Automatic detection and classification of disease in citrus fruit and leaves using a customized CNN based model / Detección y clasificación automática de enfermedades en frutas y hojas de cítricos utilizando un modelo basado en CNN personalizado

India's commercial advancement and development depend heavily on agriculture. A common fruit grown in tropical settings is citrus. A professional judgment is required while analyzing an illness because different diseases have slight variati ons in their symptoms. In order to recognize and classify diseases in citrus fruits and leaves, a customized CNN - based approach that links CNN with LSTM was developed in this research. By using a CNN - based method, it is possible to automatically differenti ate from healthier fruits and leaves and those that have diseases such fruit blight, fruit greening, fruit scab, and melanoses. In terms of performance, the proposed approach achieves 96% accuracy, 98% sensitivity, 96% Recall, and an F1 - score of 92% for ci trus fruit and leave identification and classification and the proposed method was compared with KNN, SVM, and CNN and concluded that the proposed CNN - based model is more accurate and effective at identifying illnesses in citrus fruits and leaves.

El avance y desarrollo comercial de India dependen en gran medida de la agricultura. Un tipo de fruta comunmente cultivada en en tornos tropicales es el cítrico. Se requiere un juicio profesional al analizar una enfermedad porque diferentes enfermedades tienen ligeras variaciones en sus síntomas. Para reconocer y clasificar enfermedades en frutas y hojas de cítricos, se desarrolló e n esta investigación un enfoque personalizado basado en CNN que vincula CNN con LSTM. Al utilizar un método basado en CNN, es posible diferenciar automáticamente entre frutas y hojas más saludables y aquellas que tienen enfermedades como la plaga de frutas , el verdor de frutas, la sarna de frutas y las melanosis. En términos de desempeño, el enfoque propuesto alcanza una precisión del 96%, una sensibilidad del 98%, una recuperación del 96% y una puntuación F1 del 92% para la identificación y clasificación d e frutas y hojas de cítricos, y el método propuesto se comparó con KNN, SVM y CNN y se concluyó que el modelo basado en CNN propuesto es más preciso y efectivo para identificar enfermedades en frutas y hojas de cítricos.

Biological Features and In Planta Transcriptomic Analyses of a Microviridae Phage (CLasMV1) in "Candidatus Liberibacter asiaticus".

"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.

Morphological and molecular identification of Cladosporium sphaerospermum isolates collected from tomato plant residues / Identificação morfológica e molecular de isolados de Cladosporium sphaerospermum coletados de resíduos de plantas de tomate

This study was conducted at the Agriculture College University of Karbala, Iraq to isolate and morphologically and molecularly diagnose thirteen Cladosporium isolates collected from tomato plant residues present in desert regions of Najaf and Karbala provinces, Iraq. We diagnosed the obtained isolates by PCR amplification using the ITS1 and ITS4 universal primer pair followed by sequencing. PCR amplification and analysis of nucleotide sequences using the BLAST program showed that all isolated fungi belong to Cladosporium sphaerospermum. Analysis of the nucleotide sequences of the identified C. sphaerospermum isolates 2, 6, 9, and 10 showed a genetic similarity reached 99%, 98%, 99%, and 99%, respectively, with those previously registered at the National Center for Biotechnology Information (NCBl). By comparing the nucleotide sequences of the identified C. sphaerospermum isolates with the sequences belong to the same fungi and available at NCBI, it was revealed that the identified C. sphaerospermum isolates 2, 6, 9, and 10 have a genetic variation with those previously recorded at the National Center for Biotechnology Information (NCBl); therefore, the identified sequences of C. sphaerospermum isolates have been registered in GenBank database (NCBI) under the accession numbers MN896004, MN896107, MN896963, and MN896971, respectively.

