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Article The Survival and Rate of radiata (: ) on Its Host Exposed to bassiana (: )

Luis Carlos Ramos Aguila 1,2 , Komivi Senyo Akutse 3,* , Hafiza Javaria Ashraf 1,2, Bamisope Steve Bamisile 4 , Jingyi Lin 1,2, Jiawang Dai 1,2, Huiting Wang 1,2 and Liande Wang 1,2,*

1 State Key Laboratory of Ecological Pest Control for Fujian and Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, ; [email protected] (L.C.R.A.); [email protected] (H.J.A.); [email protected] (J.L.); [email protected] (J.D.); [email protected] (H.W.) 2 Institute of Applied Ecology and Research Centre for Biodiversity and Eco-Safety, Fujian Agriculture and Forestry University, Fuzhou 350002, China 3 International Centre of Physiology and Ecology (icipe), P.O. Box 30772, Nairobi 00100, Kenya 4 Department of Entomology, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China; [email protected] * Correspondence: [email protected] (K.S.A.); [email protected] (L.W.); Tel.: +254-799-716-741(K.S.A.);  +86-133-4829-1086 (L.W.); Fax: +254-20-863-2001 (ext. 2002) (K.S.A.); +86-591-8385-3459 (L.W.) 

Citation: Ramos Aguila, L.C.; Abstract: The effect of the entomopathogenic (BB-12) on the biological Akutse, K.S.; Ashraf, H.J.; Bamisile, characteristics of parasitizing was studied under laboratory con- B.S.; Lin, J.; Dai, J.; Wang, H.; Wang, L. ditions. Twenty 3rd–5th nymphs were exposed to a single already-mated female The Survival and Parasitism Rate of (1 day old) and removed after 24 h. Subsequently, the nymphs were sprayed at 1, 24 and 48 h Tamarixia radiata (Hymenoptera: post-exposure with 1 × 108 conidial mL−1 suspension. The percentage of parasitism recorded was Eulophidae) on Its Host Exposed to 22%, 35% and 41% at 1, 24 and 48 h, respectively. The emergence rate varied between 28%, 51% and Beauveria bassiana (Ascomycota: 49% at 1, 24 and 48 h, respectively. In a subsequent experiment, nymphs of D. citri were sprayed with Hypocreales). Agronomy 2021, 11, 1 × 108 conidial mL−1 suspension and then exposed to T. radiata at 1, 24 and 48 h post-spraying to 1496. https://doi.org/10.3390/ allow for parasitism to occur. The percentage of parasitism recorded was 18%, 27% and 28% at 1, agronomy11081496 24 and 48 h, respectively, significantly lower than the parasitism rate recorded in the control (48%).

Academic Editors: Wayne B. Hunter, The percentage of emergence varied between 24%, 44% and 45% at 1, 24 and 48 h, respectively. In Liliana M. Cano and Jawwad A. both experiments, no significant difference was observed in the pre-adult duration (days) and the Qureshi 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 Received: 2 July 2021 suspension and the control. Overall, the findings of the current study revealed a negative interaction Accepted: 27 July 2021 between T. radiata and B. bassiana in controlling D. citri nymphs. This outcome is believed to be a Published: 28 July 2021 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 Publisher’s Note: MDPI stays neutral parasitism to occur over an extended period of time before the application of the fungus), T. radiata with regard to jurisdictional claims in could potentially be used in combination with B. bassiana for the successful biological control of published maps and institutional affil- D. citri. This should be carried out in order to minimize the potentially negative interactions between iations. these two biological agents.

Keywords: ; Diaphorina citri; biological control; parasitism; survival rate

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Diaphorina citri Kuwayama (: Liviidae) is the primary vector of the Attribution (CC BY) license (https:// greening disease, otherwise known as Huanglongbing (HLB) [1–3]. The disease of HLB creativecommons.org/licenses/by/ has severely affected the citrus industry worldwide [4]. Chlorpyrifos, imidacloprid, and 4.0/). bifenthrin are the synthetic most frequently used to protect the citrus plantation

Agronomy 2021, 11, 1496. https://doi.org/10.3390/agronomy11081496 https://www.mdpi.com/journal/agronomy Agronomy 2021, 11, 1496 2 of 11

