Paecilomyces lilacinus and application thereof in production of cyclosporin C and in prevention and treatment of bemisia tabaci

Abstract

This invention relates to the field of insect control, specifically to the Paecilomyces lilacinus strain XI-5, and the extraction and identification of crude toxins produced by secondary metabolites of Paecilomyces lilacinus strain XI-5. This invention confirms that the secondary metabolites of Paecilomyces lilacinus strain XI-5 produce cyclosporine C. Cyclosporine C has a stomach poison effect on adult whiteflies. Twenty-four hours after application, the LC50 concentration of cyclosporine C against adult whiteflies was 31.75 µg / ml; compared to the control group, the LC30 concentration of cyclosporine C resulted in a decrease in oviposition / fertility in female whiteflies.

Description

Technical Field

[0001] This invention relates to the field of insect control, specifically to Paecilomyces lilacinus strain XI-5, and the extraction and identification of toxins produced by secondary metabolites of Paecilomyces lilacinus strain XI-5 and their application in the control of whiteflies. Background Technology

[0002] The whitefly is a global pest that causes significant economic losses to agricultural production every year [1,2]. The whitefly-Asia Minor 1 (MEAM1) (formerly known as “Biotype B”) poses a persistent threat to field crop production worldwide [3,4]. Whitefly management programs around the world rely primarily on chemical pesticides, but the inappropriate use of these chemicals has led to the development of pesticide resistance [5,6]. Due to the rapid development of pesticide resistance in whiteflies, integrated biological control has recently gained public attention [7,8].

[0003] For over a century, entomopathogenic fungi have been considered potential agents for the biocontrol of various insects. Their diversity, complex metabolic types, and suitable levels of safety for humans and other non-target organs make them more attractive than other pest control strategies [7]. Fungi belonging to the genus *Cyclocarya* (formerly *Paecilomyces*) are pathogens of plant-borne insects and nematodes [9,10]. *Paecilomyces lilacinus* (… Purpureocilliumlilacium Paecilomyces lilacinus is a well-known insect pathogen that has been used to control various crop pests, such as aphids, whiteflies, thrips, fruit flies, and plant-parasitic nematodes [9-11]. Secondary metabolites of Paecilomyces lilacinus produce various secondary compounds / toxins known as mycotoxins

[12] . These toxins have stomach poison effects on insects and nematodes.

[0004] Cyclosporins are nonpolar cyclic oligopeptides produced by various fungal species belonging to the genera *Beauveria bassiana*, *Verticillium*, *Paecilomyces lilacinus*, and *Cyclosporium* [12,14]. Cyclosporins are secreted by fungi to kill infected insect hosts or impair their immune systems to promote fungal growth [15-17]. Cyclosporins also play a role in clearing foreign organisms from the hemolymph by blocking glycoprotein-associated pumps [14,18].

[0005] This study aimed to extract and characterize cyclosporine C produced by the secondary metabolite of Paecilomyces lilacinus strain XI-5, which has been shown to be highly pathogenic to whiteflies in our previous studies

[10] . In addition, toxicity studies were conducted on cyclosporine C to observe its effects on the survival and reproduction of whiteflies [12,13].

[0006] References

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[0034] 28. Abbott, WS A method of computing the effectiveness of aninsecticide. J. Am. Mosq. Control Assoc. 1987, 3 , 302–303.

[0035] SAS Institute. SAS User's Guide ; Statistics SAS Institute: Cary, NC, USA, 2000. Summary of the Invention

[0036] This invention first isolated the toxins from the secondary metabolites of *Paecilomyces lilacinus* strain XI-5 and confirmed that these secondary metabolites produce cyclosporine C. Further research showed that cyclosporine C has a stomach poison effect on adult whiteflies. Twenty-four hours after application, the LC50 concentration of cyclosporine C against adult whiteflies was 31.75 µg / ml; compared to the control group, the LC30 concentration of cyclosporine C resulted in a decrease in oviposition / fertility in female whiteflies. This completes the invention.

