Asian Citrus Psyllid Adults Research Articles

Evaluation of Three Commercial Biopesticides for the Reduction of Diaphorina citri (Hemiptera: Liviidae) Populations

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the most serious threat to the global citrus industry, and its management has mainly depended on the application of chemical insecticides. The use of biopesticides can play an important role in regulating this pest. In a first test, two separate experiments were conducted in two different municipalities (Apulo and Jerusalen, Cundinamarca, Colombia) to evaluate the effect of foliar Beauveria bassiana and imidacloprid sprays on ACP populations in 2015 and 2016, respectively. In a second test, two separate tests were carried out in commercial Tahiti lime orchards to evaluate the efficacy of three different commercial biopesticides (Beauveria bassiana and extracts of Sophora sp. and garlic-chili pepper). In test 1, imidacloprid-treated trees showed a reduction (60% and 80%) in cumulative ACP adults in 2016. ACP cumulative nymphs were also diminished by foliar imidacloprid and B. bassiana application, between 40% and 65% in 2015 and 2016, respectively. ACP cumulative eggs showed lower individuals in imidacloprid-treated flushes at 3 and 4 weeks after treatment 9 (WAT) in 2016. In test 2, the results obtained showed ACP adults and eggs unaffected by biopesticide treatments; nymphs were reduced 50% to 75% in trees treated with the three biopesticides in comparison with control trees. All three biopesticides tested can be considered useful tools in integrated pest management (IPM) programs for ACP, since these products reduced immature ACP individuals between 50% and 75% under field conditions.

Toxicity and enzyme inhibition activities of the essential oil and dominant constituents derived from Artemisia absinthium L. against adult Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

The extensive use of synthetic chemical pesticides has led to negative effects on the environment and human health. Therefore, scientists are in search of natural products, especially those from plant sources, which are less hazardous, eco-friendly, and effective alternatives to the conventional pesticides. Essential oils are potential alternatives to the synthetic chemical pesticides. However, the toxicity of Artemisia absinthium against Asian citrus psyllid Diaphorina citri has not been documented. Therefore, here we focused on the A. absinthium essential oil (ABEO) extracted from the population grown under the climatic conditions of Pakistan’s high plateaus, as a natural pesticide for the control of the citrus psyllid. The results indicated that the oil caused significant acute mortalities in both topical and residual contact applications, with 50% lethal dose (LD50) and 50% lethal concentration (LC50) values of 5.2 μg/insect and 24.4 mg/mL. Among the constituents tested only carvacrol, (−)-α-bisabolol and chamazulene were toxic, with LD50 values of 6.9, 8.2, and 10.3 μg/insect via topical application and LC50 values of 28.5, 33.5, and 39.4 mg/mL via residual contact exposure. Several combinations of dominant constituents were both additive and synergistic when applied topically, especially carvacrol + (−)-α-bisabolol + chamazulene (1:1:1) displayed synergistic effect, with a poison ratio of 1.39. Furthermore, the essential oil and carvacrol inhibit acetylcholinesterase activity in citrus psyllid. It has been concluded that the essential oil and some of the compounds from A. absinthium might be developed as eco-friendly approaches for the control of citrus psyllid.

Conidial Production on Different Solid Substrates and Pathogenicity ofHirsutella citriformisAgainstDiaphorina citriAdults

Conidia of four strains of H. citriformis Speare fungi grown on oat (Avena sativa L.), wheat (Triticum aestivum L.), and sorghum (Sorghum bicolor (L.) Moench) were evaluated for production and for viability and pathogenicity against D. citri Kuwayama adults. Substrates were inoculated with conidial suspensions (1 × 106 conidia/ml) of INIFAP-Hir-1, INIFAP-Hir-2, IB-Hir-1, and IB-Hir-2 strains and incubated for 35 days. Pathogenicity on Asian citrus psyllid adults also was evaluated through contact bioassay. Significantly more conidia were produced on oat (58.5-28.83 × 106 per gram) than wheat (50.9-29.4 × 106 per gram) at 21 days. Production of most conidia (8.4-6.6 × 106 per gram) required 28 days on sorghum and was less than on oat or wheat. Percentages of germination of conidia on the three substrates ranged from 96.25 to 98.0% without significant differences among them. Pathogenicity of the strains against Asian citrus psyllid were not significantly different when grown on agar (85.5–89.9%) or oat (85.8–89.3%). Results confirmed the possibility of increased production of H. citriformis conidia in natural substrates maintaining pathogenicity against Asian citrus psyllid.

Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

Evaluation of systemic neonicotinoid insecticides for the management of the Asian citrus psyllid Diaphorina citri on containerized citrus.

Studies were conducted to evaluate uptake and retention of three systemic neonicotinoid insecticides, dinotefuran, imidacloprid and thiamethoxam, in potted citrus nursery plants treated at standard label rates. Infestation of these plants placed at a field site with moderate levels of Asian citrus psyllid (ACP) was monitored for 14 weeks following treatments, and insecticide residues in leaf tissue were quantified using enzyme-linked immunosorbent assay (ELISA). Bioassays were conducted using leaves harvested on various dates post-treatment to compare the efficacies of residues against adult ACP. Residues of the three neonicotinoids were detected in leaf tissues within 1 week after treatment. Peak concentrations established at 1 week for imidacloprid and dinotefuran and at 2 weeks for thiamethoxam. Imidacloprid and thiamethoxam outperformed the control and dinotefuran treatments at protecting trees from infestations by ACP eggs and nymphs. For a given insecticide concentration in leaf tissue, thiamethoxam induced the highest mortality of the three insecticides, and dinotefuran was the least toxic. If the time needed to achieve effective thresholds of a systemic neonicotinoid is known, treatments at production facilities could be scheduled that would minimize unnecessary post-treatment holding periods and ensure maximum retention of effective concentrations after the plants have shipped to retail outlets. The rapid uptake of the insecticides and retention at effective concentrations in containerized citrus suggest that the current 30 day post-treatment shipping restriction from production facilities to retail outlets outside of quarantine could be shortened to 14 days. Thiamethoxam should be added to the list of approved nursery treatments. © 2016 Society of Chemical Industry.

Enhanced Acquisition Rates of 'Candidatus Liberibacter asiaticus' by the Asian Citrus Psyllid (Hemiptera: Liviidae) in the Presence of Vegetative Flush Growth in Citrus.

The Asian citrus psyllid preferentially feeds and exclusively reproduces on young, newly emerged flush shoots of citrus. Asian citrus psyllid nymphs feed and complete their life stages on these flush shoots. Recent studies conducted under greenhouse conditions have shown that the transmission rates of 'Candidatus Liberibacter asiaticus' (CLas), the putative causal agent of huanglongbing disease of citrus, are enhanced when flush shoots are present. However, it is unclear if CLas acquisition by migrant adult Asian citrus psyllids is similarly enhanced. To address this knowledge gap, cohorts of Asian citrus psyllid adults were allowed 1-wk acquisition access period (AAP) on flushing and nonflushing shoots of qPCR-tested symptomatic (CLas+) and asymptomatic (CLas-) 10-yr-old sweet orange trees under field conditions. After the AAP, they were tested for CLas by qPCR. Progeny Asian citrus psyllid adults that emerged 4 wk post-AAP were similarly retrieved and tested. Eighty percent of flushing and 30% of nonflushing CLas+ trees produced infective Asian citrus psyllid adults, indicating that flush shoots have greater potential to be inoculum sources for CLas acquisition. Concomitantly, 21.1% and 6.0% infective adults were retrieved, respectively, from flushing and nonflushing CLas+ trees, indicating that Asian citrus psyllid adults acquire CLas more efficiently from flush shoots relative to mature shoots. In addition, 12.1% of infective Asian citrus psyllid adult progeny were obtained from 70% of flushing CLas+ trees. Significantly lower mean Ct values were also obtained from infective adults retrieved from flushing relative to nonflushing trees. The results underscore the role of flush shoots in CLas acquisition and the need to protect citrus trees from Asian citrus psyllid infestations during flush cycles.

