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Potential of fungal metabolites as a biocontrol agent against cotton aphid, Aphis gossypii Glover and the possible mechanisms of action

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The present study investigated the insecticidal activity of the different organic extracts from the en-tomopathogenic fungi, Cladosporium cladosporioides, Metarhizium anisopliae, Purpureocillium lilacinum, and Trichoderma longibrachiatum towards
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  Contents lists available at ScienceDirect Pesticide Biochemistry and Physiology  journal homepage: www.elsevier.com/locate/pest Potential of fungal metabolites as a biocontrol agent against cotton aphid,  Aphis gossypii  Glover and the possible mechanisms of action Azza A. Elbanhawy a , Elsherbiny A. Elsherbiny b, ⁎ , Ahmed E. Abd El-Mageed a ,Gamal M. Abdel-Fattah c a  Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt  b  Plant Pathology Department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt  c  Botany Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt  A R T I C L E I N F O  Keywords: Aphis gossypii Biological controlFungal metabolitesGC – MS, biochemical characteristicsEnzymes activity A B S T R A C T The present study investigated the insecticidal activity of the di ff  erent organic extracts from the en-tomopathogenic fungi,  Cladosporium cladosporioides, Metarhizium anisopliae, Purpureocillium lilacinum , and Trichoderma longibrachiatum  towards cotton aphid,  Aphis gossypii . The methanol extracts from the mycelia andspores of   C. cladosporioides and P. lilacinum  exhibited the highest insecticidal activity against  A. gossypii  com-pared with other extracts, which LC 50  values were recorded to be 57.60 and 94.18ppm, respectively. The majorconstituents identi 󿬁 ed in both methanol extracts by GC – MS analysis were linoleic acid and palmitic acid. Themethanol extracts of   C. cladosporioides  and  P. lilacinum  caused a voluminous increase in the total carbohydratescontent of   A. gossypii  adults, while the total protein content was signi 󿬁 cantly decreased by both extracts. Theactivity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were signi 󿬁 cantly reduced bymethanol extracts. The  P. lilacinum  extract caused a considerable reduction in the activity of glutathione-S-transferase (GST),  α - and  β -esterase by 28.9, 27.9 and 23.4%, respectively. Both extracts induced a signi 󿬁 cantincrease in phenoloxidase and chitinase activity of   A. gossypii  adults. These results suggest that  C. cladosporioides and  P. lilacinum  methanol extracts could be used as a promising approach for the management of   A. gossypii  inmany economically crops. 1. Introduction  Aphis gossypii  Glover (Hemiptera: Aphididae) is a profoundly severepest worldwide that invade>200 economically important crops, in-cluding cotton, okra, cucumbers, pepper, eggplant, squash, pumpkin,asparagus, hibiscus and cantaloupes (Razmjou et al., 2012). This pestcan cause direct damages by continuous sucking of sap from the plantvascular tissues, which produces many physiological disorders in theplant, and transmission>75 plant viruses as well as inhibiting thephotosynthetic ability by secrete a large amount of honeydew thatprovides a rich medium for the growth of sooty molds (Blackman andEastop, 2000).  A. gossypii  is usually controlled with widespread appli-cation of chemical pesticides such as carbamates, neonicotinoids, pyr-ethroids, and organophosphates, but the indiscriminate and extensiveuse of these synthetic chemicals causes disruption of the biologicalcontrol system and the emergence of strains resistant to many in-secticides with several economic and health problems (Cao et al.,2008).In recent years, many researchers have focused on looking for nat-ural products extracted from plants or microorganisms as alternativesto the conventional chemical insecticides through integrated