Oleic Acid and Linoleic Acid Enhances the Biocontrol Potential of Metarhizium rileyi

Metarhizium rileyi is a wide spread insect fungi with a good biocontrol potentiality to various pests, particularly noctuid insects. However, it is characterized by its slow growth, its sensitivity to abiotic stress, and the slow speed of kill to pests, which hinder its use compared with other entomopathogenic fungi. In this study, the responses of M. rileyi to eight types of lipids were observed; among the lipids, oleic acid and linoleic acid significantly promoted the growth and development of M. rileyi and enhanced its stress tolerances and virulence. An additional mechanistic study demonstrated that exogenous oleic acid and linoleic acid significantly improved the conidial germination, appressorium formation, cuticle degradation, and cuticle infection, which appear to be largely dependent on the up-regulation of gene expression in growth, development, protective, and cuticle-degrading enzymes. In conclusion, exogenous oleic acid and linoleic acid enhanced the stress tolerances and virulence of M. rileyi via protecting conidial germination and promoting cuticle infection. These results provide new insights for the biopesticide development of M. rileyi.

Conidia are the main infection and reproductive units of entomopathogenic fungi [12].Maintaining or improving the viability, biological activity, and abundance of conidia under field conditions is the main strategy for applying entomopathogenic fungi.Many methods of improving the performance of conidia have been reported, such as regulating the culture substrate [13], adding exogenous oil or surfactants [14,15], and adapting to abiotic stress [16].Oil suspensions are one of the most common types of pesticide formulations.Oil suspension formulations of conidia made from vegetable oil or mineral oil can significantly improve the pathogenicity and abiotic stress tolerance of the conidia [14,15].Conidial oil suspensions of M. rileyi can efficiently control S. frugiperda in corn fields [17].Furthermore, vegetable oil can improve the conidial germination, yield, and thermotolerance of B. bassiana, Simplicillium lanosoniveum, and Isaria fumosorosea [18][19][20].Compared to mineral oil, vegetable oil has the function of regulating the growth and development of entomopathogenic fungi [14,15,[18][19][20], suggesting that there is a certain substance in vegetable oil that is beneficial for conidial growth and development.
The main components of vegetable oil are lipids and esters generated from glycerol [21], including palmitic acid (C16:0, 4.6-20.0%),oleic acid (C18:1, 6.2-71.1%),and linoleic acid (C18:2, 1.6-79.0%),which are present in coconut oil, castor oil, olive oil, sunflower oil, and palm oil [21].In fungal species such as Cordyceps, Metarhizium, Beauveria, and others, lipids can serve as a protective agent for conidia against stresses and signaling molecules for growth and development [10,19,22,23].Linoleic acid and oleic acid increased the conidial tolerance to heat, drought, and oxidative stresses [10,19,24], which promoted spore germination, the branching of hyphae, pseudohyphal growth, conidial production, and the transcription of growth-and development-related genes [22,23].Notably, lipids are one of the main components of oxylipins; they change the phospholipid composition, membrane homeostasis, membrane fluidity, and conidial hydrophobicity (an important indicator of the initial cuticle infection) [25][26][27].These changes are involved in improving germination, conidiogenesis effects, fungal proliferation, pathogenicity, and stress tolerance [25][26][27][28].Thus, we speculate that lipids may have important functions in the growth, development, virulence, and stress tolerance of entomopathogenic fungi.Despite many previous studies investigating the effect of lipids on the abiotic stress tolerance and conidial yield of M. rileyi, the mechanism of lipids in improving conidial quality remains poorly understood.
Therefore, the objectives of this study were as follows: (1) to investigate the effects of eight lipids on the growth and development of M. rileyi; (2) to determine the role of oleic acid and linoleic acid in the stress tolerance and pathogenicity of M. rileyi; and (3) to evaluate the mechanism through which oleic acid and linoleic acid improve the stress tolerance, growth and development, and pathogenicity of M. rileyi.

