Assessment of Plant Pathogenicity of Endophytic Beauveria bassiana in Bt Transgenic and Non-Transgenic Corn

1991). Only recently have investigations focused on relationships between B. bassiana and the corn plant. Field and greenhouse studies were conducted to determine the proBeauveria bassiana forms an endopytic relationship with clivity of Bacillus thuringiensis (Bt) (Berliner)-transgenic corn (Zea mays L.) to form an endophytic relationship with Beauveria bassiana, the corn plant and provides season-long suppression of and to evaluate the corn for possible plant pathological effects associO. nubilalis (Lewis and Cossentine, 1986; Lewis and ated with this relationship. Beauveria bassiana (Balsamo) Vuillemin Bing, 1991; Wagner and Lewis, 2000). Two areas of was applied as a granular formulation to two separate lines of corn, research must be investigated before acceptance by proexpressing Bt events MON802 and MON810, and their corresponding ducers of B. bassiana as a management tool: (i) will isolines. There were no significant differences in levels of endophytism B. bassiana colonize Bacillus thuringiensis (Bt) transbetween transgenic events or their near-isolines. In greenhouse studformed corn plants as well as non-transformed plants, ies, B. bassiana was applied as a liquid seed treatment to Bt transgenic and (ii) is the endophytic relationship void of plant corn hybrids Pioneer 34R06 (event MON810) and Ciba Max 454 pathological symptoms in transformed plants and/or (event 176) and their near isolines at a rate of 2 1010 conidia per their isolines? ml. There were no significant differences in seed germination or presence of root pathogens in transgenic or isoline seeds soaked in a This paper reports the results of research to determine B. bassiana suspension. The same lines of corn were used in field the proclivity of B. bassiana to form an endophytic relaexperiments with treatments of seeds soaked in a suspension of B. tionship with Bt transgenic corn and whether endobassiana, a foliar application of a granular formulation of B. bassiana, phytism causes a plant pathology. Field studies were and corresponding untreated checks. Plants were sampled throughout conducted in 1994 and 1995 to determine the ability of the growing season and evaluated for growth of individual plant comBt-transgenic corn to form an endophytic relationship ponents, including sheaths, leaves, stem, husk, ear, plant leaf-to-stem with B. bassiana. Greenhouse studies were performed ratio and overall plant growth. There were no significant differences in 1997 to determine the effect of B. bassiana on seed in overall plant growth between the B. bassiana treatments or in the germination and plant growth on two lines of Bt corn growth of each plant component. The results of this study indicate and their genetic isolines. Field studies in 1997 were that B. bassiana readily forms an endophytic relationship with transgenic and non-transgenic corn and causes no plant pathology. conducted to determine any possible pathogenic effects of B. bassiana on overall plant growth and dry matter accumulation. T entomopathogenic fungus Beauveria bassiana MATERIALS AND METHODS (Balsamo) Vuillemin has been used as a plant protectant to suppress populations of the European corn 1994 and 1995 Field Studies borer, Ostrinia nubilalis (Hübner) (Lepidoptera: CramA randomized complete block design with four replications bidae) on corn, Zea mays L. (Bartlett and Lefebvre, and treatments arranged as split plots was used. The whole 1934; Stirrett et al., 1937; Beall et al., 1939; York, 1958; plots were a foliar application of B. bassiana and an untreated Riba, 1984; Marcandier and Riba, 1986; Lewis and Bing, control. The split plots were corn hybrid, Bt transgenic corn (Jeremy event 802) and its near-isoline (an experimental hybrid with B73 Mo17 background). Corn was planted on an USDA-ARS, Corn Insects & Crop Genetics Research Unit, Ames, Iowa 50011. Received 30 Oct. 2000. *Corresponding author (leslewis@ iastate.edu). Abbreviations: GLM, General linear models; CFU, colony forming units; ARSEF, ARS collection of entomopathogenic fungi. Published in Crop Sci. 41:1395–1400 (2001). 