pestmanagement programs (IPM). One of the most important alternatives tosynthetic pesticides is the microbial agents that are natural insect pa-thogens and having a potential e ff  ect in pest management (Ravindranet al., 2018). More than 700 species of fungi are reported as naturalenemies of insects and only a few species are contributing to the reg-ulation of their host populations (Roy and Pell, 2010). The mode of action of entomopathogenic fungi such as  Metarhizium anisopliae, Beauveria bassiana, Isaria fumosorosea, Nomuraea rileyi, Lecanicillium le-canii, Purpureocillium  sp. and  Cladosporium  sp., involves the productionof enzymes, toxic proteins, and bioactive secondary metabolites toovercome the insect immune system and modify the host behavior(Isaka et al., 2005; Ortiz-Urquiza and Keyhani, 2013). Secondary metabolites, secreted by entomopathogenic fungi, are arich source of bioactive chemicals, including polyketides, non-ribo-somal peptides, polyketide-peptide hybrid metabolites and terpenes https://doi.org/10.1016/j.pestbp.2019.05.013Received 20 March 2019; Received in revised form 15 May 2019; Accepted 20 May 2019 ⁎ Corresponding author at: Plant Pathology Department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt.  E-mail address:  sherbiny@mans.edu.eg (E.A. Elsherbiny). Pesticide Biochemistry and Physiology 159 (2019) 34–40Available online 22 May 20190048-3575/ © 2019 Elsevier Inc. All rights reserved.    (Fox and Howlett, 2008). Several of these secondary metabolites havebeen reported to have antifeedant and insecticidal properties (Bandaniet al., 2000; Molnar et al., 2010). For example, destruxins, which are cyclic peptides, secreted by  M. anisopliae  and responsible for muscledepolarization by the direct opening of the membrane Ca 2+ channelscausing an initial tetanic paralysis of the insects (Ruiz-Sanchez et al.,2010). Also, efrapeptin peptides, extracted from  Tolypocladium  sp., areknown to have an inhibitory e ff  ect on mitochondrial ATPase (Krasno ff  et al., 1991; Hayakawa et al., 2008). Besides, cordycepin isolated from the fruiting body of   Cordyceps militaris  showed an insecticidal activityagainst  Plutella xylostella  (Kim et al., 2002). In this context, the en-tomopathogenic fungi exhibited the ability to secrete some proteintoxins. For instance, hirsutellin A was puri 󿬁 ed from the mite fungalpathogen,  Hirsutella thompsonii  (Mazet and Vey, 1995). Another toxicprotein, bassiacridin, was isolated from  B. bassiana  (Quesada-Moragaand Vey, 2004).The present study was performed to evaluate the toxicological ef-fects of secondary metabolites extracted from some entomopathogenicfungi on cotton aphid,  A. gossypii  as well as the impact on some bio-chemical characteristics and the activity of some defensive enzymes of   A. gossypii . 2. Materials and methods  2.1. Extraction of fungal metabolites Four fungal strains were obtained from Assiut University,Mycological Centre (AUMC), Assuit, Egypt. The isolates were Cladosporium cladosporioides  AUMC 5125,  Metarhizium anisopliae  AUMC5130,  Purpureocillium lilacinum  AUMC 8824, and  Trichoderma long-ibrachiatum  AUMC 5990. Fungal spores were harvested from activelygrowing cultures (7 – 15days) on potato dextrose agar (PDA) by addingsterile distilled water onto culture plates and gently scraping the cul-tures with a glass rod. The number of spores in suspension was adjustedto 1×10 6 spores/ml using a haemocytometer. One ml of a spore sus-pension of each fungus was transferred to 2l  󿬂 asks containing 1000mlof potato dextrose broth (PDB). The  󿬂 asks were incubated at 25°C inthe dark under static conditions for 40days. The liquid cultures were 󿬁 ltrated through three layers of cheesecloths, then dried to 10% of itssrcinal volume using a rotary evaporator. The concentrated liquidswere  󿬁 ltered through two layers of Whatman No.1  󿬁 lter paper using avacuum pump. The separated fungal mycelial mats were air dried andground to  󿬁 ne powders in a grinder. The concentrated culture  󿬁 ltratesof each fungus were separately extracted with ethyl acetate three timesusing separating funnel in ratio of (1:1 (v ∕ v) solvent:  󿬁 ltrate), whilepowders of mycelial mats and spores were soaked and extracted threetimes with methanol in ratio (1:3 (w ∕  v) mycelial mat: solvent). Bothorganic extracts were  󿬁 ltered over anhydrous sodium sulphate(Na 2 SO 4 ) to remove water from the solvent. The extracts were subjectedto dryness using a rotary evaporator (60 – 65°C) to remove any traces of solvents and to obtain a crude extract.  