Fungal Strain and Media
M. rileyi strain SZCY201010 was isolated from infected S. frugiperda larvae in a corn field in Shizong county, Qujing city, Yunnan province, China, and was cultured on Sabouraud maltose agar plus yeast extract medium (SMAY: 1% peptone, 1% yeast extract, 4% maltose, and 1.5% agarose; Coolaber, Beijing, China), as previously described [29].The media plates were incubated at 25 • C with a photoperiod consisting of a 12L:12D cycle in a culture chamber.Conidial suspensions were prepared from fungi grown on SMAY plates for 10 days.
Fungal stress responses: Aliquots of 5 µL of a conidial suspension (1 × 10 7 conidia/mL) were inoculated onto SMAY alone (CK) or supplemented with H 2 O 2 (3 mM) for oxidative stress; NaCl (0.5 M; Coolaber, Beijing, China), KCl (0.5 M; Coolaber, Beijing, China), glycerol (1 M; Coolaber, Beijing, China), and sorbitol (0.5 M; Coolaber, Beijing, China) for osmotic stress; and calcofluor white (5 µg/mL; Coolaber, Beijing, China) for cell wall stress.The diameters of all the colonies incubated for 10 days under the stresses at 25 • C were measured as described above.The colony response to heat shock was examined by exposing normal 2-day-old SMAY colonies to 42 • C for 1-3 h.The colonies were transferred to 25 • C for 8-day growth recovery, followed by measuring the colony diameters.This experiment was performed in triplicate, with three parallel controls each time.The percentage of relative growth inhibition (RGI) was estimated with the formula (d c − d t )/d c × 100 (d c , control colony diameter; d t , stressed colony diameter) and used as an index of hyphal sensitivity to each stress [11].
Conidial germination: Conidial suspensions (1 × 10 7 conidia/mL, 100 µL) were spread onto SMAY alone (control) or supplemented with 0.190% oleic acid, 0.175% linoleic acid, or different types of stresses.All the treatments were incubated at 25 • C and the germination percentage was determined for 100 conidia.The entire experiment was performed in triplicate.
Yeast-like blastospores: Aliquots of 5 µL of a conidial suspension (1 × 10 7 conidia/mL) were inoculated onto SMAY alone (control) or supplemented with 0.190% oleic acid or 0.175% linoleic acid.The plates were cultured at 25 • C with a photoperiod of a 12L:12D cycle in a culture chamber.The yeast-like blastospore yield of M. rileyi on the SMAY plates was measured at 3, 4, and 5 days.The entire experiment was performed five times.
Conidia production: To evaluate the conidial yield of M. rileyi, M. rileyi conidia were suspended in a 0.02% Tween 80 solution (1 × 10 6 and 1 × 10 7 conidia/mL).Aliquots of a 100 µL conidial suspension were spread onto SMAY plates with varying concentrations (0.01, 0.05, 0.1, 0.2, and 0.4%) of oleic acid and linoleic acid.The plates were cultured at 25 • C with a photoperiod of a 12L:12D cycle in a culture chamber.When the color of the colony changed from pale or olive green to malachite green, the conidial yield of M. rileyi on the SMAY plates was evaluated at 10 d.Three replicates were used for each treatment.

Fungal Virulence-Related Phenotypes
Simulation of host cuticle conidial germination: To view conidial germination on water agar plates (1.5% agarose), conidial suspensions (1 × 10 7 conidia/mL, 100 µL) were spread onto the plates.Disinfected and sterilized Locusta migratoria manilensis hindwings and water agar plates prepared in high-temperature and -pressure facilities were used to mimic the host cuticle and oligotrophic conditions, respectively.A conidial suspension (1 × 10 7 conidia/mL) was sprayed onto the locust hindwings.All the treatments were incubated at 25 • C and the germination percentage was determined for 100 conidia.Three replicates were used for each treatment.
Cuticle-degrading enzyme: Extracellular enzyme activity assays were performed according to the method described in previous reports by Gebremariam et al. [31], and appropriate modifications were made.The conidia were made into a 1 × 10 7 conidia/mL suspension, alone or supplemented with 0.190% (v/v) oleic acid or 0.175% (v/v) linoleic acid.Aliquots of 5 µL of the conidial suspension were inoculated onto 1/8 SMAY supplemented with 1.2% powdered skim milk (v/v; Yili, Huhehaote, China), 0.2% olive oil (v/v; Sangon Biotech, Shanghai, China), and 0.5% colloidal chitin (w/w; coolaber, Beijing, China) to measure the protease, chitinase, or lipase activity, respectively.The plates were incubated at 25 • C for 14 days.The determination of enzyme activity was based on the transparent hydrolysis zone around the colony.The diameter of the colonies and hydrolysis zones was measured using the cross method.The enzymatic index (EI) was calculated using the following formula: EI = (hydrolysis zone diameter)/(colony growth diameter).The entire experiment was performed five times.