1396 CROP SCIENCE, VOL. 41, SEPTEMBER–OCTOBER 2001 Iowa State University Research Farm, approximately 2 km second week after planting for three consecutive weeks. Measurements were taken from the base of the plant (vermiculite away from the nearest corn field. Experimental plantings were in rows on 0.75-m centers separated by two guard rows of a surface) to the tip of the longest leaf. After five weeks, five plants from each treatment were randomly selected for examicommercial hybrid (Garst 8543). Accepted agronomic practices of fertilizer and herbicide were used. Beauveria bassiana nation of endophytic B. bassiana. Plants were cut at their base, placed in a plastic bag, and returned to the laboratory for (ARSEF 3113, USDA-ARS, Entomopathogenic Fungi Collection, Ithaca, NY), a soil isolate passed through O. nubilalis, fungal isolation. Each plant surface was disinfected by wiping its exterior with 95% EtOH. With sterile laboratory techwas formulated on corn kernel granules and applied at 1 10 conidia per plant with a hand-held inoculator (Davis and niques, the stem of each plant was split longitudinally with the portion from the base to the growing tip excised and Oswalt, 1979). In 1994, granules containing B. bassiana were applied to plants only at V6, or at V6 and at R1 growth placed on an agar medium favoring the growth of B. bassiana (Doberski and Tribe, 1980). Plates containing the plant matestage (Ritchie et al., 1997). In 1995, B. bassiana granules were applied to plants at either the V6 or R1. Corresponding conrial were allowed to incubate in darkness at 28 C for 10 d at which time the plant tissue was examined for growth of trols which received no fungal applications were used in 1994 and 1995. B. bassiana. The remaining plants from each treatment were examined The techniques of Lewis and Bing (1991) were used to evaluate the occurrence of endophytic B. bassiana. Samples for presence of root disease. Plants were pulled from their pots and placed into a 19-L pail containing cool water. Plants of corn pith were taken with aseptic techniques and plated on agar that favors the growth of B. bassiana (Doberski and were dipped in the water repeatedly until the roots were free of vermiculite. All plants then were inspected for signs of Tribe, 1980). Five plants per treatment were sampled. If fungal applications were at V6, samples were taken at V12, R1, and disease in the root system by Dr. Gary Munkvold (Plant Pathologist, Department of Botany, Iowa State University, R6; 30, 45, and 60 d following fungal application, respectively. If application was at V6 and again at R1, samples were taken Ames, IA). at R1, R6, and senescence. If fungal application was at R1, samples were taken at R6 and senescence. Agar plates with Field Studies pith samples were incubated in total darkness at 28 C for The experiment was a randomized complete block design 10 d at which time samples were examined for the growth of with four replications. Plots were planted with a four row B. bassiana. planter on 12 May 1997. Hybrids used were the two transgenic corn lines and their near isolines used in the 1997 greenhouse Statistical Analysis studies. Whole plot treatments (10 m in length) consisted of corn seeds soaked for 3 min in an emulsifiable formulation Data were analyzed with analysis of variance using the of B. bassiana containing 2.1 10 conidia per ml or a 0.4 g General Linear Model procedure (GLM) with the B. bassiana foliar application of a granular formulation of Mycotech 726 treatment as the whole plot and genetic makeup of the corn containing 8.8 10 conidia per plant and an untreated control. as the split plot. The effects of each treatment on endophytism Plants were treated on 16 June when they reached V6 stage levels were analyzed and means separated using Student’s of development. Harvests over time served as the split plot. t-test at P 0.05 (SAS Institute, 1995). Endophytism data The two different application techniques for B. bassiana were were analyzed separately for the two granular applications for performed to confirm results found in the greenhouse and to each plating date and pooled over all plating dates. test for any adverse effects on plant growth due to granular B. bassiana application to V-stage corn. Sampling of plants began 42 d after planting at which time the corn had reached 1997 Studies the V7 growth stage. Sampling of plants was conducted every Greenhouse 2 wk for 16 wk. Plants were selected from