Evaluation of Mexican Strains ofHirsutella citriformis1AgainstDiaphorina citri2in a Semifield Bioassay

Abstract. Citrus production is threatened by Huanglongbing disease transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama. Developing strategies to control the vector is necessary. Conidial infectivity of four Mexican strains of Hirsutella citriformis Speare (INIFAP-Hir-2, INIFAP-Hir-4, IB-Hir-1, and IB-Hir-2) was evaluated in a semifield bioassay against adult Asian citrus psyllid. The bioassay was done in an orchard of Persian lime (Citrus aurantiifolia (Christm. et Panz.) Swingle var. latifolia Yu. Tanaka) at Veracruz, Mexico. The four strains showed pathogenicity, but INIFAP-Hir-2 and INIFAP-Hir-4 killed most (51.05-50.6%), with no significant difference between them. Thirty days after application in the field, several H. citriformis strains were recovered from mycosed insects and grown in different media where they varied depending on the culture medium and strain and presented morphological characteristics similar to the strains applied and to a local strain previously isolated. Mycosed inse...

Repellency of selected Psidium guajava cultivars to the Asian citrus psyllid, Diaphorina citri

Huanglongbing (HLB) is the most devastating disease of citrus worldwide. It is caused by bacteria of the genus ‘Candidatus Liberibacter’ and transmitted by two psyllid species, the Asian citrus psyllid (ACP) Diaphorina citri, and the African citrus psyllid Trioza erytreae. Considerable research has been conducted toward developing and implementing HLB and ACP management strategies. With respect to ACP control, of interest is that reports indicate guava, Psidium guajava, can be repellent to ACP. We conducted research to further evaluate repellency of guava to ACP. In one set of experiments, guava oil from five Brazilian guava cultivars (‘J3’, ‘Pedro Sato’, ‘Século XXI’, ‘Thailand’ and ‘Paluma’) was extracted from leaves (immature and mature) by hydro-distillation in a Clevenger-type apparatus and evaluated for psyllid repellency. In a second set of experiments, repellency of guava leaves to ACP was investigated using leaves (immature and mature) from two guava cultivars in Florida, ‘Pink’ and ‘Thai White’. In each set of experiments, repellency was evaluated by releasing ACP adults into a cage with two large vials, one containing a young flush shoot (= immature leaves) of Murraya exotica (a favored host plant of the psyllid, the flush of which is highly attractive to ACP) and one with M. exotica flush and the test material of interest (guava oil, immature guava leaf or mature guava leaf). The adults were free to move throughout the cage and into the vials, and the number of psyllids in each vial was counted after 24 h. The results showed that all guava materials tested had at least some repellency to ACP. Mature leaves tended to have a greater repellent effect than immature leaves. Each of the five oils exhibited repellency. A report in the literature suggested that sulfur compounds associated with guava may be responsible for ACP repellency. Interestingly, the five guava oil extracts we studied were repellent to ACP but none contained any sulfur compounds. Identification of the constituents responsible for repellency could lead to new ACP management tactics.

Impact of different temperatures on survival and energy metabolism in the Asian citrus psyllid, Diaphorina citri Kuwayama

Temperature influences the life history and metabolic parameters of insects. Asian citrus psyllid (ACP), Diaphorina citri is a tropical and subtropical pest. ACP invaded new regions around the world and threatened the citrus industry as a vector for Huanglongbing (HLB) disease. ACP is widely distributed and can survive high (up to 45°C) and low temperatures (as low as −6°C). The precise mechanism of temperature tolerance in ACP is poorly understood. We investigated adult survival, cellular energy balance, gene expression, and nucleotide and sugar-nucleotide changes under the effect of different temperature regimes (0°C to 45°C with 5°C intervals). The optimum temperatures for survival were 20 and 25°C. Low temperatures of 0°C and 5°C caused 50% mortality after 2 and 4days respectively, while one day at high temperature (40°C and 45°C) caused more than 95% mortality. The lowest quantity of ATP (3.69±1.6ng/insect) and the maximum ATPase enzyme activities (57.43±7.6μU/insect) were observed at 25°C. Correlation between ATP quantities and ATPase activity was negative. Gene expression of hsp 70, V-type proton ATPase catalytic subunit A and ATP synthase α subunit matched these results. Twenty-four nucleotides and sugar-nucleotides were quantified using HPLC in ACP adults maintained at low, high, and optimum temperatures. The nucleotide profiles were different among treatments. The ratios between AMP:ATP and ADP:ATP were significantly decreased and positively correlated to adults survival, whereas the adenylate energy charge was increased in response to low and high temperatures. Exploring energy metabolic regulation in relation with adult survival might help in understanding the physiological basis of how ACP tolerates newly invaded regions.