Bioassays for Fungal Virulence
The S. frugiperda larvae that were used for virulence determination were 3rd, 4th, and 5th instars.A Tween 80 solution (0.02%) was used alone as the control (CK) or supplemented with 0.190% (v/v) oleic acid (OA) or 0.175% (v/v) linoleic acid (LA).The conidia were made into a 1 × 10 8 conidia/mL suspension comprising M. rileyi conidia alone (Mr) or supplemented with 0.190% (v/v) oleic acid (Mr+OA) or 0.175% (v/v) linoleic acid (Mr+LA).Healthy 3rd-, 4th-, and 5th-instar larvae of the same size were selected, put into the prepared conidial suspension for 15 s, and then removed; the excess water on the surface of the insect body was removed with aseptic filter paper.Subsequently, the larvae were placed individually into a disinfected and sterilized 12-well cell culture plate with 75% alcohol.A fresh artificial diet (125 g of soybean flour, 225 g of corn flour, 225 g of corn leaf flour, 40 g of yeast extract, 20 g of casein, 0.6 g of cholesterol, 3 g of choline chloride, 6 g of sorbic acid, 6 g of methyl p-hydroxybenzoate, 36 g of agarose, 0.1 g of inositol, 7 g of vitamin C, and 1300 mL of water) was added to the holes where the larvae were placed.The above experiment was performed three times, with three treatments for each experiment and 30-36 larvae for each treatment.After the treatment, the larvae were fed the artificial diet and maintained in an artificial climate chamber at 25 • C with a relative humidity of 75% and a photocycle of 16L:8D.The number of dead insects was recorded each day, and the dead insects were kept in a Petri dish to determine whether they were infected with the test strain.
The blastospore growth was observed to examine the time it took to penetrate the cuticle.The hemolymph of five 4th-instar larvae in the Mr, Mr+OA, and Mr+LA infection groups was collected and diluted 1:1 in a sterile physiological solution (0.14 M NaCl, 0.1 M glucose, 25 mM sodium citrate, and 30 mM citric acid).The hyphal bodies were photographed and observed under a microscope every 12 h from 48 to 132 h post-inoculation of the conidia.The average of five fields was taken as one biological replicate.Five replicates were used for each treatment.

CAT, POD, and SOD Activity Assays
M. rileyi was cultured separately in the SMY culture media (1% peptone, 1% yeast extract, and 4% maltose), alone or supplemented with 0.190% (v/v) oleic acid or 0.175% (v/v) linoleic acid.These liquid culture media were inoculated with a final concentration of 10 7 conidia/mL and cultured for 6 days at 25 • C on a rotary with a speed of 200 rpm.The mycelium was filtered and washed four times with PBS.A BCA protein content assay kit, catalase (CAT) activity assay kit, peroxidase (POD) activity assay kit, and superoxide dismutase (SOD) activity assay kit (Boxbio Science & Technology Co., Ltd., Beijing, China) were used to determine the protein concentration and total enzymatic activity (U/mg protein).Three replicates were used for each treatment.

Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-qPCR)
Aliquots of 100 µL of the conidial suspension (1 × 10 7 conidia/mL) were smeared onto SMAY alone (CK) or supplemented with 0.190% oleic acid or 0.175% linoleic acid and incubated at 25 • C for 5 days for the determination of gene expression.The total RNA was isolated using the TRIzol reagent (BBI Life Sciences, Shanghai, China) by following the manufacturer's instructions.The first-strand cDNA was synthesized from the total RNA using a HiScript III All-in-One RT SuperMix Perfect for qPCR (Vazyme, Nanjing, China).The RT-qPCR analysis was carried out in 96-well 0.2 mL block plates using a QuantStudio™ 7 Flex system (Thermo Scientific, Wilmington, DE, USA).Each reaction contained 1.0 µL of the template (1 ng/µL), 10.0 µL of 2 × Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), and 0.4 µL of each primer (10 µM), and 8.2 µL of EDPC-ddH 2 O, with a final volume of 20 µL.The RT-qPCR condition was pre-denaturation at 95 • C for 30 s, and then 40 cycles at 95 • C for 5 s and at 60 • C for 20 s.After each reaction, a melting curve analysis (denatured at 95 • C for 15 s, annealed at 60 • C for 60 s, and denatured at 95 • C for 15 s) was conducted to ensure the consistency and specificity of the amplified product.Three biological replicates were used for each treatment in the RT-qPCR analyses.The β-tubulin gene (TUB) and transcription elongation factor (TEF) genes were employed as internal references [32,33].The 2 −∆∆CT technique was utilized to determine the relative expression of every gene [34].Table S1 contains a list of primers utilized in the RT-qPCR tests.

