Received: 31 October 2017 | Revised: 18 January 2018 | Accepted: 19 January 2018 DOI: 10.1002/mbo3.607 ORIGINAL RESEARCH Susceptibilities of Candidatus Liberibacter asiaticus-infected and noninfected Diaphorina citri to entomopathogenic fungi and their detoxification enzyme activities under different temperatures Mubasher Hussain1,2,3,4 | Komivi Senyo Akutse1,2,4,5 | Yongwen Lin1,2,4 | Shiman Chen1,2,4 | Wei Huang1,2,4 | Jinguan Zhang1,2,4 | Atif Idrees1,6 | Dongliang Qiu3 | Liande Wang1,2,4 Plant Protection College, Fujian Agriculture and Forestry University, Fuzhou, China 1 Abstract State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 350002, China Some entomopathogenic fungi species, Isaria fumosorosea, and Hirsutella citriformis College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, China psyllid infected with Candidatus Liberibacter asiaticus (Las), which is transmitted by 2 3 Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fuzhou, China 4 were found to be efficient against the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). However, the susceptibility to these fungi increases when the 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), International Centre of Insect Ecology and Physiology, Nairobi, Kenya they showed less susceptibility to entomopathogenic fungi as compared with control Institute of Beneficial Insects, Fujian Agriculture and Forestry University, Fuzhou, China served between temperature and percentage mortality caused by different isolates 5 6 Correspondence Liande Wang, Plant Protection College, Fujian Agriculture and Forestry University, Fuzhou China. Email: liande_wang@126.com Funding information The Key Projects of Science and Technology of Fujian Province, Grant/Award Number: 2016N0005; The Research Fund for the International Collaborative Program, Grant/Award Number: CXZX2017211 and KXGH17004; Fujian Agriculture and Forestry University (27°C). Irrespective of infection with Las, a significantly positive correlation was obof 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. KEYWORDS asian citrus psyllid, bacterial infection, detoxification enzymes, entomopathogenic fungi, microbial ecology, temperature effects This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. MicrobiologyOpen. 2018;7:e607. https://doi.org/10.1002/mbo3.607 www.MicrobiologyOpen.com | 1 of 9 2 of 9 | 1 | I NTRO D U C TI O N HUSSAIN et Al. In this study, susceptibility of D. citri to entomopathogenic fungi under cold stress (5°C) was investigated and compared with control The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Liviidae), (27°C). Furthermore, experiments were conducted to assess Las- is native to Asia and southeastern Florida and has invaded several infected and Las-uninfected D. citri detoxification enzymes activity regions of the world (Capoor & Viswanath, 1967; Grafton-Cardwell, levels; glutathione S-transferase (GST), cytochrome P450, and gen- Stelinski, & Stansly, 2013; Huang, Tsai, & Wang, 1984; Nava et al., eral esterase, at different temperature regimes. Tiwari et al. (2011) 2010; Yan, Zeng, & Zhong, 2015). Diaphorina citri primarily attacks have previously reported that these detoxifying enzyme systems young flush of citrus trees but can also attack stressed citrus trees were correlated with D. citri insecticide resistance. The aim of this if the pest population density is high. It has become the most im- study was also to investigate the effect of different entomopatho- portant insect pest of citrus in southeastern Florida (Halbert, 1997) genic fungi, temperature and pathogenicity correlations in Las- and has recently threatened native citrus plants in China (Yan et al., infected and Las-uninfected psyllids. 