as natural enemies of Diaphorina citri (Hemiptera: Liviidae), vector of the bacteria Candidatus Liberibacter spp. causing Huanglongbing disease. To develop an effective biological control program, the establishment of the genetic structure of the biocontrol agent species is mandatory. Consequently, the goal of this work was to obtain reliable DNA barcoding regions of the two species, and then by sequence analysis of the mitochondrial gene cytochrome c oxidase subunit I, evaluate the genetic structure of Mexican lime populations of C. cubana and C. valida from the state of Colima. This research produced the first barcode region of C. cubana and C. valida with morphological and molecular confirmation. The genetic parameters revealed the presence of 15 and 10 haplotypes, and haplotype diversity values of 0.889 and 0.838 for C. cubana and C. valida, respectively. The populations showed high diversity and gene flow, and AMOVA analysis demonstrated no genetic structure in the two populations. Consequently, these single populations of C. cubana and C. valida could be used as unique genetic source for mass production and release in the Mexican lime-producing state of Colima to control D. citri.

Chitin synthase is a critical enzyme that catalyzes N -acetylglucosamine to form chitin, which plays an important role in the growth and development of insects. In this study, we identified a chitin synthase gene ( CHS ) with a complete open reading frame (ORF) of 3180 bp from the genome database of Diaphorina citri , encoding a protein of 1059 amino acid residues with the appropriate signature motifs (EDR and QRRRW). Reverse transcription-quantitative PCR (RT-qPCR) analysis suggested that D. citri CHS ( DcCHS ) was expressed throughout all developmental stages and all tissues. DcCHS had the highest expression level in the integument and fifth-instar nymph stage. Furthermore, the effects of diflubenzuron (DFB) on D. citri mortality and DcCHS expression level were investigated using fifth-instar nymph through leaf dip bioassay, and the results revealed that the nymph exposed to DFB had the highest mortality compared with control group (Triton-100). Silencing of DcCHS by RNA interference resulted in malformed phenotypes and increased mortality with decreased molting rate. In addition, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) also revealed corresponding ultrastructural defects. Our results suggest that DcCHS might play an important role in the development of D. citri and can be used as a potential target for psyllid control.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of 'Candidatus Liberibacter asiaticus' (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), 'Candidatus Carsonella ruddii,' and 'Candidatus Profftella armatura.' Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem.

Citrus greening or huanglongbing (HLB) is the main threat to the European citrus industry since one of its vectors, the African citrus psyllid, Trioza erytreae, has recently become established in mainland Europe. In this context, classical biological control programmes should be implemented to reduce the spread of the psyllid. The aims of this study were to: i) disentangle the parasitoid complex of T. erytreae combining morphological and molecular characterization; and ii) to study the biology of its main parasitoids in its area of origin in South Africa for their future importation into Europe. The main citrus producing areas of South Africa were surveyed during 2017. In contrast to previous studies, the parasitoid complex of T. erytreae included three species of primary parasitoids: Tamarixia dryi, Psyllaephagus pulvinatus and another parasitoid of the genus Tamarixia. Molecular analysis showed that it is a new species closely related to T. dryi. Tamarixia dryi was the most abundant parasitoid but its relative abundance varied among sampling sites. The sex ratio (males/females) of T. dryi and Tamarixia sp. decreased with T. erytreae size and became female biased when psyllid nymphs were larger than 0.6 and 1.2 mm 2 , respectively. These parasitoids were attacked by three species of hyperparasitoids, Aphidencyrtus cassatus, Marietta javensis and a species of the genus Aphanogmus. Aphidencyrtus cassatus, the most abundant hyperparasitoid, tended to emerge from large nymphs, and adult females lived as long as those of T. dryi. The implications of these results are discussed within the framework of the introduction of T. dryi into Europe.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most serious pest of citrus worldwide. It acts as a vector for a group of phloem-limited bacteria (Candidatus Liberibacter spp.) that causes Huanglongbing (HLB) disease. Thus, D. citri management is an important strategy against HLB, and biological control is currently considered as the most effective method because of the unsustainable and negative side effects of chemical control. Here, we isolated a new strain of entomopathogenic fungus, Cordyceps javanica (GZQ-1), from one cadaver of D. citri adult based on its morphological and phylogenetic data. Five conidial concentrations of the C. javanica pathogen (1 × 10 3 , 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 conidia/ml) were assessed against six life stages of D. citri (1st-5th instar nymphs and adults). Results showed that C. javanica GZQ-1 was highly pathogenic to D. citri nymphs (69.49%-90.87% mortality) and adults (69.98% mortality). The LC 50 and LT 50 values of C. javanica against 1st-2nd instar (younger), 3rd-4th instar (middle aged), 5th instar (older), and adults were 1.20 × 10 5 , 1.10 × 10 6 , 4.47 × 10 6 , 8.12 × 10 6 conidia/ml and 4.25, 4.51, 5.17, 5.49 days, respectively. Moreover, glasshouse experiments indicated that this C. javanica GZQ-1 caused higher infection rates of D. citri adults compared to two other fungal strains we previously isolated in the laboratory, Cordyceps fumosorosea (IF010) and Metarhizium anisopliae (CNGD7)., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Diaphorina citri is a vector of 'Candidatus Liberibacter asiaticus,' (CLas), associated with Huanglongbing, (HLB, or citrus greening) disease in citrus. D. citri exhibits three different color morph variants, blue, gray and yellow. Blue morphs have a greater capacity for long-distance flight as compared to non-blue morphs, but little else is known about how color morphology influences vector characteristics. In this study, we show that the color morphology of the insect is derived from pigmented cells of the fat body. Blue morphs acquire a lower level of CLas in their bodies from infected trees as compared to their gray and yellow conspecifics, referred to in this paper collectively as non-blue morphs. Accordingly, CLas titer in citrus leaves inoculated by non-blue insects was 6-fold higher than in leaves inoculated by blue insects. Blue color morphs harbored lower titers of Wolbachia and 'Candidatus Profftella armatura,' two of the D. citri bacterial endosymbionts. Expression of hemocyanin, a copper-binding oxygen transport protein responsible for the blue coloration of hemolymph of other arthropods and mollusks, was previously correlated with blue color morphology and is highly up-regulated in insects continuously reared on CLas infected citrus trees. Based on our results, we hypothesized that a reduction of hemocyanin expression would reduce the D. citri immune response and an increase in the titer of CLas would be observed. Surprisingly, a specific 3-fold reduction of hemocyanin-1 transcript levels using RNA silencing in blue adult D. citri morphs had an approximately 2-fold reduction on the titer of CLas. These results suggest that hemocyanin signaling from the fat body may have multiple functions in the regulation of bacterial titers in D. citri, and that hemocyanin is one of multiple psyllid genes involved in regulating CLas titer., Competing Interests: The authors have declared that no competing interests exist.