Behavioral Assay on Asian Citrus Psyllid Attraction to Orange Jasmine

The Asian citrus psyllid (ACP) is an important pest because it transmits a bacterium putatively responsible for huanglongbing, a devastating citrus disease. Research on ACP chemical ecology is of interest with respect to identifying attractants and repellents for managing the psyllid. We report on an assay for investigating ACP attraction to the foliar odor of one of its host plants, orange jasmine (Murraya exotica L.). Flush shoots from this plant were placed into 25 dram vials covered with a paper wrapper, a lid with a small entrance hole was snapped onto each vial, the vials were placed into a small cage, adult ACP were released into the cage, and the location of the adults was determined 24 h later. A positive response required an adult to find and enter a vial. When single males or females were released, they were attracted into vials with flush 64 % of the time. At release rates of 25, 50 or 100 adults with 30 repetitions for each rate, relatively large mean percentages of adults (ca. 83 %) positively responded to flush shoots. ACP can escape from the assay vial and will do so if flush is not present. Working with ACP in empty vials, fluoropolymer resin applied to the inside of vials reduced escapes but did not eliminate them. An entrapment funnel for reducing escapes was tested, but it reduced ACP response rates. A good method of preventing escapes remains to be developed. When two vials of flush were placed in the assay cage and one was treated with a candidate repellent, only 8 % of adults settled in the vial with the repellent.

Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the insect vector of the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease, or Huanglongbing (HLB). The widespread invasiveness of the psyllid vector and HLB in citrus trees worldwide has underscored the need for non-traditional approaches to manage the disease. One tenable solution is through the deployment of RNA interference technology to silence protein-protein interactions essential for ACP-mediated CLas invasion and transmission. To identify psyllid interactor-bacterial effector combinations associated with psyllid-CLas interactions, cDNA libraries were constructed from CLas-infected and CLas-free ACP adults and nymphs, and analyzed for differential expression. Library assemblies comprised 24,039,255 reads and yielded 45,976 consensus contigs. They were annotated (UniProt), classified using Gene Ontology, and subjected to in silico expression analyses using the Transcriptome Computational Workbench (TCW) (http://www.sohomoptera.org/ACPPoP/). Functional-biological pathway interpretations were carried out using the Kyoto Encyclopedia of Genes and Genomes databases. Differentially expressed contigs in adults and/or nymphs represented genes and/or metabolic/pathogenesis pathways involved in adhesion, biofilm formation, development-related, immunity, nutrition, stress, and virulence. Notably, contigs involved in gene silencing and transposon-related responses were documented in a psyllid for the first time. This is the first comparative transcriptomic analysis of ACP adults and nymphs infected and uninfected with CLas. The results provide key initial insights into host-parasite interactions involving CLas effectors that contribute to invasion-virulence, and to host nutritional exploitation and immune-related responses that appear to be essential for successful ACP-mediated circulative, propagative CLas transmission.

Further investigations on colonization of Poncirus trifoliata by the Asian citrus psyllid

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) vectors a bacterium, ‘Candidatus Liberibacter asiaticus’ (CLas), associated with one of the world's most serious diseases of citrus, huanglongbing (HLB) (also known as citrus greening disease). There is no known cure for this disease, which severely reduces tree productivity and fruit quality and promotes tree decline and death. Most growers apply insecticides to control ACP as a tactic to prevent or reduce the incidence of HLB. Plant resistance to ACP may be a viable tactic for managing ACP and CLas. Resistance to colonization by ACP populations has been reported in the genotype Poncirus trifoliata L., which is cross compatible with citrus. The Citrus Research Center (CRC) in Riverside, California, maintains a large number of accessions of P. trifoliata. We conducted four free-choice experiments with 29 CRC accessions of P. trifoliata and found that ACP deposited fewer eggs on 19 of those accessions compared with susceptible sweet orange, Citrus sinensis (L.) Osbeck. Subsequent infestation densities of nymphs generally reflected reductions in oviposition but there were notable exceptions. Some accessions may be susceptible to oviposition but contain traits that confer antibiosis to nymphs. In no-choice experiments with seedling plants representing eight P. trifoliata accessions, oviposition rates were reduced compared with sweet orange and it appeared that factors associated with mature leaves were involved. In no-choice experiments with adult ACP confined to flush cuttings, large reductions were observed in the number of eggs and nymphs on each of five P. trifoliata accessions compared with sweet orange. Based on these results in conjunction with published reports, accessions were identified that consistently showed resistance to oviposition. However, the level of resistance in these accessions has been variable possibly due to environmental conditions, plant age, proximity of plants to susceptible germplasm, and other factors.

