In Vitro Effects of Secondary Plant Compounds on Germination ofBlastospores of the Entomopathogenic Fungus Paecilomyces

fumosoroseus (Deuteromycotina: Hyphomycetes)

Fernando E. Vega,*,1 Patrick F. Dowd,* Michael R. McGuire,* Mark A. Jackson,† and Terry C. Nelsen‡*Bioactive Agents Research Unit, †Fermentation Biochemistry Research Unit, and ‡Biostatistician–Midwest Area, National Center for

Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 North University Street, Peoria, Illinois 61604

Received August 19, 1996; accepted June 4, 1997

Seven secondary plant compounds (catechol, chloro-genic acid, gallic acid, salicylic acid, saponin, sinigrin,and tannic acid) mixed with Noble agar at three concen-trations (100, 500, and 1000 ppm) were tested for theireffects on germination of blastospores of the fungalentomopathogen Paecilomyces fumosoroseus. With in-dividual allelochemicals incorporated at 100 ppm inNoble agar, significant differences in time to 95% germi-nation were found between two allelochemicals (cat-echol and salicylic acid) and the control. Blastosporesin media containing 100 ppm catechol took twice aslong (10 hr) to reach 95% germination as the control.Germination of blastospores in medium containingcatechol, salicylic acid, or tannic acid at 500 was 55, 56,and 46%, respectively, in contrast to less than 10%when the concentration was 1000 ppm. These resultsindicate that the presence of allelochemicals on asubstrate (e.g., insect cuticle or leaf) may be an addi-tional constraint to the survival of entomopathogenicfungi. r 1997 Academic Press

Key Words: germination; blastospores; allelochemi-cals; entomopathogens; Paecilomyces fumosoroseus.

INTRODUCTION

Although examples abound on how secondary plantcompounds or allelochemicals might affect the thirdtrophic level (Price et al., 1980; Barbosa, 1988), theeffect of host plant chemistry on insect susceptibility toinfection by entomopathogenic fungi has not beenextensively studied. Several studies have examinedchemical components of an insects’ diet and their effecton fungal entomopathogens (Hare and Andreadis, 1983;Costa and Gaugler, 1989a; Boucias et al., 1984; Ra-moska and Todd, 1985; Fargues and Maniania, 1992;

Gopalakrishnan and Narayanan, 1989; Gallardo et al.,1990; Hajek and Renwick, 1993; Hajek et al., 1995),with a few studies aimed at in vitro effects (Raghavaiahand Jayaramaiah, 1987; Costa and Gaugler, 1989b;Gallardo et al., 1990; Storey et al., 1991; Guirard et al.,1995).

The results from in vitro studies indicate a variableresponse with plant allelochemicals tested on the growthof fungal insect pathogens. For example, Raghavaiahand Jayaramaiah (1987) found cases of reduced growthof Beauveria bassiana when tested against extractsfrom 10 plants (betelvine, garlic, onion, turmeric, peri-winkle, ginger, datura, tulasi, madder, and lantana).Costa and Gaugler (1989b) reported that the alkaloidstomatine and solanine were inhibitory to B. bassiana,depending upon the concentration tested (1, 5, 14, 40,and 100 ppm) and whether the medium was buffered.Gallardo et al. (1990) reported that tomatine at 500 and1000 ppm inhibited growth of Nomuraea rileyi. How-ever, Storey et al. (1991) determined that the presencein the egg of a pyrrolizidine alkaloid sequestered by anarctiid moth did not defend against B. bassiana orPaecilomyces lilacinus. When the alkaloid was tested invitro at concentrations ranging from 80 to 5000 ppm, ithad no effects on germination of B. bassiana, Metarhiz-ium anisopliae, P. lilacinus, or P. fumosoroseus. In astudy of 88 fungi, Guirard et al. (1995) found thatcatechol at 300 ppm resulted in 50% inhibition of B.bassiana.

Our objective was to determine the in vitro effect ofseveral different allelochemicals on the growth andgermination of blastospores of P. fumosoroseus (Wize)Brown & Smith (Deuteromycotina: Hyphomycetes).This fungus is receiving widespread attention as abiological control agent due to its broad host range ofover 41 insect species (see review, Smith, 1993) and afaster germination of blastospores on the insect cuticlethan of conidia (Vega et al., unpublished). We felt that itwould be important to assess the effects of plantdefensive chemicals on the fungus as a means of

1To whom correspondence and reprint requests should be ad-dressed at European Biological Control Laboratory, USDA, ARS,Parc Scientifique Agropolis, 34397 Montpellier Cedex 5, France.