Citrus Huanglongbing (HLB), also known as greening, is a destructive disease caused by the fastidious, phloem-colonizing bacteria Candidatus Liberibacter spp.; 'Ca. Liberibacter asiaticus' (Las) is the most prevalent of the species causing HLB. The Asian citrus psyllid (ACP, Diaphorina citri) transmits Las. HLB is threatening citrus production worldwide, and there is no cure for infected trees. Management strategies targeting diseased trees at different stages of colonization by Las are needed for sustainable citrus production in HLB-endemic regions. We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost on mildly diseased trees. We tested thermotherapy on severely diseased trees and assessed tree protectors to prevent feeding by ACP, thus preventing Las from being transmitted to new plantings that replaced HLB-moribund trees. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control. Thermotherapy treatment (55°C for 2 min) exhibited a suppressive effect on growth of Las and progress of HLB in severely diseased trees for 2 to 3 months after treatment. The tree protectors prevented feeding by ACP, and therefore young replant trees remained healthy and free from infection by Las over the 2-year duration of the experiment. Taken together, these results may contribute to a basis for developing a targeted approach to control HLB based on stage of host colonization, application of plant defense elicitors, N fertilizer, compost, thermotherapy, and tree protectors. There is potential to implement these strategies in conjunction with other disease control measures to contribute to sustainable citrus production in HLB-endemic regions.