2.2. Toxicity bioassay  Cotton aphids,  A. gossypii  adults were collected from infested un-sprayed leaves of cotton plants in the Plant Protection ResearchInstitute, Mansoura, Egypt. The aphids were reared on seedlings of cotton var. Giza 86 grown in plastic pots in a greenhouse at 25 ± 2°C.The insect colonies were kept on rearing seedlings without any ex-posure to insecticides until reaching the adult stage. To determinewhether aphids were directly a ff  ected by the fungal extracts, the slide-dipping technique as described by Stribley et al. (1983) was used. Apart of the double face scotch tape was  󿬁 xed tightly to the glass slide.Ten adult aphids were a ffi xed to the tape on their backs. The slides weredipped in the fungal organic extracts, dissolved in sterile distilled waterwith Tween 80, at di ff  erent concentrations for 10s. The treatmentwithout fungal extracts was used as a control. The aphids were main-tained at 25 ± 2°C and 70% relative humidity. The mortality per-centage was recorded after 24h. The insects were considered to be alivewhen aphids were responded to touching with the  󿬁 ne brush. Eachconcentration contained three replicates (10 adults per replicate) andthe experiments were conducted twice. The corrected mortality (%) wascalculated using Abbott ʼ s formula (Abbott, 1925). The toxicity indexwas determined as follow: Toxicity Index (%)=(LC 50  of the most ef-fective fungal extract / LC 50  of the tested fungal extract)×100.  2.3. Gas chromatography   –  mass spectrometry (GC   –   MS) analysis The methanol extracts of   C. cladosporioides  and  P. lilacinum  wereanalysed using an Agilent 6890 gas chromatography (Agilent, USA)prepared with fused silica capillary column PAS-5MS(30m×0.32mm×0.25 μ m  󿬁 lm thickness), because these extractswere the most e ff  ective against  A. gossypii  according to the results ob-tained after the toxicity experiments. The carrier gas was helium at aratio of 1ml/min., pulsed splitless mode. The solvent delay was at3min, and the size of the injection was 1 μ l. The mass spectrometricdetector was operated in electron impact ionization mode with EI of 70eV ionization voltage over the range of   m /  z   50 to 500. The EMvoltage was adjusted at 1650V. The ionization temperature was 230°C.The program temperature was initially adjusted at 60°C (2min) thenraised to 280°C at 5°C/min. The injector and detector temperatureswere kept at 250 and 280°C, respectively. Identi 󿬁 cation of the com-pounds was made by comparing their mass spectral patterns with thoseof WILEY/NIST mass spectral database.  2.4. E   ff  ect on total carbohydrates, total proteins and triglycerides The total carbohydrates content was determined with the phenol-sulfuric acid method using glucose as a standard. One gram of aphidstreated with LC 50  of   C. cladosporioides  and  P. lilacinum  methanol ex-tracts was homogenized at 0°C in 5ml of 0.3N HClO 4  for 1min sepa-rately, and then the insects were preserved in ice for 10min. The ob-tained insoluble residues were separated by centrifugation at 12000  g  for 3min. The supernatants were washed twice with 5ml of ice-coldHClO 4  to obtain the acid extract. One hundred  μ l of acid extract weremixed with 0.5ml of 20% phenol, and then 5ml of sulfuric acid wereadded quickly with shaking for 10min. The mixture was incubated at25 – 30°C for 10 – 20min with shaking. The absorbance was measured