from D. citri [5–7]. Their overuse, however, has led to developed resurgence problems, residues and resistance in D. citri populations [8,9]. The current food market preferences increasingly require farmers growing horticulture to use pesticide-free products and to reduce the use of conventional broad spectrum chemical [10]. Consequently, a suitable alternative to chemical insecticides is the use of biological control agents, mainly entomopathogenic fungi [11–14] and the primary-associated ectoparasitoid Tamarixia radiata Waterston (Hymenoptera: Eulophidae) [15]. In the past 15 years, the development and use of entomopathogens as classical, con- servational and augmentative biological control agents have included a number of suc- cesses and some setbacks [10]. Several studies suggested the use of entomopathogenic fungi against D. citri, including fumosorosea [14,16,17]; anisopliae and bassiana [18]; and citriformis [19,20]. The ectoparasitoid T. radiata is a well-known and principal natural enemy used for the biological control of the major citrus pest D. citri [21–23]. T. radiata has been extensively used in many countries as a classical biological control agent due to its high parasitism capacity and field adaptation [24–27]. Other control agents include predators such as lady beetles, lacewings and spiders [28]. In integrated pest management control, including the use of in combi- nation with entomopathogenic fungi may improve pest management efficacy. However, interspecific competition between two or more species of natural enemies, interacting in the same host, could result in complex multitrophic interactions and outcomes which can be synergistic, addictive or antagonistic with respect to host mortality [29–31]. A pathogen with a broader host range in favorable environmental conditions can easily infect the natural enemies [30] but, if the pathogen has a specific host range, it could interact with the parasitoid and enhance the efficacy of controlling the host pests [32,33]. Therefore, the strategies that incorporate fungi in combination with predators and para- sitoids need to be defined/explored to ensure compatibility and maximize efficacy. The present study provides information to better understand the potential interaction of the entomopathogenic fungus Beauveria bassiana on the survival and parasitism rate of the ectoparasitoid T. radiata, by controlling D. citri under controlled conditions.

2. Materials and Methods 2.1. Culture Diaphorina citri and Tamarixia radiata were obtained from the laboratory of Insect Ecology and the Biological Control of Fujian Agricultural and Forestry University (FAFU), Fuzhou, China. Both populations were maintained for about ten generations, prior to the commencement of the experiments on jasmine (Murraya paniculata) (Sapindales: Rutaceae), in mesh cages (0.60 m wide, 0.50 m deep, 0.50 m high) grown in a climate room at 25 ± 2 ◦C, 75 ± 5% relative humidity and under 14 h of photoperiod (from 6:00–20:00).

2.2. Fungal Strain and Conidia Suspension Preparation Beauveria bassiana strain 12 (BB-12) was obtained from the fungal culture bank of the Insect Ecology and Biological Control laboratory of Fujian Agriculture and Forestry University, Fuzhou, China, based on the source and the findings of preliminary stud- ies conducted in the laboratory [34–36]. The strain was identified using morphological description through the keys of Humber [37,38]. To confirm the identity of the isolate, molecular identification was carried out in addition to the morphological structures us- ing the sequence data from the ITS region of nuclear rRNA (GenBank accession number MG844429). Fungal strain BB-12 was sub-cultured on Potato Dextrose Agar (PDA) culture media in 90 mm × 15 mm Petri dishes at a constant temperature of 25 ± 2 ◦C, 70–75% relative humidity (RH) in complete darkness for 18–20 days to obtain conidia. Under sterile conditions, conidia were harvested by scraping the fungal mycelia using a sterile spatula and then suspending in sterile distilled water (SDW) containing the surfactant Tween 80 (0.01%). Later on, conidia were removed from the hyphal and homogenized by vortexing the mixture for 4 min, then filtered using a sterile syringe and cotton wool to remove Agronomy 2021, 11, 1496 3 of 11

hyphal debris. Finally, conidial concentration was adjusted to 1 × 108 conidia mL−1 under the microscope using a Neubauer hemocytometer. Conidia viability was determined by plating 100 µL of the first serial dilution on 2.5% water agar and then incubated at 25 ◦C for 24 h. The viability was assessed by counting the number of germinated conidia. A was considered to be viable when it developed and projected a germ tube that was longer than half of its diameter.

2.3. Diaphorina Citri First Parasitized by Tamarixia radiata and Afterward Treated with Beauveria bassiana Suspension The study investigated the effects of the entomopathogenic fungus B. bassiana on the survival and developmental rate of T. radiata feed on D. citri nymphs. The parasitized nymphs were treated by spraying 1 × 108 conidial ml−1 suspension of B. bassiana at 1, 24, and 48 h after exposure to the parasitoids. For the control, sterile distilled water containing the surfactant Tween 80 (0.01%) was sprayed. For each treatment, 10 rearing bottles made of plastic (20 cm in height and 8 cm in diameter) were used. To permit air circulation, perforated openings were made, and then covered with a fine mesh. For each bottle, M. paniculata shoots, containing 20 3rd–5th instar nymphs of D. citri each, were used. Shoots were 15 cm in height and placed in small 25 mL Polypropylene Deli Containers containing cotton and socked with water to keep the shoots fresh. Afterward, one female of T. radiata (1 day old), previously mated, was released in the individual container to allow parasitism for 24 h, after which the parasitoid was removed. Subsequently, potentially parasitized nymphs were sprayed with the suspension at 1, 24, and 48 h after parasitism. Thereafter, containers were kept in climate chambers at 25 ± 2 ◦C, 70 ± 5% RH, and 14 h:10 h dark photoperiod to enable an evaluation of the emergence of adult parasitoids. Each treatment was replicated 10 times, with each replicate consisting of 20 D. citri nymphs per each M. paniculata shoot. In total, 200 nymphs were examined per treatment. The developmental parameters estimated were: percent of parasitism, preadult duration, percent of emergence, sex ratio, survival and longevity. The percentage of parasitism was evaluated on the 6th day based on the mummies formation. The sex ratio was determined by counting the total female individuals and dividing this by the total offspring for each replicate. For the longevity of T. radiata, emerged females and males per treatment were each individually kept in 50 mL Polypropylene Deli Containers with the openings covered with a fine mesh for air circulation. Honey was provided as food and placed in a climate chamber at 25 ± 2 ◦C, 70 ± 5% RH, for 14 h of photoperiod, and the longevity was monitored until the death of all individuals.

