Siddharth Tiwari

Insecticide resistance in field populations of Asian citrus psyllid in Florida.

BACKGROUND Asian citrus psyllid (ACP), Diaphorina citri, is a major pest of citrus because it vectors the putative causal agent of huanglongbing disease. Insecticides are currently the basis of psyllid management programs, and the number of annual insecticide applications has increased significantly. In this paper, a series of investigations of insecticide resistance among field populations of adult and immature ACP in Florida is described. RESULTS In 2009, the highest level of resistance for adult ACP, as compared with the laboratory susceptible (LS) population, was found with imidacloprid with an LD(50) resistance ratio (RR(50) ) of 35 in one population. This was followed by chlorpyriphos (RR(50) = 17.9, 13.3, 11.8 and 6.9), thiamethoxam (RR(50) = 15 and 13), malathion (RR(50) = 5.4 and 5.0) and fenpropathrin (RR(50) = 4.8). In 2010, mortality of adults from all five sites sampled was lower than with the LS population at three diagnostic concentrations of each insecticide tested. Among nymph populations, indications of resistance were observed with carbaryl (RR(50) = 2.9), chlorpyriphos (RR(50) = 3.2), imidacloprid (RR(50) = 2.3 and 3.9) and spinetoram (RR(50) = 4.8 and 5.9). General esterase, glutathione S-transferase and monooxygenase levels were also elevated in field-collected adult and nymph ACP as compared with the LS population. CONCLUSION The present results suggest that varying levels of insecticide susceptibility exist in ACP populations across the citrus-growing areas of Florida. Increased levels of detoxifying enzymes in these populations may partially explain these differences. The present results indicate that insecticide resistance may become an emerging problem for ACP control if effective resistance management is not practiced.

Induced Release of a Plant-Defense Volatile 'Deceptively' Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.

Incidence of Candidatus Liberibacter asiaticus Infection in Abandoned Citrus Occurring in Proximity to Commercially Managed Groves

ABSTRACT Huanglongbing is one of the most devastating diseases of citrus (Citrus spp.). One management tactic against huanglongbing is aggressive management of the vector, the Asian citrus psyllid (Diaphorina citri Kuwayama), with insecticide applications. However, D. citri in abandoned groves are not controlled and therefore pose a risk of reinfestation for nearby commercial citrus. These abandoned groves could serve as a reservoir for the vector, as well as a source of the presumed causal agent for huanglongbing in Florida, Candidatus Liberibacter asiaticus (Las). The current study was conducted to determine the degree to which Las is present in abandoned Florida citrus groves and to compare relative inoculum levels in nearby managed and abandoned groves during times of the year when D. citri are abundant (June, July, and August). In addition, the movement of Las by dispersing D. citri adults from inner and edge rows of abandoned grove plots to the corresponding rows of managed plots was quantified during the same 3 mo. The results of the current study confirmed the presence of Las in both D. citri and plant tissue in abandoned groves at statistically equivalent levels to those in nearby managed groves. The mean number of D. citri adults dispersing from abandoned to managed grove plots ranged from 7.25 ± 1.70 to 70.25 ± 21.25 per 4-d intervals. Of those, the mean number of dispersing D. citri adults that were carrying the Las pathogen ranged from 1.00 ± 0.58 to 1.50 ± 0.50. Our results indicate that abandoned citrus groves are a significant source of Ca. Las and that dispersing D. citri move this pathogen into nearby managed groves.

Characterization of five CYP4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus-infected and uninfected psyllids

Previously, we reported that Candidatus Liberibacter asiaticus (Las)‐infected Diaphorina citri are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In the present study, we investigated expression levels of family 4 cytochrome P450 (CYP4) genes in Las‐infected and uninfected D. citri adults. Five novel CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) were identified. Four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las‐infected males, whereas only one was expressed at significantly higher levels in uninfected than Las‐infected females. These results suggest that levels of cytochrome P450 monooxygenases in D. citri may be linked to expression levels of these CYP4 genes. Expression of all five CYP4 genes was induced by exposure of D. citri to imidacloprid, suggesting their possible involvement in metabolism of this toxin. Higher expression of the five CYP4 genes was found in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA‐interference to silence overexpression of genes associated with insecticide resistance in D. citri. These newly identified genes may also serve as DNA‐based screening markers for cytochrome P450‐mediated insecticide resistance in field populations of D. citri.

Glutathione Transferase and Cytochrome P450 (General Oxidase) Activity Levels in Candidatus Liberibacter Asiaticus-Infected and Uninfected Asian Citrus Psyllid (Hemiptera: Psyllidae)

ABSTRACT Candidatus Liberibacter asiaticus (Las) has been reported to increase the susceptibility of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), to selected insecticides. Reduced general esterase activity in Las-infected, compared with uninfected, D. citri has been proposed as a possible explanation for this difference in insecticide susceptibility. The current study was conducted to quantify glutathione transferase (GST) and cytochrome P450 (general oxidase) activities in Las-infected D. citri to further explain the possible mechanisms for altered susceptibility to insecticides due to Las infection. GST and cytochrome P450 activities (indirectly through general oxidase levels) were quantified in Las-infected and uninfected D. citri nymphs and adults. Mean (±SEM) GST activity was significantly lower in Las-infected (468.23 ± 26.87 /µmol/min/mg protein) than uninfected (757.63 ± 59.46 µmol/min/mg protein) D. citri adults. Likewise, mean cytochrome P450 activity was significantly lower in Las-infected (0.23 ± 0.02 equivalent units [EU] cytochrome P450/mg protein) than uninfected (0.49 ± 0.05 EU cytochrome P450/mg protein) D. citri adults. Immature stages (second and fifth instars) were characterized by significantly lower GST activity than adults for uninfected D. citri. However, cytochrome P450 activity was significantly higher in second instar nymphs than adults and fifth-instar nymphs for uninfected D. citri. Lower activities of GST and general oxidase in Las-infected D. citri indicate that infection with Las alters D. citri physiology in a manner that could increase insecticide susceptibility. The reduced activities of these detoxifying enzymes due to Las infection may be explained by examining expression levels of associated genes in Las-infected and uninfected D. citri.