at490nm using a T80 UV/VIS spectrophotometer (PG Instruments Ltd.,Leicestershire, England), and the total carbohydrate was expressed asmilligrams per gram of insects.For total protein content, the insects treated with LC 50  of bothmethanol extracts were homogenized in distilled water (50mg /1ml),and then centrifuged at 12000  g   for 15min at 2°C. The supernatant wascollected as a crude enzyme extract. Protein content in samples wasdetermined according to Bradford (1976) using bovine serum albumin(BSA) as standard. Protein reagent was prepared by dissolving 100mgof Coomassie Brilliant blue G-250 in 50ml of 95% ethanol and 100mlof 85% phosphoric acid. The obtained solution was diluted to a  󿬁 nalvolume of 1l. Fifty  μ l of crude enzyme extract was mixed with 5ml of protein reagent. The absorbance was recorded after 2min at 595nmusing a spectrophotometer. The total protein was expressed as milli-grams per gram of insects.Triglycerides concentration was assayed using Stanbio kit (StanbioLaboratory Inc., San Antonio, Texas, USA). The reaction mixture con-tained 10 μ l of crude enzyme extract and 1ml of kit reagent. The re-action mixture was incubated at 37°C for 5min. The absorbance wasmeasured at 550nm using a spectrophotometer. Triglycerides wereexpressed as milligrams per gram of insects. The experiment was con-ducted twice with three replicates per treatment.  A.A. Elbanhawy, et al.  Pesticide Biochemistry and Physiology 159 (2019) 34–40 35   2.5. E   ff  ect on enzymes activity  The aphids treated with methanol extracts of   C. cladosporioides  and  P. lilacinum  at LC 50  were homogenized in distilled water (50mg /1ml)separately. The mixture was centrifuged at 12000  g   for 15min (2°C),and then the supernatant was collected as a crude enzyme extract.Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)activities were determined according to Valero and Garcia-Carmona(1998). The reaction mixture consisted of 0.2ml of crude enzyme ex-tract mixed with 1ml of 0.1M phosphate bu ff  er (pH7.4), 29mg of 0.2mM  α -ketoglutaric acid and 2.6g of 200mM DL aspartate per liter.After incubation for 30min, 1ml of 2,4-dinitrophenylhydrazine reagentwas added. Ten ml of 0.4N NaOH was added after 30min. The ab-sorbance was measured after 5min at 505nm. The activity of the en-zymes was expressed as U/min/mg protein.The  α -esterase and  β -esterase activities were determined accordingto the method of  Zheng et al. (2014) using  α - and  β -naphthyl acetate assubstrates, respectively. Five ml of substrate solution (120 μ l of 3.0mM α - or  β -naphthyl acetate dissolved in 1% acetone and 12ml of 0.1Mphosphate bu ff  er, pH7) was mixed with 20 μ l of crude enzyme extract.The reaction mixture was incubated at 27°C for 30min. One ml of diazoblue laurylsulphate solution (DBLS) was added. The absorbancewas recorded at 600 and 555nm for  α -esterase and  β -esterase, re-spectively. The  α - and  β -esterase activities were expressed as  μ g/min/mg protein.The activity of glutathione-S-transferase (GST) was estimated ac-cording to Vontas et al. (2000). The reaction mixture consisted of 1mlof phosphate bu ff  er (pH6.5), 100 μ l of reduced glutathione (GSH), and20 μ l of crude enzyme extract. The GST reaction was started by adding25 μ l of 1mM 1-chloro 2,4-dinitrobenzene (CDNB) as a substrate. Thereaction mixture was incubated for 5min at 30°C. The absorbance wasmeasured at 540nm. The enzyme activity was expressed as  μ M/min/mg protein.For phenoloxidase, 20 μ l of crude enzyme extract was mixed with0.5ml of 0.2M phosphate bu ff  er (pH7), 0.5ml of H 2 O, and 200 μ lcatechol solution (2%) according to the modi 󿬁 ed method of  Laughtonand Siva-Jothy (2011). The enzyme reaction was started when the ca-techol solution was added. After 1min, the absorbance was recorded at420nm. The enzyme activity was expressed as U/min/mg protein.For chitinase, 200 μ l of crude enzyme extract were mixed with 1mlof 0.2M phosphate bu ff  er (pH6.5) and 200 μ l of 