[0037] This invention provides a method for extracting crude toxins from Paecilomyces lilacinus strain XI-5, comprising the following steps:

[0038] 1) Culture Paecilomyces lilacinus strain XI-5 with shaking;

[0039] 2) Obtain the bacterial solution by vacuum filtration, adjust the pH of the bacterial solution to 2.5-4.0, and add ethyl acetate for extraction;

[0040] 3) The secondary metabolites of the strain, namely crude toxins, were obtained by separating the organic phase and distilling ethyl acetate using a rotary evaporator.

[0041] Preferably, the method further includes: dissolving the obtained crude toxin in isopropanol, filtering it through a 0.22 μm filter membrane, and then analyzing it using ESI positive and negative ion analysis.

[0042] More preferably, the spore suspension of *Paecilomyces lilacinus* strain XI-5 was inoculated into CM liquid medium with shaking culture to achieve a concentration of 1.0 × 10⁻⁶. 6 Spores / mL, then placed in a shaking incubator at 27°C and 125 rpm for 2 weeks.

[0043] Specifically, the pre-culture steps include:

[0044] The Paecilomyces lilacinus strain XI-5 was inoculated onto PDA medium and cultured for 10 days at 26 ± 1℃ with an L:D ratio of 16:8 until it was fully mature.

[0045] After conidia are produced, the mycelium is scraped into a 0.05% Tween-80 solution using a small spatula, and then thoroughly stirred with a magnetic stirrer to ensure even dispersion of the conidia. The final solution is prepared as a 1.0 × 10⁻⁶ solution. 8 A suspension with a spore / mL concentration was then inoculated into CM liquid culture medium.

[0046] Preferably, in step 2), the bacterial solution is obtained by vacuum filtration, the pH of the bacterial solution is adjusted to 3.0, the same volume of ethyl acetate is added for extraction for 24 h, and the solution is placed in a 4°C low-temperature refrigerator and stirred thoroughly every 4 h.

[0047] More preferably, the method further includes performing LC-MS analysis on the extracted crude toxin to determine the cyclosporine C content.

[0048] The present invention also provides a toxin obtained by the method, and its application in the control of whiteflies.

[0049] This also provides information on the application of cyclosporine C in the control of whiteflies.

[0050] This invention first isolated the toxins from the secondary metabolites of *Paecilomyces lilacinus* strain XI-5, which can be used to control whiteflies. Furthermore, cyclosporine C is widely used in the medical field, and this invention also found the presence of cyclosporine C in the strain, which exhibits high toxicity to whiteflies. Therefore, using cyclosporine C to control whiteflies contributes to the study of the pathogenicity of entomopathogenic fungi to whiteflies and slows down the increase in drug resistance in whiteflies. Attached Figure Description

[0051] Figure 1 LC-MS identification spectrum of crude toxin from Paecilomyces lilacinus strain XI-5.

[0052] Figure 2 LC-MS analysis of cyclosporin C produced from secondary metabolites of Paecilomyces lilacinus strain XI-5.

[0053] Figure 3 Infrared spectral analysis of ethyl acetate mycelial extract of Paecilomyces lilacinus.

[0054] Figure 4 The active compounds produced by the secondary metabolites of Paecilomyces lilacinus strain XI-5 1 HNMR analysis.

[0055] Figure 5 The cumulative mortality rate of adult whiteflies 24 hours after being fed XI-5 crude toxin.

[0056] Figure 6 Corrected mortality rate of adult whiteflies 24 h after feeding with cyclosporine C toxin.

[0057] Figure 7 The effect of cyclosporine C (22 µg / ml) treatment at different time intervals on the reproductive capacity of whiteflies. Detailed Implementation

[0058] The present invention will be further illustrated below through specific embodiments.