the center two rows of each four row plot on each of eight sample dates. Plants The experiment was a randomized complete block design within 0.1 m of the beginning and end of each plot were not with four replications and a factorial arrangement of the four sampled to eliminate any effect that the alleyways may have lines of corn (Pioneer 34R06 event MON810, and Ciba Max had on plant growth. The remaining 9.8 m of row in each of 454 event 176, and their genetic isolines Pioneer 3489 and Ciba the center two rows of the plot were sampled approximately 4494, respectively) and three seed treatments: seeds soaked in every 1 m in succession along the length of the plot. One plant a 0.1% tween and distilled water suspension of B. bassiana was selected from each of the center two rows that was evenly (2 10 conidia per ml), seeds soaked in sterile 0.1% tween spaced with the adjacent plants. The distance from the selected and distilled water, and unsoaked seeds. Seeds were soaked plant to the two adjacent plants was measured and divided for 10 min, placed on sterile filter paper, and air-dried under by two to determine the amount of row space that the selected a bioflow hood. The dry seeds were placed into sterile packets plant occupied. The selected plant was cut at the soil surface and taken to the greenhouse for planting. The unsoaked seed and its height measured from the base to the longest outwas taken directly from the commercial bag and also placed stretched leaf or tassel. Plants were folded and placed into a in sterile packets before planting. A replication consisted of plastic bag and returned to the laboratory for processing. ten seeds of each hybrid planted into individual 10 cm plastic Plants were held in a cold room at 4 C until processed. pots containing sterile vermiculite. Pots were watered at plantLeaf blades were removed at the junction of the sheath and ing and thereafter when the surface of the vermiculite was blade. Sheaths were circumcised at their base around the stalk. dry to touch. After the first week of plant growth, pots were As the plants matured and developed ears, the ears and ear fertilized weekly with liquid soluble fertilizer (N-P-K, 20-20shoots were removed. The husk, shank, and silks were sepa20). Conditions in the greenhouse were maintained at photorated from the cob and kernels. The leaves, sheaths, stalks, period 14:10 (L:D), 80% RH, and 27 C during the day and ears, and husk were placed in a brown paper bag. Bags con21 C at night. taining the fresh material were weighed. Samples were placed Emergence was recorded 1 wk after planting and weekly for five weeks. Plant heights were measured beginning the in a drying oven for a minimum of 4 d at 57.2 C or until they LEWIS ET AL.: ENDOPHYTIC B. BASSIANA IN BT AND NON-BT CORN 1397 reached a constant dry weight. After drying, the samples were 4.4, N 8), R1 (Transgenic X 21.9 9.9, N 8; reweighed to obtain the dry weight and calculate dry matter Isoline X 20.6 7.3, N 8), or R6 (Transpercentage for each plant part. genic X 72.5 9.2, N 8; Isoline X 57.5 14.4, To determine the amount of endophytic B. bassiana, the N 8). There were no significant interactions in endostalk surface was disinfected and split longitudinally, from the phytism levels between B. bassiana application and the base to the sixth node (Lewis and Bing, 1991). Nodes were genetic makeup of the corn for samples taken at V12, excised using sterile techniques and placed on an agar media R1, or R6 (Table 1). Results of pooling all sample dates favoring the growth of B. bassiana (Doberski and Tribe, 1980). resulted in the same trends; i.e., an overall significant Plates containing the plant material were allowed to incubate difference between B. bassiana applications (F 10.70; in total darkness at 28 C for 10 d at which point the plant tissue was examined for B. bassiana growth. df 1, 3; P 0.05) and no significant difference between plant types or a plant type B. bassiana interaction. Whorl-stage and pollen shed (R1) application of B. Statistical Analysis bassiana in 1994 did not result in significant differences Greenhouse data were analyzed as repeated measures using in whole plot effects when the plants were sampled at GLM with the line of corn as the whole plot and seed treatment R1 (Treated X 26.3 7.4, N 8; Check X as the split