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 bacte- 2 | M ATE R I A L S A N D M E TH O DS ria (Candidatus Liberibacter spp.) that cause citrus greening disease (huanglongbing = HLB) (Hall & Rohrig, 2015). Currently, many biocontrol agents have been used against D. citri The original population of D. citri was initially collected from Fuzhou (FzP, 26.07877° N, 119.2969° E), Fujian China from Murraya pan- (Khan, Arif, Hoddle, & Hoddle, 2014; Qureshi, Rogers, Hall, & Stansly, iculata (L.) Jacq. (Sapindales: Rutaceae) Plants (Orange Jasmine). 2009) but the most effective agents are entomopathogenic fungi, The stock populations were maintained for about nine generations Isaria fumosorosea Wize (= Paecilomyces fumosoroseus) (Hypocreales: prior to experiments on the same host plants kept in mesh cages Cordycipitaceae) and Hirsutella citriformis Spear (Hypocreales: (50 × 50 × 50 cm) in greenhouse at a temperature of 27 ± 1°C, a Ophiocordycipitaceae) (Avery et al., 2013; Speare 1920). These bio- photoperiod of 14:10 light/dark (L/D; 14 hr light 6:00–20:00), and control agents have received interest for use in the management of 75 ± 5% relative humidity (RH), with no insecticide exposure. To ob- D. citri in Florida (Avery et al., 2011; Hall et al., 2012). During sum- tain clean and homogenous colony of Las-infected and uninfected mer, D. citri adults disperse, and females lay eggs on suitable citrus psyllids colonies for the experiments, the apparently clean above trees (Capoor & Viswanath, 1967; Lewis-Rosenblum, Martini, Tiwari, stock populations were transferred onto Orange jasmine plants & Stelinski, 2015; Tsai & Liu, 2000). Diaphorina citri is under good grown from seeds in an insect-proof greenhouse at 28°C, 40% RH biocontrol program in warmer locations on citrus trees (Dahlsten and L16: D8 for 4 months in mesh cages (50 × 50 × 50 cm). The et al., 1998). Entomopathogenic fungi then applied to these citrus 4-month-old seedlings were infected by grafting of Orange jas- trees, where they infect D. citri nymphs and adults (Avery et al., mine with four pieces of bud wood sticks from a PCR-positive HLB 2013). source. The infection was determined and confirmed using PCR as However, the susceptibility of insects and mites to pesticides described by Tatineni et al. (2008). Infected and uninfected orange has changed due to some abiotic factors such as temperature, rain- jasmine were used in this rearing of the psyllids, which were also fall, and humidity, as well as nonenvironmental factors including tested by real-time PCR for the presence or absence of Las infec- pesticide coverage, host plants, and host infection status (Grafton- tion before the bioassays (Lin et al., 2016). Before each experiment, Cardwell et al., 2013; Musser & Shelton, 2005; Tiwari, Mann, Rogers, the purity of cultures HLB-free or HLB-infected established colonies & Stelinski, 2011; Xie et al., 2011; Yang, Margolies, Zhu, & Buschman, was further checked regularly by the random amplified polymorphic 2001). Insecticides toxicity depends on the target pest and appli- DNA polymerase chain reaction (RAPD-PCR) technique and further cation method at given temperature (Musser & Shelton, 2005). In tested with mtCOI sequencing as described by Tatineni et al. (2008) tropical and sub-tropical areas where D. citri are present, the vari- for infection (established colony). ation in toxicity of insecticides is caused by different temperature In this study, five entomopathogenic fungal isolates from variations ranging between 5°C and 40°C (Boina, Onagbola, Salyani, two genera; Isaria fumosorosea isolates 3A Ifr, 5F Ifr, PS Ifr, and & Stelinski, 2009). In some areas of the world, like Florida D. citri are Hirsutella citriformis isolates HC3D and 2H were used