Este estudo foi conduzido na Agriculture College University of Karbala, Iraque, para isolar e diagnosticar morfológica e molecularmente treze isolados de Cladosporium coletados de resíduos de plantas de tomate presentes nas regiões desérticas das províncias de Najaf e Karbala, no Iraque. Diagnosticamos os isolados obtidos por amplificação por PCR usando o par de primers universais ITS1 e ITS4 seguido de sequenciamento. A amplificação por PCR e a análise de sequências de nucleotídeos usando o programa BLAST mostraram que todos os fungos isolados pertencem a Cladosporium sphaerospermum. A análise das sequências de nucleotídeos dos isolados 2, 6, 9 e 10 de C. sphaerospermum identificados mostrou similaridade genética de 99%, 98%, 99% e 99%, respectivamente, com aqueles previamente registrados no National Center for Biotechnology Informações (NCBl). Ao comparar as sequências de nucleotídeos dos isolados de C. sphaerospermum identificados com as sequências pertencentes aos mesmos fungos e disponíveis no NCBI, foi revelado que os isolados 2, 6, 9 e 10 de C. sphaerospermum identificados têm variação genética com aqueles anteriormente registrados no National Center for Biotechnology Information (NCBl). Portanto, as sequências identificadas de isolados de C. sphaerospermum foram registradas no banco de dados GenBank (NCBI) sob os números de acesso MN896004, MN896107, MN896963 e MN896971, respectivamente.

Lime sulfur to control citrus flat mite and its interactions with the entomopathogenic fungus Lecanicillium muscarium

Lime sulfur is one of the few products indicated to control Brevipalpus yothersi in Brazilian organic citrus orchards. Other strategies, such as the use of entomopathogenic fungi should be evaluated, and Lecanicillium muscarium is one of the basic choices for pest management. Knowledge of the interactions between lime sulfur and this entomopathogen is critical for developing control strategies. With this goal, it was conducted the toxicological characterization of lime sulfur to B. yothersi and the compatibility evaluation with L. muscarium. Finally, the effects of L. muscarium and lime sulfur mixtures on B. yothersi control were evaluated. Product evaluation for B. yothersi was done through direct and residual contact bioassay, and different concentrations of lime sulfur mixed in potato dextrose agar culture medium were used to evaluate compatibility with L. muscarium. Lime sulfur was effective against adults of B. yothersi and caused eggs unviability of up to 71.0%, at a dose of 80 L per 2,000 L of H2O. The lethal concentration (LC50 and LC99) of lime sulfur estimated for mite adults were 246.62 and 858.5 µg of sulfur per mL of H2O (ppm a.i.). Lime sulfur concentrations of 180 to 560 ppm a.i. showed promise for use in combination with L. muscarium. However, concentrations of 1,000 and 5,600 ppm significantly reduced colony size and the number of spores/colony. The mixture of 100 and 180 ppm a.i. of lime sulfur with L. muscarium (108 conidia·mL­1) was not able to reduce the lethal time of entomopathogen on B. yothersi.

Sublethal effects of bifenazate on biological traits and enzymatic properties in the Panonychus citri (Acari: Tetranychidae).

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: LC10 and LC30, 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 (R0), mean generation (T) were decreased, intrinsic rate of increase (rm), finite rate (λ) were decreased in LC30, 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 LC30 than control and LC10, 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.

Field Experiment Effect on Citrus Spider Mite Panonychus citri of Venom from Jellyfish Nemopilema nomurai: The Potential Use of Jellyfish in Agriculture.

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.

'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, 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 IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (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 IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Host-specific gene expression as a tool for introduction success in Naupactus parthenogenetic weevils.

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.

Modeling and dynamics of physiological and behavioral resistance of Asian citrus psyllid.

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.

Environmental variables influence the developmental stages of the citrus leafminer, infestation level and mined leaves physiological response of Kinnow mandarin.

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 cm2 in size while observing no entrance above 3 cm2 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.

Using micro-computed tomography to reveal the anatomy of adult Diaphorina citri Kuwayama (Insecta: Hemiptera, Liviidae) and how it pierces and feeds within a citrus leaf.

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.

Comparative proteomics provides insights into diapause program of Bactrocera minax (Diptera: Tephritidae).

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.

Complete genome sequence and analysis of Alcaligenes faecalis strain Mc250, a new potential plant bioinoculant.

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.

Asian citrus psyllid adults inoculate huanglongbing bacterium more efficiently than nymphs when this bacterium is acquired by early instar nymphs.

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.

Metabolic response of Citrus limon to Asian citrus psyllid infestation revealed by EESI-MS and HPLC.

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.

Pheromone Chemistry of the Citrus Borer, Diploschema rotundicolle (Coleoptera: Cerambycidae).

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.

De Novo Assembly of the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Psyllidae) Transcriptome across Developmental Stages.

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.

First report of the occurrence of Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae), an invasive species in Nigeria, West Africa.

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.

Mitochondrial genetic variation reveals phylogeographic structure and cryptic diversity in Trioza erytreae.

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.

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

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