Statistical Analysis
All the data are presented as the mean ± standard deviation.The data were analyzed for the normality of their distribution using the Shapiro-Wilk test and for homoscedasticity using Levene's test.When these assumptions were not fulfilled, the data were subjected to a log or square root transformation.If the data met the assumptions of normality and homogeneity, two groups were analyzed using an unpaired Student's t-test and data among the various groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.If the data were not normal and/or not homogenous, two groups were analyzed using a Wilcox test and the data among the various groups were analyzed using the non-parametric Kruskal-Wallis test, followed by the Games-Howell test.The survival data were subjected to a Kaplan-Meier survival log-rank analysis.The median lethal time (LT 50 ) for certain treatments was calculated using linear regression in Microsoft Excel, version 2019.p < 0.05 was considered to indicate statistical significance.

Effects of Lipids Addition on M. rileyi Growth and Development
The growth and development rhythm of M. rileyi, including conidial germination, yeast-like blastospore production, mycelial growth, and sporulation, was determined (Figure S1).We used a heat map clustering analysis based on the yeast-like blastospore formation time, hyphal growth time, sporulation time, and relative growth rate data to compare the effects of different lipids.As shown in Figure 1A, oleic acid and linoleic acid clustered into one branch, while other lipids (SA, eicosane, cetane, PA, tetracosane, and octacosanol) clustered into another.Exogenous oleic acid and linoleic acid significantly increased the colony diameter in the conidial suspensions with concentrations of 10 5 , 10 6 , and 10 7 conidia/mL (Figure 1B; Table S2).Next, we measured the conidial production of M. rileyi at different concentrations of oleic acid and linoleic acid and used curve fitting (y = c + bx + ax 2 ) to determine the optimal concentration, where y is the conidial production and x is the concentration.The optimal concentration of oleic acid was 0.199% and 0.190% for concentrations of 10 6 and 10 7 conidia/mL, respectively (Figure 1C; Table S3).The optimal concentration of linoleic acid was 0.224% and 0.175% for concentrations of 10 6 and 10 7 conidia/mL, respectively (Figure 1D; Table S3).Thus, the concentrations of 0.190% oleic acid and 0.175% linoleic acid were selected for further study.

Exogenous Oleic Acid and Linoleic Acid Improved the Virulence of M. rileyi
The laboratory bioassays revealed a high virulence for the 0.190% oleic acid and 0.175% linoleic acid treatments against the 3rd instars of S. frugiperda, with death beginning on the first day and survival reaching 34.3 ± 1.3% and 35.2 ± 1.3% after 6 days (Figure 5A,B; Table S4), respectively.The virulence of M. rileyi against the 3rd, 4th, and 5th instars of S. frugiperda was determined, with survival reaching 13.6 ± 3.7%, 60.0 ± 1.8%, and 68.7 ± 2.9% (Figure 5; Table S4), respectively, 10 days after the treatment using suspensions of 1 × 10 8 conidia/mL.The LT 50 of the 3rd instars treated with M. rileyi was 5.7 ± 0.1 d.However, the death rate of the 4th and 5th instars due to M. rileyi did not reach 50%, so the LT 50 could not be calculated.The combined treatment of M. rileyi and oleic acid against the 3rd, 4th, and 5th instars of S. frugiperda resulted in survival rates of 0.9 ± 1.3%, 45.6 ± 2.3%, and 51.5 ± 4.3%, which were significantly lower than those resulting from the 0.190% oleic acid treatment alone or the M. rileyi suspensions alone (Figure 5A,C,E; Table S4).Similarly, the combined treatment of M. rileyi and linoleic acid against the 3rd, 4th, and 5th instars of S. frugiperda resulted in survival rates of 0.9 ± 1.3%, 47.8 ± 3.4%, and 51.5 ± 4.3%, which were significantly lower than those resulting from the 0.175%  treatment alone or the M. rileyi suspensions alone (Figure 5A,C,E; Table S4).Similarly, the combined treatment of M. rileyi and linoleic acid against the 3rd, 4th, and 5th instars of S. frugiperda resulted in survival rates of 0.9 ± 1.3%, 47.8 ± 3.4%, and 51.5 ± 4.3%, which were significantly lower than those resulting from the 0.175% linoleic acid treatment alone or the M. rileyi suspensions alone (Figure 5B,D,F; Table S4).The LT50 for 4th-instar S. frugiperda with M. rileyi+OA and M. rileyi+LA was 7.6 ± 0.2 d and 7.6 ± 0.3 d, respectively.