against D. citri. occasionally exposed to cold stress below −6.5°C and −8°C; and it The fungal isolates were cultured on potato dextrose agar (PDA) was reported that D. citri adults and nymphs tolerate these cold tem- plates and were maintained at 25 ± 2°C in complete darkness. For peratures (Hall, Wenninger, & Hentz, 2011; Hussain, Lin, & Wang, bioassay, entomopathogenic fungi conidia were scraped off from 2017; Hussain, Akutse, et al., 2017). Thus, lethal concentration (LC50) 2-week-old plates with a sterilized spatula and suspended in 20 ml of values of chemical insecticides for D. citri, based on the tempera- autoclaved deionized water containing 0.03% Tween 80. The conid- ture regimes have been previously investigated (Boina et al., 2009). ial suspensions were vortexed in for 5 min to produce homogenous However, this lethal effect as regards temperatures has not been conidial suspensions and then filtered through Miracloth. Fungal studied with entomopathogenic fungi. Additionally, the effect of un- conidia were counted using a Neubauer Hemacytometer, and con- favorable temperature stress on entomopathogenic fungi pathoge- centrations were determined through dilution. The required conidial nicity has not been investigated for D. citri. suspension with a standard concentration of 1 × 108 conidia/ml was | HUSSAIN et Al. obtained for the five entomopathogenic fungal isolates by serial dilutions containing 0.03% Tween 80 (Fluka) as a wetting agent. 3 of 9 The enzymes were prepared by following previously described methods (Gao & Zhu, 2000; Smith et al., 1985; Zhu & Gao, 1999) and then recording the absorbance at 490 nm with a 96-well plate 2.1 | Diaphorina citri susceptibility to entomopathogenic fungi at cold temperature reader (ELISA plates, FEP-100- 096, JET BIOFIL, China) at 25 ± 1°C. General esterase activity was calculated using α-naphthyl acetate (α-NA) (Sigma-Aldrich, China) as a substrate (Tiwari et al., 2011). Prior to experiments, psyllids were carefully collected from the above GST activity was calculated using 1-chloro-2,4-dinitrobenzene tested pure (HLB-free) and HLB-infected colonies of D. citri and (CDNB) (Sigma-Aldrich) (Habig, Pabst, & Jakoby, 1974; Tiwari, Pelz- transferred onto clean citrus plants in mesh cages (50 × 50 × 50 cm). Stelinski, Mann, & Stelinski, 2011) as substrate. Cytochrome P450 The mesh cages containing D. citri were shifted to climate box set activity was measured by calculating heme peroxidase activity at 5°C, 55% RH, and at a photoperiod of 14:10 hr L:D or were main- (Tiwari et al., 2011; William & Janet, 1997). As heme consists of cy- tained at 27°C (control) for 1 or 2 weeks before the experiment. tochrome P450 in nonblood feeding arthropods, the quantification Psyllids from 5°C temperature were assessed using a leaf-dip Petri of heme activity can be used in comparing the levels of cytochrome dish method described by Kumar, Poehling, & Borgemeister (2005)) P450 (Casimiro, Coleman, Hemingway, & Sharp, 2006; Penilla et al., and Tiwari et al. (2011). Plastic disposable Petri dishes consisted 2007; William & Janet, 1997). Heme peroxidase activity was cal- of 60-mm diameter and 1.5% agar solution (2 mm solidified layer) culated by using 3,3,5,5-tetramethylbenzidine (TMBZ) substrate is used for bioassay. Leaf disks from fresh citrus leaves were cut (Sigma-Aldrich). (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 2.3 | Susceptibility of Las-infected and Lasuninfected Diaphorina citri to entomopathogenic fungi under different temperatures layers after 1 hr, and 20–30 adult psyllids of mixed gender were Entomopathogenic fungi bioassays were conducted using a leaf- shifted to the dish using a soft camel hair brush. Psyllids were an- dip Petri dish