Effect of Candidatus Liberibacter asiaticus infection on susceptibility of Asian citrus psyllid, Diaphorina citri, to selected insecticides

BACKGROUND In the present investigation, the effect of Candidatus Liberibacter asiaticus (Las), a bacterium considered to be responsible for causing huanglongbing (HLB) disease in citrus, on the physiology of its vector, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama, was determined. Specifically, the effects of Las infection on the susceptibility of ACP to selected insecticides were determined. Furthermore, total protein content and general esterase activity were quantified in Las-infected and uninfected ACP to gain insight into the possible mechanism(s) responsible for altered susceptibility to insecticides owing to Las infection. RESULTS LC(50) values were significantly lower in Las-infected than in uninfected ACP adults for chlorpyrifos and spinetoram. Furthermore, there was a general trend towards lower LC(50) values for three other insecticides for Las-infected ACP; however, the differences were not statistically significant. Total protein content (µg mL(-1)) was significantly lower in Las-infected (23.5 ± 1.3 in head + thorax; 27.7 ± 1.9 in abdomen) than in uninfected (29.7 ± 2.1 in head + thorax; 35.0 ± 2.3 in abdomen) ACP. Likewise, general esterase enzyme activity (nmol min(-1) mg(-1) protein) was significantly lower in Las-infected (111.6 ± 4.5 in head + thorax; 109.5 ± 3.7 in abdomen) than in uninfected (135.9 ± 7.5 in head + thorax; 206.1 ± 23.7 in abdomen) ACP. CONCLUSION Susceptibility of ACP to selected insecticides from five major chemistries was greater in Las-infected than in uninfected ACP. The lower total protein content and reduced general esterase activity in Las-infected than in uninfected ACP may partly explain the observed higher insecticide susceptibility of Las-infected ACP.

Seasonal Movement Patterns and Long-Range Dispersal of Asian Citrus Psyllid in Florida Citrus

ABSTRACT The Asian citrus psyllid, Diaphorina citri Kuwayama, is the vector of the bacterial pathogen, Candidatus Liberibacter asiaticus, which is the causal agent of huanglongbing (HLB) in the United States. Both short-range and long-range dispersal of D. citri adults affect the spread of HLB; however, little is known about the long-range dispersal capabilities of D. citri in the field or the seasonality of flight behavior. In the present study, an in situ protein marking technique was used to determine the dispersal of D. citri by trapping marked adults under natural field conditions. D. citri movement from abandoned citrus groves to adjacent managed citrus groves was greatest during the spring and summer months and decreased significantly during the colder months (September–March). D. citri were able to traverse potential geographic barriers such as roads and fallow fields. In an experiment conducted to determine long-range dispersal capacity in the absence of severe weather events, D. citri were able to disperse at least 2 km within 12 d. Wind direction was not correlated with the number of marked psyllids captured, indicating substantial flight capability by D. citri. Finally, the number of marked psyllids captured increased with the density of emerging young leaves on surrounding trees. The results confirm that abandoned citrus groves in Florida serve as reservoirs for D. citri, which can disperse across long distances despite geographical barriers.

Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.

Sexual Transmission of a Plant Pathogenic Bacterium, Candidatus Liberibacter asiaticus, between Conspecific Insect Vectors during Mating

Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 µm in length and 0.19 to 0.39 µm in width. The spherical structures measured from 0.61 to 0.80 µm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees.

Effects of cyantraniliprole, a novel anthranilic diamide insecticide, against Asian citrus psyllid under laboratory and field conditions

BACKGROUND The Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), is the most destructive pest of citrus in Florida. The development of insecticide resistance in several populations of D. citri has been documented. There is an urgent need to develop and integrate novel tools for the successful management of D. citri and also to prevent the development of insecticide resistance. RESULTS The effects of a relatively newer chemistry, cyantraniliprole, against D. citri were investigated. The contact toxicity of cyantraniliprole was 297-fold higher against D. citri than its primary parasitoid, Tamarixia radiata (Hymenoptera: Eulophidae). D. citri settled and fed less on cyantraniliprole-treated plants than controls at concentrations as low as 0.025 and 0.125 µg AI mL⁻¹ respectively. D. citri egg production, first-instar emergence and adult emergence were significantly reduced on plants treated with 0.25, 0.02 and 0.25 µg AI mL⁻¹ of cyantraniliprole, respectively, when compared with control plants. Under field conditions, foliar and drench treatments with cyantraniliprole (1436.08 g ha⁻¹) reduced numbers of D. citri adults and nymphs, as well as of a secondary pest, citrus leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae), more than a standard insecticide. CONCLUSIONS These results suggest that cyantraniliprole should be a valuable new tool for rotation into D. citri management programs. For insecticide resistance management, cyantraniliprole may be particularly useful for rotation with neonicotinoids. In addition, cyantraniliprole was much less toxic to T. radiata than to D. citri and thus may have less impact on biological control than other currently used broad-spectrum insecticides, such as organophosphates and pyrethroids.