2.4. Diaphorina Citri First Sprayed with Beauveria bassiana and Afterward Exposed to Tamarixia radiata For this experiment, D. citri nymphs were treated first with the suspension of B. bassiana by spraying 1 × 108 conidial mL−1 and at 1, 24, and 48 h before spraying, T. radiata was released to allow parasitism for 24 h. For this study, the methodology previously described above was used, where 20 D. citri nymphs were used per one female of T. radiata, with 10 replicates per treatment and sterile distilled water was used as the control. Similar developmental parameters, as described above, were evaluated. The study was set up using a complete randomized design (CRD); consisting of three treatment groups (1, 24 and 48 h) and 10 replicates per treatment.

2.5. Statistical Analysis Raw data obtained were subjected to a test of the normality and homoscedasticity of the variance before the statistical analysis. To test normality, the Anderson Darling test was used (p > 0.05); where raw data was abnormal (p < 0.05), the Bliss transformation was used. To test homoscedasticity, the Bartlet test was used when the data were normal, while the Levene test was used when the data were not normal. For all the analyses, the statistical software, Minitab 16, was used. Survival statistics were performed using Kaplan-Meier- Agronomy 2021, 11, x FOR PEER REVIEW 4 of 11

was used (p > 0.05); where raw data was abnormal (p < 0.05), the Bliss transformation was used. To test homoscedasticity, the Bartlet test was used when the data were normal, while the Levene test was used when the data were not normal. For all the analyses, the statis- Agronomy 2021, 11, 1496 4 of 11 tical software, Minitab 16, was used. Survival statistics were performed using Kaplan- Meier-analysis and subsequent Log-Rank-test for the F1-progeny. Data were analyzed us- ing analysis of variances (ANOVA, α = 0.05), followed with pairwise comparison of means postanalysis hoc with and subsequentTukey (p < 0.05) Log-Rank-test. These statistical for the analyses F1-progeny. were performed Data were using analyzed SPSS using 19.0 (SPSSanalysis Inc., of Chicago, variances IL (ANOVA,, USA). α = 0.05), followed with pairwise comparison of means post hoc with Tukey (p < 0.05). These statistical analyses were performed using SPSS 19.0 3.(SPSS Results Inc., Chicago, IL, USA). 3.1. Diaphorina Citri Parasitized by Tamarixia radiata and Subsequently Treated with Beauveria 3. Results bassiana (BB-12) 3.1. Diaphorina Citri Parasitized by Tamarixia radiata and Subsequently Treated with Beauveria bassianaThe (BB-12)parasitism rate showed significant differences between the control and the treat- ment consisting of spraying the fungal suspension 1 h after parasitism (F3.36 = 38.92; p < The parasitism rate showed significant differences between the control and the treat- 0.0001), Figure 1A. The parasitism rates at 24 and 48 h were 39% and 42%, respectively. ment consisting of spraying the fungal suspension 1 h after parasitism (F3.36 = 38.92; Thep < 0.0001fungal), suspension Figure1A. The application parasitism after rates parasitism at 24 and 48had h wereno influence 39% and on 42%, the respectively. emergence rateThe of fungal T. radiata suspension F1 generation. application The afteremergence parasitism rates had at 1, no 24, influence and 48 h on were the emergence29%, 60%, and rate 55%,of T. respectively. radiata F1 generation. However, The significant emergence differences rates at were 1, 24, observed and 48 h betwee were 29%,n the 60%, control and and55%, the respectively. treatment However,consisting significantof spraying differences the fungal were suspension observed (F between3.36 = 63.18; the p control < 0.0001). and In addition, no significant differences (F3.36 = 63.18; p = 0.6722) were observed in the emer- the treatment consisting of spraying the fungal suspension (F3.36 = 63.18; p < 0.0001). In gence rate in the treatment consisting of spraying the fungal suspension 24 and 48 h after addition, no significant differences (F3.36 = 63.18; p = 0.6722) were observed in the emer- parasitismgence rate in(Figure the treatment 1B). The consistingnymphs that of spraying were not the parasitized fungal suspension during the 24 and24 h 48 that h after the parasitizationparasitism (Figure was 1allowed,B). The nymphswere infected that were by B. not bassiana parasitized; some duringof them the die 24d because h that the of fungalparasitization infection was while allowed, others wereemerge infectedd as adults by B. and bassiana died ;a somefew days of them later. died because of fungal infection while others emerged as adults and died a few days later.

