PertanikaJ. Trop. Agric. Sci. 25(2): 115 -120 (2002) ISSN: 1511-3701© Universiti Putra Malaysia Press

Free Fatty Acids on the Integument of the Striped Flea Beetle, Phyllotretastriolata F., and Their Effects on Conidial Germination of the

Entomopathogenic Fungi Metarhiziutn anisopiiae, Beauveriabassiana and Paecilomyces fumosoroseus

T.P. PRIYATNO & Y.B. IBRAHIMDepartment of Plant Protection,

Faculty of Agriculture,Universiti Putra Malaysia

43400 UPM, Serdang, Selangor, Malaysia

Keywords: Beauveria bassiana, conidial germination, free fatty acids, Metarhiziutn anisopliae,Paecilomyces fumosoroseus, Phyllotreta striolata

ABSTRAK

Asia1 lemak betas dart integumen kumbang lenting berjalur, Phyllotreta striolata F., dan kesannya he ataspercambahan konidium Metarhizium anisopliae (Metch.) Sorokin, Beauveria bassiana (Bals.) Vuill danPaecilomyces fumosoroseus (Wise) Brown & Smith telah dikaji. Analisis kromatograf gas ekstrak kutikelkumbang lenting dewasa menunjukkan dua puncak yang memaparkan jangka penahanan yang bersesuaiandengan asid butirik dan asid nonanoik. Lain-lain asid lemak yang telah dikesan ialah kaproik, heptanoik dankaprilik asid. Toksisiti asid lemak ini ke atas percambahan conidium M. anisopliae, B. bassiana dan P.fumosoroseus adalah bergantung kepada jenis kepekatannya. Butirik ialah yang paling kurang toksik, hanyatoksik ke atas B. bassiana dan P. fumosoroseus pada kepekatan melebihi 0.125%. Begitupun, pada kepekatanmelebihi 0.015%, kesemua asid lemak didapati terlalu toksik. Asid nonanoik didapati paling toksik dan iamerencatkan kesemua percambahan konidium pada kepekatan terendah 0.025%, terutamanya ke atas M.anisopliae dan P. fumosoroseus. Asid heptanoik didapati amat toksik ke atas B. bassiana denganmembenarkan hanya 12.5% percambahan pada kepekatan terendah 0.025%. Dirumuskan bahatoa asid lemakrantai lurus di atas integumen kumbang lenting melindungi kumbang itu daripada serangan kulat entomopatogen.

ABSTRACT

Free fatty acids on the integument of the striped flea beetle, Phyllotreta striolata F., and their effects on theconidial germination of Metarhizium anisopliae (Metch.) Sorokin, Beauveria bassiana (Bals.) Vuill. andPaecilomyces fumosoroseus (Wise) Brown & Smith were investigated. Gas chromatographic analysis of adultflea beetle cuticular extracts showed two peaks exhibiting a retention time corresponding to those of butyric acidand nonanoic add. Other fatty acids detected were caproic, heptanoic and caprylic acids. Toxicity of these fattyacids to conidial germinaton ofM. anisopliae, B. bassiana and P. fumosoroseus was dependent on their typeand concentration. Butyric was the least toxic-only toxic to B. bassiana and P. fumosoroseus at aconcentration higher than 0.125%. However, at concentrations greater than 0.075%, all of the fatty acids werevery toxic. Nonanoic acid was the most toxic and it completely inhibited conidial germination at the lowestconcentration of 0.025%, especially to M. anisopliae and P. fumosoroseus. Heptanoic acid was very toxicto B. bassiana, allowing only 12.5% germination at the lowest concentration of 0.025%. It is surmised thatstraight-chain fatty acids on the flea beetle integument protected the beetles against the invasion of entomopathogenicfungi.

INTRODUCTION ring via penetration of the integument was influ-Invasion of the striped flea beetle, Phyllotreta e n c e d bY b o t h chemical and physical propertiesstriolata F., by entomopathogenic fungi occur- of the integument (Samson ^oZ. 1988). Prior to

T.P. PRIYATNO & Y.B. IBRAHIM

such penetration it is necessary for the fungalconidia to germinate on the surface of the bee-tle. Epicuticular compounds such as fatty acids,amino acids and glucosamines were thought toplay a significant role in the germination proc-ess (Smith and Grula 1982; Saito and Aiko 1983;Boucisa and Pendland 1984; Kerwin 1984; Woodsand Grula 1984). Successful germination of thefungi requires the presence of utilisable nutri-ents and that either toxic components not bepresent in amounts sufficient to inhibit germina-tion, or that the organism be able to ignore orovercome them (Smith and Grula 1982).