Exogenous Oleic Acid and Linoleic Acid Enhanced Cuticle Infection of M. rileyi
Exogenous oleic acid and linoleic acid improved the virulence-related phenotypes and increased the cuticle-degradation-related enzyme activity and gene expression in M. rileyi, suggesting that exogenous oleic acid and linoleic acid enhance host cuticle infection by M. rileyi.After fungal invasion, the host hemocytes flocked together and formed melanic dots.Then, the time for host melanic dots to be created and blastospores to be generated during M. rileyi infection was observed.The times of melanic dot formation were 96, 72, and 72 h in the CK, oleic acid, and linoleic acid groups, respectively.The CK, oleic acid, and linoleic acid treatments started to generate blastospores from the host hemocoel at 108, 84, and 96 h, respectively (Figure 7A).The number of blastospores gradually increased from 72 to 132 h, and significantly increased in the oleic acid and linoleic acid treatment groups at multiple time points compared to the CK group (Figure 7B; Table S5).Finally, we observed 30 larvae in each treatment group at 132 h.The infection rates in the CK, oleic acid, and linoleic acid groups were 47, 67, and 63%, respectively (Figure 7C).

Exogenous Oleic Acid and Linoleic Acid Enhanced Cuticle Infection of M. rileyi
Exogenous oleic acid and linoleic acid improved the virulence-related phenotypes and increased the cuticle-degradation-related enzyme activity and gene expression in M. rileyi, suggesting that exogenous oleic acid and linoleic acid enhance host cuticle infection by M. rileyi.After fungal invasion, the host hemocytes flocked together and formed melanic dots.Then, the time for host melanic dots to be created and blastospores to be generated during M. rileyi infection was observed.The times of melanic dot formation were 96, 72, and 72 h in the CK, oleic acid, and linoleic acid groups, respectively.The CK, oleic acid, and linoleic acid treatments started to generate blastospores from the host hemocoel at 108, 84, and 96 h, respectively (Figure 7A).The number of blastospores gradually increased from 72 to 132 h, and significantly increased in the oleic acid and linoleic acid treatment groups at multiple time points compared to the CK group (Figure 7B; Table S5).Finally, we observed 30 larvae in each treatment group at 132 h.The infection rates in the CK, oleic acid, and linoleic acid groups were 47, 67, and 63%, respectively (Figure 7C).