The citrus leaf-mining beetle, Podagricomela weisei Heikertinger, is an important citrus pest that ingests the mesophyll and new shoots. The mechanism underlying the xenobiotic metabolism of P. weisei is not well understood, in part because of a lack of available genomic and transcriptomic data, which has hampered the development of novel pest management approaches [e.g., RNA interference (RNAi)]. In this study, we completed the deep sequencing of the P. weisei transcriptome to identify factors potentially involved in xenobiotic metabolism and the core RNAi machinery. The sequencing of the P. weisei transcriptome generated >27 million clean reads, ultimately yielding 90,410 unigenes with an N50 of 1065 bp. The unigenes were used as queries to search the Nr database, which revealed that 21,847 unigenes were homologous to known genes in various species. Transcripts encoding genes involved in xenobiotic metabolism were identified, including genes encoding cytochrome P450 monooxygenase (P450, 47 unigenes), glutathione S-transferase (GST, 12 unigenes), esterase (EST, 25 unigenes), and the ATP-binding cassette transporter (ABC transporter, 32 unigenes). A parallel sequencing of small RNAs detected 30 conserved miRNAs, with the most abundant being Pwe-miR-1-3p, with an expression level reaching 517,996 reads in the prepared library, followed by Pwe-miR-8-3p (149,402 reads). Genes encoding components of the miRNA, siRNA, and piRNA pathways were also identified, and the results indicated that P. weisei possesses only one of each gene in all three pathways. In summary, this is the first detailed analysis of the transcriptome and small RNAs of P. weisei. The datasets presented herein may form the basis for future molecular characterizations of P. weisei as well as the development of enhanced pest control strategies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The African citrus triozid (ACT) Trioza erytreae Del Guercio (Hemiptera: Triozidae) is one of the most devastating pests of citrus with a well-known role as a vector of the phloem-limited bacteria (Candidatus Liberibacter africanus) associated with huanglongbing (citrus greening disease), currently considered the world's most serious disease of citrus. Although the pest can successfully develop and reproduce on non-citrus host plants, there is no documented information on the geometric morphometry of ACT. We determine the effect of host plants on ACT morphometry under controlled laboratory conditions using traditional and geometric analysis., Results: ACT reared on C. limon and Citroncirus spp. was significantly larger than when reared on the other host plant species. ACT reared on C. anisata and C. tangelo was consistently smaller than that reared on M. koenigii and C. sinensis. Based on warped outline drawings, ACT reared on Citroncirus spp. and Murraya koenigii had narrower wings than when reared on C. anisata, C. limon, and C. sinensis with slightly broader wing patterns., Conclusion: This study clearly demonstrates that host plant species affect morphometric variation in ACT, which might have a direct impact on fitness parameters of the pest as well as its potential for dispersion. Wing shape and size appear to be useful in separating populations of ACT into different groups. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Novel, suitable and sustainable alternative control tactics that have the potential to reduce migration of Diaphorina citri into commercial citrus orchards are essential to improve management of huanglongbing (HLB). In this study, the effect of orange jasmine (Murraya paniculata) as a border trap crop on psyllid settlement and dispersal was assessed in citrus orchards. Furthermore, volatile emission profiles and relative attractiveness of both orange jasmine and sweet orange (Citrus × aurantium L., syn. Citrus sinensis (L.) Osbeck) nursery flushes to D. citri were investigated. In newly established citrus orchards, the trap crop reduced the capture of psyllids in yellow sticky traps and the number of psyllids that settled on citrus trees compared to fallow mowed grass fields by 40% and 83%, respectively. Psyllids were attracted and killed by thiamethoxam-treated orange jasmine suggesting that the trap crop could act as a 'sink' for D. citri. Additionally, the presence of the trap crop reduced HLB incidence by 43%. Olfactometer experiments showed that orange jasmine plays an attractive role on psyllid behavior and that this attractiveness may be associated with differences in the volatile profiles emitted by orange jasmine in comparison with sweet orange. Results indicated that insecticide-treated M. paniculata may act as a trap crop to attract and kill D. citri before they settled on the edges of citrus orchards, which significantly contributes to the reduction of HLB primary spread.

The impact of light on reproductive rates of Asian citrus psyllid (Diaphorina citri Kuwayama) was assessed in an air-conditioned, polycarbonate greenhouse. This psyllid is an important pest because it transmits a bacterium presumed responsible for a serious citrus disease known as Asiatic huanglongbing. Numbers of psyllids produced were compared among rearing cages subjected to different amounts of light provided by natural sunlight and light-emitting diode floodlights. Light to some rearing cages was purposely reduced by shading. The cages received a daily mean of 12 h of light (range 7 to 14 h) during immature development. Irradiance during daylight hours in the cages during a 24-h oviposition period varied from 2 to 145 (mean 66) W/m2 and during immature development to the adult stage from 3 to 169 (mean 71) W/m2. Estimates of illuminance during immature development ranged from 354 to 73,500 (mean 22,409) lumens/m2. Oviposition rates were not correlated with these light variables. Numbers of adults produced were positively correlated with daily hours of light (r = 0.57, P = 0.002), irradiance (r = 0.39, P = 0.05), and illuminance (r = 0.59, P = 0.001). For producing large numbers of adults, optimal targets for these light variables as measured in this study were projected to be 14 or more hours of daylight, 60 or more W/m2, and 20,000 or more lumens/m2. Comparisons of oviposition rates and resulting numbers of adults produced in a cage indicated that increasing these light variables increased survival of immatures to the adult stage, possibly because the quality of host plants increased as these light variables increased.