[0059] I. Collect soil samples and isolate fungi

[0060] Soil samples for fungal isolation were collected from Chebaling Forest in Jiangxi Province, People's Republic of China in 2009. Sample collection: The soil samples were dug up 10 cm below the soil surface and placed in plastic bags. The collected samples were stored at 4°C. Fungal isolation was performed using the methods of Imoulan et al. (2011) and Du et al. (2019). Briefly, a mixture of soil samples (3 g) and 30 mL of 0.05% Tween-80 prepared with ddH2O was stirred for 15 minutes. The mixture was then pipetted (1 mL) onto PDA plates, followed by 25 mL pipettes and 80 mL pipettes. A 0.1:8 h (light:black) pipette was used. After 7 days of observation on PDA plates, the well-grown fungi were re-inoculated onto fresh PDA plates. The inoculation process was repeated until a pure fungal culture was obtained. Based on phenotypic characteristics and fungal morphology (Saito and Brownbridge 2016), the cultured fungi were purified until only one colony, numbered XI-5, was found on the PDA plate, which was identified as Paecilomyces lilacinus. Purpureocillium lilacinum(This has been published in Tingfei Sun, Jianhui Wu and Shaukat, Morphological and molecular identification of four) Purpureocillium isolatesandevaluating their efficacy against the sweetpotato whitefly, Bemisiatabaci (Genn.)(Hemiptera: Aleyrodidae). Egyptian Journal of Biological Pest Control(2021) 31:27(https: / / doi.org / 10.1186 / s41938-021-00372-y).

[0061] I. Extraction of Cyclosporine C Toxin

[0062] 1. Insect culture:

[0063] Whiteflies were bred on upland cotton (maintained at 26±1°C, 70±10% relative humidity, with a photoperiod of 14 hours: 10 hours of darkness) as outlined by Sun et al.

[10] .

[0064] 2. Fungal inoculum and culture conditions

[0065] The strain used was Paecilomyces lilacinus strain XI-5, isolated from soil. Following the method of Ali et al.

[26] , this fungal strain was cultured in potato dextrose agar (PDA) medium. The basic fungal suspension used in this experiment was obtained from PDA plates (cultured for 10 days) containing 0.05% Tween-80 in ddH2O, filtered through filter paper (Whatman No.; Scientific Kit and Bore Al Laboratory, New York, USA) and placed in sterile vials. The spore suspension was prepared to 1 × 10⁻⁶ spores by counting with a hemocytometer and observing under a microscope. 7 The concentration of conidia per ml.

[0066] 6 ml of conidial suspension (1×10) 7 Conidia ( / ml) were added to a 500ml Erlenmeyer flask containing 150ml of sterile culture medium (per liter) consisting of 30g sucrose, 5g tryptone, 5g yeast extract, 10g Na2CO3, 1g K2HPO4 and 0.2g MgSO4·7H2O, at pH 8.5, and then incubated at 125 rpm and 27°C for 2 weeks.

[0067] 3. Extraction of secondary metabolites from Paecilomyces lilacinus

[0068] Fungal cultures (after 2 weeks of growth) were subjected to vacuum filtration to remove mycelia, and the pH of the supernatant was adjusted to 3.0. Bioactive compounds were extracted using an equal volume of ethyl acetate as solvent by ultrasonic extraction at room temperature for 20 min, followed by incubation at 4°C for 24 h. The organic phase (pale yellow) was separated from the fermentation broth, and ethyl acetate was then separated from the extract by rotary vacuum evaporation at 65°C. The dried extract, appearing as a brown paste, was used for further analytical studies.

[0069] 4. Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis

[0070] LC-MS analysis was performed using an LC-MS / MS system comprising an Agilent 1290 LC-MS / MS system with a binary pump, an autoinjector, a binary high-pressure mixing gradient pump, four solvent selection valves, a vacuum degasser, a plunger cleaning device, a thermostat, and a DAD UV detector, coupled with an Agilent 6540BQ-TOF equipped with AJSESI and APCI. The obtained toxins were dissolved in isopropanol and filtered through a 0.22 μm filter. Chromatographic separation of the compounds was performed on an ECLIPSPLUS C18 (1.8 μm, 100 × 2.1 mm) column. The mobile phases were as follows: methanol as phase A, and water (0.2% acetic acid and 10 mM ammonium acetate) as phase B. The flow rate was 100 µL min−1 in all steps. The detection wavelength was 215 nm. The injection volume was 5 µL, and ESI positive and negative analyses were performed using the Agilent 6540BQ-TOF.

[0071] The chromatogram of the ethyl acetate extract of *Paecilomyces lilacinus* strain XI-5 showed that most of the detected peaks had retention times of 6–11 minutes. Due to the high sample concentration, the chromatogram also showed overlapping peaks. Figure 1 The sample was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) to collect one fraction within 7.8–7.9 minutes. The collected fraction was further analyzed by LC-MS, such as… Figure 2 As shown, the amount of chemical components present was determined. Cyclosporine C was identified as having a molecular weight of 1216.82 (…). Figure 2 ).