Discussion
Fatty acids have previously been identified as protectors of M. rileyi conidia against abiotic stresses, such as dehydration and thermotolerance [10,23,37].In this study, exogenous oleic acid and linoleic acid protected the M. rileyi conidial germination and improved various abiotic stress tolerances, including the tolerance to oxidative, osmotic, cell wall, and heat shock stresses.However, fatty acids can also inhibit the spore germination, hyphal growth, and pathogenicity of entomopathogenic fungi (e.g., B. bassiana and Conidiobolus coronatus) [38][39][40].The mechanism for this involves a lipid-droplet protein (BbLar1) that maintains the intracellular homeostasis of fatty acids and is crucial to the fungal tolerance to linoleic acid stress, which significantly contributes to fungal compatibility with fatty acids [41].We found that exogenous oleic acid and linoleic acid significantly increased the activity of protective enzymes and the expression of genes encoding the CAT and SOD isoenzymes.The cumulative evidence proves that the protective enzymes of fungi are crucial to resisting biological and abiotic stress processes [36,42,43], implying that exogenous oleic acid and linoleic acid protect the conidial germination of M. rileyi and improve its stress tolerance by increasing the expression of protective enzymes.The cell wall integrity pathway senses hypotonic conditions or multi-stresses, including heat shock, oxidative, osmotic, and cell wall stresses, by controlling cell wall biosynthetic enzymes and the expression of cell-wall-related genes [36,44].In this study, the transcription level of chitin synthesis genes was significantly up-regulated by the oleic acid and linoleic acid treatments, suggesting that oleic acid and linoleic acid may be signal molecules of the cell wall integrity pathway.These results indicate that fatty acids may function as protector agents of biological and abiotic stresses for M. rileyi.
Oil suspension formulations of conidia significantly improve the pathogenicity of entomopathogenic fungi [14,15], among which fatty acids may play an important role [26].In this study, exogenous oleic acid and linoleic acid significantly improved the virulence of M. rileyi against the 4th and 5th instars of S. frugiperda.Exogenous oleic acid and linoleic acid significantly elevated the conidial germination on water agar plates and locust hindwings and the appressorium formation rates.After adhering to the cuticle of insects, a fungal conidium germinates and differentiates into a specialized infection structure called an appressorium [12,30,45].Appressorium-mediated cuticle penetration is accompanied by the combined action of cuticle-degrading enzymes (protease, lipase, and chitinase) [31,46].Our results also showed that the protease activity was significantly elevated after the oleic acid and linoleic acid treatments.Similarly, protease genes were significantly up-regulated in the oleic acid and linoleic acid groups.No differences were found for extracellular lipase or chitinase.However, the expression of the extracellular lipase gene, secreted lipase gene, and chitinase genes was significantly up-regulated in the oleic acid and linoleic acid groups.This may have been due to the insufficient sensitivity of the plate enzyme activity detection method.Furthermore, vegetable oil has the function of regulating the growth and development of entomopathogenic fungi [14,15,[18][19][20].Triacylglycerides is one of the main components of oil suspension, and is hydrolyzed into fatty acids and glycerol by lipases [47].In addition, exogenous fatty acids also can enter cells through fatty acid-binding protein.Degradation of fatty acids by the β-oxidation pathway results in the formation of acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle for the production of ATP.TCA cycle intermediates may be used as key sources of carbon molecules for biosynthesis of nucleotides, amino acids, and lipids [48].Notably, the oleic acid and linoleic acid treatments improved the conidial germination rate on water agar plates and locust hindwings and increased the growth and development of M. rileyi [23], suggesting that exogenous oleic acid and linoleic acid provide energy substance for M. rileyi's growth and development [23,30].Combined with the results of this study, we speculate that oleic acid and linoleic acid may improve the ability of M. rileyi to infect S. frugiperda via enhancing the process of conidial germination, appressorium formation, and cuticle penetration.
When the conidia attach to the host cuticle under the appropriate environmental conditions and signals, they germinate and invade the host [49].Lipids are one of the main compo-nents of the body wall, and they may be signaling molecules for conidial germination [22].Oleic acid and linoleic acid have a generic structure of lipo-chitooligosaccharides (LOCs), and LOCs are perceived at the plasma membrane by receptor-like kinases with extracellular LysM domains [50,51].For example, LOCs act as regulatory signals of fungal growth and development, and an LOC treatment increased the differentially expressed genes of A. fumigatus from 91 to 152 at 30 min and 130 min, respectively [22].Fungi possess a significant number of LysM-containing proteins [22], suggesting that oleic acid and linoleic acid can be recognized by LysM-containing proteins.Furthermore, fatty acids can be oxidized to produce oxylipins through enzymatic and non-enzymatic reactions [52].Oxylipin may act as ligands through specific G-protein-coupled receptors (GPCRs), and GPCRs are abundant filamentous fungi [53,54].For example, oleic acid promotes GPCR membrane docking and activity as well as related signaling molecules [55]; oleic acid increases the expression of growth-and development-related genes (Mrap1, MrNsdD, MrPbs2, MrSwi6, MrSte12, and MrMsn2) in M. rileyi [23]; and oxylipin increases the level of chitin in filamentous fungi [54].In this study, exogenous oleic acid and linoleic acid up-regulated the expression of growth-and development-related genes, CAT genes, SOD genes, chitin synthase family genes, and cuticle-degrading enzyme-related genes, suggesting that oleic acid and linoleic acid function as signaling molecules for M. rileyi conidia.Notably, fatty acid is converted to polyunsaturated fatty acid [56].Polyunsaturated fatty acids can be metabolized to multiple oxylipins by the cyclooxygenase, lipoxygenase, and/or cytochrome P450 pathways [57].Oxylipins induce fungal cellular differentiation, including lateral branching, appressorium formation, the yeast-to-hyphal transition, sexual development, and spore release [52,54], suggesting that oleic acid-and linoleic acid-derived oxylipins play a critical role in the regulation of growth and development in M. rileyi.
Our and others' previous work has demonstrated that oleic acid and linoleic acid increase the conidial tolerance to heat, drought, oxidative, osmotic, and cell wall stresses [10,19,24].These abiotic stresses strongly limit the application of entomopathogenic fungi as biological insecticides [14,58].Conidial oil suspensions of M. rileyi, M. anisopliae, and B. bassiana can efficiently prevent and control pests in fields [17,59,60].These results indicate that oleic acid and linoleic acid can be added to oil suspensions of conidia.

Conclusions
Exogenous oleic acid and linoleic acid improve the stress tolerances and virulence of M. rileyi.Their favorable biological characteristics appear to be largely mediated through protecting conidial germination and promoting cuticle infection.These results provide new insights for the development of biopesticides based on M. rileyi.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof10080521/s1, Figure S1: Chemical structures for oleic acid, linoleic acid, stearic acid, eicosane, cetane, palmitic acid, tetracosane, and octacosanol.Figure S2: Growth and development of M. rileyi, including conidial germination at 48 h, yeast-like blastospore production at 4 days, mycelial growth at 5 days, and sporulation at 7 days; Table S1: qRT-PCR primers used in this study; Table S2: Colony diameter for all the conidial suspension concentrations; Table S3: Conidial production of M. rileyi at different concentrations of oleic acid and linoleic acid; Table S4: The mortality rate of M. rileyi against the 3rd, 4th, and 5th instars of S. frugiperda; Table S5: The number of M. rileyi hyphal bodies in the hemolymph from 72 to 132 h.