JOURNAL OF INVERTEBRATE PATHOLOGY 70, 209–213 (1997)ARTICLE NO. IN974693

209 0022-2011/97 $25.00Copyright r 1997 by Academic Press

All rights of reproduction in any form reserved.

understanding more about the ecology of this entomo-pathogen. The chemicals tested were five phenolics,catechol, chlorogenic acid, gallic acid, salicylic acid, andtannic acid; a triterpenoid (saponin); and a glucosino-late (sinigrin). These allelochemicals have been re-ported to occur in a wide variety of plants (Harborneand Baxter, 1993.)

MATERIALS AND METHODS

Chemicals were purchased from Sigma Chemical Co.(St. Louis, MO): catechol (Cat. No. C-9510, pyrocatechol),chlorogenic acid (Cat. No. C-3878, 1,3,4,5-tetrahydroxycy-clohexanecarboxylic acid 3-[3,4-dihydroxycinnamate]), gal-lic acid (Cat. No. G-7384, 3,4,5-trihydroxybenzoic acid),salicylic acid (Cat. No. S-3007, 2-hydroxybenzoic acid,sodium salt), saponin (Cat. No. S-1252), sinigrin (mono-hydrate, Cat. No. S-1647), and tannic acid (Cat. No.T-0125).

P. fumosoroseus was grown in a liquid medium for 4days at 28°C and 300 rpm (Jackson et al., 1997). Liquidmedium results in the production of blastospores; theseare larger than conidia (Inch et al., 1986). Blastosporeswere filtered twice in the laminar flow hood through adouble layer of sterile cheesecloth to remove hyphalfragments and centrifuged for 10 min at 10,000 rpm.The supernatant was discarded, the pellet was resus-pended in 100 ml of sterile distilled water, and theprocess was repeated. Allelochemicals were mixed withNoble agar (Difco Laboratories, Detroit, MI) to obtainconcentrations equal to 100, 500, and 1000 ppm; theseare well within the range reported for some plants(Table 1). Noble agar is a highly purified solidifyingagent essentially free of impurities. The purity of thisagar reduces the possibility of allelochemicals bindingto some medium components as might happen withother complex media. Ten milliliters of Noble agarcontaining 100, 500, or 1000 ppm of each allelochemicalwas poured in each of two 100 3 15-mm petri plates(VWR, West Chester, PA), allowed to dry in the laminarflow hood for 5–10 min and stored overnight in thedark. A 1 3 106 blastospores/ml dilution was sprayed oneach plate using an aerosol sprayer (Fisher Scientific,Cat. No. 15-233, Pittsburgh, PA). The sprayed solutionwas allowed to dry in the laminar flow hood, and theplates were incubated at 28°C in the dark (VWR lowtemperature incubator, VWR Products, West Chester,PA). Groups of 100 blastospores from each plate wereexamined 2, 4, 8, 12, and 24 hr after spraying, using anOlympus IMT-2 inverted research microscope (Olym-pus America Inc., Lake Success, NY) to assess germtube formation. Germination was deemed positive whenobvious germ tube elongation was observed. For all ofthe treatments which did attain 95% or greater germi-nation, a dose–response curve (DeLean et al., 1978) wasfit for each replication of each concentration of each

allelochemical using the NLIN procedure of SAS soft-ware (SAS Institute, 1989). The equation was

Y 5 A 1B

1 1 1XC2D

,

where Y is germination percentage, X is time in hours,A is the lowest level of Y, B is the highest level of Y(minus X ), C is the approximate time at inflection (X atY 5 A 1 B/2), and D is a function of the slope of theline. The derived equations were used to estimate timeat 95% germination (H95) for each treatment–concen-tration–replication. These H95 values were then ana-lyzed by analysis of variance and allelochemicals werecompared separately for each concentration.

The pH of the medium was determined on threereplicates per allelochemical concentration. The me-dium was kept at ca. 45°C and a VWRbrand BenchtoppH/ISE meter (West Chester, PA) was used, adjustingfor temperature. Analysis was conducted within eachallelochemical concentration using Dunnett’s T test(SAS Institute, 1989).

RESULTS

Blastospores grown in media containing three of theallelochemicals did not attain 95% germination whenthey were at 500 or 1000 ppm. The three allelochemi-cals and the maximum germination reached at 500 and

TABLE 1Examples of Allelochemical Concentration in Plants

Allelochemical PlantConcentration

ppm Reference

Catechol Psorospermumguineense (Gut-tiferae)

95,400 Karrer (1958)

Chlorogenic Tobacco 3,000–5,000 Karrer (1958)acid Sunflower 17,500 Karrer (1958)

Sunflower 27,000 Liener (1980)Gallic acid Beech 200 Janes and

Morgan (1960)Rheum maximow-

iczii (Polygona-ceae)

7,000 Hegnauer (1973)

Salicylic acid Salix babylonica 2,000 Hegnauer (1973)Salix caesia 6,000 Hegnauer (1973)

Saponin Sugarbeet 3,000 Applebaum andBirk (1979)

Sinigrin Brassica oleracea(cabbage)

17–584 Tookey et al.(1980)

Armoracia lapathi-folia (relative ofhorseradish)

5,000 Tookey et al.(1980)

Black mustard 11,000–12,000 Karrer (1958)Tannin Sorghum 50,000 Liener (1980)

210 VEGA ET AL.

1000 ppm, respectively, were salicylic acid, 56 and 9%;tannic acid, 46 and 7%; and catechol, 55 and 7%.