[0072] 5. Fourier Transform Infrared Spectroscopy (FTIR)

[0073] Secondary metabolites were further characterized by Fourier transform infrared spectroscopy (FTIR). The Fourier transform infrared spectroscopy results were measured using a MIR8035 FTIR spectrometer (Thermo Fisher, Germany). To obtain the functional group structures of the active substances, all measurements were performed at a resolution of 4 cm⁻¹ in the frequency range of 400 to 4000 cm⁻¹. Liquid samples were used for analysis, and spectra were recorded at room temperature.

[0074] FTIR analysis confirmed the major functional groups of the composite material ( Figure 3 (See Table 6). FTIR analysis of the secondary metabolites showed a significant band at 3380 cm⁻¹ (due to OH alcohols or phenols), 1713.67 cm⁻¹ (C=O carboxylic acids), 1566.20 cm⁻¹ (C=C alkanes), 1414.53 cm⁻¹ (C=O trans alkenes), 1266.43 cm⁻¹ (C=C ester carbonyls), 1059.64 cm⁻¹ (CO trans alkenes), and small amounts of C=C aromatics at 885.96, 820.35, 779.88, 618.76, and 456.61 cm⁻¹.

[0075] Table 6. Infrared spectra of ethyl acetate extract of Paecilomyces lilacinus strain XI-5.

[0076] <![CDATA[Observed wave number (cm −1 )]]> Functional groups Key mode 3380.60 OH stretch alcohols or phenols Strong, very broad 1713.07 C=O carboxylic acid Strong, sharp 1566.20 C=C stretch alkane Weak, sharp 1414.53 C=O trans alkenes Weak, broad 1266.43 C=C ester carbonyl group Weak, broad 1059.64 CO trans-alkenes Strong, sharp 885.96 C=C aromatics Weak, broad 820.35 C=C aromatics Strong, sharp 779.88 C=C aromatics Strong, sharp 618.78 C=C aromatics (Ring) Weak, sharp 456.61 C=C Aromatics (Ring) Weak, sharp

[0077] 6. Nuclear Magnetic Resonance (NMR)

[0078] Following the method of Wu et al.

[22] , nuclear magnetic resonance (NMR) was performed using a Bruker advance III-HD 600 NMR spectrometer (Bruker, Karlsruhe, Germany). The sample was dissolved in dimethyl sulfoxide (DMSO), and NMR spectra were obtained at 25 °C. The working frequency for protons (¹H) was 500 MHz, and the working frequency for carbon (¹³C) was 125 MHz. A 5 mm triple resonance reverse probe was used.

[0079] 1H NMR analysis of metabolites produced by secondary metabolites of *Paecilomyces lilacinus* strain XI-5 revealed low-resonance bands in the aliphatic region within the range of 0.97–1.23 ppm and high-resonance bands in the aliphatic region within the range of 1.87 ppm. Low-resonance bands were observed in the allyl, pyrrolidine ring and NeCH (2.3–2.5 ppm), diethyl ether bond (3.2–3.8 ppm), allyl proton (5.72 ppm), and amide proton (6.66–7.27 ppm) regions. These findings further confirm the production of metabolites detected by LC-MS analysis. Figure 4 ).

[0080] The crude extract and purified cyclosporine C were diluted in isopropanol and subjected to high-performance liquid chromatography (HPLC) at 35 °C using an LC-20AD HPLC system (Shimadzu Chromatography Instruments, Kyoto, Japan) and an Ultimate XB-C18 column (4.6 × 150 mm, 5 µm). Acetonitrile was used as the mobile phase at a flow rate of 1.0 mL / min. The injection volume was 5 µL. The compound peak was detected at a wavelength of 220 nm. Cyclosporine C was used as a standard. The amount of specific compounds similar to the standard is expressed in mg / mL.

[0081] II. Toxicity determination of crude toxin and cyclosporine C toxin against whiteflies

[0082] 1. Pathogenicity test for whiteflies

[0083] The crude toxin extracted above was dissolved in isopropanol and filtered through a 0.22 μm filter membrane. Then, it was prepared into five concentration gradients of 2, 4, 8, 16, 32.5, 75, and 150 μg / mL using a feeding solution (15% sucrose water). The feeding solution was then transferred to the feeding experimental device.