Figure 1 .Figure 1 .
Figure 1.Effects of lipids addition on M. rileyi growth and development.(A) Cluster heat map and clustering through the level of growth and development.Red and blue boxes indicate increased and decreased levels, respectively.LA, linoleic acid; OA, oleic acid; SA, stearic acid; PA, palmitic acid; GR, relative growth rate; YG, yeast cell formation time; HG, hyphal growth time; ST, sporulation time; e5, 10 5 conidia/mL; e6, 10 6 conidia/mL; e7, 10 7 conidia/mL.(B) Colony diameter.The conidial production of M. rileyi at different concentrations of oleic acid (C) and linoleic acid (D) was determined and curve fitting (y = c + bx + ax 2 ) was used to determine the optimal concentration.The conidial yield for the 10 6 conidia/mL suspension is shown by the red line and the conidial yield for the 10 7 conidia/mL suspension is shown by the green line.GT50, conidial median germination time.The data are presented as the mean ± standard deviation.SDs of the means are from three independent replicates.* p < 0.05 and ** p < 0.01 when the OA group is compared to the water group.# p ##

Figure 2 .
Figure 2. Exogenous oleic acid and linoleic acid enhanced the growth and development of M. rileyi.(A) Conidial germination curve.(B) Conidial median germination time.(C) Yeast-like blastospore formation time.(D) Hyphal growth time.(E) Sporulation time.(F) The number of yeast-like blastospores after 4, 5, and 6 days.(G) The conidial yield at 10 days.(H) The expression of growthand development-related genes.LA, linoleic acid; OA, oleic acid; Pbs2, mitogen-activated protein kinase kinase Pbs2; Msn2, zinc finger DNA-binding protein Msn2; Swi6, cell cycle box-binding transcription factor MrSwi6; NsdD, GATA-type transcription factor NsdD; Ste12, transcription factor Ste12; TEF, transcription elongation factor; TUB, β-tubulin.The data are presented as the mean ± standard deviation.A-E, G, and H: SDs of the means from three independent replicates.F: SDs of the means from five independent replicates.* p < 0.05 and ** p < 0.01 when the OA group is compared to the control (CK) group; ## p < 0.01 when the LA group is compared to the control (CK).

Figure 3 .
Figure 3. Exogenous oleic acid and linoleic acid enhanced multiple stress tolerances of M. rileyi.Images (A) and relative growth inhibition percentages (B) of fungal colonies incubated at 25 • C for 10 days on SMAY plates or supplemented with the indicated concentrations of H 2 O 2 , KCl, NaCl, sorbitol (SBT), glycerol (GLY), and calcofluor white (CFW) and of SMAY colonies incubated at 25 • C for 8 days of growth recovery after 2-day-old colonies were exposed to a 42 • C heat shock (HS) for 1, 2, or 3 h.Scale bars = 1.0 cm; LA, linoleic acid; OA, oleic acid; RGI, relative growth inhibition; HS, heat shock.The data are presented as the mean ± standard deviation.SDs of the means are from three independent replicates.* p < 0.05 and ** p < 0.01 compared to the control (CK) response.

Figure 4 .
Figure 4. Exogenous oleic acid and linoleic acid protected conidial germination of M. rileyi and improved potential for stress tolerance.Conidial germination curve with H2O2 (A), GLY (B), and NaCl (C) stresses.(D) Conidial median germination time.The activity of CAT (E), SOD (F), and POD (G).The expression of genes encoding the CAT (H) and SOD (I) isoenzymes.(J) The expression of genes encoding the chitin synthase isoenzymes.LA, linoleic acid; OA, oleic acid; GT50, conidial median germination time; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Chs, chitin synthase.The data are presented as the mean ± standard deviation.SDs of the means are from three independent replicates.* p < 0.05 and ** p < 0.01 when compared to the control (CK).