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0 0 l l h h h o h h h o 1 4 8 tr 1 4 8 tr 2 4 n 2 4 n o o C C Figure 1. Percentage of parasitism (A) and emergence (B) of Tamarixia radiata from nymphs where Diaphorina citri nymphs Figurewere first 1. Percentage parasitized of by parasitismTamarixia ( radiataA) andand emergence subsequently (B) of Tamarixia treated with radiata Beauveria from nymphs bassiana where (BB-12) Diaphorina at 1, 24,and citri48 nymphs h after wereparasitism. first para Meanssitized followed by Tamarixia by the radiata same letterand subsequently are not significantly treated with different Beauveria (Tukey, bassianap < 0.05). (BB-12) at 1, 24, and 48 h after parasitism. Means followed by the same letter are not significantly different (Tukey, p < 0.05). There was no statistical difference in the pre-adult duration of the parasitoid (days) amongThere the was treatments no statistical and thedifference control in (p the> 0.05). pre-adult The duration sex ratio of of the the parasitoid F1 generation (days) of amongT. radiata thewas treatments statistically and similar the control between (p > treatment 0.05). The and sex controlratio of ( pthe> 0.05)F1 generation (Table1). of T. radiata was statistically similar between treatment and control (p > 0.05) (Table 1). Table 1. Means and SE of the pre-adult duration, sex ratio and longevity of Tamarixia radiata when Diaphoria citri nymphs

were first parasitized by Tamarixia radiata and subsequently treated with Beauveria bassiana (BB-12).

Pre-Adult Duration Treatment n◦ Nymphs Exposed Sex Ratio Female Male 1 h 20 10.35 ± 0.11a 9.15 ± 0.10a 0.48 ± 0.06a 24 h 20 10.85 ± 0.25a 9.45 ± 0.15a 0.56 ± 0.04a 48 h 20 10.90 ± 0.10a 9.60 ± 0.16a 0.53 ± 0.04a Control 20 10.20 ± 0.16a 9.40 ± 0.12a 0.58 ± 0.01a Means in the same column with the same letter are not significantly different (Tukey, p < 0.05). Agronomy 2021, 11, x FOR PEER REVIEW 5 of 11

Table 1. Means and SE of the pre-adult duration, sex ratio and longevity of Tamarixia radiata when Diaphoria citri nymphs were first parasitized by Tamarixia radiata and subsequently treated with Beauveria bassiana (BB-12).

Pre-Adult Duration Treatment n° Nymphs Exposed Sex Ratio Female Male 1 h 20 10.35 ± 0.11a 9.15 ± 0.10a 0.48 ± 0.06a 24 h 20 10.85 ± 0.25a 9.45 ± 0.15a 0.56 ± 0.04a 48 h 20 10.90 ± 0.10a 9.60 ± 0.16a 0.53 ± 0.04a Control 20 10.20 ± 0.16a 9.40 ± 0.12a 0.58 ± 0.01a Agronomy 2021, 11, 1496Means in the same column with the same letter are not significantly different (Tukey, p ˂ 0.05). 5 of 11

The values of the survival rate of T. radiata females and males are plotted in Figure 2. Results showed that when applying B. bassiana after parasitism, the median survival time of T.The radiata values females of the was survival significantly rate of T. reduced radiata females (p < 0.0001), and malesfrom 31 are days plotted in the in Figurecontrol2 .to Results14, 20 and showed 27 days that at when 1, 24 applyingand 48 h,B. respectively bassiana after (Figure parasitism, 2A). The the median median survival survival time time of T. radiata p ofT. radiata malesfemales was was also significantly significantly reduced reduced ( (p< < 0.0001), 0.0001), from from 31 19 days days in in the the control control to to 14, 20 and 27 days at 1, 24 and 48 h, respectively (Figure2A). The median survival time of 11 and 14 days at 1 and 24 h, respectively. Meanwhile there were no significant differences T. radiata males was also significantly reduced (p < 0.0001), from 19 days in the control to 11 (p = 0.3208) between the male median survival in the control and at 48 h of parasitism, and 14 days at 1 and 24 h, respectively. Meanwhile there were no significant differences with the median survival time of 19 days seen in both treatments (Figure 2B). (p = 0.3208) between the male median survival in the control and at 48 h of parasitism, with the median survival time of 19 days seen in both treatments (Figure2B). A B

