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The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease. [ABSTRACT FROM AUTHOR]

Asian citrus psyllid (Diaphorina citri) feeding behaviors play a significant role in the transmission of the phloem-limited Candidatus Liberibacter asiaticus (CLas) bacterium that causes the economically devastating citrus greening disease. Sustained phloem ingestion by D. citri on CLas infected plants is required for pathogen acquisition and transmission. Recent studies have shown a fibrous ring of thick-walled sclerenchyma around the phloem in mature, fully expanded citrus leaves that is more prominent on the abaxial compared with the adaxial side. The composition and thickness of this fibrous ring may have an important role in selection of feeding sites by D. citri based on leaf age and leaf surface, which in turn can affect pathogen acquisition and transmission. We measured feeding behavior using electrical penetration graph (EPG) recordings of individual D. citri adults placed on abaxial or adaxial surfaces of young or mature Valencia orange leaves to study the role of the sclerenchymatous ring in modifying D. citri feeding behavior. Feeding sites on the same leaf tissues were then sectioned and examined by epifluorescence microscopy. The duration of phloem ingestion (E2 waveform) by psyllids was significantly reduced on mature compared with young leaves, and on abaxial compared with adaxial leaf surfaces. The longest duration of phloem ingestion was observed from psyllids placed on the adaxial side of young leaves that had the least developed sclerenchyma. Bouts of phloem salivation (E1 waveform), however, were significantly longer on mature leaves compared with young leaves. D. citri adults made consecutive phloem feeding attempts (bouts) on the abaxial side of mature leaves and those bouts resulted in unsuccessful or shorter periods of phloem ingestion. Adults also made more frequent and longer bouts of xylem ingestion on mature leaves compared with adult psyllids placed on young leaves. Epifluorescence microscopy showed that the fibrous ring in young leaves was thinner and autofluoresced in red whereas the ring in mature leaves was thicker and autofluoresced in blue, indicating changes in structure and composition (e.g., lignification) of sclerenchyma correlated with leaf age. Our results support the hypothesis that the presence of a thick, well-developed fibrous ring around phloem tissues of mature leaves acts as a barrier to frequent or prolonged phloem ingestion by D. citri from citrus leaves. This may have an important role in limiting or preventing CLas acquisition and/or transmission by D. citri, and could be used for identification and development of resistant citrus cultivars. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) implicated as causative agent of citrus huanglongbing (citrus greening), currently the most serious citrus disease worldwide. Las is transmitted by D. citri in a persistent-circulative manner, but the question of replication of this bacterium in its psyllid vector has not been resolved. Thus, we studied the effects of the acquisition access period (AAP) by nymphs and adults of D. citri on Las acquisition, multiplication and inoculation/transmission. D. citri nymphs or adults (previously non-exposed to Las) were caged on Las-infected citrus plants for an AAP of 1, 7 or 14 days. These ‘Las-exposed’ psyllids were then transferred weekly to healthy citrus or orange jasmine plants, and sampled via quantitative polymerase chain reaction (qPCR) analysis 1–42 days post-first access to diseased plants (padp); all tested nymphs became adults 7–14 days padp. Our results indicate that following 1 or 7 day AAP as nymphs 49–59% of Las-exposed psyllids became Las-infected (qPCR-positive), whereas only 8–29% of the psyllids were infected following 1–14 day AAP as adults. Q-PCR analysis also indicated that Las titer in the Las-exposed psyllids (relative to that of the psyllid S20 ribosomal protein gene) was: 1) significantly higher, and increasing at a faster rate, following Las acquisition as nymphs compared to that following Las acquisition as adults; 2) higher as post-acquisition time of psyllids on healthy plants increased reaching a peak at 14–28 days padp for nymphs and 21–35 days padp for adults, with Las titer decreasing or fluctuating after that; 3) higher with longer AAP on infected plants, especially with acquisition as adults. Our results strongly suggest that Las multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for Las to reach higher levels in the vector. However, under the conditions of our experiments, only D. citri that had access to infected plants as nymphs were able to inoculate Las into healthy citrus seedlings or excised leaves. The higher probability of Las inoculation into citrus by psyllids when they have acquired this bacterium from infected plants during the nymphal rather than the adult stage, as reported by us and others, has significant implications in the epidemiology and control of this economically important citrus disease. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the principal vector of 'Candidatus Liberibacter asiaticus' (CLas) associated with huanglongbing (HLB), the most serious citrus disease worldwide. New control measures including pesticides are urgently needed to combat HLB, especially to protect young or newly planted citrus trees from CLas-inoculation by vector psyllids. Here, we tested CLas-inoculation by D. citri adults (CLas-exposed, reared on infected plants) by feeding them for 7 d on excised healthy citrus leaves with dry residues of cyantraniliprole (Exirel), a novel insecticide, in comparison with fenpropathrin (Danitol 2.4EC), an insecticide commonly used against D. citri. Fewer adults settled (putatively feeding or probing) on leaves treated with cyantraniliprole than those treated with fenpropathrin or water controls. Also, psyllid adults died at a slower rate on leaves treated with cyantraniliprole than those treated with fenpropathrin, although the final cumulative mortality did not differ between the two treatments. In quantitative real-time polymerase chain reaction tests, 59.0-65.3% of the CLas-exposed psyllid adults were proven to be CLas-positive. Inoculation rates of CLas (using 10 adults per leaf) into untreated healthy citrus leaves (47.5-85%) were significantly higher than rates into leaves treated with cyantraniliprole or fenpropathrin (2.5-12.5%). Reduced inoculation rates to leaves treated with cyantraniliprole probably occurred as a result of reduced feeding or probing by D. citri. The excised leaf assay method, which took only a few weeks compared with up to a year or longer using whole plants, can be an effective tool for testing the effect of new pesticides or other treatments in reducing CLas inoculation or transmission by psyllid vectors. [ABSTRACT FROM AUTHOR]

The ultrastructure and development of new stylets was studied in pre-molting first instar nymph of Diaphorina citri Kuwayama (Hemiptera: Liviidae). Two oval-shaped masses of cuboidal hypodermal cells, located in the cephalic region, had long extensions that ended with developing pairs of mandibular and maxillary stylets, apparently coiled around these masses. A new structure, probably composed of softer cuticle, was found on the ventral side of each developing stylet suggesting that this structure may work as a mold during formation of the new stylets. Other organs of 1st instar nymphs, including the filter chamber and bacteriome, are also ultrastructurally described. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) associated with huanglongbing, or citrus greening, the most devastating citrus (Citrus spp.) disease worldwide. Here, we developed a new "excised-leaf assay" that can speed up Las-inoculativity tests on Asian citrus psyllid from the current 3-12 mo (when using whole citrus seedlings for inoculation) to only 2-3 wk. Young adults of Asian citrus psyllid that had been reared on Las-infected plants were caged on excised healthy sweet orange [Citrus sinensis (L.) Osbeck] leaves for a I-2-wk inoculation access periods (IAP), and then both psyllids and leaves were tested later by quantitative polymerase chain reaction (PCR). When single adults were tested per leaf, percentages of Las-positive leaves averaged 2-6% by using HLBaspr primers and 10-20% by using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves were obtained with 1) higher densities of inoculating psyllids (5-10 adults per leaf), 2) longer IAPs, and 3) incubation of leaves for 1 wk postinoculation before PCR. Logistic regression analysis indicated a positive correlation between Las titer in Asian citrus psyllid adults tested singly and the probability of detecting Las in the inoculated leaves, correlations that can be very useful in epidemiological studies. Comparison between excised leaves and whole seedlings, inoculated consecutively for I wk each by one or a group of psyllids, indicated no significant difference between Las detection in excised leaves or whole plants. This new excised-leaf assay method saves considerable time, materials, and greenhouse space, and it may enhance vector relation and epidemiological studies on Las and potentially other Liberibacter spp. associated with huanglongbing disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Asian citrus psyllid, Diaphorina citri, transmits Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing disease. Although they primarily feed on the phloem of Citrus and related plants, when grove or host conditions are unfavorable, D. citri may be able to use weed species as alternate food sources for survival. To explore this possibility, electrical penetration graph (EPG) recordings (18 h) were performed to investigate the feeding behavior of psyllid adults and nymphs on three common south Florida weeds (Bidens alba, Eupatorium capillifolium, and Ludwigia octovalvis). EPG recordings revealed that the proportion of time spent by D. citri feeding on xylem was similar on all tested weed species (19%–22%) and on the positive control (20%), the preferred host, Citrus macrophylla. Very little to no phloem feeding was observed on weed species by either nymphs or adults. Histological studies using epifluorescence microscopy showed that salivary sheaths were branched and extended into xylem of weed species, whereas they ended in phloem on citrus plants. No choice behavioral assays showed that adults can obtain some nutrition by feeding on weed species (xylem feeding) and they may be able to survive on them for short intervals, when host conditions are unfavorable. [ABSTRACT FROM AUTHOR]