Yongwen Lin

Effects of different temperature regimes on survival of Diaphorina citri and its endosymbiotic bacterial communities

The Asian citrus psyllid, Diaphorina citri, is a major pest of citrus and vector of citrus greening (huanglongbing) in Asian. In our field-collected psyllid samples, we discovered that Fuzhou (China) and Faisalabad (Pakistan), populations harbored an obligate primary endosymbiont Candidatus Carsonella (gen. nov.) with a single species, Candidatus Carsonella ruddii (sp. nov.) and a secondary endosymbiont, Wolbachia surface proteins (WSP) which are intracellular endosymbionts residing in the bacteriomes. Responses of these symbionts to different temperatures were examined and their host survival assessed. Diagnostic PCR assays showed that the endosymbionts infection rates were not significantly reduced in both D. citri populations after 24 h exposure to cold or heat treatments. Although quantitative PCR assays showed significant reduction of WSP relative densities at 40°C for 24 h, a substantial decrease occurred as the exposure duration increased beyond 3 days. Under the same temperature regimes, Ca. C. ruddii density was initially less affected during the first exposure day, but rapidly reduced at 3-5 days compared to WSP. However, the mortality of the psyllids increased rapidly as exposure time to heat treatment increased. The responses of the two symbionts to unfavorable temperature regimes highlight the complex host-symbionts interactions between D. citri and its associated endosymbionts.

Volatiles from Plants Induced by Multiple Aphid Attacks Promote Conidial Performance of Lecanicillium lecanii.

Herbivore-induced plant volatiles (HIPVs) are clues that help predatory insects search for food. The hypothesis that entomopathogenic fungi, which protect plants, benefit from the release of HIPVs was tested. The plant Arabidopsis thaliana was used as the source of HIPVs. The insect herbivore Lipaphis erysimi (Kaltenbach) was used as the inducer, and the fungal pathogen of the aphid Lecanicillium lecanii was exposed to HIPVs to test our hypothesis. When exposed to aphid-induced A. thaliana volatiles, the mortality of aphids pre-treated with a conidial suspension of L. lecanii, the conidial germination and the appressorial formation were significantly increased compared with the control. The decan-3-ol and 4-methylpentyl isothiocyanate that were detected in the headspace seemed to have positive and negative affection, respectively. Moreover, HIPVs generated from groups of eight aphids per plant promoted significantly increased conidial germination and appressorial formation compared with HIPVs from groups of one, two and four aphids per plant. Our results demonstrated that the pathogenicity of the entomopathogenic fungus L. lecanii was enhanced when exposed to HIPVs and that the HIPVs were affected by the number of insect herbivores that induced them.

Diaphorina citri Induces Huanglongbing-Infected Citrus Plant Volatiles to Repel and Reduce the Performance of Propylaea japonica

Transmission of plant pathogens through insect vectors is a complex biological process involving interactions between the host plants, insects, and pathogens. Simultaneous impact of the insect damage and pathogenic bacteria in infected host plants induce volatiles that modify not only the behavior of its insect vector but also of their natural enemies, such as parasitoid wasps. Therefore, it is essential to understand how insects such as the predator ladybird beetle responds to volatiles emitted from a host plant and how the disease transmission alters the interactions between predators, vector, pathogens, and plants. In this study, we investigated the response of Propylaea japonica to volatiles from citrus plants damaged by Diaphorina citri and Candidatus Liberibacter asiaticus through olfactometer bioassays. Synthetic chemical blends were also used to determine the active compounds in the plant volatile. The results showed that volatiles emitted by healthy plants attracted more P. japonica than other treatments, due to the presence of high quantities of D-limonene and beta-ocimene, and the lack of methyl salicylate. When using synthetic chemicals in the olfactory tests, we found that D-limonene attracted P. japonica while methyl salicylate repelled the predator. However, beta-ocimene attracted the insects at lower concentrations but repelled them at higher concentrations. These results indicate that P. japonica could not efficiently search for its host by using volatile cues emitted from psyllids- and Las bacteria-infected citrus plants.

Phylogenetic relationship and genetic diversity of citrus psyllid populations from China and Pakistan and their associated Candidatus bacterium

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a severely devastating pest of Rutaceae plants, mainly citrus, across the globe, and causal agent of Huanglongbing (HLB) disease. To find out the genetic relationship and diversity among the populations of ACP and associated Candidatus Liberibacter asiaticus (CLas) from two countries (China and Pakistan), sequence data of three different genes, cytochrome oxidase subunit I (COI), Cu-transporting protein (ATOX1) and 16S rRNA, were used to characterize all populations. In the present study, MEGA-7 and statistical parsimony software (TCS-1.2) were used to depict the phylogenetic relationship among all populations under both genes, whereas diversity was calculated by DnaSP v5. All analyses were done for country wise and overall relationship among all populations. For ACP populations, both genes presented a significant strong intermingled relationship among all populations and put all population into a single haplotype (Dcit-2), which proved similarity between Chinese and Pakistani populations. Moreover, for CLas strains, 16S gene also presented strong relationship for all sampled populations. All three genes of ACP and CLas populations elucidated more than 95% resemblance to each other. On the other hand, a significant genetic variation was observed by three genes for overall populations, although, country wise variation was non-significant between all collected populations. ATOX1 gene presented higher diversity through Fus Fs test (π = 0.01081, p < 0.003) whereas COI gene gave less diversity under Fus Fs and Tajimas D test (π = 0.00512, p < 0.000 and 0.05, respectively). Similarly, nucleotide mismatch distribution also had shown enough genetic variation among all ACP populations, under both genes. Our sequence data for both genes proved the invasion of the Chinese ACP population (Dcit-2) into Pakistan, through all phylogenetic relationship, which proved a similar genetic makeup among all ACP populations from both countries. Therefore, these results can be helpful to utilize any novel designed control measure equally for both countries.