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Figure 2. Survival rate for females (A) and males (B). Survival curves of Tamarixia radiata by log-rank analysis were generatedFigure 2. where SurvivalDiaphorina rate for citrifemalesnymphs (A) and were males firstparasitized (B). Survival by curvesTamarixia of Tamarixia radiata and radiata subsequently by log-rank treated analysis with Beauveriawere gen- bassianaerated (BB-12)where Diaphorina (Females n citri= 23, nymphs 32, 34 and were 70 atfirst 1 h, paras 24 h,itized 48 h andby Tamarixia control, respectively) radiata and (Malessubsequentlyn = 26, treated 25, 30 and with 51 Beauveria at 1, 24, 48bassiana h and control, (BB-12) respectively).(Females n = 23, (** p32,< 34 0.001). and 70 at 1 h, 24 h, 48 h and control, respectively) (Males n = 26, 25, 30 and 51 at 1, 24, 48 h and control, respectively). (** p < 0.001). There were statistically significant differences between group means per sample in the longevityThere of w F1ere generation statistically of femalessignificant and differences males of T. between radiata, asgroup determined means per by one-waysample in ANOVAthe longevity (F3.13 = of 57.59; F1 generationp < 0.0001) of and females (F3,11 and= 55.45; malesp < of 0.0001), T. radiata respectively., as determined However, by one no - statisticallyway ANOVA significant (F3.13 = 57.59; differences p < 0.0001) were and observed (F3,11 = 55.45; in the p longevity < 0.0001), ofrespectively. females (p =Howeve 0.3502)r, andno males statistically (p = 0.9190) significant in the treatmentdifferences group were consisting observed of in spraying the longevityB. bassiana of femalessuspension (p = 480.3502) h after and parasitism males ( andp = 0.9190) the control in the group treatment (Figure group2A,B). consisting of spraying B. bassiana suspension 48 h after parasitism and the control group (Figure 2A,B). 3.2. Diaphorina Citri Treated with Beauveria bassiana (BB-12) and Parasitized by Tamarixia radiata 3.2. Diaphorina Citri Treated with Beauveria bassiana (BB-12) and Parasitized by Tamarixia radiataThe parasitism rate by T. radiata showed a significant difference between the con- trol and the treatment consisting of spraying the fungal suspension at 1, 24, and 48 h The parasitism rate by T. radiata showed a significant difference between the control before parasitism (F = 62.71; p < 0.0001) (Figure3A). However, no significant differences and the treatment 3.36consisting of spraying the fungal suspension at 1, 24, and 48 h before were observed when T. radiata was parasitized at 1, 24, and 48 h after the fungal suspen- parasitism (F3.36 = 62.71; p < 0.0001) (Figure 3A). However, no significant differences were sion application (p = 0.9526). The parasitism rates at 1, 24 and 48 h were 16%, 20% and 22%,observed respectively. when T. radiata was parasitized at 1, 24, and 48 h after the fungal suspension The parasitoid releasing time after fungal suspension application had no influence on the emergence rate of the T. radiata F1 generation. The emergence rates at 1, 24, and 48 h F were 13%, 40.18%, and 45.43%, respectively. However, significant differences ( 3.36 = 70.60; p < 0.0001) were observed between the control and the treatment where D. citri was first treated with B. bassiana and subsequently parasitized by T radiata, while no significant differences (p = 0.7096) were observed at 24 and 48 h (Figure3B). Pre-adult duration of the parasitoid (days) among the treatment groups and the control were statistically similar. There was no statistical difference between treatment and control concerning the sex ratio of the F1 generation of T. radiata.(p < 0.005) (Table2). Agronomy 2021, 11, x FOR PEER REVIEW 6 of 11

application (p = 0.9526). The parasitism rates at 1, 24 and 48 h were 16%, 20% and 22%, Agronomy 2021, 11, 1496 6 of 11 respectively.

