The inoculation methods, the fungal strains, and several other factors are known to influent the success of fungal entomopathogens colonization in plants. The physiological status of the plant could also be another determinant. In the present study, the ability of three strains of Beauveria bassiana and one strain of Metarhizium anisopliae to successfully colonize Citrus limon plants and the influence of seedling age on endophytic colonization success was examined. Three, 4, and 6 months old seedlings were inoculated with 10 mL of 1 × 108 conidial·mL−1 suspensions of each of the four fungal strains via foliar spraying. All fungal strains successfully colonized citrus seedlings and were sustained up to 2 months in colonized plants irrespective of the seedling age, with differences in the mean percentage colonization recorded at various post-inoculation periods among the fungal strains. The highest percent endophytic fungi recovery rate was recorded in the 3 months old seedlings, where fungal mycelia of inoculated fungi were successfully re-isolated from 65.6% of the untreated newly developed leaf and stem tissues. One strain of B. bassiana, BB Fafu-12, significantly improved seedling height and leaf number. The study demonstrates the influence of seedling age on B. bassiana and M. anisopliae successful colonization in the citrus plant.

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Microbiome analysis in a host-parasitoid interaction network was conducted to compare the taxonomic composition of bacterial communities of Diaphornia citri, Tamarixia radiata, and Diaphorencyrtus aligarhensis. The comparative analysis revealed differences in the composition and diversity of the symbiont populations across the host and its associated parasitoids. Proteobacteria was the most dominant phylum, representing 67.80% of the total bacterial community, while Candidatus Profftella armature and Wolbachia were the dominant genera across the host and parasitoids. There were clear differences observed in alpha and beta diversity of microbiota through the host and its associated parasitoids. The function prediction of bacterial communities and Pearson correlation analysis showed that specific bacterial communities displayed positive correlations with the carbohydrate metabolism pathway. Furthermore, when symbiotic bacteria were eliminated using a broad-spectrum antibiotic, tetracycline hydrochloride, the parasitoids' median survival time and longevity were significantly reduced. We confirmed the physiological effects of symbiotic bacteria on the fitness of parasitoids and demonstrated the effect of antibiotics in decreasing the food intake and measurement of amino acids in the hemolymph. This study sheds light on basic information about the mutualism between parasitoids and bacteria, which may be a potential source for biocontrol strategies for citrus psyllid, especially D. citri.

The parasitism rate and host-feeding rate of Tamarixia radiata (Hymenoptera: Eulophidae), an ectoparasitoid of Diaphorina citri (Hemiptera: Liviidae), were evaluated at 20, 27.5, 30, and 35°C, at 70 ± 5% RH, and 14 h of photoperiod. The biological control efficacy of T. radiata was evaluated by linking the age-stage predation rate with the two-sex life table. The net host-feeding rate (C0) by T. radiata was 32.05, 54.40, 17.25, and 1.92 nymphs per female parasitoid at 20, 27.5, 30, and 35°C, respectively. The total net nymphs killing rate (Z0) was 103.02, 223.82, 72.95, and 6.60 nymphs per female parasitoid at 20, 27.5, 30, and 35°C, respectively. Noneffective parasitism rate was observed at 35°C because of high mortality at this temperature. Our results indicated that temperature had meaningful effects on parasitism and host-feeding rate parameters in the laboratory, and may affect biological control efficiency of the parasitoid in the field. The highest host-feeding rate and total biological control efficiency of T. radiata were recorded at 27.5°C. Most importantly, we found that host-feeding activity of the parasitoid is temperature-dependent, and changed across temperature regimes: the host-feeding rate increased as the temperature increased up to 30°C, started to decrease after this temperature and declined to its minimum level at 35°C. This information is valuable for developing biological control and integrated pest management techniques for Asian citrus psyllid management.

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Tamarixia radiata is one of the established biocontrol pests against the major Asian citrus psyllid, Diaphorina citri, a vector of Candidatus Liberibacter that is a causal agent of citrus Huanglongbing (HLB) disease. Updated information and regional exploration on biocontrol pests are important elements for effective disease management strategies. In this study, the diversity and parasitism rate of T. radiata populations were evaluated. Due to the importance of the host–parasitoid relationship, the presence of Wolbachia as an endosymbiont was also investigated. The parasitism rate of various T. radiata populations from Ecuador and China ranged between 57.27% and 66.32%, respectively, with a non-significant emergence rate and a statistically similar sex ratio. Sequence analysis of ITS and COI from T. radiata populations was consistent with the morphological hypothesis that the collections represent a single species, whereas phylogeny of the wsp gene confirmed the presence of Wolbachia pipientis as an endosymbiont within T. radiata populations. Based on partial COI sequences, the maximum genetic diversity such as total haplotype diversity (Hd = 0.788), nucleotide, diversity (π = 0.2439), and average nucleotide difference (k = 171.844) was also estimated for different T. radiata populations. Furthermore, neutrality tests based on COI sequences indicated an overall contraction in T. radiata populations, whereas an expansion trend was observed in associated W. pipientis strains. This study clearly demonstrated the presence of genetically diverse T. radiata populations that were able to parasitize D. citri effectively, and these can be further explored as promising biocontrol candidates in integrated pest management strategies to solve citriculture economic loss caused by D. citri.

The effect of the entomopathogenic fungus Beauveria bassiana (BB-12) on the biological characteristics of Tamarixia radiata parasitizing Diaphorina citri was studied under laboratory conditions. Twenty 3rd–5th instar nymphs were exposed to a single already-mated female parasitoid (1 day old) and removed after 24 h. Subsequently, the nymphs were sprayed at 1, 24 and 48 h post-exposure with 1 × 108 conidial mL−1 suspension. The percentage of parasitism recorded was 22%, 35% and 41% at 1, 24 and 48 h, respectively. The emergence rate varied between 28%, 51% and 49% at 1, 24 and 48 h, respectively. In a subsequent experiment, nymphs of D. citri were sprayed with 1 × 108 conidial mL−1 suspension and then exposed to T. radiata at 1, 24 and 48 h post-spraying to allow for parasitism to occur. The percentage of parasitism recorded was 18%, 27% and 28% at 1, 24 and 48 h, respectively, significantly lower than the parasitism rate recorded in the control (48%). The percentage of emergence varied between 24%, 44% and 45% at 1, 24 and 48 h, respectively. In both experiments, no significant difference was observed in the pre-adult duration (days) and the sex ratio of F1 generation. Meanwhile, significant differences were observed in the longevity of the F1 generation of females and males of T. radiata in a treatment consisting of spraying the fungal suspension and the control. Overall, the findings of the current study revealed a negative interaction between T. radiata and B. bassiana in controlling D. citri nymphs. This outcome is believed to be a result of the antagonistic effects of B. bassiana on the developmental process of the pre-adult stages of the parasitoid. However, our results also show that with a properly timed application (allowing parasitism to occur over an extended period of time before the application of the fungus), T. radiata could potentially be used in combination with B. bassiana for the successful biological control of D. citri. This should be carried out in order to minimize the potentially negative interactions between these two biological agents.

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