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

Abstract 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.


| INTRODUC TI ON
The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Liviidae), is native to Asia and southeastern Florida and has invaded several regions of the world (Capoor & Viswanath, 1967;Grafton-Cardwell, Stelinski, & Stansly, 2013;Huang, Tsai, & Wang, 1984;Nava et al., 2010;Yan, Zeng, & Zhong, 2015). Diaphorina citri primarily attacks young flush of citrus trees but can also attack stressed citrus trees if the pest population density is high. It has become the most important insect pest of citrus in southeastern Florida (Halbert, 1997) and has recently threatened native citrus plants in China (Yan et al., 2015). Diaphorina citri greatly reduces the production, destroys the economic value of the fruits and eventually kills citrus trees when inoculates healthy citrus plants with phloem-limited bacteria (Candidatus Liberibacter spp.) that cause citrus greening disease (huanglongbing = HLB) (Hall & Rohrig, 2015).
However, the susceptibility of insects and mites to pesticides has changed due to some abiotic factors such as temperature, rainfall, and humidity, as well as nonenvironmental factors including pesticide coverage, host plants, and host infection status (Grafton-Cardwell et al., 2013;Musser & Shelton, 2005;Tiwari, Mann, Rogers, & Stelinski, 2011;Xie et al., 2011;Yang, Margolies, Zhu, & Buschman, 2001). Insecticides toxicity depends on the target pest and application method at given temperature (Musser & Shelton, 2005). In tropical and sub-tropical areas where D. citri are present, the variation in toxicity of insecticides is caused by different temperature variations ranging between 5°C and 40°C (Boina, Onagbola, Salyani, & Stelinski, 2009). In some areas of the world, like Florida D. citri are occasionally exposed to cold stress below −6.5°C and −8°C; and it was reported that D. citri adults and nymphs tolerate these cold temperatures (Hall, Wenninger, & Hentz, 2011;Hussain, Akutse, et al., 2017). Thus, lethal concentration (LC 50 ) values of chemical insecticides for D. citri, based on the temperature regimes have been previously investigated (Boina et al., 2009).
However, this lethal effect as regards temperatures has not been studied with entomopathogenic fungi. Additionally, the effect of unfavorable temperature stress on entomopathogenic fungi pathogenicity has not been investigated for D. citri.
In this study, susceptibility of D. citri to entomopathogenic fungi under cold stress (5°C) was investigated and compared with control (27°C). Furthermore, experiments were conducted to assess Lasinfected and Las-uninfected D. citri detoxification enzymes activity levels; glutathione S-transferase (GST), cytochrome P450, and general esterase, at different temperature regimes. Tiwari et al. (2011) have previously reported that these detoxifying enzyme systems were correlated with D. citri insecticide resistance. The aim of this study was also to investigate the effect of different entomopathogenic fungi, temperature and pathogenicity correlations in Lasinfected and Las-uninfected psyllids.

| MATERIAL S AND ME THODS
The original population of D. citri was initially collected from Fuzhou source. The infection was determined and confirmed using PCR as described by Tatineni et al. (2008). Infected and uninfected orange jasmine were used in this rearing of the psyllids, which were also tested by real-time PCR for the presence or absence of Las infection before the bioassays (Lin et al., 2016). Before each experiment, the purity of cultures HLB-free or HLB-infected established colonies was further checked regularly by the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique and further tested with mtCOI sequencing as described by Tatineni et al. (2008) for infection (established colony).
The fungal isolates were cultured on potato dextrose agar (PDA) plates and were maintained at 25 ± 2°C in complete darkness. For bioassay, entomopathogenic fungi conidia were scraped off from 2-week-old plates with a sterilized spatula and suspended in 20 ml of autoclaved deionized water containing 0.03% Tween 80. The conidial suspensions were vortexed in for 5 min to produce homogenous conidial suspensions and then filtered through Miracloth. Fungal conidia were counted using a Neubauer Hemacytometer, and concentrations were determined through dilution. The required conidial suspension with a standard concentration of 1 × 10 8 conidia/ml was obtained for the five entomopathogenic fungal isolates by serial dilutions containing 0.03% Tween 80 (Fluka) as a wetting agent.

| Diaphorina citri susceptibility to entomopathogenic fungi at cold temperature
Prior to experiments, psyllids were carefully collected from the above tested pure (HLB-free) and HLB-infected colonies of D. citri and transferred onto clean citrus plants in mesh cages (50 × 50 × 50 cm).
The mesh cages containing D. citri were shifted to climate box set at 5°C, 55% RH, and at a photoperiod of 14:10 hr L:D or were maintained at 27°C (control) for 1 or 2 weeks before the experiment.
Psyllids from 5°C temperature were assessed using a leaf-dip Petri dish method described by Kumar, Poehling, & Borgemeister (2005)) and Tiwari et al. (2011). Plastic disposable Petri dishes consisted of 60-mm diameter and 1.5% agar solution (2 mm solidified layer) is used for bioassay. Leaf disks from fresh citrus leaves were cut (60 mm), dipped for 1-3 min in entomopathogenic fungal conidial suspensions prepared in 0.03% Tween 80 as described above, and left for air dry under a hood for 1 hr before bioassays. Leaf disks were dipped in 0.03% Tween 80, as the control treatment. Leaf disks (Grafton-Cardwell et al., 2013;Yan et al., 2015) were placed on agar layers after 1 hr, and 20-30 adult psyllids of mixed gender were shifted to the dish using a soft camel hair brush. Psyllids were anesthetized shortly (10 sec) with cold temperature for easy handling and transfer. Petri dishes were sealed with parafilm (Laboratory film, PM-996, USA) to block the psyllids. Sealed Petri dishes with adult insects were transferred into a growth chamber (Safe, China) set at 26 ± 1°C, 55% RH, and at a photoperiod of 14:10 hr L:D. Psyllids mortality was recorded 48 hr after placing the Petri dishes into the growth chamber. Psyllids were considered dead when seemed their sides or backs and not able to move when touched with a soft camel hair brush. Each fungal isolate was replicated three times, and all bioassays were repeated three times at each temperature over time.
In addition, mycosis test was also conducted with the cadavers to assess fungal growth and confirm if the mortality is due to the fungal isolates infection in the various treatments.