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0 0 l l h h h o h h h o 1 4 8 tr 1 4 8 tr 2 4 n 2 4 n o o C C Figure 3. Percentage of parasitism (A) and emergence (B) of Tamarixia radiata from nymphs where Diaphorina citri first were Figuretreated 3. with PercentageBeauveia bassianaof parasitism(BB-12) (A and) and subsequently emergence exposed(B) of Tamarixia to Tamarixia radiata radiata from(parasitoids nymphs where were releasedDiaphorina at 1,citri 24, first and were48 h aftertreatedBeauberia with Beauveia bassiana bassianaapplication). (BB-12) Means and subsequently followed by the exposed same letterto Tamarixia are not significantlyradiata (parasitoids different were (Tukey, releasedp < 0.05).at 1, 24, and 48 h after Beauberia bassiana application). Means followed by the same letter are not significantly different (Tukey, pTable ˂ 0.05). 2. Means and SE of the pre-adult duration, sex ratio and longevity of Tamarixia radiata when Diaphorina citri nymphs were first treated with Beauveria bassiana (BB-12) and subsequently parasitized by Tamarixia radiata. The parasitoid releasing time after fungal suspension application had no influence on the emergence rate of the T. radiataPre-Adult F1 generation. Duration The emergence rates at 1, 24, and 48 Treatment n◦ Nymphs Exposed Sex Ratio h were 13%, 40.18%, and Female 45.43%, respectively. However, Male significant differences (F3.36 = 70.60; p < 0.0001) were observed between the control and the treatment where D. citri was 1 h 20 10.35 ± 0.16a 9.25 ± 0.13a 0.28 ± 0.13a 24 hfirst treated 20 with B. bassiana 10.80 and± 0.20asubsequently parasitized 9.30 ± 0.13a by T radiata, while 0.31 no± 0.10asignificant 48 hdifferences 20 (p = 0.7096) were 10.50 observed± 0.19a at 24 and 48 9.40 h ±(Figure0.12a 3B). 0.41 ± 0.11a ControlPre 20-adult duration of 10.40 the± parasitoid0.16a (days) among 9.45 ± 0.11athe treatment groups 0.51 ±and0.02a the con- Means introl the same were column statistically with the samesimilar. letter There are not significantlywas no statistical different (Tukey,differencep < 0.05). between treatment and control concerning the sex ratio of the F1 generation of T. radiata. (p < 0.005) (Table 2). In the experiment where T. radiata were released after B. bassiana spraying, the median Table 2. Means and SE of thesurvival pre-adult rate duration, of T. radiata sex ratiofemales and longevity was significantly of Tamarixia reduced radiata when (p < 0.0001) Diaphorina to 9, citri 12, nymphs and 11 days were first treated with Beauveriaat 1, bassiana 24, and (BB 48-12) h, and respectively, subsequently in comparisonparasitized by to Tamarixia 29 days radiata in the. control; however, there n° Nymphswere no significant differencesPre-Adult (Durationp = 0.1984) between the different parasitoid’s realizing Treatment time (Figure4A). The median survival rate of T. radiata males was significantlySex Ratio reduced Exposed Female Male (p < 0.0001) to 6, 10, and 10 days at 1, 24, and 48 h, respectively, from 16 days in the control; 1 h 20 10.35 ± 0.16a 9.25 ± 0.13a 0.28 ± 0.13a however, there were no differences (p = 0.5817) between the median survival at 24 and 48 h 24 h 20 (Figure4B). 10.80 ± 0.20a 9.30 ± 0.13a 0.31 ± 0.10a 48 h 20 There were10.50 statistically ± 0.19a significant differences9.40 ± 0.12a between group means0.41 per± 0.11a sample in the Control 20 longevity of the10.40 F1 generation ± 0.16a of females and9.45 males ± 0.11a of T. radiata, as determined0.51 ± 0.02a by one-way ˂ Means in the sameANOVA column (F 3.80with= the 122.9; samep letter< 0.0001) are not and significantly (F3.69 = 35.66; differentp < 0.0001),(Tukey, p respectively. 0.05). Meanwhile, no significant differences (p = 0.6389), (p = 0.7708) and (p = 0.9890) were observed in the longevityIn the experiment of females where between T. radiata the treatmentwere released groups after at B. 1, bassiana 24 and spraying, 48 h, respectively. the me- dianAdditionally, survival rate the maleof T. populationradiata females showed was significantly no significant reduced differences (p < (0.0001)p = 0.9794) to 9, between 12, and 1124 days and 48at h.1, 24, and 48 h, respectively, in comparison to 29 days in the control; however, there were no significant differences (p = 0.1984) between the different parasitoid’s realiz- ing time (Figure 4A). The median survival rate of T. radiata males was significantly re- duced (p < 0.0001) to 6, 10, and 10 days at 1, 24, and 48 h, respectively, from 16 days in the control; however, there were no differences (p = 0.5817) between the median survival at 24 and 48 h (Figure 4B).

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0 0 0 10 20 30 40 0 10 20 30 40 Age (day) Age (day)