0.5% colloidal chitin.After incubation at 37°C for 60min, the enzyme activity was termi-nated by adding 1.2ml of DIVSA reagent. The reaction mixture washeated for 5min, and then cooled in ice. Then, the undigested chitinwas removed by centrifugation at 12000  g   for 15min as described byKoga et al. (1997). The supernatant was adjusted to 1ml with 0.2Mphosphate acetate bu ff  er (pH6). Series concentrations of   N  -acet-ylglucoseamine standards were prepared and mixed with 1ml of thebu ff  er and 0.3ml saturated sodium borate solution. The mixture washeated for 10min and transferred rapidly to cold water. Eight ml of glacial acetic acid was added with 1ml of Ehrlich reagent (1g of   p -dimethylaminobenzoate dissolved in 50ml of glacial acetic acid and2.5ml of HCl). After 30min, the absorbance was measured at 550nm,and the enzyme activity was expressed as  μ g/min/mg protein. Theexperiments were repeated twice with three replicates for each treat-ment.  2.6. Statistical analysis The data were subjected to one-way analysis of variance (ANOVA).The analysis was conducted using SAS software (version 9.1, SASInstitute, Cary, NC, USA). The signi 󿬁 cant di ff  erence of the means wasdetermined by Fishers' least signi 󿬁 cant di ff  erence (LSD) test at  P   < .05. The LC 50  and LC 90  values of all fungal organic extracts weredetermined using probit analysis. 3. Results 3.1. Toxicological e  ff  ects of fungal organic extracts The methanol extract of   C. cladosporioides  was the most e ff  ectivefungal extract against  A. gossypii  adults followed by  P. lilacinum  me-thanol extract, which recorded 100 and 61.16% toxicity index, re-spectively (Table 1). In contrast,  T. longibrachiatum  methanol extractexhibited only 3.70% toxicity index. Also,  C. cladosporioides  methanolextract recorded 57.60 and 1431.86ppm for LC 50  and LC 90 , respec-tively (Table 1). Among ethyl acetate extracts,  T. longibrachiatum  wasthe most e ff  ective extract on  A. gossypii  adults with toxicity index by39.03% (Table 2), while  P. lilacinum  extract had the lowest toxicityindex by 4.04%. The LC 50  and LC 90  values were recorded to be 147.55and 4526.00ppm for ethyl acetate extract of   T. longibrachiatum , re-spectively (Table 2). 3.2. GC   –   MS analysis The compounds identi 󿬁 ed from  C. cladosporioides  and  P. lilacinum methanol extracts are listed in Tables (3) and (4), respectively. Theanalysis of   C. cladosporioides  methanol extract led to the identi 󿬁 cationof 26 di ff  erent compounds, representing 92.60% of the total extract(Table 3). The main constituents detected were 9,12-octadecadienoicacid (Z,Z)- (linoleic acid) (30.70%), n-hexadecanoic acid (palmitic acid)(14.73%), 9,12-octadecadienoic acid, methyl ester, (E,E)- (14.04%),and hexadecanoic acid, methyl ester (10.96%). Octadecanoic acid(stearic acid) (4.19%), octadecanoic acid, methyl ester (methyl stea-rate) (2.84%), and ergosterol (2.37%) were also detected in the extract.On the other hand, the components identi 󿬁 ed in  P. lilacinum  methanolextract were 19 di ff  erent compounds, representing 92.34% of the totalextract (Table 4). The major compounds detected were 9,12-Octade-cadienoic acid (Z,Z)- (linoleic acid) (32.68%), n-hexadecanoic acid(palmitic acid) (31.68%), and ergosterol (8.01%). Table 1 Toxicity of fungal methanol extracts on  Aphis gossypii  adults after 24h. Methanol extract No. aphids LC 50  (ppm)(95% CI)LC 90  (ppm)(95% CI)Slope ± SE Toxicity Index (% ± SE)  χ 2  P- value Cladosporium cladosporioides  300 57.60(51.2 – 64.6)1431.86(1387.4 – 1514.6)0.92 ± 0.1 100 ± 0.0 1.90 0 0.38  Metarhizium anisopliae  300 196.96(189.6 – 211.7)1191.75(1176.3 – 1312.7)1.6 ± 0.2 29.24 ± 1.7 5.48 0.06  Purpureocillium lilacinum  300 94.18(87.4 – 117.4)7282.74(7142.5 – 7423.7)0.68 ± 0.1 61.16 ± 1.2 6.15 0.10 Trichoderma longibrachiatum  300 1553.81(1524.6 – 1583.4)8221.08(7653.7 – 9275.2)1.7 ± 0.2 3.70 ± 1.5 5.15 0.16 95% CI, con 󿬁 dence interval at 95%; SE, standard error.  