plot. The effects of each seed treatment on germina27.5 7.4, N 8), R6 (Treated X 69.4 11.3, N tion, plant growth, and endophyte formation were analyzed 8; Check X 63.8 6.5, N 8), or senescence separately for each line of corn so as not to attribute differences between seed treatments to differences between hybrids. (Treated X 74.4 7.0, N 8; Check X 58.2 Means were separated using Student’s t-test at P 0.05 (SAS 12.5, N 8). Genetic makeup of the plant played no Institute, 1995). Field data were analyzed using GLM with significant role in the levels of endophytism observed the line of corn and B. bassiana treatment as the whole plot when samples were taken at R1 (Transgenic X and the harvests over time the split plot. Differences in overall 25.0 6.7, N 8; Isoline X 28.8 8.1, N 8), R6 whole-plant dry weight, sheath dry weight, leaf dry weight, (Transgenic X 62.5 11.6, N 8; Isoline X stem dry weight, husk dry weight, ear dry weight, and the 70.6 5.7, N 8), or senescence (Transgenic X dry leaf-to-stem ratio were determined between B. bassiana 62.5 12.3, N 8; Isoline X 70.0 8.2, N 8). application methods along with the hybrid B. bassiana treatThere were no significant interactions in endophytism ment interactions. Means were separated using Student’s t-test at P 0.05 (SAS Institute, 1995). levels between the B. bassiana application and the genetic makeup of the corn for samples taken at R1, R6, or senescence (Table 1). Results of pooling all sample RESULTS dates resulted in the same overall trends with no signifi1994 and 1995 Field Studies cant difference between B. bassiana applications, plant types, or a plant type B. bassiana interaction. Whorl-stage (V6) application alone of B. bassiana in Application of B. bassiana at V6 alone in 1995 did 1994 resulted in significant differences in whole plot not result in significant differences in when the plants effects when the plants were sampled at V12 (F 60.51; were sampled at V12 (Treated X 14.3 3.4, N df 1, 3; P 0.004), with treated plants having signifi8; Check X 17.5 19.8, N 8), R1 (Treated X cantly more endophytism (X 36.9 10.6, N 8) than 25.0 10.5, N 8; Check X 10.0 5.3, N 8), or the untreated check (X 0.0 0.0, N 8). This was R6 (Treated X 7.5 5.3, N 8; Check X 7.5 not the case for plants sampled for endophytism at R1 5.3, N 8). Genetic makeup of the plant played no (Treated X 25.0 10.5, N 8; Check X 17.5 significant role in the levels of endophytism observed 6.1, N 8) or R6 (Treated X 82.5 7.0, N 8; when sampled at V12 (Transgenic X 17.1 4.9, N Check X 47.5 13.1, N 8). Genetic makeup of 8; Isoline X 15.0 6.3, N 8), R1 (Transthe plant played no significant role in the levels of engenic X 15.0 9.8, N 8; Isoline X 20.0 7.6, dophytism observed when sampled at V12 (Transgenic X 28.1 12.8, N 8; Isoline X 10.0 N 8), or R6 (Transgenic X 5.0 4.9, N 8; Table 1. Mean ( SEM) percentage of plants with endophytic B. bassiana from the 1994 field studies. Percentage of plants with an endophyte ( SEM) B. bassiana Time of Hybrid† Treatment‡ Application (V12)§ (R1)§ (R6)§ Transgenic Treated V6 17.5 6.0a 25.0 17.7a 85.0 9.6a Isoline Treated V6 56.3 15.7a 25.0 14.4a 80.0 11.5a Transgenic Control V6 0.0 0.0a 18.8 11.9a 60.0 14.1a Isoline Control V6 0.0 0.0a 16.3 5.5a 35.0 22.2a Percentage of plants with an endophyte ( SEM) Time of Application (R1)§ (R6)§ (Senescence)§ Transgenic Treated V6 and R1 25.0 10.2a 55.0 5.0a 46.3 21.7a Isoline Treated V6 and R1 30.0 12.2a 72.5 11.1a 70.0 12.9a Transgenic Control V6 and R1 25.0 10.2a 70.0 23.8a 78.8 8.3a Isoline Control V6 and R1 27.5 12.3a 68.8 5.2a 70.0 12.2a † Hybrid: Transgenic Jeremy event 802; Isoline B73 Mo17. ‡ B. bassiana Treatment: Treated ARSEF 3113 at 1 106 conidia per plant; control untreated. § Means from the same growth stage and time of fungal application with the same letter are not significantly different P 0.05 (SAS Institute, 1995). 