Radial Growth, Sporulation, and Virulence of Mexican Isolates ofHirsutella citriformis1againstDiaphorina citri2

Abstract. Hirsutella citriformis Speare is an important entomopathogenic fungus that infects diverse species of hemipteran insects, including the vector of huanglongbing, Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). The objective of this study was to determine the growth and conidiation of eight isolates of H. citriformis as well as virulence of the conidia on Asian citrus psyllid adults and nymphs. The strains were isolated from infected D. citri in states of Mexico where citrus, Citrus spp., are grown. Optimum radial growth was obtained at 25°C for the eight isolates incubated in three agar media (potatodextrose agar, potato dextrose agar with 0.5% yeast extract, and Sabouraud dextrose agar). Most daily radial growth was by six strains and most production of conidia (1.833 × 107 per milliliter) was by strain IB-Hir-2 after 34 days of incubation on potato dextrose agar with 0.5% yeast extract. In a preliminary bioassay, strains INIFAP-Hir-1, IB-Hir-2, and IB-Hir-1 killed most (8...

Identification and entomopathogenicity of newly-isolated fungi infecting Diaphorina citri Kuwayama (Homotera: Psyllidae) in Murraya orchards of Fujian, China

Among fungal isolates obtained from Murraya panciculata L. groves in Fujian, China, seven were tested pathogenic against the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Homoptera: Psyllidae). In the present paper, the isolates were identified for their taxonomic ranks and compared on their entomopathogenicity against ACP adults. Based on the analysis of conidia morphological data and ITS sequences of 18 S rDNA, the fungal isolates FJAT-9620, FJAT-9621, FJAT-9622, FJAT-9624 and FJAT-9719 were identified as Beauveria bassiana, FJAT-9623 as B. asiatica and FJAT-9720 as Lecanicillium attenuatum. Bioassays revealed that fungal isolates FJAT-9622, FJAT-9623, FJAT-9719 and FJAT-9720 infected adult psyllids with mortality of 95.00-98.33% at 27±1oC and 100% relative humidity (RH) in the laboratory. Meanwhile, isolates FJAT-9620, FJAT-9621 and FJAT-9624 induced significantly lower mortality (3.33-40.00%) on the psyllids.

Effect of time and storage methods on the detection of Candidatus Liberibacter asiaticus in Diaphorina citri by qPCR

The assessment of bacterialiferous Asian citrus psyllid (ACP) frequency is important for (i) studies of bacteria acquisition and inoculation by ACP, (ii) disease detection in disease free areas but with ACP presence, (iii) efficiency evaluation of inoculum reduction strategies, (iv) evaluation of frequency of Candidatus Liberibacter asiaticus (Las)-positive ACP and the abundance of inoculum sources or putative new HLB infections relationships. Depending on the conditions and time of storage of collected psyllids, Las DNA in ACP could degrade and Las-false negative results might occur. Thus, this study was conducted to evaluate the detection of Las in ACP adults submitted to different storage methods and time of storage by real-time PCR (qPCR). Two 2x3x7 factorial experiments were conducted. Factors were ‘Ethanol’ (with or without 70% ethanol), ‘Temperature’ (-20°C, 4°C and 26°C) and ‘Time’ (0, 3, 7, 14, 21, 28 and 35 days). For each treatment, 20 samples with 3 ACP adults from nymphs reared on Las infected trees were tested for Las presence by qPCR. No significant differences in percentages of psyllids samples positive for Las were observed among the storage methods up to 35 days, except a slight trend of decline in Las detection in samples storage without ethanol at 26°C after 14 days of storage.

