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Huanglongbing (HLB) is a destructive disease of citrus primarily transmitted by the Asian citrus psyllid (ACP). Biocontrol of ACP is an environmentally sustainable alternative to chemicals. However, the risk of parasitoid rational application in ACP biocontrol has never been evaluated. Here we show, the dominant parasitoid of ACP, Tamarixia radiata, can acquire the HLB pathogen Candidatus Liberibacter asiaticus (CLas) and transmit it horizontally when probing ACP nymphs. If these ACP nymphs survive the probing, develop to adults and move to healthy plants, CLas can be transmitted to citrus leaves during feeding. We illustrate the formerly unrecognized risk that a parasitoid can potentially serve as a phoretic vector of the pathogen transmitted by its host, thus potentially diminishing some of the benefits it confers via biocontrol. Our findings present a significant caution to the strategy of using parasitoids in orchards with different infection status of insect-vectored pathogens., Guo et al. demonstrate the possibility of a parasitoid to contract and transmit a bacterial pathogen via its insect host to the citrus plant it is used to protect. This reduces the efficacy of the biological control agent to protect the plant from ill effects.

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.

Tamarixia radiata (Waterston) is a predominant parasitoid of the Asian citrus psyllid (ACP), a destructive citrus pest and vector of huanglongbing (HLB) disease in the fields of southern China. To explore the functioning of target genes in T. radiata, the screening of specific reference genes is critical for carrying out the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) under different experimental conditions. However, no reference gene(s) for T. radiata has yet been reported. Here, we selected seven housekeeping genes of T. radiate and evaluated their stability under the six conditions (developmental stage, sex, tissue, population, temperature, diet) by using RefFinder software, which contains four different programs (geNorm,&Delta, Ct, BestKeeper, and NormFinder). Pairwise variation was analyzed by geNorm software to determine the optimal number of reference genes during the RT-qPCR analysis. The results reveal better reference genes for differing research foci: 18S and EF1A for the developmental stage, PRS18 and EF1A for sex, PRS18 and RPL13 for different tissues (head, thorax, abdomen), EF1A and ArgK between two populations, &beta, tubulin and EF1A for different temperatures (5, 15, 25, 35 &deg, C), and ArgK and PRS18 for different feeding diets. Furthermore, when the two optimal and two most inappropriate reference genes were chosen in different temperatures and tissue treatments, respectively, the corresponding expression patterns of HSP70 (as the reporter gene) differed substantially. Our study provides, for the first time, a more comprehensive list of optimal reference genes from T. radiata for use in RT-qPCR analysis, which should prove beneficial for subsequent functional investigations of target gene(s) in this natural enemy of ACP.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most serious pest of citrus worldwide. It acts as a vector for a group of phloem‐limited bacteria (Candidatus Liberibacter spp.) that causes Huanglongbing (HLB) disease. Thus, D. citri management is an important strategy against HLB, and biological control is currently considered as the most effective method because of the unsustainable and negative side effects of chemical control. Here, we isolated a new strain of entomopathogenic fungus, Cordyceps javanica (GZQ‐1), from one cadaver of D. citri adult based on its morphological and phylogenetic data. Five conidial concentrations of the C. javanica pathogen (1 × 103, 1 × 104, 1 × 105, 1 × 106, and 1 × 107 conidia/ml) were assessed against six life stages of D. citri (1st‐5th instar nymphs and adults). Results showed that C. javanica GZQ‐1 was highly pathogenic to D. citri nymphs (69.49%–90.87% mortality) and adults (69.98% mortality). The LC50 and LT50 values of C. javanica against 1st‐2nd instar (younger), 3rd‐4th instar (middle aged), 5th instar (older), and adults were 1.20 × 105, 1.10 × 106, 4.47 × 106, 8.12 × 106 conidia/ml and 4.25, 4.51, 5.17, 5.49 days, respectively. Moreover, glasshouse experiments indicated that this C. javanica GZQ‐1 caused higher infection rates of D. citri adults compared to two other fungal strains we previously isolated in the laboratory, Cordyceps fumosorosea (IF010) and Metarhizium anisopliae (CNGD7).

Wolbachia is a group of intracellular bacteria that infect a wide range of arthropods including the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. This insect is the vector of Candidatus Liberibacter asiaticus (CLas), the causal pathogen of Huanglongbing or citrus greening disease. Here, we investigated the localization pattern and infection dynamics of Wolbachia in different developmental stages of ACP. Results revealed that all developmental stages of ACP including egg, 1st–5th instar nymphs, and adults of both gender were infected with Wolbachia. FISH visualization of an ACP egg showed that Wolbachia moved from the egg stalk of newly laid eggs to a randomly distributed pattern throughout the egg prior to hatching. The infection rate varied between nymphal instars. The titers of Wolbachia in fourth and fifth instar nymphs were significantly higher than those in the first and second instar nymphs. Wolbachia were scattered in all nymphal stages, but with highest intensity in the U‐shaped bacteriome located in the abdomen of the nymph. Wolbachia was confined to two symmetrical organizations in the abdomen of newly emerged female and male adults. The potential mechanisms of Wolbachia infection dynamics are discussed.