A.A. Elbanhawy, et al.  Pesticide Biochemistry and Physiology 159 (2019) 34–40 36  3.3. E   ff  ect on total carbohydrates, total proteins and triglycerides The treatment of   A. gossypii  adults with LC 50  of   C. cladosporioides and  P. lilacinum  methanol extracts exhibited signi 󿬁 cantly increase(  P   < .05) in the total carbohydrates content compared with the control(Table 5). Both methanol extracts caused a signi 󿬁 cant decrease(  P   < .05) in the total protein content of   A. gossypii  adults. However,there was no signi 󿬁 cant di ff  erence between the control and the treat-ment of   A. gossypii  by  P. lilacinum  methanol extract (Table 5). The LC 50 for  C. cladosporioides  and  P. lilacinum  methanol extracts exhibited non-signi 󿬁 cant decrease (  P   < .05) in triglycerides content with the reduc-tion by 2.58 and 2.75%, respectively compared with the control(Table 5). 3.4. E   ff  ect on enzymes activity  The methanol extracts of   C. cladosporioides  and  P. lilacinum  at LC 50 caused a signi 󿬁 cant reduction (  P   < .05) in the activity of alanineaminotransferase (ALT) by 75.93 and 10.49%, respectively in  A. gossypii adults. Likewise, both extracts caused 60.01 and 76.02% reduction,respectively, in the activity of Aspartate aminotransferase (AST) com-pared with the control (Table 6). A signi 󿬁 cant reduction (  P   < .05) in α -esterase activity was obtained with LC 50  of C . cladosporioides  and  P.lilacinum  methanol extracts by 14.41% and 27.85%, respectively(Table 6). Similarly, the activity of   β -esterase was signi 󿬁 cantly de-creased (  P   < .05) by both methanol extracts at LC 50 , while no sig-ni 󿬁 cant di ff  erence was found between the control and the methanolextract of C . cladosporioides . In contrast, C . cladosporioides  extractcaused a signi 󿬁 cant increase (  P   < .05) in the activity of glutathione-S-transferase (GST) by 9.03%, while,  P. lilacinum  extract signi 󿬁 cantlyreduced the enzyme activity by 28.86% compared with the control(Table 6). The phenoloxidase activity in  A. gossypii  adults was sig-ni 󿬁 cantly increased (  P   < .05) by 76.94% and 35.92% with the treat-ment by LC 50  of C . cladosporioides  and  P. lilacinum  methanol extracts,respectively (Table 6). Both extracts exhibited signi 󿬁 cantly increase(  P   < .05) in the activity of chitinase by 34.85% and 9.82%, respec-tively (Table 6). However, there was no signi 󿬁 cant di ff  erence between  P. lilacinum  methanol extract and control. 4. Discussion Entomopathogenic fungi produce a diverse array of bioactive com-pounds known as secondary metabolic compounds. The fungal meta-bolites have shown to possess potential insecticidal activity and might Table 2 Toxicity of fungal ethyl acetate extracts on  Aphis gossypii  adults after 24h. Ethyl acetate extract No. aphids LC 50  (ppm)(95% CI)LC 90  (ppm)(95% CI)Slope ± SE Toxicity Index (% ± SE)  χ 2  P- value Cladosporium cladosporioides  300 480.34(463.8 – 511.5)15,966.05(15,682.7 – 17,497.4)0.84 ± 0.2 11.99 ± 1.2 6.74 0.08  Metarhizium anisopliae  300 490.40(477.3 – 541.5)9392.96(9037.9 – 9965.6)0.97 ± 0.2 11.74 ± 1.5 4.87 0.18  Purpureocillium lilacinum  300 1423.89(1396.5 – 1512.7)696,228.63(663,795.6 – 779,475.5)0.48 ± 0.2 4.04 ± 1.5 2.56 0.46 Trichoderma longibrachiatum  300 147.55(130.1 – 166.6)4526.00(4254.7 – 5198.8)0.86 ± 0.2 39.03 ± 1.3 6.42 0.09 95% CI, con 󿬁 dence interval at 95%; SE, standard error. Table 3 GC – MS analysis of methanol extract of the mycelia and spores of   Cladosporium cladosporioides . No. Rt a Compound b Molecular formula Molecular weight Peak area (%)1 3.50 2,3-Butanediol C 4 H 10 O 2  90.12 0.182 10.97 L-Homoserine lactone,  N  ,  N  -dimethyl- C 6 H 11 NO 2  129.15 1.063 14.03 Glycerin C 3 H 8 O 3  92.09 1.574 16.11 Benzeneacetic acid C 8 H 8 O 2  136.14 0.505 24.45 Tridecanoic acid, 12-methyl-, methyl ester C 15 H 30 O 2  242.39 0.196 25.26 Methyl tetradecanoate C 15 H 30 O 2  242.39 0.167 26.34 Tetradecanoic acid C 14 H 28 O 2  228.37 