Figure 4. Survival rate for females (A) and males (B). Survival curves of Tamarixia radiata by log-rank analysis were generatedFigure 4. whereSurvivalDiaphorina rate for females citri were (A first) and treated males with (B). SurvivalBeauveria curves bassiana of(BB-12) Tamarixia and radiata subsequently by log-rank exposed analysis to Tamarixia were gen- radiataerated(Females where Diaphorinan = 7, 12, 15 citri and were 57 at first 1 h, 24treated h, 48 hwith and Beauveria control, respectively) bassiana (BB (Males-12) andn =subsequently 6, 15, 16 and exposed 50 at 1, 24, to 48Tamarixia h and control,radiata respectively).(Females n = 7 (**, 12,p <15 0.001). and 57 at 1 h, 24 h, 48 h and control, respectively) (Males n = 6, 15, 16 and 50 at 1, 24, 48 h and control, respectively). (** p < 0.001). 4. Discussion TheThere results were indicated statistically a lower significant percentage differences of parasitism between and emergencegroup means in the per treatments sample in wherethe longevity the fungal of suspension the F1 generation was sprayed of females immediately and males after parasitismof T. radiata (1, has post determined treatment). by Inone concordance-way ANOVA with (F3.80 our = 122.9; results, p < Martins0.0001) and et al.(F3.69 [39 =] 35.66; reported p < 0.0001), that the respectively. parasitism Mean- and emergencewhile, no significant of the differences parasitoid (pDiaeretiella = 0.6389), rapae(p = 0.7708)McIntoch and (Hymenoptera:(p = 0.9890) were Braconidae) observed in wasthe significantlylongevity of females lower in between the treatments the treatment where groupsD. rapae at and1, 24 B.and bassiana 48 h, respectively.were exposed Ad- withinditionally 0 and, the 24 h.male Rashki population et al. [40 showed] also reported no significant a reduction differences in the ( numberp = 0.9794) of mummiesbetween 24 andand the 48 percentageh. emergence of the F1 generation of Aphidius matricariae (Hymenoptera: Braconidae) when the time interval was short between application of the fungus and exposure4. Discussion to the parasitoid; likewise for the exposure to the parasitoid and subsequent fungalThe application. results indicated However, a accordinglower percentage to Lord [of41 parasitism] parasitoids and evade emergence direct competition in the treat- withments entomopathogens where the fungal by suspension detecting infectedwas sprayed hosts immediately using their antennae.after parasitism According (1 h post to Manntreatment). et al [42 In] concordanceT. radiata perceives with our the results, odor of Martins,D. citri nymphset al. [39] before reported deciding that the to parasit- either feedism or and oviposit. emergence In the of same the way, aphid Chien parasitoid et al [ 43D]iaeretiella reported rapae that femalesMcIntoch of T. (Hymenoptera radiata used : theBraconidae) ovipositor w toas search significantly for suitable lower hosts in the for treatments oviposition. where D. rapae and B. bassiana were exposedOur resultswithin showed0 and 24 a h. negative Rashki, effect et al. of[40]B. also bassiana reported(BB-12) a reduction on the development in the number and of survivalmummies of theand parasitoid the percentage larva emergenc when D. citrie of nymphsthe F1 generation were first of exposed Aphidius to matricariaeT. radiata and (Hy- thenmenoptera: treated immediatelyBraconidae) when with thethe fungaltime interval suspension. was short This between may suggest application that the of eggs the fun- or prematuregus and exposure larval to the ofparasitoid;T. radiata likewisehave vulnerability for the exposure to B. bassiana to the parasitoid. Additionally, and subse- our resultsquent showedfungal application. that by extending However, the according time interval to Lord between [41] parasitoids parasitoid evade parasitism direct andcom- fungalpetition infection, with entomopathogens the survival of by the detecting parasitoid infected larva ishosts enhanced. using their Similar antennae. trends Accord- were reporteding to Mann, by Rashki et al. et[42] al [T.40 radiata] and Martins perceives et althe [39 odor], where of D. theycitri foundnymphs that before the numberdeciding of to mummieseither feed formation, or oviposi emergencet. In the same rate, way, survival Chien, and et al. parasitoid [43] reported behavior that females are dependent of T. radiata on theused time the interval ovipositor between to search parasitism for suitable and fungal hosts spraying. for oviposition. This indicates that the number of host mummiesOur results obtained showed is directlya negative related effect to of the B. timebassiana interval (BB- between12) on the parasitioid development release and andsurvival fungal of application. the parasitoid Additionally, larva when Chow D. citri et alnymphs [44] reported were first that exposed when a parasitoidto T. radiata and and anthen entomopathogen treated immediately were attacking with the thefungal same suspension. host, the time This and may order suggest among that parasitism the eggs or andpremature infection larval commanded instars of the T. interaction radiata have and vulnerability consequence. to B. bassiana. Additionally, our resultsIn the showed current that study, by extending where the the fungal time interval suspension between was parasitoid sprayed at parasitism 24 and 48 and h after fun- potentialgal infection, parasitism, the survival the parasitoid of the parasitoid emergence larva rate is enhanced. was higher, Similar but when trends the were fungal reported sus- pension was sprayed immediately after potential parasitism, the emergence rate was at by Rashki, et al. [40] and Martins, et al. [39], where they found that the number of mum- the minimum level. Similarly, van Lenteren and Fransen [45] reported that the fungal mies formation, emergence rate, survival and parasitoid behavior are dependent on the infection level decreased as the time interval between parasitization and treatment with time interval between parasitism and fungal spraying. This indicates that the number of fungi increased. Additionally, Mesquita and Lacey [46] reported that if the parasitism had host mummies obtained is directly related to the time interval between parasitioid release 48 h before exposure to the fungus, the parasitoid could escape the lethal action of the