method as described above. Petri dishes containing esthetized shortly (10 sec) with cold temperature for easy handling the sprayed leaves at 1 × 108 conidia/ml of conidial suspensions of and transfer. Petri dishes were sealed with parafilm (Laboratory film, each fungal isolate and adult psyllids were wrapped with parafilm PM-996, USA) to block the psyllids. Sealed Petri dishes with adult and transferred into temperature-controlled growth chambers set insects were transferred into a growth chamber (Safe, China) set at different temperatures: 5, 10, 20, 27, 35, or 40°C with 55 ± 5% at 26 ± 1°C, 55% RH, and at a photoperiod of 14:10 hr L:D. Psyllids RH and 14:10 hr L:D photoperiod. For all entomopathogenic fungal mortality was recorded 48 hr after placing the Petri dishes into the isolates, treatments (Las-infected and uninfected D. citri) were rep- growth chamber. Psyllids were considered dead when seemed their licated three times, where each replicate comprised of n = 80–128 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. TA B L E 1 Mean mortality (%) of Diaphorina citri adults by entomopathogenic fungi after preexposure periods of 1 or 2 weeks at cold stress (5°C) or control temperature (27°C) Entomopathogenic fungus (5°C) (27°C) p value 1-week exposure period 2.2 | 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). 3A Ifr 70.0 ± 0.5 80.4 ± 4.6 .146 5F Ifr 56.5 ± 7.0 84.7 ± 2.4 .0001* PS Ifr 93.1 ± 0.1 87.2 ± 1.0 .226 HC3D 78.1 ± 1.1 83.5 ± 0.4 .249 2H 81.6 ± 3.5 87.8 ± 0.4 .322 2-week exposure period Each entomopathogenic fungal isolate was repeated three times at each temperature, and for each replication 80–100 adult psyllids 3A Ifr 88.8 ± 2.1 80.1 ± 5.0 .144 were tested. Psyllids of mixed gender were transferred onto leaves 5F Ifr 83.0 ± 3.4 92.1 ± 1.0 .121 sprayed with entomopathogenic fungal isolate suspensions at the PS Ifr 96.4 ± 1.5 91.1 ± 1.1 .824 concentration of 1 × 10 8 conidia/ml, using the Petri dish method as HC3D 76.6 ± 3.0 81.3 ± 4.4 .201 described above. About 80–100 adult psyllids were shifted to each 2H 71.0 ± 4.2 89.1 ± 1.2 .024* Petri dish. Psyllids which survived from each treatment after 48 hr exposure were collected and used immediately for detoxifying enzymes expression levels assays. *Represent significant differences in mortality between entomopathogenic fungi and temperature interactions at different exposure period, at p < .05. 4 of 9 | HUSSAIN et Al. F I G U R E 1 Cytochrome P450 (a), general esterase (b) and glutathione S-transferase (c) activity levels comparison in Las-uninfected Diaphorina citri at different temperatures regimes. Bars represent means with standard errors (SE), and means followed by different letters indicate a significant difference at p < 0.05 psyllids. Each entomopathogenic fungal treatment and the control two bioassays conducted with psyllids were collected for subse- were replicated three times, and the whole bioassay was repeated quent analyses. twice. Psyllid mortality was calculated 48 hr after shifting to 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. 2.4 | Statistical analysis The mean mortality (%) among psyllids exposed to various en- For bioassays using Las-infected psyllids, each live or dead tomopathogenic fungi was analyzed using Analysis of Variance D. citri was transferred into a sterile 2 ml microcentrifuge tube (ANOVA) test, and correlation analyses were conducted at p < .05, (Promega, China) consisting of 80% ethanol, and kept at −20°C for between Las-infected and Las-uninfected psyllid treatments, en- further analysis. After mortality data were calculated, DNA was tomopathogenic fungi, and temperature regimes. Las-infected and Las- again extracted from the exposed Las-infected psyllids, just for