7864· JOURNAL OF L"IVERTEBRATE PATHOLOGY 70,209-213 (19971 ARTICLE NO. IN974693

In Vitro Effects of Secondary Compounds on Germination of Blastospores of the Entomopathogenic Fungus Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Fernando E. Vega,*,l Patrick F. Dowd,* Michael R. McGuire,* MarkA. Jackson,t and Terry C. Nelsen:;: *Bioactive Agents Research Unit, tFermentation Biochemistry Research Unit, and Wiostatistician-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

Gopalakrishnan and Narayanan, 1989; Gallardo et ai., Seven secondary plant compounds (catechol, chIoro· 1990; Hajek and Renwick, 1993; Hajek et al., 1995), genic acid, gallic acid, , , sinigrin, with a few studies aimed at in vitro effects (Raghavaiah andtannic acid) mixedwithNoble agaratthree concen· and Jayaramaiah, 1987; Costa and Gaugler, 1989b; trations (100, 500, and 1000 ppm) were tested for their Gallardo et al., 1990; Storey et al., 1991; Guirard et ai., effects on germination of blastospores of the fungal 1995). entomopathogen Paecilomyces fumosoroseus. With in· The results from in vitro studies indicate a variable dividual allelochemicals incorporated at 100 ppm in response with plant allelochemicals tested on the growth Noble agar, significant differences intime to 95% germi. of fungal insect pathogens. For example, Raghavaiah nation were found between two allelochemicals (cat· echol and salicylic acid) and the control. Blastospores and Jayaramaiah (1987) found cases ofreduced growth in media containing 100 ppm catechol took twice as of Beauveria bassiana when tested against extracts long (10 hr) to reach 95% germination as the control. from 10 (betelvine, garlic, onion, turmeric, peri­ Germination of blastospores in medium containing winkle, ginger, datura, tulasi, madder, and lantana). catechol, salicylic acid, ortannic acid at 500 was 55, 56, Costa and Gaugler (l989b) reported that the alkaloids and 46%, respectively, in contrast to less than 10% tomatine and solanine were inhibitory to B. bassiana, when the concentration was 1000 ppm. These results depending upon the concentration tested (1, 5, 14,40, indicate that the presence of allelochemicals on a and 100 ppm) and whether the medium was buffered. substrate (e.g., insect cuticle or leaf) may be an addi· Gallardo et al. (1990) reported that tomatine at 500 and tional constraint to the survival of entomopathogenic 1000 ppm inhibited growth of Nomuraea rileyi. How­ fungi. '" 1997Academic Pre•• ever, Storey et al. (1991) determined that the presence Key Words: germination; blastospores; allelochemi· in the egg of a pyrrolizidine alkaloid sequestered by an cals; entomopathogens;Paecilomyces fumosoroseus. arctiid moth did not defend against B. bassiana or Paecilomyces lilacinus. When the alkaloid was tested in vitro at concentrations ranging from 80 to 5000 ppm, it INTRODUCTION had no effects on germination ofB. bassiana, Metarhiz­ ium anisopliae, P. lilacinus, or P. fumosoroseus. In a Although examples abound on how secondary plant study of 88 fungi, Guirard et al. (1995) found that compounds or allelochemicals might affect the third catechol at 300 ppm resulted in 50% inhibition of B. trophic level (Price et al., 1980; Barbosa, 1988), the bassiana. effect ofhost plant chemistry on insect susceptibility to Our objective was to determine the in vitro effect of infection by entomopathogenic fungi has not been several different allelochemicals on the growth and extensively studied. Several studies have examined germination of blastospores of P. fumosoroseus (Wize) chemical components ofan insects' diet and their effect Brown & Smith