Figure 4 .
Figure 4. Exogenous oleic acid and linoleic acid protected conidial germination of M. rileyi and improved potential for stress tolerance.Conidial germination curve with H 2 O 2 (A), GLY (B), and NaCl (C) stresses.(D) Conidial median germination time.The activity of CAT (E), SOD (F), and POD (G).The expression of genes encoding the CAT (H) and SOD (I) isoenzymes.(J) The expression of genes encoding the chitin synthase isoenzymes.LA, linoleic acid; OA, oleic acid; GT 50 , conidial median germination time; CAT, catalase; SOD, superoxide dismutase; POD, peroxidase; Chs, chitin synthase.The data are presented as the mean ± standard deviation.SDs of the means are from three independent replicates.* p < 0.05 and ** p < 0.01 when compared to the control (CK).

Figure 5 .
Figure 5. Exogenous oleic acid and linoleic acid improved the virulence of M. rileyi.(A,B) The survival of 3rd instars of S. frugiperda exposed to M. rileyi.(C,D) The survival of 4th instars of S. frugiperda exposed to M. rileyi.(E,F) The survival of 5th instars of S. frugiperda exposed to M. rileyi.LA, linoleic acid with a 0.175% concentration; The dashed line represents a survival rate of 50%; OA, oleic acid with a 0.190% concentration; Mr, M. rileyi conidial suspension with 10 8 conidia/mL; Mr+OA, combined treatment of M. rileyi and oleic acid; Mr+LA, combined treatment of M. rileyi and linoleic acid.The data are presented as the mean ± standard deviation.SDs of the means are from three independent replicates.

Figure 5 .
Figure 5. Exogenous oleic acid and linoleic acid improved the virulence of M. rileyi.(A,B) The survival of 3rd instars of S. frugiperda exposed to M. rileyi.(C,D) The survival of 4th instars of S. frugiperda exposed to M. rileyi.(E,F) The survival of 5th instars of S. frugiperda exposed to M. rileyi.LA, linoleic acid with a 0.175% concentration; The dashed line represents a survival rate of 50%; OA, oleic acid with a 0.190% concentration; Mr, M. rileyi conidial suspension with 10 8 conidia/mL; Mr+OA, combined treatment of M. rileyi and oleic acid; Mr+LA, combined treatment of M. rileyi and linoleic acid.The data are presented as the mean ± standard deviation.SDs of the means are from three independent replicates.

Figure 6 .
Figure 6.Exogenous oleic acid and linoleic acid increased fungal virulence-related phenotypes.Conidial germination rate on water agar plates (A) and locust hindwings (B).Appressorium formation rates (C) and images (D) at 48 and 72 h.The red arrow represents the appressorium.Ap, appressorium; Co, conidia.Extracellular enzymatic index (E) and images (F) at 14 days.(G) The expression of extracellular lipase gene and secreted lipase gene.(H) The expression of genes encoding the protease isoenzymes.(I) The expression of genes encoding the chitinase isoenzymes.LA, linoleic acid; OA, oleic acid; Chit, chitinase; Pr, protease; EL, extracellular lipase; SL, secreted lipase.The data are presented as the mean ± standard deviation.A, C, and G-I: SDs of the means are from three independent replicates.E: SDs of the means are from five independent replicates.* p < 0.05 and ** p < 0.01 compared to the control (CK).

Figure 6 .
Figure 6.Exogenous oleic acid and linoleic acid increased fungal virulence-related phenotypes.Conidial germination rate on water agar plates (A) and locust hindwings (B).Appressorium formation rates (C) and images (D) at 48 and 72 h.The red arrow represents the appressorium.Ap, appressorium; Co, conidia.Extracellular enzymatic index (E) and images (F) at 14 days.(G) The expression of extracellular lipase gene and secreted lipase gene.(H) The expression of genes encoding the protease isoenzymes.(I) The expression of genes encoding the chitinase isoenzymes.LA, linoleic acid; OA, oleic acid; Chit, chitinase; Pr, protease; EL, extracellular lipase; SL, secreted lipase.The data are presented as the mean ± standard deviation.A, C, and G-I: SDs of the means are from three independent replicates.E: SDs of the means are from five independent replicates.* p < 0.05 and ** p < 0.01 compared to the control (CK).

Figure 7 .
Figure 7. Exogenous oleic acid and linoleic acid enhanced cuticle infection of M. rileyi.(A) Hemocyte encapsulation was examined under a microscope at an interval of 12 h.Scale bars = 20 µm.The red arrow represents the fungal blastospore in the hemolymph.(B) The number of M. rileyi hyphal bodies in the hemolymph from 72 to 132 h.(C) The relative percentage of infection at 132 h.LA, linoleic acid; OA, oleic acid.The data are presented as the mean ± standard deviation.SDs of the means are from five independent replicates.* p < 0.05 and ** p < 0.01 when OA is compared to the control (CK); ## p < 0.01 when LA is compared to the control (CK).