Butt et al (1984) recently reported that fleabeetles and aphids are differentially susceptibleto the entomopathogenic M. anisopliae. Theaphids Myzus persicae and Lipaphis erysimi werehighly susceptible and died more quickly thanthe mustard beetle, Phaedon cochleariae, and theflea beetle, Psylliodes chrysocephala. The stripedflea beetle adult is also more resistant to Manisopliae, B. bassiana and P. fumosaroseus thanthe larvae (Priyatno and Ibrahim 2002). Differ-ences in susceptibility reflect the response of thepathogen to cuticular cues. According to Butt etal (1995), M anisopliae conidia germinate fasteron the surface of live aphids and at sites underthe elytra than on the dorsal elytra and theventral thorax of live beetles. Presence offungistatic compounds in the dorsal elytra andventral thorax could probably be responsible forprotect ing the insect against potentialentomopathogenic fungi, as attributed by poorconidial germination (Butt et al 1995). Themost toxic components of the insect cuticle arethe straight-chain saturated fatty acids such asbutyric, valeric, caproic, heptanoic, caprylic and

nonanoic acids (Koidsumi 1957; Smith and Grula1982; Saito and Aoki 1983). However, accordingto Samson et al (1988), straight-chain fatty acidsare not present in all insect orders; they areabsent from some Coleoptera and Homoptera(Brey et al 1985).

The aims of this study were to identify thefatty acids on the surface of the striped fleabeetle adults and to determine the effects ofsuch compounds on the conidial germination ofM anisopliae, B. bassiana and P. fumosoroseus,

MATERIALS AND METHODS

Fungus

The original, hosts and countries of origin forthe isolates of entomopathogenic fungi used inthis study are listed in Table 1. All isolates weremaintained at room temperature of 28 ± 2°C onpotato dextrose agar (PDA, Oxoid, Basingstoke,UK) containing 0.5% yeast extract (Difco) whichhad been sterilised for 20 minutes at 121°C at1.05 kg/cm2. To prepare fungal inocula, conidiafrom 15 day-old sporulating cultures were scrapedfrom the surface of the plates with a sterilescalpel and suspended in a 0.05% aqueous solu-tion of Tween 80. A Neubauer haemocytometerwas used to estimate the conidial concentrationand subsequent appropriate dilutions were made.

Insect

Field-collected flea beetles from Chinese mus-tard, Brassica juncea, in the vegetable plot at theUPM Research Park were used for variousbioassays. They were maintained at an ambientenvironment of 28 ± 2°C, 12:12 (L:D) h pho-toperiod and 80 ± 10% R.H. in plastic contain-

TABLE 1Isolates of M. anisopliae, B. bassiana and P. fumosoroseus, their original hosts and countries

of origin

Species

M. anisopliae

B. bassiana

P. fumosoroseus**

Code

Cy3

MPs

Wls

Pf

Insect host

Cylas formicariu(Curculionidae)

Phylbtreta striolata(Chrysomelidae)

Leptocorisa oratorius(Alydidae)

Pteroma pendula(Psychidae)

Country of origin

Indonesia

Malaysia

Indonesia

Malaysia

116 PERTANIKA J. TROP. AGRIC. SCI. VOL. 25 NO. 2, 2002

FREE FATTY ACIDS ON THE INTEGUMENT OF THE STRIPED FT .FA BEETLE

ers and provided with the same host plant as inthe field. The beetles were collected with anaspirator.

Lipid Extraction

Fatty acids were extracted from the adult beedesaccording to the method described by Smithand Grula (1982). A sample of 0.5 g of beetleswas placed in a beaker of distilled water andcontinuously agitated for 15 min with a mag-netic stirrer to remove any dirt. Insects werethen placed in a beaker containing 5 mLmethanolic-HCl (2.5% v/v, methanol/HCl) andshaken for 30 min. The extract was then filteredusing a vacuum suction pump through WhatmanNo. 1 filter paper using a rotary evaporator.The residue was then methylated for gaschromatography by the addition of 4 ml sodium-dried benzene, 4 ml 2,2-dimethoxypropane and0.5 ml of methanolic-HCl (2.5% v/v, me-thanol.HCl). The solution was kept at roomtemperature overnight after which time the re-sulting methyl esters would have evaporated.

Gas chromatography was accomplished us-ing HITACHI 263-50 equipped with a DEGScapillary column, with hydrogen as the carriergas. The temperature was held at 150°C forthree minutes after injection and then increasedto 250°C. The column was connected to aHITACHI D-2000 reader.

Bioassays

The medium used for conidial germination ofthe fungal pathogen was water agar (1.5% w/v,Bacto). A 5 ml sample of the medium wasplaced in a tube and autoclaved for 20 min at121°C at 1.05 kg/cm2. The medium was left tocool to 40°C. The hydrophobic butyric, caprylic,caproic, heptanoic and nonanoic acids as deter-mined through gas chromatography earlier, wereneutralised and made water soluble by an addi-tion of 0.2M Na2HPO4. Each of these fatty acidswas added to the medium in concentrations of0.025, 0.050, 0.075, 0.10 and 0.125% (v/v). Themedium was then left to cool in a Petri dish (8cm diameter). A 50 ml of conidial suspensioncontaining 1 x 10r> conidia mL'1 was applied tothe medium using a Gilson pipetman and spreadwith a bent glass rod. Conidial germination wasrecorded under a microscope 24 h after incuba-tion at room temperature.

