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Endophytic fungi are increasingly studied for their ability to enhance plant performance in field crops, yet there are few equivalent studies in floricultural crops. Given the economic importance of these crops and pressures faced by growers to produce plants of high aesthetic quality, we surveyed the natural occurrence of foliar fungal endophytes in Knock Out roses to identify candidate beneficial isolates. We also tested the effects of entomopathogenic fungal inocula on marigold and zinnia plant growth using different application approaches. Our survey of Knock Out rose foliage collected from five sites within central Texas revealed at least 24 different fungal genera and 30 probable species, including some isolates providing plant stress tolerance and pathogens or antagonists of insects and nematode pests. The effects of entomopathogen inocula on plant growth varied with host plant (marigold vs. zinnia) and inoculation method (soil drench vs. seed soak). Plant responses were complex, but inoculation with Isaria fumosorosea Wize tended to have a negative effect on plant performance characteristics whereas Beauveria bassiana (Bals.-Criv.) Vuill. tended to have positive effects. When applied to marigold as a seedcoating, I. fumosorosea reduced germination, seedling fresh weight, and produced seedlings with a less compact form. By contrast, seeds inoculated with B. bassiana required less time to germinate, had higher germination rates, and increased the plant compactness. These results show that the impact of fungal entomopathogens applied as endophytes depends on the specific fungi-plant combination being examined. The effect of plant inoculation with entomopathogenic fungi within a pest management context requires further evaluation. Fungi or bacteria causing unapparent or asymptomatic infections entirely within plant tissues are known as endophytes (Stone et al., 2000; Wilson. 2000). Gurulingappa et al. (2010) demonstrated that fungal insect pathogens (entomopathogens) can also be inoculated as endophytes within various monocot and dicot crops, including bean, wheat, corn, pumpkin, tomato, and cotton. Endophytic fungi enhance plant growth in some field crops (Jaber and Enkerli, 2017; Vega et al., 2009); however, few studies have examined how fungal endophytes or entomopathogen inocula may affect floricultural crops. Floricultural crops are a significant component of U.S. agriculture, with a 2015 wholesale value of $4.37 billion from 15 states (U.S. Department of Agriculture, 2016). Annual bedding plants contribute the greatest proportion (30.4%) of the total wholesale value (United States Department of Agriculture, 2016). Among bedding plants, marigolds (Tagetes erecta L.) and zinnia (Zinnia elegans L.) are included in the top 15 flowering and foliar annuals in terms of annual sales (U.S. Department of Agriculture, 2014). Marigolds are one of the premiere garden annuals that perform well in dry, hot conditions and are frequently used as a border, pot, or cut flower. Zinnias are popular garden flowers that come in a wide range of flower colors and shapes, can withstand hot summer temperatures, and are easy to grow from seeds. Babu et al. (2014) report roses are among the most popular flowering shrubs in the United States, with a total wholesale value of U.S. $194 million per year. In Florida and Texas alone, 304,000 potted roses were sold in 2015 (U.S. Department of Agriculture, 2016). The double Knock Out rose, Rosa hybrida L. ‘Radtko’ is a popular hybrid landscape shrub rose that is shade tolerant, capable of growing in a wide range of soils, is very heat and drought tolerant once established, and is resistant to fungal diseases (black spot, powdery mildew, and rust), and aphids (Harp et al., 2009; Martin, 2010). Rose, marigold, and zinnia are ideal host plants for endophyte research due to their economic importance, well-known propagation, and finishing guidelines (Ball Seed, 2019; Harp et al., 2009). Investigations into the use of fungal endophytes for enhancing herbaceous ornamentals are primarily limited to mycorrhizal fungi. Marigolds inoculated with an arbuscular mycorrhizal fungus ‘‘AMF’’ (Glomus constrictum Trappe) displayed an increase in height, shoot biomass, and flower diameter compared with untreated plants (Asrar and Elhindi, 2011). Greater root biomass has also been documented in petunia, marigold, and aster (Gaur and Adholeya, 2005), and higher shoot biomass in orchids (Hou andGuo, 2009) and geraniums (Biermann and Linderman, 1983) treated with mycorrhizal fungi. Some entomopathogens are also capable of colonizing plants as endophytes and studies have shown they can also enhance plant growth (Jaber and Enkerli, 2017). Cotton plants treated with Beauveria bassiana (Bals.