re- uninfected mean mortalities for the various entomopathogenic fungi confirming Las infection by using quantitative real-time PCR follow- and at the different temperatures were compared using Chi-square. ing the previously described protocol (Tiwari, Lewis-Rosenblum, Mortality percentages in all treatment were corrected using Abbott’s Pelz-Stelinski, & Stelinski, 2010). Mortality data obtained from the formula (Abbott, 1925). To calculate the effects of temperature and | HUSSAIN et Al. entomopathogenic fungi on mean mortality (%) of psyllids, ANOVA 5 of 9 entomopathogenic fungus had a significant effect on the mean mor- tests and Fisher’s LSD mean separation tests were performed. The tality (%) of psyllids (Table 2). In Las-infected psyllids, entomopatho- effect of temperature on detoxifying enzyme levels was measured in- genic fungi (F4,50 = 63.40, p = .0034) and temperature (F5,50 = 13.10, dividually for each entomopathogenic fungi by ANOVA test (p < .05), p = .0011) had each a significant effect on D. citri mortality; however, whereas the correlation analyses between detoxifying enzymes ex- the relationship effects between these factors was nonsignificant pression levels and temperature were determined separately for each (F20,50 = 0.70, p = .5102). Data analysis showed that the tempera- entomopathogenic fungi and enzyme combination. SPSS 19.0 statis- ture regimes had significant effects on the mortality of psyllids for tics software was used to perform all the data analysis. 3A Ifr (F5,117 = 12.41, p = .0021), 5F Ifr (F5,117 = 49.41 p = .0012), PS Ifr (F5, 117 = 3.22, p = .013), HC3D (F5,117 = 4.24, p = .0133) and 2H 3 | R E S U LT S 3.1 | 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 entomopath- (F5,117 = 3.10, p = .001) (Table 3). Likewise, a significantly positive correlation was observed between temperature and psyllids mortality for 5F Ifr (r = 0.8131, p = .0001), PS Ifr (r = 0.6101, p = .0001), HC3D (r = 0.8224, p = .0023), and 2H (r = 0.6501, p = .0024). In contrast, there was a significantly negative correlation between temperature and psyllids mortality for 3A Ifr (r = −0.6002, p = .0001). For the Las-uninfected psyllids, entomopathogenic fungi ogenic fungi showed that when D. citri were exposed to cold stress (F5,117 = 15.23, p = .0001) and temperature (F4,117 = 2.52, (5°C) for 1 week, they were significantly less susceptible to 5F Ifr than p = .0063) and the interaction between these factors had signif- control psyllids at 27°C (Table 1). Similarly, psyllids that were exposed icant effects on D. citri mortality (F20,117 = 7.13, p = .0001). For to cold stress (5°C) for 2 weeks were less susceptible to 2H than psyl- all entomopathogenic fungi tested, temperature had a significant lids maintained at 27°C. However, when mortalities were compared effect on psyllids mortality for 3A Ifr (F5,17 = 8.52, p = .0002), 5F between psyllids exposed to cold temperature (5°C) and the control Ifr (F5,17 = 9.17, p = .0002), HC3D (F4,17 = 5.51, p = .0002) and 2H (27°C), no significant differences were observed for the other tested (F5,17 = 9.12, p = .0001). In contrast, temperature had no signifi- entomopathogenic fungi (3A Ifr, PS Ifr, and HC3D; Table 1). cant effect for PS Ifr (F5,17 = 1.42, p = .1010) (Table 3). In addition, a significantly positive correlation was observed between tempera- 3.2 | Effect of temperature on different enzymes levels Psyllids treated with 5F Ifr (F5,10 = 1.0; p = .1451) or HC3D ture and psyllids morality for, 5F Ifr (Pearson correlation coefficient; r = 0.6010, p = .0001), PS Ifr (r = 0.3154, p = .0121), HC3D (r = 0.7112, p = .0001), and 2H (r = 0.8031, p = .0001). It was also observed that a significantly negative correlation exists be- (F5,10 = 