Targeting juvenile hormone metabolic genes in the Asian citrus psyllid (Diaphorina citri Kuwayama) as a strategy to reduce the spread of citrus greening disease

Author(s): Van Ekert, Evelien; Borovsky, D.; Powell, C. A.; Cave, R. D.; Alessandro, R. T.; Shatters, Jr., R. G. | Abstract: Diaphorina citri Kuwayama, the Asian citrus psyllid (ACP), is a devastating citrus pest due to its transmission of a phloem-limited bacterial pathogen, Candidatus Liberibacter asiaticus, that causes citrus greening. Psyllid control is a major part of effective greening disease management, and our research targets perturbation of insect juvenile hormone metabolism as a new psyllid control strategy.n Previous studies have shown that application of a juvenile hormone (JH) analogue, pyriproxyfen, is known to produce ovicidal/nymphicidal effects, morphological abnormalities, and reduced fecundity in ACP adults. These observations prompted us to identify JH biosynthetic and degradative pathways as targets for biologically-based control strategies, including RNA interference, as alternatives to heavy reliance on broad-spectrum pesticides. First, candidate genes/cDNAs encoding the JH metabolic enzymes, juvenile hormone acid methyl transferase (JHAMT) and juvenile hormone esterase, were identified through computational analysis of the D. citri genome. Second, JHAMT cDNA was cloned, expressed in E. coli and a functional protein was purified. This JHAMT had a high affinity for substrates leading to JHI and JHIII synthesis, making it plausible that both juvenoids are present in the ACP. Results are discussed with respect to mechanism(s) of juvenile hormone biosynthesis/catabolism in the ACP and targeting this process as an interdiction point for a bio-rational ACP control strategy.

Huanglongbing management on bearing groves based on favorable periods for symptomatic-trees removal and vector control

Author(s): Bassanezi, R. B.; Montesino, L. H.; Bergamin Filho, A. | Abstract: In Brazil, Huanglongbing (HLB) has been managed by removal of symptomatic-trees and Asian citrus psyllid (ACP) control. Although new HLB-affected trees and ACP can be detected all year long, visual detection of HLB-affected trees has been more pronounced from March to August while ACP population densities are higher from September to February. Therefore, the aim of this work was to compare the efficiency of applying the control strategies only during the higher occurrence periods of new HLB affected trees and ACP adults with the control application during all year long. The experiment was carried out in a 4-yr old sweet orange Valencia/Rangpur lime grove and had a 2 by 3factorial design with 3 replications (1.4 ha plots). The factor “HLB-tree elimination” had 2 treatments: monthly elimination all year long; and monthly elimination from March to August, both based on visual inspection. The factor “Vector control” had 3 treatments: monthly ACP control all year long; monthly ACP control from September to February; and ACP control when 10% of 48 yellow sticky traps (YST) placed in the center of plots had at least one adult psyllid. All Treatments of ACP control were done alternating foliar sprays of Provadoâ, Dimetoatoâ, Trebonâ and Marshalâ+Micromiteâ. After 5 years, no significant differences were detected among different treatments for the variables mean cumulative HLB incidence and disease progress rate estimated by linear regression of the last 4 years cumulative disease incidence. The mean cumulative HLB incidence increased from 0.4% to 14.2% (Yr1 4.9%, Yr2 1.9%, Yr3 2.3%, Yr4 1.7%, and Yr5 3.0%). The number of caught ACP per YST per assessment and the area under the curve of percentage of YST with ACP were significantly higher for monthly ACP control from September to February (total of 34 sprays), but did not differed between monthly ACP control all year long (total of 65 sprays) and control based on ACP monitoring with YST (total of 21 sprays). We believe that HLB management wasn’t better because there was a significant amount of new HLB-symptomatic trees (25.2%) found from December to February, and 12.3% of ACP caught in August. In conclusion, with some adjusts the management of HLB could be optimized according to the favorable periods for HLB-symptomatic trees detection and ACP populations.