-Criv.) Vuill. exhibited increased biomass andmedian number of flower buds (Lopez and Sword, 2015). A commercial formulation of B. bassiana (BotaniGard) was shown to endophytically colonize strawberry and cabbage plants and promote their growth (Dara et al., 2013, 2017). Further research is needed to determine whether plant growth benefits from inoculation with fungal entomopathogens can be applied to ornamental plants. We first conducted a field survey of fungal endophytes naturally associated with roses to identify candidate beneficial fungal endophytes. We also tested the effects of commercial formulations of entomopathogenic fungal inocula applied as soil drenches and seed soaks on T. erecta and Z. elegans plant growth. Since commercial T. erecta seed is often coated to facilitate sowing in automatic seeders, we Received for publication 10 July 2018. Accepted for publication 26 Sept. 2018. This research was financially supported by the American Floral Endowment and gifts-in-kind from Ernst Benary of America, Inc. (EBA). We are grateful to Doug Holden (EBA Global Head of Breeding) and Norbert M€uller (EBA Global Head of Supply Chain) for sharing their expertise on the seed coating chemistry and for providing horticultural expertise. We also thank Cesar Valencia, lab manager for G.A. Sword, for his guidance in developing and performing the endophyte culturing techniques required for this study. A previous draft of this manuscript was improved greatly by comments provided by S.P. Arthurs and H.B. Pemberton. ASHS member. Corresponding author. E-mail: kmheinz@tamu. edu. HORTSCIENCE VOL. 53(12) DECEMBER 2018 1791 tested for the effects of seedcoating to mediate marigold performance when treated with entomopathogenic fungi. Materials and Methods Survey of Fungal Endophytes in Knock Out Rose. Foliar fungal endophytes in Knock Out roses (Rosa hybrida L. ‘Radtko’) were sampled in June 2013 from five geographically distinct locations within the Brazos (Site 1: lat. 30.608685 N, long. 96.288763 W; Site 2: lat. 30.655523 N, long. 96.343588 W;Site 3: lat. 30.596378 W, long. 96.348997 N; and Site 5: lat. 30.634555 W, long. 96.35853 N) and Washington (Site 4: lat. 30.316068 N, long. 96.343081 W) counties of central Texas. At each site, 10 leaves from 10 randomly selected healthy plants 5–7 m apart were collected and returned to the laboratory. Leaves were surfacesterilized, cut into 1cm fragments and plated on potato dextrose agar (PDA) plates in 9 cm diameter petri dishes under sterile conditions. Presumed endophytic fungi growing from the leaf fragments were subcultured, tentatively identified using morphology (Barnett and Hunter, 1998), and the identifications confirmed by sequencing the ribosomal DNA internal transcribed sequence (ITS) region as a DNA barcode (see Ek-Ramos et al., 2013 for methods). Briefly, a 200–300 base pair ITS1 region was PCR amplified using primers ITS1 (5# TCC GTA GGT GAA CCT GCG G 3#) and ITS2 (5# GCT GCG TTC ATC GAT GC 3#). Once purified, these sequences were sequenced at Macrogen Corp. facilities (Rockville, MD). GenBank and UNITE databases were used to identify the species level based on a >98% similarity percentage. Marigold and Zinnia responses to fungal entomopathogen inoculation. The effects of two commercially-available fungal entomopathogens, B. bassiana strain GHA (BotaniGard WP; BioWorks, Inc., Victor, NY) and Isaria fumosoroseaWize (NoFly WP; Novozymes BioAg Inc., Houston, TX), were tested on marigold (‘Discovery Yellow’) and zinnia (Giant Mix) provided by Ernst Benary of America, Inc. (DeKalb, IL). Inocula for tests were obtained by culturing fungi on sabouraud dextrose agar (SDA) and incubated for 5 weeks at 25 C. Mycelia and conidia were harvested by flooding the media with 6 mL of autoclaved 0.1% Triton X-100, scraping the surface, pipetting the contents, and vortexing for 1 min. The liquid was strained with a steel sieve of mesh size 0.001 inches into a second tube. The suspensions were diluted in 2% methyl cellulose to a concentration of 108 conidia/mL using a haemocytometer. A germination test was performed on each fungal suspension to confirm the viability of conidia before treatment applications as described by Gurulingappa et al. (2010). Before use, seeds were surface sterilized in 0.5% sodium hypochlorite for 2 min, followed by 2 min in 70% ethanol, and rinsed three times in sterile water and dried on a sterile paper towel (Posada and Vega, 2006). Two inoculation protocols were used for each fungal treatment in a full factorial design: 1) soaking surface-sterilized seeds for 12 h in aqueous spore suspensions (10 spores/mL) or the water control, and 2) applying a soil drench (1 mL of the 10 spore solution or water control) directly to the seed and surrounding soil at planting. Suspensions of conidia rather than formulated products were used to standardize dosage of a single, pure strain of conidia. Percent conidial germination from seeds soaked for 12 h in suspensions of either of the two fungal isolates was always in excess of 80%. Seeds from all plant/fungi treatment combinations were planted individually in 8.9 cm wide-by-8.9 cm deep, thinwall square pots (Dillen Products, Middlefield

Fungal Endophytes in Knock Out® Rose and Performance Effects of Entomopathogens on Marigold and Zinnia