RESULTS AND DISCUSSIONGas chromatographic analysis of adult flea bee-tle cuticular extract showed that several fattyacids were present in differing amounts. Therewere two peaks (Fig. 1). These peaks exhibitedretention times corresponding to those of bu-tyric and nonanoic acids. Three other fatty acidsdetected were caproic, heptanoic and caprylicacids (Table 2). According to Smith and Grula(1982), chromatography of inhibitory com-pounds from the surface of the corn earworm,Heliothis zea, showed only one peak that exhib-ited a retention time corresponding to caprylicacid, indicating that besides the sclerotised cuti-cle, fungistatic straight-chain fatty acid couldplay an important role in the establishment ofan entomopathogenic fungal infection.

Fig. 2 shows that depending on the type andconcentration, fatty acids inhibited the conidialgermination of M. anisopliae, B. bassiana and P.fumosoroseus. Gershon et al (1973) reported thattoxicity of fatty acids to fungi was affected by thechain length of the fatty acid, pH of the mediumand the presence in the test medium of anadsorbent such as serum albumen. At concen-trations between 0.025 - 0.125, all the fatty acidsdemonstrated varying levels of toxicity to M.anisopliae. Butyric acid was the least toxic-only toxic to B. bassiana and P. fumosoroseus atconcentrations higher than 0.125%, i.e. the con-centration at which all the fatty acids were verytoxic. Nonanoic acid was toxic to both M.anisopliae and P. fumosoroseus with no conidialgermination at the lowest concentration of0.025%, while B. bassiana managed to germinateat between 3 -18%. The conidial germination ofB. bassiana was especially inhibited by heptanoicacid with only 12.5% germination at the lowestconcentration of 0.025%. Results from this studyconcur with the conclusion made by Smith andGrula (1982) who reported that at concentra-tions between 0.02 and 0.04% saturated fattyacids with 10 carbons or less were highly inhibi-tory to the conidial germination of B. bassiana.

Samson et al. (1988) reported that moststraight-chain saturated fatty acids were toxic tothe conidia of entomogenous fungi. The fattyacids detected on the surface of corn earwormsidentified as caprylic, valeric and nonanoic acidswere also reported to inhibit the conidialgerminaton of B. bassiana (Smith and Grula

PERTANIKAJ. TROP. AGRIC. SCI. VOL. 25 NO. 2, 2002 117

T.P. PRTYATNO & Y.B. IBRAHIM

V/V

2 . 3 4 5Retention time (minute)

Fig. 1: GC tracing ofhexane extract of adult striped flea beetle, P. striolata F., integument

TABLE 2Concentration of fatty acids extracted from the

integument of the adult Phyllotreta striolata

Fatty acid

Butyric (C4)Caproic (C6)Caprylic (C7)Heptanoic (C8)Nonanoic (C9)

Concentration (%)

0.08810.00140.00630.00120.0157

1982), while Koidsumi (1957) reported thatcuticular lipids of the silkworm and the ricestem borer, presumably caprylic or caproic ac-ids, inhibited the germination and growth ofAspergillus flaws.

Smith and Grula (1982) suggested that thetoxicity of fatty acids to B. bassiana conidia invitro was dependent on the nutritional condi-tions present, since the fungus could grow inthe presence of different carbon, nitrogen andenergy sources. On the contrary, Boucias andPendland (1984) observed that isolates of

Nomuraea rileyi could use epicuticular fatty acidsto germinate. In other cases, these moleculesinduced penetration of germ tubes on the hostcuticle (Kerwin 1984; Latge et al 1987). Whileaccording to Lecuona et al (1997), a pentane-extract of the cuticular lipids of M. meblonthalarvae did not affect germination and hyphalgrowth of the virulent isolate of B. bassiana.However, it acted as an inhibitor to an avirulentisolate. Similar results were reported for thecuticular lipids of Acyrthosiphon pisum to the ag-gressive strains of Conidiobolus abscurus (Bouciasand Latge 1988). This study has shown thatstraight-chain fatty acids on the flea beetle in-tegument were inhibitory to conidial germina-tion and thus protected the beetles against theinvasion of entomopathogenic fungi. Therefore,the use of entomopathogenic fungi to controlflea beetles, and perhaps other ground dwellinginsect pests too, could probably be made moreeffective if nutrients were added into a conidialformulation of virulent strain so as to stimulateconidial germination.

118 PERTANIKA J. TROP. AGRIC. SCI. VOL. 25 NO. 2, 2002

FREE FATTY ACIDS ON THE INTEGUMENT OF THE STRIPED FLEA BEETLE

100 -

8 0 -

40

20-

£ 100 -

§ 80-

.8 60 1

I -"rt 20 -

0100

80 Ji

8C -j

40-I

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00.025 0.050 0.075

Concentration of fatty acids

0.100 0.125

Fig. 2. Effect of fatty acids (C4 S , C6 L , C7 S , C8 Id , C9 1 ) on percentage ofconidial germination of M. anisopliae (A), B. bassiana (B) and P. fumosoroseus (C) on water agar

ACKNOWLEDGEMENT

The authors would like to record their gratitudeand appreciation to Universiti Putra Malaysia fortheir facilities and technical support.

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(Received: 2 May 2001)(Accepted: 28 February 2002)

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