1398 CROP SCIENCE, VOL. 41, SEPTEMBER–OCTOBER 2001 Table 2. Mean ( SEM) percentage of plants with endophytic B. bassiana from the 1995 field studies. Percentage of plants with an endophyte ( SEM) B. bassiana Time of Hybrid† Treatment‡ Application (V12)§ (R1)§ (R6)§ Transgenic Treated V6 15.0 9.6a 5.0 5.0a 10.0 10.0a Isoline Treated V6 20.0 11.5a 15.0 9.6a 5.0 5.0a Transgenic Control V6 20.0 10.0a 25.0 18.9a 0.0 0.0a Isoline Control V6 10.0 5.8a 25.0 12.3a 15.0 9.6a Percentage of plants with an endophyte ( SEM) Time of Application (R6)§ (Senescence)§ Transgenic Treated R1 0.0 0.0a 40.0 18.3a Isoline Treated R1 50.0 10.0b 45.0 18.9a Transgenic Control R1 60.0 14.1b 45.0 20.6a Isoline Control R1 5.0 5.0a 30.0 10.0a † Hybrid: Transgenic Jeremy event 802; Isoline B73 Mo17. ‡ B. bassiana Treatment: Treated ARSEF 3113 at 1 106 conidia per plant; control untreated. § Means from the same growth stage and time of fungal application with the same letter are not significantly different P 0.05 (SAS Institute, 1995). Isoline X 10.0 5.3, N 8). There were no signififive weeks (Table 3). There were significant differences in plant growth due to seed treatment within three of cant interactions in endophytism levels between the B. bassiana application and the genetic makeup of the the hybrids (Table 3). Endophytism levels within each hybrid did not vary significantly between treatments. corn for samples taken at V12, R1, or R6 (Table 2). Results of pooling all sample dates for the V6 applicaNo disease causing organisms were observed on roots in any of the treatments. tion resulted in the same trends with no overall significant difference between B. bassiana applications, plant types, or a plant type B. bassiana interaction. 1997 Field Studies Application of B. bassiana at R1 alone in 1995 did There were no differences in whole plant dry weight, not result in a significant difference in endophytism dry sheath weight, dry leaf weight, dry stem weight, dry levels when plants were sampled at R6 (Treated X husk weight, dry ear weight, or in the dry leaf-to-stem 17.5 9.6, N 8; Check X 50.0 9.3, N 8), ratio between B. bassiana treatments. There were also early senescence (Treated X 25.0 10.5, N 8; no hybrid B. bassiana treatment interactions in whole Check X 32.5 12.5, N 8), or dry down plant dry weight, dry sheath weight, dry leaf weight, dry (Treated X 42.5 12.2, N 8; Check X 37.5 stem weight, dry husk weight, or in the dry leaf-to10.9, N 8). Genetic makeup of the plant also played stem ratio. There was a significant hybrid B. bassiana no significant role in the levels of endophytism observed treatment interaction in dry ear weight when analyzed when samples were taken at R6 (Transgenic X over all harvest dates (F 4.54; df 6, 18; P 0.006). 35.0 11.2, N 8; Isoline X 32.5 11.2, N 8), However, when the plants reached physiological matuearly senescence (Transgenic X 30.0 13.1, N 8; rity at harvest dates seven and eight, there was no longer Isoline X 27.5 9.9, N 8), or dry down (Transa significant interaction between B. bassiana treatment genic X 42.5 12.8, N 8; Isoline X 37.5 and hybrid when harvest dates seven and eight are 10.3, N 8). There were no significant interactions in combined. endophytism levels between the B. bassiana application and the genetic makeup of the corn for samples taken Table 3. Mean ( SEM) plant height (cm) at week three and the at early senescence or dry down (Table 2). There was, mean ( SEM) number of 10 seeds germinated from four corn however, a significant interaction between endophytism hybrids with the following treatments in the 1997 greenhouse study. levels and genetic makeup of the plants for samples taken at early senescence (F 33.92; df 1, 6; P Pioneer Ciba 0.001), with the transgenic corn with no B. bassiana Treatment† 3489 34R06 4494 Max 454 applied (6%) and the B. bassiana granules applied to the B.b. isoline (50%) having significantly higher endophytism Plant height 39.8 2.3a‡ 40.7 0.9a 38.3 2.1b 36.2 2.3b levels than the plants subjected to the other two treatSeeds germinated 9.0 0.41a‡ 10.0 0.0a 9.3 0.48a 8.8 1.3a ments (Table 2). Results of pooling all sample dates Unsoaked showed no significant difference between B. bassiana Plant height 40.9 1.5a 35.1 2.6a 40.8 1.8b 43.7 1.6a applications, or between plant types but a significant Seeds germinated 9.8 0.3a 9.5 0.3a 9.5 0.5a 9.8 0.3a plant type B. bassiana interaction (F 6.68; df 1, Water 6; P 0.04). Plant height 35.3 2.1a 36.2 2.0a 45.8 1.1a 36.3 2.3b Seeds germinated 9.0 0.71a 9.0 0.6a 10.0 0.0a 9.3 0.8a † Treatments: B.b. seeds soaked in 0.1% Tween 80 suspension of B. 1997 Greenhouse Studies bassiana (2 1010 conidia per ml) for 10 minutes; unsoaked seeds not soaked and planted directly out of seed bag; water seeds soaked in a There were no significant differences in the total numsterile 0.1% tween suspension for 10 minutes. ber of germinated seeds between treatments in Pioneer ‡ Treatment means of plant height and seed germination from a hybrid with the same letter are not significantly different P 0.05. 