Susceptibilities of Candidatus Liberibacter asiaticus-infected and noninfected Diaphorina citri to entomopathogenic fungi and their detoxification enzyme activities under different temperatures

Some entomopathogenic fungi species, Isaria fumosorosea, and Hirsutella citriformis were found to be efficient against the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). However, the susceptibility to these fungi increases when the psyllid infected with Candidatus Liberibacter asiaticus (Las), which is transmitted by D. citri and causes citrus greening disease. In this study, we examined the Las‐infected and Las‐uninfected D. citri susceptibility to entomopathogenic fungi at different temperature regimes (5–40°C). When D. citri adults exposed to cold temperature (5°C), they showed less susceptibility to entomopathogenic fungi as compared with control (27°C). Irrespective of infection with Las, a significantly positive correlation was observed between temperature and percentage mortality caused by different isolates of I. fumosorosea, 3A Ifr, 5F Ifr, PS Ifr, and H. citriformis isolates, HC3D and 2H. In contrast, a significantly negative correlation was found between temperature and percentage mortality for 3A Ifr for both Las‐infected and Las‐uninfected psyllids. Detoxification enzymes, Glutathione S‐transferase levels in D. citri showed a negative correlation, whereas cytochrome P450 and general esterase levels were not correlated with changes in temperature. These findings revealed that detoxification enzymes and general esterase levels are not correlated with altered susceptibility to entomopathogenic fungi at the different temperature regimes. Conclusively, temperature fluctuations tested appear to be a significant factor impacting the management strategies of D. citri using entomopathogenic fungi.

Effects of flower thrips (Thysanoptera: Thripidae) on nutritional quality of banana (Zingiberales: Musaceae) buds

The abundance of banana flower thrips (Thrips hawaiiensis Morgan) in a banana (Musa acuminata Colla “Williams” cultivar) plantation was investigated using yellow sticky traps (29.70 cm × 21.00 cm) in 2015. Banana flower thrips occurred throughout the year with monthly variation, and the maximum occurrence was observed in October and November during the bud burst (73.80 ± 6.32 adults/trap) and young fruit (70.06 ± 5.69 adults/trap) periods. The damage rates were as follows: interior flowers >3rd-layer flowers > 2nd-layer flowers > 1st-layer flowers > young fruits. This result indicates that thrips migrated to lower bracts, young fruits, and other flower buds as bracts gradually opened. Results also showed that the reducing sugar, vitamin C, protein and ash contents in thrips-damaged flowers were all significantly lower than those in undamaged flowers, while there was no significant difference between damaged and undamaged young banana fruit. Our results indicated that the abundances of banana flower thrips were closely associated with the growing stage of banana. Thrips mainly infested flower buds and caused a reduction in nutrients for the host plant, especially the reducing sugar and vitamin C contents, which reduced the nutritional quality of banana fruits and the quality of flower bud by-products of banana.

Endophytic entomopathogenic fungi enhance the growth of Phaseolus vulgaris L. (Fabaceae) and negatively affect the development and reproduction of Tetranychus urticae Koch (Acari: Tetranychidae)

Entomopathogenic fungi (EPF) have primarily been applied as an inundative approach to manage pests. However, in recent decade multifunctional role of EPF have been documented which provide multiple benefits to host plants when colonized as an endophyte. In this study five fungal isolates from the genus Beauveria (three), Isaria (one) and Lecanicillium (one) were evaluated for their ability to colonize common bean, Phaseolus vulgaris and to assess their effects in planta on plant growth promotion and possible negative effects on the two-spotted spider mites, Tetranychus urticae. All the tested isolates in this study were able to endophytically colonize root, stem and even leaves of inoculated plants examined at 7 and 14 days post inoculation, indicating the systemic colonization of EPF. Colonized plants showed increased plant heights, fresh shoot and root weights compared to plants without inoculation. Survivorship of T. urticae significantly differed among the treatments with higher survival probability in control plants. Significant reduction in larval development, adult longevity and female fecundity of spider mites were observed when fed on treated plants compared to control plants. The negative effects were found to be carried over the second generation fed on fresh plants. Overall, our results show (i) the positive effects of fungal endophytes on plant growth, (ii) reduction in population growth rate and (iii) negative effects of endophytes on growth and reproduction of spider mites in successive generations. The study presents reports on the endophytic management of plant-feeding mites and highlights the possibility of utilizing entomopathogenic fungal endophytes in the integrated pest management program.