0.358 26.81 Tetradecanoic acid, 12-methyl-, methyl ester C 16 H 32 O 2  256.42 1.009 27.41 Pentadecanoic acid, methyl ester C 16 H 32 O 2  256.42 0.3410 29.77 Hexadecanoic acid, methyl ester C 17 H 34 O 2  270.45 10.9611 30.20 9-Octadecenoic acid (  Z  )- (Oleic acid) C 18 H 34 O 2  282.46 0.4312 30.93 Octadecanoic acid (Stearic acid) C 18 H 36 O 2  284.47 4.1913 32.65 n-Hexadecanoic acid (Palmitic acid) C 16 H 32 O 2  256.42 14.7314 33.40 9,12-Octadecadienoic acid, methyl ester, (E,E)- C 19 H 34 O 2  294.47 14.0415 33.74 Octadecanoic acid, methyl ester (Methyl stearate) C 19 H 38 O 2  298.50 2.8416 35.99 9,12-Octadecadienoic acid (Z,Z)- (Linoleic acid) C 18 H 32 O 2  280.44 30.7017 36.60 11,14-Eicosadienoic acid, methyl ester C 21 H 38 O 2  322.52 1.4618 37.06 Hexadecanoic acid, 15-methyl-, methyl ester C 18 H 36 O 2  284.47 0.5719 37.38 8-Octadecenoic acid, methyl ester C 19 H 36 O 2  296.48 0.4120 38.07 9,17-Octadecadienal C 18 H 32 O 264.44 0.7121 40.30 2-Hydroxy-1-(hydroxymethyl)ethyl palmitate C 19 H 38 O 4  330.50 0.6122 40.54 Bis (2-ethylhexyl) phthalate C 24 H 38 O 4  390.55 0.5423 41.62 Tricosanoic acid, methyl ester C 24 H 48 O 2  368.63 0.0924 43.04 9-Octadecenoic acid (Z)-, 2-hydroxyethyl ester C 20 H 38 O 3  326.51 2.2425 46.71 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester C 21 H 40 O 4  356.53 0.3626 50.99 Ergosterol C 28 H 44 O 396.64 2.37 a Rt, retention time (min). b Compounds are listed in the order of their elution.  A.A. Elbanhawy, et al.  Pesticide Biochemistry and Physiology 159 (2019) 34–40 37  be used as biopesticides (Wang et al., 2007). In the present study, themethanol extracts of   C. cladosporioides  and  P. lilacinum  were more ef-fective against  A. gossypii  adults after 24h compared with other fungalmethanol and ethyl acetate extracts. There are no previous studies onusing organic extracts of   C. cladosporioides  and  P. lilacinum  for thecontrol of   A. gossypii . Nevertheless, many reports are showing the e ff  ectof some fungal organic extracts on other insects. For example, themethanol extract of   P. lilacinus  caused 100% mortality against  Aedescaspius  larvae after 24h (AL-Mekhla 󿬁 , 2017). Similarly, the ethylacetate extract of   Lecanicillium lecanii  exhibited 70% mortality at 2%and LC 50  value was 0.65% against  A. gossypii , while the methanol ex-tract of L.  lecanii  caused the insect mortality ranged from 24% to 82%(Gurulingappa et al., 2011). Also, spore suspension of   C. cladosporioides and  M. anisopliae  caused 81.6 and 98% mortality in spotted spider mite, Tetranychus urtica e after 7days at the concentration of 1×10 10 spores/ml, while the mortality was 72.5 and 87.1% for  T. cinnabarinus , re-spectively at the same concentration (Habashy et al., 2016).The results of this study showed that the fatty acids or their deri-vatives, fatty acid methyl esters (FAME) and fatty acid ethyl esters(FAEE), were the characteristic constituents of the methanol extracts of  C. cladosporioides  and  P. lilacinum  by 82.80 and 81.59%, respectively.The insecticidal activity of both methanol extracts could be due to themajor compounds or by the synergistic e ff  ects between the major andminor components. Several studies have shown the insecticidal andnematicidal properties of fatty acids. For instance, the fatty acidsidenti 󿬁 ed from Australian  P. lilacinus  isolates were linoleic, oleic, andlinolenic acids. Those fatty acids exhibited nematicidal activity against Caenorhabditis elegans  (Park et al., 2004). The unsaturated fatty acidlinoleic acid exhibited an insecticidal activity towards second andfourth instar larvae of   Spodoptera littoralis  with LC 50  values of 4.78 and9.11g/100ml, respectively (Yousef et al., 2013). In this context, themajor fatty acids extracted from the ripe fruits of   Melia azedarach  werelinoleic acid methyl ester, oleic acid methyl ester, oleic acid, stearicacid methyl ester, and palmitic acid. These fatty acids caused 100 