| Effect of temperature on enzymes levels
The effect of temperature on three detoxifying enzymes expression levels was studied by using the Las-uninfected colony. Treatments consisted of 5F Ifr, or HC3D-treated adults maintained for 48 hr at six different temperature regimes (5, 10, 20, 27, 35, and 40°C).
Each entomopathogenic fungal isolate was repeated three times at each temperature, and for each replication 80-100 adult psyllids were tested. Psyllids of mixed gender were transferred onto leaves sprayed with entomopathogenic fungal isolate suspensions at the concentration of 1 × 10 8 conidia/ml, using the Petri dish method as described above. About 80-100 adult psyllids were shifted to each Petri dish. Psyllids which survived from each treatment after 48 hr exposure were collected and used immediately for detoxifying enzymes expression levels assays.

| Susceptibility of Las-infected and Lasuninfected Diaphorina citri to entomopathogenic fungi under different temperatures
Entomopathogenic fungi bioassays were conducted using a leafdip Petri dish method as described above. Petri dishes containing

| Statistical analysis
The mean mortality (%) among psyllids exposed to various entomopathogenic fungi was analyzed using Analysis of Variance (ANOVA) test, and correlation analyses were conducted at p < .05, between Las-infected and Las-uninfected psyllid treatments, entomopathogenic fungi, and temperature regimes. Las-infected and Lasuninfected mean mortalities for the various entomopathogenic fungi and at the different temperatures were compared using Chi-square.
Mortality percentages in all treatment were corrected using Abbott's formula (Abbott, 1925

| Susceptibility of Diaphorina citri to entomopathogenic fungi at cold temperature regime
Percentage mortality comparisons which were performed between cold temperature and control psyllids for each period and entomopathogenic fungi showed that when D. citri were exposed to cold stress (5°C) for 1 week, they were significantly less susceptible to 5F Ifr than control psyllids at 27°C (Table 1). Similarly, psyllids that were exposed to cold stress (5°C) for 2 weeks were less susceptible to 2H than psyllids maintained at 27°C. However, when mortalities were compared between psyllids exposed to cold temperature (5°C) and the control (27°C), no significant differences were observed for the other tested entomopathogenic fungi (3A Ifr, PS Ifr, and HC3D; Table 1).

| D ISCUSS I ON
The results of the study showed that psyllids exposed to cold stress were more tolerant to entomopathogenic fungi than controls, seems to be possible effects that could be observed during winter compared to summer temperature variations. However, the cold temperature did not affect the susceptibility of D. citri to PS Ifr, 3A Ifr, and HC3D.
Further studies are warranted to understand the mechanism underlying decreased susceptibility of cold stress psyllids populations to entomopathogenic fungi. Some studies reported that decrease in susceptibility might influence the management of psyllids under field conditions using I. fumosorosea and H. citriformis (Lezama-Gutiérrez et al., 2012;Pérez-González, Sandoval-Coronado, & Maldonado-Blanco, 2016).
Temperature affects the rate of metabolism (Hussain, Akutse, et al., 2017;, the binding of the enzyme with its substrate (Hochachka & Somero, 1984) and the rate of enzymatic activity (Hoffmann, 1984). Therefore, we assumed that the different levels of susceptibility of psyllids to entomopathogenic fungi due to temperature changes observed in this study might comprise altered levels of different enzyme activities. The data analysis showed that temperature affected the levels of GST,  Wang et al., 2013), the proportion of Las-infected psyllids is in some cases more than 95% (Coy & Stelinski, 2015). However Las-infected psyllids were more susceptible to some synthetic insecticides than Lasuninfected psyllids (Tiwari, Pelz-Stelinski, & Stelinski, 2011). Our results showed that fluctuation in temperature is correlated with Las-infected and uninfected D. citri susceptibility to entomopathogenic fungi. A general trend in Las-infected and uninfected D. citri was observed; with a positive correlation between temperature and mortality for PS Ifr, 5F Ifr, HC3D and 2H, and a negative correlation for 3A Ifr.
Although previous studies have been conducted to show the effect of temperature on synthetic insecticides susceptibility, the alteration in entomopathogenic fungi pathogenicity due to changes in temperature was not understood (Deng, Zhang, Wu, Yu, & Wu, 2016;Garcia et al., 2016;Lasa, Williams, & Caballero, 2008;Zhang et al., 2015). However, susceptibility to entomopathogenic fungi was increased in Las-infected as compared to uninfected D. citri, but Las infection did not affect the correlation coefficients between temperature and psyllids mortality.
Recently, biopesticides are developed as the best tool for management of herbivores like D. citri; thus the knowledge of interactions between abiotic factors (temperature) and herbivore susceptibility to biopesticides may help to understand and design better management strategies against the pests. Our results showed that alterations in psyllids susceptibility to entomopathogenic fungi are due to changes in temperature, which are not related to changes in detoxifying enzymes expression levels. However, psyllids exposed to cold stress are less susceptible to entomopathogenic fungi I. fumosorosea and H. citriformis than control. Thus, seasonal variations in temperature and entomopathogenic fungi may have a major impact on the management of D. citri under field conditions.

ACK N OWLED G M ENTS
All authors are thankful to Ru Xinhui and Bao Lu for their kind as-

CO N FLI C T O F I NTE R E S T
None declared.