[0084] Similarly, cyclosporin C was diluted with dimethyl sulfoxide (DMSO) to a concentration of 5 mg / mL to prepare a stock solution, which was stored at -20°C for later use. The cyclosporin C stock solution was diluted with feeding solution (15% sucrose solution) to five concentration gradients of 10, 20, 30, 40, and 50 μg / mL, filtered through a 0.22 μm filter membrane, sterilized, and then transferred to the feeding experimental apparatus.

[0085] The experimental setup consisted of a transparent double-ended glass tube wrapped in black paper, with one end aligned with the bottom of the tube and the other end 1 cm from the top to encourage whiteflies to gather towards the feed-containing end. The top of the tube was sealed with parafilm, and 200 μl of cyclosporine C at different concentration gradients was added to the top using a pipette. The tube was then covered with the sealing film again, forming a "sandwich" structure. During the experiment, 50 adult whiteflies, approximately 7 days after emergence, were transferred from the bottom to the experimental setup. The sealing film was immediately closed, and small holes were punctured with a dissecting needle to maintain ventilation. The feeding setup was placed in a climate chamber at 26±1℃, relative humidity 55±10%, and photoperiod L:D = 14:10 h. Each treatment was replicated in triplicate, with a control group fed a diet without cyclosporine C. After 24 h, the number of dead whiteflies in each treatment was counted, and the LC50 of cyclosporine C on adult whiteflies over 24 h was calculated. 50 .

[0086] The results showed that 24 hours after application, the mortality rate of adult whiteflies induced by the crude toxin increased with increasing concentration. Figure 5 The mortality rate of adult whiteflies induced by different concentrations of cyclosporine C increased with increasing concentration. Figure 6 ).

[0087] 2. Effects of cyclosporine C on oviposition in adult whiteflies

[0088] The LC30 concentration of cyclosporine C (20.00 µg / mL) against adult whiteflies, calculated using the methods described above, was used to investigate the effect of cyclosporine C on the oviposition capacity of whiteflies. A transparent double-ended glass tube, wrapped in black paper, was used, with one end aligned with the bottom of the tube and the other end 1 cm from the top, to encourage whiteflies to gather towards the end containing feed. The top of the tube was sealed with parafilm, and 200 μl of cyclosporine C at different concentration gradients was added to the top using a pipette. The tube was then covered with the sealing film again, forming a "sandwich" structure. During the experiment, 50 adult whiteflies, approximately 7 days after emergence, were inhaled and transferred from the bottom to the experimental setup. The sealing film was immediately replaced, and small holes were punctured with a dissecting needle to maintain ventilation. The feeding device was placed in a climate chamber at 26±1℃, relative humidity of 55±10%, and a photoperiod of L:D = 14:10 h. Twenty-four hours later, surviving whitefly adults were transferred to fresh cotton leaves to lay eggs. The number of eggs laid was observed every 5 days using transmission electron microscopy. Data analysis was performed using the Abbott formula to calculate insect mortality (%), which was then corrected for against control mortality

[28] . LC30 and LC50 values ​​were calculated using probability analysis. SAS 9.2 was used for all statistical analyses

[29] .

[0089] The LC30 concentration of cyclosporine C significantly affected the reproductive capacity of whiteflies at different time intervals after application. After 15 days of cyclosporine C treatment, the average egg production of female whiteflies was 16.54 eggs / female, while the reproductive capacity of female whiteflies in the control treatment was 29.5 eggs / female. Figure 7 ).

[0090] In summary, this invention studies the characteristics of cyclosporine C produced by secondary metabolites of Paecilomyces lilacinus strain XI-5 and its toxicity to whiteflies. LC-MS analysis showed that cyclosporine C (molecular weight = 1216.13) was detected within a retention time of 7.866 minutes. This finding differs from that of Burhan et al.

[20] . Burhan et al. showed that different amino acids and other secondary metabolites could be produced within a retention time of 5–16 minutes.