0.22; p = .4236) had nonsignificant effect on cytochrome tween temperature and psyllids mortality for 3A Ifr (r = −0.9105, P450 activity at all the temperature regimes (Figure 1a). Similarly, p = .0001). correlation analysis showed nonsignificant association between The comparison of the correlation coefficients for Las-infected temperature and cytochrome P450 activity for psyllids treated and Las-uninfected psyllids showed no significant differences with 5F Ifr (r = −0.2520, p = .1340) and HC3D (r = 0.0100, among Las-infected and uninfected psyllids for 3A Ifr (z = −0.40, p = .6226). General esterase activity levels of psyllids treated with p = .5023), 5F Ifr (z = −2.00, p = .202), PS Ifr (z = 0.13, p = .7001), 5F Ifr (F5,10 = 0.43; p = .4202) or HC3D (F5,10 = 1.12; p = .2073) had positive effect of temperature (Figure 1b). Temperature and general esterase activity in psyllids treated with 5F Ifr (r = 0.0623, p = .5226) or HC3D (r = 0.1356, p = .2207) had nonsignificant relationship. However, when comparing the cytochrome P450 and TA B L E 2 Interaction effects of temperature and Las infection on the susceptibility of Diaphorina citri to entomopathogenic fungi general esterase activities (Figures 1a, b), temperature had sig- Factors df, residuals nificant effect on the GST activity levels for psyllids treated with Las infection 1, 214 19.60 .0035* 5F Ifr (F5,10 = 2.11; p = .0213) or HC3D (F5,10 = 8.11; p = .0034) Temperature 5, 214 13.17 .0045* (Figure 1c). A negative correlation was observed between temper- Entomopathogenic fungi 4, 214 10.27 .0020* Las infection × temperature 5, 214 1.15 .144 Las infection × entomopathogenic fungus 4, 214 5.63 .0023* Temperature × entomopathogenic fungus 20, 214 15.81 .0049* Las infection × temperature × entomopathogenic fungus 20, 214 0.32 .6115 ature and GST activity for psyllids treated with 5F Ifr (r = −0.6020, p = .0024) or HC3D (r = −0.5345, p = .0206). 3.3 | Susceptibility of Las-infected and Lasuninfected Diaphorina citri to entomopathogenic fungi under different temperatures Our results showed that the interactions between psyllids treatments (Las-infected and Las-uninfected psyllids), temperature and F value *Represent significant factors and interactions at p < .05. p value 6 of 9 | HUSSAIN et Al. Treatment Mean mortality % (± SE) Entomopathogenic fungus Temperature (°C) 3A Ifr 5 93.0 ± 2.1aA 85.3 ± 1.6aA 10 85.1 ± 3.4aA 73.0 ± 1.2abA 20 79.0 ± 2.2aA 65.0 ± 1.1abB 27 85.2 ± 4.7aA 66.0 ± 1.0bcB 35 55.4 ± 4.7bA 62.8 ± 4.0cdA 5F Ifr HC3D 2H Las-uninfected 40 59.3 ± 1.0bA 54.7 ± 4.2dA 5 73.5 ± 0.3cA 50.3 ± 0.4cC 10 74.0 ± 1.2cA 56.0 ± 8.3bcC 20 63.0 ± 1.3cA 49.0 ± 6.1bcC 27 84.6 ± 1.5bA 65.1 ± 6.0bcC 35 90.0 ± 0.01aA 72.0 ± 2.0abB 40 PS Ifr Las-infected 5 100.0 ± 0.0aA 87.4 ± 1.5aA 72.5 ± 2.4bA 58.0 ± 1.1bC 10 74.6 ± 8.4bA 59.1 ± 2.1bC 20 68.5 ± 6.2bA 50.0 ± 2.0bB 27 88.0 ± 2.0aA 65.0 ± 2.8bC 35 91.2 ± 1.5aA 66.8 ± 4.1bC 40 97.0 ± 0.5aA 88.8 ± 3.0aA 5 59.8 ± 1.4cB 65.7 ± 0.6cA 10 69.3 ± 4.7bcA 70.0 ± 3.0bcA 20 62.2 ± 3.5bcA 60.0 ± 2.1bcA 27 78.7 ± 3.0bcA 79.3 ± 1.0bA 35 83.5 ± 7.0bA 79.0 ± 1.7bA 40 99.0 ± 0.2aA 91.5 ± 1.2aA 5 65.4 ± 7.3bA 52.0 ± 6.0cB 10 68.0 ± 3.6bA 64.1 ± 2.5cA 20 61.0 ± 2.4bA 58.0 ± 2.2cA 27 82.8 ± 8.1abA 70.0 ± 4.1bcA 35 93.0 ± 1.2aA 85.6 ± 3.6abA 40 97.1 ± 1.2aA 91.1 ± 1.6aA TA B L E 3 Effect of temperature on pathogenicity of various entomopathogenic fungal isolates to Diaphorina citri Means with the same lower case letters within a column of each fungal isolate at the different temperatures are not significantly different at p < .05. And means with the same upper case letters within rows are not significantly different. HC3D (z = 0.23, p = .625) and 2H (z = 0.61, p = .220) at the various entomopathogenic fungi. Some studies