209 0022-2011/97 $25.00 Copyright ,j) 1997 by Academic Press All rights ofreproduction in any form reserved. 210 VEGAETAL. understanding more about the ecology of this entomo­ TABLE 1 pathogen. The chemicals tested were five phenolics, Examples ofAllelochemical Concentration in Plants catechol, chlorogenic acid, gallic acid, salicylic acid, and ; a triterpenoid (saponin); and a glucosino­ Concentration Allelochemical Plant ppm Reference late (sinigrin). These allelochemicals have been re­ ported to occur in a wide variety of plants (Harborne Catechol Psorospermum 95,400 Karrer (19581 guineense (Gut- and Baxter, 1993.) tiferae) Chlorogenic Tobacco 3,000-5,000 Karrer (1958 ) acid Sunflower 17.500 Karrer (1958) MATERIALS AND METHODS Sunflower 27,000 Liener (1980) Gallic acid Beech 200 Janes and Chemicals were purchased from Sigma Chemical Co. Morgan (1960) (St. Louis, MO): catechol (Cat. No. C-9510, pyrocatechol), Rheum maximow- 7,000 Hegnauer (19731 chlorogenic acid (Cat. No. C-3878, 1,3,4,5-tetrahydroxycy­ iczii (Polygona- ceae) clohexanecarboxylic acid 3-[3,4-dihydroxycinnamate]), gal­ Salicylic acid Salix babylonica 2,000 Hegnauer (19731 lic acid (Cat. No. G-7384, 3,4,5-), Salix caesia 6,000 Hegnauer (19731 salicylic acid (Cat. No. S-3007, 2-hydroxybenzoic acid, Saponin Sugarbeet 3,000 Applebaum and sodium salt), saponin (Cat. No. S-1252), sinigrin (mono­ Birk (1979) Sinigrin Brassica oleracea 17-584 Tookey et al. hydrate, Cat. No. S-1647), and tannic acid (Cat. No. (cabbage) (1980) T-0125). Armoracia lapathi- 5,000 Tookey et at. P. fumosoroseus was grown in a liquid medium for 4 folia (relative of (1980) days at 28°C and 300 rpm (Jackson et at., 1997). Liquid ) medium results in the production ofblastospores; these Black 1l,00(}-'12,000 Karrer (1958) Sorghum 50,000 Liener (1980) are larger than conidia (Inch et al., 1986). Blastospores were filtered twice in the laminar flow hood through a double layer of sterile cheesecloth to remove hyphal fragments and centrifuged for 10 min at 10,000 rpm. allelochemical using the NUN procedure of SAS soft­ The supernatant was discarded, the pellet was resus­ ware (SAS Institute, 1989). The equation was pended in 100 ml of sterile distilled water, and the process was repeated. Allelochemicals were mixed with B Noble agar Wifco Laboratories, Detroit, MI) to obtain Y=A + (Xf' concentrations equal to 100, 500, and 1000 ppm; these are well within the range reported for some plants 1 + \e) (Table 1). Noble agar is a highly purified solidifying agent essentially free of impurities. The purity of this where Y is germination percentage, X is time in hours, agar reduces the possibility of allelochemicals binding A is the lowest level of Y, B is the highest level of Y to some medium components as might happen with (minus X), C is the approximate time at inflection (X at other complex media. Ten milliliters of Noble agar Y = A + B/2), and D is a function of the slope of the containing 100, 500, or 1000 ppm ofeach allelochemical line. The derived equations were used to estimate time was poured in each of two 100 x 15-mm petri plates at 95% germination (H95) for each treatment-<:oncen­ (VWR, West Chester, PA), allowed to dry in the laminar tration-replication. These H95 values were then ana­ flow hood for 5-10 min and stored overnight in the lyzed by analysis ofvariance and allelochemicals were dark. A 1 X 106 blastospores/ml dilution was sprayed on compared separately for each concentration. each plate using an aerosol sprayer (Fisher Scientific, The pH of the medium was determined on three Cat. No. 15-233, Pittsburgh, PA). The sprayed solution replicates per allelochemical concentration. The me­ was allowed to dry in the laminar flow hood, and the dium was kept at ca. 45°C and a VWRbrand Benchtop plates were incubated at 28°C in the dark (VWR low pHiiSE meter (West Chester, PA) was used, adjusting temperature incubator, VWR Products, West Chester, for temperature. Analysis was conducted within each PA). Groups of 100 blastospores from each plate were allelochemical concentration using Dunnett's T test examined 2, 4, 8, 12, and 24 hr after spraying, using an (SAS Institute, 1989). Olympus IMT-2 inverted research microscope (Olym­ pus America Inc., Lake Success, NY) to assess germ RESULTS tube formation. Germination was deemed positive when obvious germ tube elongation was observed. For all of Blastospores grown in media containing three of the the treatments which did attain 95% or greater germi­ allelochemicals did not attain 95% germination when nation, a dose-response curve (DeLean et at., 1978) was they were at 500 or 1000 ppm. The three allelochemi­ fit for each replication of each concentration of each cals and the maximum germination reached at 500 and EFFECTS OF SECONDARY PLANT COMPOUNDS ON BLASTOSPORES 211