and77.5% mortality of second and fourth instars of   S. littoralis , respectively(Farag et al., 2011).Carbohydrates are the main source of energy in most insects. Theyare required for many physiological processes such as synthesis of chitin, synthesis of sugar alcohols and may be converted to lipids withthe contribution to the production of amino acids (Kunieda et al.,2006). Our  󿬁 ndings showed that both the methanol extracts caused asigni 󿬁 cant increase in the levels of total carbohydrates of   A. gossypii adults. This increase may be a natural phenomenon because the tre-halose levels respond to the physiological conditions such as infection,starvation or applied some toxic material such as fungal extracts. Table 4 GC – MS analysis of methanol extract of the mycelia and spores of   Purpureocillium lilacinum (  formerly  Paecilomyces lilacinus) . No. Rt a Compound b Molecular formula Molecular weight Peak area (%)1 24.01 2(3H)-Furanone, dihydro-5-(2-octenyl)-, (Z)- C 12 H 20 O 2  196.28 0.202 26.99 Tetradecanoic acid C 14 H 28 O 2  228.37 0.213 29.05 Pentadecanoic acid C 15 H 30 O 2  242.39 0.974 29.45 Hexadecanoic acid, methyl ester C 17 H 34 O 2  270.45 1.875 30.74 Hexadecanoic acid, ethyl ester C 18 H 36 O 2  284.47 1.266 31.94 n-Hexadecanoic acid (Palmitic acid) C 16 H 32 O 2  256.42 31.687 32.79 9,12-Octadecadienoic acid, methyl ester, (E,E)- C 19 H 34 O 2  294.47 2.888 32.87 11-Octadecenoic acid, methyl ester C 19 H 36 O 2  296.48 1.069 32.99 Heptadecanoic acid C 17 H 34 O 2  270.45 0.9310 33.29 Octadecanoic acid, methyl ester (Methyl stearate) C 19 H 38 O 2  298.50 0.5011 33.92 Linoleic acid ethyl ester C 20 H 36 O 2  308.49 0.5812 34.02 Octadecanoic acid (Stearic acid) C 18 H 36 O 2  284.47 2.0913 35.15 9,12-Octadecadienoic acid (Z,Z)- (Linoleic acid) C 18 H 32 O 2  280.44 32.6814 37.81 Linoleic acid ethyl ester C 20 H 36 O 2  308.49 1.1015 40.20 Methyl dehydroabietate C 21 H 30 O 2  314.46 0.9616 40.38 Bis (2-ethylhexyl) phthalate C 24 H 38 O 4  390.55 0.5017 42.96 9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl) ethyl ester C 21 H 38 O 4  354.52 3.7818 46.03 9-Octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester C 21 H 40 O 4  356.53 1.0819 50.99 Ergosterol C 28 H 44 O 396.64 8.01 a Rt, retention time (min). b Compounds are listed in the order of their elution. Table 5 Total carbohydrates, total protein and triglycerides content of   Aphis gossypii adults treated with LC 50  of fungal methanol extracts. Methanol extract Totalcarbohydrates(mg/g)Total proteins(mg/g)Triglycerides(mg/g) Cladosporiumcladosporioides 4.82 ± 0.3 b ⁎  5.19 ± 0.2 b 188.66 ± 0.4 a  Purpureocillium lilacinum  5.23 ± 0.5 a 7.51 ± 0.3 a 188.33 ± 0.3 aControl 4.41 ± 0.3 c 8.23 ± 0.3 a 193.66 ± 0.2 a ⁎ Results represent mean ± standard error (SE). Values with di ff  erent lettersin the same column are signi 󿬁 cantly di ff  erent according to Fisher's test at  P   < .05. Table 6 Activity of defensive enzymes of   Aphis gossypii  adults treated with LC 50  of fungal methanol extracts. Methanol extract ALT(U/min/mgprotein)AST(U/min/mgprotein) α -esterase( μ g/min/mgprotein) β -esterase( μ g/min/mgprotein)GST( μ M/min/mgprotein)Phenoloxidase(U/min/mgprotein)Chitinase( μ g/min/mgprotein) Cladosporiumcladosporioides 39.0 ± 0.3 c ⁎  3720.0 ± 0.7 b 87.9 ± 0.4 b 77.6 ± 0.3 a 3448.3 ± 0.9 a 7.3 ± 0.3 a 366.3 ± 0.5 a  Purpureocillium lilacinum  145.0 ± 0.3 b 2230.3 ± 0.5 c 74.1 ± 0.3 c 61.2 ± 0.3 b 2250.0 ± 0.7 c 5.6 ± 0.5 b 298.3 ± 0.2 bControl 162.0 ± 0.2 a 9302.6 ± 0.3 a 102.7 ± 0.4 a 79.9 ± 0.3 a 3162.6 ± 0.4 b 4.1 ± 0.2 c 271.6 ± 0.3 b ALT, alanine aminotransferase; AST, aspartate aminotransferase; GST, glutathione-S-transferase. ⁎ Results represent mean ± standard error (SE). Values with di ff  erent letters in the same column are signi 󿬁 cantly di ff  erent according to Fisher's test at  P   < .05.  A.A. Elbanhawy, et al.  Pesticide Biochemistry and Physiology 159 (2019) 34–40 38
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