Analysis of variance on time to 95% germination(obtained using the derived equations) indicated thatallelochemical was a significant source of variation(F 5 6.3, df 5 7, 8, P , 0.01) at 100 ppm, but was notsignificant at 500 ppm (F 5 1.3, df 5 4, 5, P 5 0.39) or1000 ppm (F 5 4.4, df 5 4, 5, P 5 0.07). At 100 ppm,catechol and salicylic acid took a significantly longertime to reach 95% germination than the control (Table2). When the means at 1000 ppm were compared byBonferroni’s method, gallic acid and chlorogenic acidtook a significantly longer time to reach 95% germina-tion than sinigrin, saponin, or the control (Table 2).

A second analysis of variance was done on the fourallelochemicals (gallic acid, chlorogenic acid, sinigrin,and saponin) plus the controls where H95 estimateswere available at all three concentrations. This secondmodel included allelochemical and concentration andtheir interactions as sources of variation. Allelochemi-cal (F 5 5.6, df 5 4, 15, P , 0.01) and concentration(F 5 7.6, df 5 2, 15, P , 0.01) were significant and theinteraction was not significant (F 5 0.6, df 5 8, 15,P 5 .76). The overall allelochemical and concentrationdifferences in means were examined by t tests ofleast-squares means and the differences among concen-trations within each allelochemical were examined byBonferroni’s adjustment of the t test procedure. Theoverall effects of concentrations were that 100 and 500

ppm had similar effects and 1000 ppm delayed 95%germination. The comparisons within each allelochemi-cal are shown in Table 2.

There were significant pH differences between someallelochemical concentrations and the control (Table 3).The pH values for tannic, chlorogenic, and gallic acid atall concentrations were significantly lower than thecontrol. Catechol and sinigrin showed no significantdifferences from the control at any concentration, withsaponin having a significantly higher pH than thecontrol at 500 and 1000 ppm and salicylic acid having asignificantly higher pH at 1000 ppm.

DISCUSSION

The results clearly indicate an inhibitory allelochemi-cal effect on germination rates depending upon theconcentration and the sampling time. For example,catechol, salicylic acid, and tannic acid reduced germi-nation rates at concentrations of 500 or 1000 ppm. Thisdelayed germination could have important implica-tions in the field, where additional factors such asmoisture and temperature also influence establish-ment. The rate of fungal spore germination has beenassociated with improved efficacy in infecting weedsand insects. Schisler et al. (1991) reported that conidiaof Colletotrichum truncatum produced in medium con-taining a 10:1 C:N ratio germinated faster and pro-vided better control of the weed Sesbania exaltata thanthose in media with other C:N ratios. Similarly, Far-gues et al. (1994) demonstrated that presoaking conidiaof P. fumosoroseus in water (in order to allow forgermination) prior to use in bioassays resulted inhigher mortality of Spodoptera frugiperda when com-pared to ungerminated conidia.

Overall, the germination results suggest that pH didnot have an effect on germination. For example, chloro-genic acid and gallic acid exhibited significantly lower

TABLE 2Hours to 95% Germination for Each Allelochemical at Three

Concentrations

Allelochemical

Concentration (ppm)1

1002 5002 10002 Comparisons3

Catechol 10.0a n nSalicylic acid 7.8b n nTannic acid 7.1b,c n nGallic acid 7.1b,c 6.5a 9.2a XY-X-YChlorogenic acid 6.9b,c 7.8a 9.3a X-XY-YSinigrin 6.0c 6.4a 7.1b X-X-XSaponin 5.7c 5.8a 6.4b X-X-XControl 5.1c 6.2a 6.9b X-X-X

Note. n, not calculated because the treatment never reached 95%germination.

1 Values were calculated from dose–response fits to each of tworeplicates.

2 Values within a column followed by the same letter are notsignificantly different as determined by t tests of least-squares meansat P , 0.05.

3 Values within rows were compared by Bonferroni’s adjustment oft test of least-squares means at P , 0.03 in an ANOVA which includedonly those allelochemicals with values at all three concentrations.X-X-X denotes no differences; XY-X-Y denotes a difference between500 and 1000 ppm but neither different from 100 ppm; X-XY-Ydenotes a difference between 100 and 1000 ppm with 500 ppmintermediate.