1000 ppm, respectively, were salicylic acid, 56 and 9%; TABLE 3 tannic acid, 46 and 7%; and catechol, 55 and 7%. pH Levels in Three Concentrations ofSevenAllelochemicals Analysis of variance on time to 95% germination and the Control (Noble Agar) (obtained using the derived equations) indicated that allelochemical was a significant source of variation ppm (F = 6.3, df= 7, 8, P < 0.01) at 100 ppm, but was not Allelochemical 100 500 1000 significant at 500 ppm (F = 1.3, df = 4, 5, P = 0.39) or Catechol 4.77 4.75 4.71 1000 ppm (F = 4.4, df = 4, 5, P = 0.07). At 100 ppm, Salicylic acid 4.71 4.79 4.90* catechol and salicylic acid took a significantly longer Tannic acid 4.26* 3.95* 3.64* time to reach 95% germination than the control (Table Gallic acid 4.11* 3.82* 3.64* Chlorogenic acid 4.12* 3.67* 3,43* 2). When the means at 1000 ppm were compared by Sinigrin 4.59 4.69 4.64 Bonferroni's method, gallic acid and chlorogenic acid Saponin 4.72 4.82* 4.92* took a significantly longer time to reach 95% germina­ Control 4.70 tion than sinigrin, saponin, or the control (Table 2). A second analysis of variance was done on the four * Numbers followed by an asterisk indicate significant differences from the control using Dunnett's t test (ex = 0.05). Sample size is allelochemicals (gallic acid, chlorogenic acid, sinigrin, three for each allelochemical concentration and nine for the control. and saponin) plus the controls where H95 estimates were available at all three concentrations. This second model included allelochemical and concentration and their interactions as sources ofvariation. Allelochemi­ ppm had similar effects and 1000 ppm delayed 95% cal (F = 5.6, df = 4, 15, P < 0.01) and concentration germination. The comparisons within each allelochemi­ (F = 7.6, df= 2, 15, P < 0.01) were significant and the cal are shown in Table 2. interaction was not significant (F = 0.6, df = 8, 15, There were significant pH differences between some P = .76). The overall allelochemical and concentration allelochemical concentrations and the control (Table 3). differences in means were examined by t tests of The pH values for tannic, chlorogenic, and gallic acid at least-squares means and the differences among concen­ all concentrations were significantly lower than the trations within each allelochemical were examined by control. Catechol and sinigrin showed no significant Bonferroni's adjustment of the t test procedure. The differences from the control at any concentration, with overall effects of concentrations were that 100 and 500 saponin having a significantly higher pH than the control at 500 and 1000 ppm and salicylic acid having a significantly higher pH at 1000 ppm. TABLE 2 DISCUSSION Hours to 95% Germination for Each Allelochemical at Three Concentrations The results clearlyindicate an inhibitory allelochemi­ Concentration (ppm)l cal effect on germination rates depending upon the concentration and the sampling time. For example, 2 2 2 3 Allelochemical 100 500 1000 Comparisons catechol, salicylic acid, and tannic acid reduced germi­ Catechol 10.0" n n nation rates at concentrations of500 or 1000 ppm. This Salicylic acid 7.8b n n delayed germination could have important implica­ b Tannic acid 7.1 ,e n n tions in the field, where additional factors such as Gallic acid 7.1b,e 6.5" 9.2" xy-x-y Chlorogenic acid 6.9b,e 7.8" 9.3" X-xy-y moisture and temperature also influence establish­ Sinigrin 6,oe 6,4" 7.1b X-X-X ment. The rate of fungal spore germination has been Saponin 5.7e 5.8" 6,4b X-X-X associated with improved efficacy in infecting weeds Control 5.1e 6.2" 6.9b X-X-X and insects. Schisler et al. (1991) reported that conidia Note. n, not calculated because the treatment never reached 95% of Colletotrichum truncatum produced in medium con­ germination. taining a 10:1 C:N ratio germinated faster and pro­ 1 Values were calculated from dose-response fits to each of two vided better control ofthe weed Sesbania exaltata than replicates. those in media with other C:N ratios. Similarly, Far­ 2 Values within a column followed by the same letter are not significantly different as