[0091] FTIR analysis of ethyl acetate mycelial extracts showed distinct bands for OH alcohols or phenols, C=O carboxylic acids, C=C alkanes, C=O trans-olefins, C=C ester carbonyl groups, CO trans-olefins, C=C aromatics, and aromatic rings. The functional groups observed in this study differed from the FTIR spectra of secondary compounds of Cordyceps fumarate observed by Wu et al.

[22] . The FTIR spectra of Cordyceps fumarate contained NH alkanes (methyl), CH alkanes (methylene), C≡ stretching nitrile, C=O carboxylic acids, C=C ester carbonyl groups, C=C ethers / alcohols, and C=C vinyl olefins, in addition to the functional groups observed in this study. These changes may be related to the differences in the molecular structure and molecular weight of the compounds detected in the two studies.

[0092] Metabolic compounds produced by secondary metabolites of Paecilomyces lilacinus strain XI-5 1 HNMR analysis showed that the resonance bands in the aliphatic region were low (0.97-1.23 ppm) and high (1.87 ppm). Low resonance bands were also observed in the aliphatic, allyl, pyrrolidine ring and NeCH (an ether bond) and amide proton regions. These results are consistent with those of Metarhizium anisopliae

[23] . 1 Similar to the HNMR spectrum, and comparable to that of *Rhizopus rosenbergii*

[22] . 1 The HNMR spectra are different.

[0093] Cyclosporine C exhibits dose-dependent stomach poisoning in adult whiteflies. 24 hours after application, the LC50 concentration of cyclosporine C in adult whiteflies was 31.75 µg / ml, lower than the LC50 observed by Wang et al. for Verticillium spp. toxin (649 µg / ml)

[24] . The results further indicate that, compared with the control group, LC30 concentrations of cyclosporine C resulted in reduced oviposition / reproductive capacity in female whiteflies. The inhibition of whitefly fertility may be related to the ovarian destructive effect of cyclosporine C

[25] .

Claims

1. A method for extracting crude toxin containing cyclosporine C from Paecilomyces lilacinus strain XI-5, comprising the following steps: 1) Inoculate a spore suspension of Paecilomyces lilacinus strain XI-5 into CM liquid medium and culture with shaking; 2) Obtain the bacterial solution by vacuum filtration, adjust the pH of the bacterial solution to 2.5-4.0, and add the same volume of ethyl acetate for extraction; 3) The organic phase was separated and the ethyl acetate was distilled using a rotary evaporator to obtain the secondary metabolites of the strain, which contained crude toxin C cyclosporine.

2. The method for extracting toxins from Paecilomyces lilacinus strain XI-5 as described in claim 1, characterized in that, Further includes: The crude toxin obtained was dissolved in isopropanol, filtered through a 0.22 μm filter membrane, and then analyzed by ESI positive and negative ion analysis.

3. The method for extracting toxins from Paecilomyces lilacinus strain XI-5 as described in claim 1, characterized in that, The spore suspension of Paecilomyces lilacinus strain XI-5 was inoculated into CM liquid medium with shaking culture to achieve a concentration of 1.0 × 10⁻⁶. 6 Spores / mL, then placed in a shaking incubator at 27°C and 125 rpm for 2 weeks.

4. The method for extracting toxins from Paecilomyces lilacinus strain XI-5 as described in claim 3, characterized in that, The steps before shaking culture include: The Paecilomyces lilacinus strain XI-5 was inoculated onto PDA medium and cultured for 10 days at 26 ± 1℃ and L:D = 16:8 until it was fully mature. After conidia are produced, the mycelium is scraped into a 0.05% Tween-80 solution using a small spatula, and then thoroughly stirred with a magnetic stirrer to ensure even dispersion of the conidia. The final solution is prepared as 1.0 × 10⁻⁶ ppm. 8 A suspension with a spore / mL concentration was then inoculated into CM liquid culture medium.

5. The method for extracting toxins from Paecilomyces lilacinus strain XI-5 as described in claim 1, characterized in that, Step 2) Obtain the bacterial solution by vacuum filtration, adjust the pH of the bacterial solution to 3.0, add the same volume of ethyl acetate for extraction for 24 h, place in a 4℃ low temperature freezer, and stir thoroughly every 4 h.

6. The method for extracting toxins from Paecilomyces lilacinus strain XI-5 as described in claim 1, characterized in that, Further, the extracted crude toxins were analyzed by LC-MS.

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