Neuropeptides are endogenous active substances that widely exist in multicellular biological nerve tissue and participate in the function of the nervous system, and most of them act on neuropeptide receptors. In insects, neuropeptides and their receptors play important roles in controlling a multitude of physiological processes. In this project, we sequenced the transcriptome from twelve tissues of the Asian citrus psyllid, Diaphorina citri Kuwayama. A total of 40 candidate neuropeptide genes and 42 neuropeptide receptor genes were identified. Among the neuropeptide receptor genes, 35 of them belong to the A-family (or rhodopsin-like), four of them belong to the B-family (or secretin-like), and three of them are leucine-rich repeat-containing G-protein-coupled receptors. The expression profile of the 82 genes across developmental stages was determined by qRT-PCR. Our study provides the first investigation on the genes of neuropeptides and their receptors in D. citri , which may play key roles in regulating the physiology and behaviors of D. citri .

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ' Candidatus Liberibacter asiaticus' ( CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a severely devastating pest of Rutaceae plants, mainly citrus, across the globe, and causal agent of Huanglongbing (HLB) disease. To find out the genetic relationship and diversity among the populations of ACP and associated Candidatus Liberibacter asiaticus (CLas) from two countries (China and Pakistan), sequence data of three different genes, cytochrome oxidase subunit I (COI), Cu-transporting protein (ATOX1) and 16S rRNA, were used to characterize all populations. In the present study, MEGA-7 and statistical parsimony software (TCS-1.2) were used to depict the phylogenetic relationship among all populations under both genes, whereas diversity was calculated by DnaSP v5. All analyses were done for country wise and overall relationship among all populations. For ACP populations, both genes presented a significant strong intermingled relationship among all populations and put all population into a single haplotype (Dcit-2), which proved similarity between Chinese and Pakistani populations. Moreover, for CLas strains, 16S gene also presented strong relationship for all sampled populations. All three genes of ACP and CLas populations elucidated more than 95% resemblance to each other. On the other hand, a significant genetic variation was observed by three genes for overall populations, although, country wise variation was non-significant between all collected populations. ATOX1 gene presented higher diversity through Fu's Fs test (π = 0.01081, p < 0.003) whereas COI gene gave less diversity under Fu's Fs and Tajima's D test (π = 0.00512, p < 0.000 and 0.05, respectively). Similarly, nucleotide mismatch distribution also had shown enough genetic variation among all ACP populations, under both genes. Our sequence data for both genes proved the invasion of the Chinese ACP population (Dcit-2) into Pakistan, through all phylogenetic relationship, which proved a similar genetic makeup among all ACP populations from both countries. Therefore, these results can be helpful to utilize any novel designed control measure equally for both countries., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Asian citrus psyllid (ACP), Diaphorina citri, is a major vector of the bacteria Candidatus Liberibacter asiaticus and C.L. americanus, which cause Huanglongbing disease (HLB) (aka Citrus greening disease), considered the most serious bacterial disease of citrus trees. As part of a multidisciplinary project on psyllid biology (www.citrusgreening.org), the results presented here concern a detailed anatomical study of the male reproductive system (testes, seminal vesicles, accessory glands, sperm pump, connecting ducts, and aedeagus) using micro-computed tomography (micro-CT). The study summarizes current knowledge on psyllids male reproductive system and represents significant advances in the knowledge of ACP anatomy., Competing Interests: The authors have declared that no competing interests exist.

Fruit flies (Diptera: Tephritidae) represent a threat to fruit growing worldwide, mainly the citrus culture, however, biological studies show that fruit flies are not perfectly adapted to this host. This study investigated oviposition of Anastrepha fraterculus (Wiedemann, 1830) and Ceratitis capitata (Wiedemann, 1824) and its relation with the pericarp of citrus fruits. We evaluated the relationship between depth of oviposition of A. fraterculus and C. capitata and epicarp thickness of orange [Citrus sinensis (L.) Osbeck)] 'Navelina' and tangerine [C. reticulata (L.)] 'Clemenules' and the influence of fruit mesocarp of tangerine 'Clemenules' on oviposition of these species. The study was conducted under controlled conditions of temperature (25 ± 2 °C), relative humidity (70 ± 10% RH) and photophase (12 h). A. fraterculus and C. capitata laid their eggs in the flavedo region of orange 'Navelina' and between the albedo and flavedo of tangerine 'Clemenules'. When fruits with mesocarp exposed were offered, there was no oviposition by both fruit fly species. The results show that epicarp thickness of citrus fruits did not influence oviposition of A. fraterculus and C. capitata as oviposition did not occur only in the presence of the mesocarp, suggesting that other factors are involved in oviposition of these species.