reported that decrease in temperature regimes (Table 3). susceptibility might influence the management of psyllids under field conditions using I. fumosorosea and H. citriformis (Lezama-Gutiérrez 4 | D I S CU S S I O N et al., 2012; Pérez-González, Sandoval-Coronado, & MaldonadoBlanco, 2016). Temperature affects the rate of metabolism (Hussain, Akutse, The results of the study showed that psyllids exposed to cold stress et al., 2017; Hussain, Lin, et al., 2017), the binding of the enzyme were more tolerant to entomopathogenic fungi than controls, seems with its substrate (Hochachka & Somero, 1984) and the rate of to be possible effects that could be observed during winter compared enzymatic activity (Hoffmann, 1984). Therefore, we assumed that to summer temperature variations. However, the cold temperature the different levels of susceptibility of psyllids to entomopatho- did not affect the susceptibility of D. citri to PS Ifr, 3A Ifr, and HC3D. genic fungi due to temperature changes observed in this study Further studies are warranted to understand the mechanism under- might comprise altered levels of different enzyme activities. The lying decreased susceptibility of cold stress psyllids populations to data analysis showed that temperature affected the levels of GST, | HUSSAIN et Al. 7 of 9 but not of cytochrome P450 and general esterase activity. A sig- I. fumosorosea and H. citriformis than control. Thus, seasonal varia- nificant reduction in levels of GST was observed at 40°C in psyllids tions in temperature and entomopathogenic fungi may have a major treated with 5F Ifr and HC3D. The decline in GST activity does not impact on the management of D. citri under field conditions. induce a decrease in mortality of the psyllids treated with 3A Ifr at 40°C. However, GST reduction would be expected to increase psyllids susceptibility to entomopathogenic fungi at high tempera- AC K N OW L E D G M E N T S tures rather than decreasing susceptibility. Our results showed All authors are thankful to Ru Xinhui and Bao Lu for their kind as- that, in response to temperature changes, susceptibility of psyllids to entomopathogenic fungi was not correlated with detoxifying enzyme activity. Therefore, the temperature-related temperature changes, in the psyllid susceptibility to entomopathogenic fungi may be due to other factors. However, many studies have investigated the effect of temperature on detoxifying enzymes that are sistance in culturing of entomopathogenic fungi. This project was funded by the Key Projects of Science and Technology of Fujian Province (2016N0005), the Research Fund for the International Collaborative Program (KXGH17004) with (CXZX2017211) from Fujian Agriculture and Forestry University (FAFU). due to the effect of synthetic insecticides (Chandler, King, Jewess, & Reynolds, 1991; Hodjati & Curtis, 1999; Wadleigh, Koehler, Preisler, Patterson, & Robertson, 1991). Our results also underlined the susceptibility correlation ef- C O N FL I C T O F I N T E R E S T None declared. fects of Las-infected and uninfected psyllids to entomopathogenic fungi as regards to the changes in temperatures, which was poorly understood previously. Boina et al. (2009) reported pos- ORCID itive correlation between temperature and synthetic insecticide Mubasher Hussain susceptibility for Las-uninfected D. citri. As the detection of Las Liande Wang http://orcid.org/0000-0003-0833-7399 http://orcid.org/0000-0002-0506-3655 infection has increased in Asia and some other parts of the world (Bove & Ayres, 2007; Chen et al., 2010; Islam et al., 2012; Morris, Erick, & Estes, 2009; Puttamuk et al., 2014; 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. 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