Agronomy 2021, 11, 1496 8 of 11

entomopathogen and complete its development to the adult stage. According to Ibarra- Cortés et al [47], to reduce the negative interaction between these two biocontrol agents, it would be advisable to first release the parasitoids to allow parasitism and host feeding. Then, when a significant number of D. citri nymphs were carrying pupae, the fungi could be applied to infect remaining nymphs that were not parasitized. Moreover, according to Rashki et al [40] the percentage of parasitoid emergence was also influenced by competition for food between parasitoid’s larvae and entomopathogenic fungi, especially when the time among oviposition and fungal spraying was less than 96 h. To cope with this competition, the parasitoid emitted fungicidally active substances within the host, thus preventing the development of mycosis via pathogens, allowing the normal development and emergence of their offspring [45,48]. In the experiment where D. citri nymphs were first treated with B. bassiana and afterward exposed to T. radiata, the parasitism rate, at the three times it was interval tested (1, 24, and 48 h), was at its minimum level, which was significantly lower than the parasitism rate recorded in the control. In concordance with our results, Mesquita and Lacey [46] reported the reduced parasitism of Aphelinus asychis (Hymenoptera: Aphelinidae) as a function of time between treatment with the entomopathogenic fungus fumosoroseus (Deuteromycotina: ). This was because A. asychis could easily detect the presence of the hyphal from the fungi on the surface or the fungal metabolites into the hemolymph of the host and thus avoid the infected insect for oviposition. Brobyn et al [32] proved that parasitoids avoid ovipositing in sprayed with fungi even after 72 h of being treated, while in fungi-free aphids the oviposition rate was high because the percentage of parasitism was lower compared with healthy aphids. According to Lord [41], this was because parasitoids could detect infected hosts by entomopathogens and avoid them for oviposition. This was probably the case for T. radiata, although parasitoid behavior toward treated hosts was not monitored in this experiment. Our results have shown significant differences in both the longevity and reproductive potential of T. radiata among the treatment groups and the control in the experiment where D. citri nymphs were first exposed to T. radiata and afterward treated with B. bassiana suspensions. Similar results were reported by Rashki et al [40] and Martins et al [39]. These findings may suggest that, due to the exposure of the natural enemies of the pests to the entomopathogenic fungi, the emerged adult could become infected by B. bassina resulting in its early death. Similar trends were observed in the longevity of females where D. citri nymphs were first treated with B. bassiana and then exposed to T. radiata.

5. Conclusions Our results showed a negative interaction between T. radiata and B. bassiana in control- ling D. citri nymphs due to the antagonistic effects generated by B. bassiana on the parasitism percentage and normal development of parasitoid larvae. However, according to our find- ings, the application/interaction between T. radiata and B. bassiana could potentially be combined and applied for the management of D. citri, wherein effective time management would be required in order to minimize the potentially negative interactions between these two biological control agents; the most suitable strategy would be to allow parasitism to occur over an extended period of time (>48 h) before the application of B. bassiana. This was evident in the significant positive correlations between the time interval, the parasitism percentage and adults’ emergence obtained in this study. The results from this study show the potential to design sustainable biological control programs to control Asian citrus psyllid and other economic pests, when combining the entomopathogenic fungi B. bassiana and parasitoids. The findings also demonstrate the importance of providing accurate information to farmers and stakeholders for the optimal release and application times for both biocontrol agents. Although the present study was carried out in controlled conditions, it provides significant information that can be fundamental for future field trial assessment and validation. Further studies are therefore warranted to verify conclusions made from the controlled environment where these experiments were conducted and to Agronomy 2021, 11, 1496 9 of 11

evaluate the effectiveness of the combined use of these two biocontrol agents under field or greenhouses conditions. Large-scale field trials in citrus are needed to demonstrate the effective release/application rates and timing for effective integration into current D. citri management programs.

Author Contributions: Conceptualization, L.C.R.A., K.S.A., H.J.A., B.S.B., L.W.; formal analysis, L.C.R.A., K.S.A., H.J.A., B.S.B., J.L., H.W., J.D., L.W.; funding acquisition, L.W., K.S.A.; investigation, L.C.R.A., K.S.A., H.J.A., B.S.B., J.D., J.L., H.W.; writing—original draft, L.C.R.A., H.W., J.L., J.D.; writing—review and editing, K.S.A., H.J.A., B.S.B., L.W. All authors have read and agreed to the published version of the manuscript. Funding: The research was financially supported by grants from the National Key Projects of R & D of China (2018YFD0201502), and the Research Fund for the International Collaborative Program (KXGH17004) and grants (KF2015068, CXZX2016134) from Fujian Agriculture and Forestry University (FAFU) and the UK’s Foreign, Commonwealth & Development Office (FCDO) (B2329A-DFID-FAW and B2291A- FCDO -) through the International Centre of Insect Physiology and Ecology (icipe). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We are grateful to Xiaofang Li, Wenjie Wang, Shi-Shi Cui and Xue Jiang at the Key Laboratory of Integrated Pest Management for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, China for their assistance with the experiments. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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