3489, Pioneer 34RO6, Ciba 4494, or Ciba Max 454 after LEWIS ET AL.: ENDOPHYTIC B. BASSIANA IN BT AND NON-BT CORN 1399 Table 4. Mean ( SEM) percentage of plants with endophytic DISCUSSION B. bassiana from the 1997 field studies summed over hybrids. Results of studies with transgenic plants in 1994–1997 Treatment† % of plants with an endophyte consistently showed that the incorporation of a gene for Seed 13.7 2.4a‡ the production of the toxin from B. thuringiensis had Leaf 11.7 2.0ab no effect on the plants ability to form an endophyte with Control 8.2 1.9b B. bassiana. The significant differences in endophytism † Treatments: Seed seeds soaked for three minutes in a 0.1% tween observed in these studies occurred as a result of plots suspension of B. bassiana (2.1 108 conidia per ml); Leaf 8.8 107 conidia applied to whorl of V7-stage corn; control untreated control. receiving an application of B. bassiana at V6 stage in ‡ Means in the same column with the same letter are not significantly 1994 having significantly higher endophytism levels redifferent P 0.05. gardless of plant type. There were unusual interactions in 1995 with the untreated transgenic plants (X 60.0 The percentage of nodes sampled with endophytic B. 14.14, N 4) and the treated isoline (X 50.0 10.0, bassiana was not significantly different between hybrids, N 4) having significantly higher percentage endophybut was significantly different between B. bassiana aptism levels than the treated transgenics and the unplication methods (F 6.58; df 2, 6; P 0.03). Seed treated isoline. Although the differences were signifitreatment with B. bassiana resulted in a significantly cant, they show that transgenic plants are as well suited larger percentage of the plants forming an endophytic to form an endophytic relationship with B. bassiana as relationship compared with untreated plants (Table 4). nontransgenic corn. While B. bassiana application did The overall percentage of plants with endophytic B. not generally increase the percentage of plants with an bassiana among all hybrids increased steadily from no endophyte, the intensity of the endophytic relationship endophyte present on the first two harvest dates to may have been greater in the treated than the untreated 35% of all of the plants collected endophytic at harvest plants (Bing, 1990). eight. The percentage of endophytism increased in a Greenhouse studies demonstrated that seed treatsimilar fashion for each hybrid individually, although ment with B. bassiana had no detrimental impact on there was a drop during the time of seed formation (Fig. seed germination or seedling growth, and did not result 1). Levels of endophytism did not drop at the same in the formation of root disease. These results were harvest date, but a decline occurred during one reproconfirmed when plants were allowed to grow to maturity ductive stage or another in all hybrids. The decline ocunder field conditions with no significant differences in curred during harvest 6 for Ciba 4494 (R5, dent stage), plant growth between B. bassiana treatments for any of harvest 7 for Max 454 (R5, dent stage), harvest 5 for the hybrids. There were no ill effects of a B. bassiana Pioneer 3489 (R4, dough stage), and harvest 4 for Pioseed treatment on transgenic corn or their isoline. Seed treatment did result in a significant increase in endoneer 34RO6 (R3, milk stage). Fig. 1. Percentage endophytism for each hybrid over the course of the growing season in 1997 field studies. Hybrids: Ciba 4494; Ciba Max 454; x Pioneer 3489; * Pioneer 34R06. 1400CROP SCIENCE, VOL. 41, SEPTEMBER–OCTOBER 2001 nomics Experiment Station, Ames, Iowa. Project No. 3543.phytism. The reason for the lack of endophytism in theNames are necessary to report factually on available data;seeds treated with B. bassiana in the greenhouse studyhowever, neither the USDA nor Iowa State University guaran-are unclear.tees or warrants the standard of the product, and the use ofNo significant differences were observed between hy-the name implies no approval of the product to the exclusionbrids in their ability to form an endophytic relationshipof others that may be suitable.with B. bassiana in 1997. Differences in plant growthon a dry-weight basis between hybrids were found butwill not be discussed because they represent differencesREFERENCESbetween hybrids only and not treatment effects. TheBartlett, K., and C. Lefebvre. 