TABLE 3pH Levels in Three Concentrations of Seven Allelochemicals

and the Control (Noble Agar)

Allelochemical

ppm

100 500 1000

Catechol 4.77 4.75 4.71Salicylic acid 4.71 4.79 4.90*Tannic acid 4.26* 3.95* 3.64*Gallic acid 4.11* 3.82* 3.64*Chlorogenic acid 4.12* 3.67* 3.43*Sinigrin 4.59 4.69 4.64Saponin 4.72 4.82* 4.92*Control 4.70

* Numbers followed by an asterisk indicate significant differencesfrom the control using Dunnett’s t test (a 5 0.05). Sample size isthree for each allelochemical concentration and nine for the control.

211EFFECTS OF SECONDARY PLANT COMPOUNDS ON BLASTOSPORES

pH levels than the control (Table 3) but they bothreached 95% germination. Similarly, saponin had asignificantly higher pH at 500 and 1000 ppm (4.82 and4.92, respectively, vs 4.70 for the control) but time to95% germination at these levels was not significantlydifferent from the control. Tannic acid had lower pHlevels than the control at all concentrations. At 100ppm, time to 95% germination was not different fromthe control. In contrast, germination levels at 500 and1000 ppm remain consistently at significantly lowerlevels than the control. The pH for tannic acid wassimilar or higher (depending on allelochemical concen-tration) than the pH for chlorogenic acid and gallic acid,and germination in the latter reached levels that werenot significantly different from the control, indicatingthat the spore will germinate at lower pH levels thanthose found in tannic acid; thereby it is likely that thelower germination in tannic acid is due to toxic effect ofthe chemical and not to pH. To further assess thepossible pH effects, we conducted germination tests onpotato dextrose broth (pH 5.4) with pH adjusted to 3, 4,5, or 6. At 8 hr after initiation, there were no significantdifferences in germination rates, ranging from 97 to100% (data not shown).

Even though there are several examples on theeffects of secondary plant compounds on predators andparasitoids (Duffey, 1980; Duffey et al., 1986; Barbosaand Letourneau, 1988), the role that allelochemicalsplay in the infection process of entomopathogenic fungiis not as well understood. Allelochemicals have beenshown to prevent insect infection by viruses (Felton etal., 1987; Young et al., 1995) or bacteria (Reichelderfer,1991) as well as to facilitate infection by bacteria(Barbosa, 1988; Ludlum et al., 1991). Phenolics purport-edly produced by a bacterium (Pantoea (Enterobacter)agglomerans) present in the midgut of desert locustsinhibit growth of the fungal entomopathogen M. aniso-pliae (Dillon and Charnley 1995). Similarly, phenolicspresent in lichen are inhibitory to fungal growth(Lawrey et al., 1994). Our data suggest that germina-tion of fungal spores coming in contact with allelochemi-cals can be delayed in a manner similar to the detrimen-tal effect that constitutive antifungal compoundspresent on the leaf surface (e.g., leaf exudates) have onplant fungal pathogens (Wippich and Wink 1985; Tomas-Barberan et al., 1988; Brownlee et al., 1990; Grayer andHarborne, 1994).

In addition to the leaf surface, contact betweenspores and allelochemicals is possible on the insectcuticle or in the hemolymph. However, available infor-mation on concentrations of allelochemicals in thecuticle or hemolymph is very limited. Self et al. (1964)reported that of the total nicotine consumed by tobaccohornworms, 5.2% could be detected in the hemolymph 4hr after ingestion and 0% 24 hr after ingestion. Duffeyand Scudder (1974) reported that very small amounts

of cardenolides could be detected in the hemolymph ofOncopeltus fasciatus Dallas (Hemiptera: Lygaeidae)and that high concentrations were present in thedorsolateral spaces of the thorax and abdomen. Someinformation on carotenes and flavonoids in the cuticlehas been published (see Duffey, 1980), but it dealsmostly with their protective coloration role.

In conclusion, our results suggest that in addition tomoisture levels, temperature, and UV light, allelochemi-cals might present a constraint (via delayed germina-tion) in the infection process of fungal entomopathogenspores.

ACKNOWLEDGMENTS

We thank Pedro Barbosa (University of Maryland), Ann E. Hajek(Cornell University), Robert A. Norton (NCAUR), and two anony-mous reviewers for comments on an earlier draft of this paper andAngela R. Payne (NCAUR) for growing blastospores. Also KarenKester (University of Arizona) for providing important informationrelated to the presence of allelochemical in insects. The mention offirm names or trade products does not imply that they are endorsedor recommended by the U.S. Department of Agriculture over otherfirms or similar products not mentioned. Names are necessary toreport factually on data; however, the USDA neither guarantees norwarrants the standard of the product, and the use of the name byUSDA implies no approval of the product to the exclusion of othersthat may be suitable.

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213EFFECTS OF SECONDARY PLANT COMPOUNDS ON BLASTOSPORES