Effect of Temperature on Lighted Sticky Traps (TransTrap®) used to Detect Asian Citrus Psyllids in Shipping Containers

The Asian citrus psyllid (ACP) is an insect native to tropical and subtropical regions in Asia, which has spread into most citrus producing regions around the world.  The main concern with ACP is the spread of Huanglongbing (HLB), a disease vectored by the psyllid. The volume of citrus shipments from Mexico into the U.S. prompted questions about the risk of moving ACP and HLB on this pathway.  Our goal was to determine temperatures at which ACP would be active and thus fly to lighted sticky traps inside a transport trailer.Our results found that temperature significantly affected the number of adults captured.  No psyllids were recovered on the traps at 12˚C (53˚F), which is the average temperature of citrus shipments crossing the border.  This is compare with an average capture of 43, 47, and 42% of the released adults at 24, 28, and 32°C (75, 82, and 90°F), respectively.  Only 1%, 2% and 18% were captured at 18, 20 and 22°C (64, 68, and 72˚F), respectively.  Given that the average temperature of limes arriving at the border is 12˚C, it is unlikely that ACP adults would fly to a lighted sticky trap placed inside a refrigerated trailer.  In addition, the majority of the packing houses in Mexico place the fruit into coolers prior to loading. We believe a lighted sticky trap would not be effective in detecting ACP within refrigerated citrus shipments for the period of time from loading at the packing house to trap recovery at the border crossing.

Detection of Candidatus Liberibacter asiaticus in Diaphorina citri caught on yellow sticky traps during the winter and summer of Sao Paulo State Brazil

The assessment of bacterialiferous Asian citrus psyllid (ACP) frequency is important in epidemiological and management studies because it can be related with the abundance of inoculum sources and with putative new HLB infections. For that, ACP can be collected directly or on yellow sticky traps (YST) commonly used by Brazilian growers to monitor psyllid population. The YST are usually left in the field for 2 weeks after which time YST are visually evaluated for the ACP presence, and if present, the psyllids are removed from the YST and tested by real-time PCR (qPCR) for liberibacter presence. Previous studies in Florida showed that the incidence of Las-positive ACP declined with increasing time on the YST (Irey et al., 2011). Thus, the objective of this work was to determine if time ACP is keep on YST affects qPCR results for Las and if it was related to weather conditions during winter and summer of Araraquara-SP (Brazil). ACP adults from nymphs reared on Las infected trees were placed on YST (BUG-Agentes Biologicos) in the field and 20 samples with 3 individuals were tested after 0, 1, 3, 9, 12 and 15 days. The results were compared with samples directly collected without trap glue. Experiments were done in June, July and August (winter) and in January, February and March (summer). In contrast with previous report in Florida, no difference on the incidence of Las-positive ACP samples was observed up to 15 days on the YST in both seasons.

Stylet Morphometrics and Ultrastructure in Relation to Feeding Behavior and Pathogen Transmission by Nymphs and Adults of the Asian Citrus Psyllid Diaphorina citri, Vector of Citrus Huanglongbing Bacterium

The feeding behavior and stylet morphometrics were studied in nymphs and adults of the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera, Psyllidae), vector of Candidatus Liberibacter asiaticus (Las) associated with citrus huanglongbing (HLB) disease. The stylet length of first instar nymphs averaged 266 µm (83% of body length) whereas that of 5th instar nymphs was 615 µm (34% of body length). Younger ACP nymphs feed only on young citrus leaves on smaller veins or on the sides of the midrib, whereas adults can feed anywhere on the veins of young or old citrus leaves. Epifluorescence microscopy of cross sections in citrus leaves indicated that the thick-walled fibrous layer around the phloem is much more prominent in older than in younger leaves. Additionally, first instar nymphs can reach the phloem because the distance to the phloem is shorter from the sides of the midrib compared to that from the top, and is considerably shorter in younger than in older/mature leaves. Ultrastructural studies on ACP stylets show that the width of the maxillary food canal in first instar nymphs is wide enough for Las bacteria to go through during food ingestion (and Las acquisition). However, the width of the maxillary salivary canal in first instar nymphs may not be wide enough for Las bacteria to go through during salivation (and inoculation of Las bacterium) into host plants. This may explain previous studies indicating that older ACP nymphs and adults can transmit HLB bacterium whereas younger nymphs probably cannot.