1934. Field experiments with Beauveriaproportion of plants exhibiting an endophytic relation-bassiana (Bals.) Vuill., a fungus attacking the European corn borer.ship over all hybrids increased steadily from the thirdJ. Econ. Entomol. 27:1147–1157.harvest date. The same pattern was true for each hybrid Beall, G., G.M. Stirrett, and I.L. Conners. 1939. A field experimentindividually although a drop in the proportion of plantson the control of the European corn borer, Pyrausta nubilalisexhibiting an endophytic relationship occurred duringHübn., by Beauveria bassiana Vuill. Sci. Agric. 19:531–534. Bing, L.A. 1990. The entomopathogen, Beauveria bassiana (Balsamo)ear set for each hybrid before endophytism levels reVuillemin: Colonization and movement in Zea mays L. and poten-bounded (Fig. 1). A similar trend in a nontransgenictial for Ostrinia nubilalis (Hübner) suppression. M.S. thesis, Iowahybrid was reported by Bing and Lewis (1992a,b). WhenState University, Ames.plants reached the point of physiological maturity the Bing, L.A., and L.C. Lewis. 1992a. 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Isolation of entomogenous fungito produce grain.from elm bark and soil with reference to ecology of Beauveria bassiana and Metarhizium anisopliae. Trans. Br. Mycol. Soc. 74:95–100.Over the entire growing season, application of B.Lewis, L.C., and L.A. Bing. 1991. Bacillus thuringiensis Berliner andbassiana either as a seed or foliar application did notBeauveria bassiana (Balsamo) Vuillemen for European corn borerresult in significant differences in dry matter accumula-control: Potential for immediate and season-long suppression. Can.tion. The corn plant grows normally and shows no signsEntomol. 123:387–393.that B. bassiana is acting as a plant pathogen. The corn Lewis, L.C., and J.E. Cossentine. 1986. Season long intraplant epizoot-plant benefits by having an insect defense system, andics of entomopathogens, Beauveria bassiana and Nosema pyrausta, in a corn agroecosystem. Entomophaga 31:363–369.B. bassiana is able to survive in the moist, humid, nutri-Marcandier, S., and G. Riba. 1986. Endemisme de la mycose a Beau-ent-rich environment within the corn plant.veria bassiana (Bals.) Vuillemin dans les populations geograph-Results of this research are significant in terms ofiques de la pyrale du mais, Ostrinia nubilalis (Hübner). Acta Ecol.resistance managment and concerns of transgenic plantsApplic. 7:39–46.forming a “biological control vacuum,” i.e., incorporaRiba, G. 1984. Application en essais parcellaires de plein champ d’untion of the gene for production of the toxin from B.mutant artificiel du champignon entomopathogene Beauveria bassiana [Hyphomycete] contre la pyrale du mais, Ostrinia nubilalisthuringiensis into the corn plant had no adverse effect[Lep.: Pyralidae]. Entomophaga 29:41–48.on the ability of B. bassiana to form an endophyticRitchie, S.W., J.J. Hanway, and G.O. Benson. 1997. How a corn plantrelationship. As a result B. bassiana innoculum loadsdevelops. Iowa State Univ. Sci. Tech. Spec. Rep. 48.will most likely remain stable in the environment even SAS Institute, Inc. 1995. JMP statistics and graphic guide, version 3.1.if large acreages of transgenic corn are planted. If O.SAS Institute, Inc., Cary, NC.nubilalis resistance to Bt occurs, naturally occurring or Stirrett, G.M., G. Beall, and M. Timonin. 1937. A field experiment on the control of the European corn borer, Pyrausta nubilalisapplied B. bassiana could be a significant tool in managHübn., by Beauveria bassiana Vuill. Sci. Agric. 17:587–591.ing resistant O. nubilalis populations.Wagner, B.L., and L.C. Lewis. 2000. Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana. Appl. Envi-ACKNOWLEDGMENTSron. Microbiol. 66:3468–3473.This article is a joint contribution: USDA–ARS and Journal York, G.T. 1958. Field tests with the fungus Beauveria sp. for control of the European corn borer. Iowa State J. Sci. 33:123–129.Paper No. J-18799 of the Iowa Agriculture and Home Eco-