determined byt tests ofleast-squares means gues et al. (1994) demonstrated that presoaking conidia at? < 0,05. of P. fumosoroseus in water (in order to allow for 3 Values within rows were compared by Bonferroni's adjustment of germination) prior to use in bioassays resulted in t test ofleast-squares means at? < 0.03 in anANOVA which included higher mortality of Spodoptera frugiperda when com­ only those allelochemicals with values at all three concentrations. pared to ungerminated conidia. X-X-X denotes no differences; XY-X-Y denotes a difference between 500 and 1000 ppm but neither different from 100 ppm; X-XY-Y Overall, the germination results suggest that pH did denotes a difference between 100 and 1000 ppm with 500 ppm not have an effect on germination. For example, chloro­ intermediate. genic acid and gallic acid exhibited significantly lower 212 VEGAETAL. pH levels than the control (Table 3) but they both of could be detected in the hemolymph of reached 95% germination. Similarly, saponin had a Oncopeltus fasciatus Dallas (Hemiptera: Lygaeidae) significantly higher pH at 500 and 1000 ppm (4.82 and and that high concentrations were present in the 4.92, respectively, vs 4.70 for the control) but time to dorsolateral spaces of the thorax and abdomen. Some 95% germination at these levels was not significantly information on carotenes and in the cuticle different from the control. Tannic acid had lower pH has been published (see Duffey, 1980), but it deals levels than the control at all concentrations. At 100 mostly with their protective coloration role. ppm, time to 95% germination was not different from In conclusion, our results suggest that in addition to the control. In contrast, germination levels at 500 and moisture levels, temperature, and UV light, allelochemi­ 1000 ppm remain consistently at significantly lower cals might present a constraint (via delayed germina­ levels than the control. The pH for tannic acid was tion) in the infection process offungal entomopathogen similar or higher (depending on allelochemical concen­ spores. tration) than the pH for chlorogenic acid and gallic acid, and germination in the latter reached levels that were ACKNOWLEDGMENTS not significantly different from the control, indicating We thank Pedro Barbosa (University of Marylandl, Ann E. Hajek that the spore will germinate at lower pH levels than (Cornell University), Robert A Norton (NCAUm, and two anony­ those found in tannic acid; thereby it is likely that the mous reviewers for comments on an earlier draft of this paper and lower germination in tannic acid is due to toxic effect of Angela R. Payne (NCAUR) for growing blastospores. Also Karen Kester (University ofArizona) for providing important information the chemical and not to pH. To further assess the related to the presence of aJlelochemical in insects. The mention of possible pH effects, we conducted germination tests on firm names or trade products does not imply that they are endorsed potato dextrose broth (pH 5.4) with pH adjusted to 3, 4, or recommended by the U.S. Department of Agriculture over other 5, or 6. At 8 hr after initiation, there were no significant firms or similar products not mentioned. Names are necessary to report factually on data; however, the USDA neither guarantees nor differences in germination rates, ranging from 97 to warrants the standard of the product, and the use of the name by 100% (data not shown). USDA implies no approval of the product to the exclusion of others Even though there are several examples on the that may be suitable. effects of secondary plant compounds on predators and parasitoids (Duffey, 1980; Duffey et al., 1986; Barbosa REFERENCES and Letourneau, 1988), the role that allelochemicals Applebaum, S. W., and Birk, Y. 1979. .In "Herbivores: Their play in the infection process ofentomopathogenic fungi Interactions with Secondary Plant Metabolites" (G. A Rosenthal is not as well understood. Allelochemicals have been and D. H. Janzen, Eds.), pp. 539-566. Academic Press, New York. shown to prevent insect infection by viruses (Felton et Barbosa, P., and Letourneau, D. K (Eds.) 1988. 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