Characterization and microsatellite marker development for a common bark and ambrosia beetle associate, Geosmithia obscura.

Symbioses between Geosmithia fungi and wood-boring and bark beetles seldom result in disease induction within the plant host. Yet, exceptions exist such as Geosmithia morbida, the causal agent of Thousand Cankers Disease (TCD) of walnuts and wingnuts, and Geosmithia sp. 41, the causal agent of Foamy Bark Canker disease of oaks. Isolates of G. obscura were recovered from black walnut trees in eastern Tennessee and at least one isolate induced cankers following artificial inoculation. Due to the putative pathogenicity and lack of recovery of G. obscura from natural lesions, a molecular diagnostic screening tool was developed using microsatellite markers mined from the G. obscura genome. A total of 3256 candidate microsatellite markers were identified (2236, 789, 137 di-, tri-, and tetranucleotide motifs, respectively), with 2011, 703, 101 di-, tri-, and tetranucleotide motifs, respectively, containing markers with primers. From these, 75 microsatellite markers were randomly selected, screened, and optimized, resulting in 28 polymorphic markers that yielded single, consistently recovered bands, which were used in downstream analyses. Five of these microsatellite markers were found to be specific to G. obscura and did not cross-amplify into other, closely related species. Although the remaining tested markers could be useful, they cross-amplified within different Geosmithia species, making them not reliable for G. obscura detection. Five novel microsatellite markers (GOBS9, GOBS10, GOBS41, GOBS43, and GOBS50) were developed based on the G. obscura genome. These species-specific microsatellite markers are available as a tool for use in molecular diagnostics and can assist future surveillance studies.

Microsatellite Loci Reveal High Genetic Diversity, Mutation, and Migration Rates as Invasion Drivers of Callery Pear (Pyrus calleryana) in the Southeastern United States.

Pyrus calleryana Decne. (Callery pear) is a deciduous tree native to China, Japan, Korea, and Taiwan. It is a popular ornamental tree in the United States (US) with early spring blooms and vibrant fall color. There are at least 26 cultivars of P. calleryana available in the US of which "Bradford" is the most well-known. Open-pollinated P. calleryana escapees are becoming one of the most common invasive tree species in the eastern United States. Developing better management practices for invasive P. calleryana requires detailed knowledge about reproductive biology and genetic diversity of the species, however, little is currently known about genetic variability within those open-pollinated populations. We investigated genetic diversity and population structure of non-cultivated, escaped P. calleryana populations within a ∼177 km radius in the southeastern United States. Because P. calleryana exhibits a range of morphological variation with great evolutionary potential, we hypothesized that a high genetic diversity would be manifested among escaped P. calleryana. Using 15 previously developed microsatellite loci, we genotyped 180 open-pollinated P. calleryana individuals that were collected across six naturally occurring sites in Tennessee, Georgia, and South Carolina, United States. Our results demonstrated the presence of a population structure with high genetic diversity, high gene flow, and high genetic differentiation between individuals across collection sites. Our results revealed that P. calleryana populations had differentiated shortly after the introduction to the US, most likely from specimens imported from Asia, consistent with historical records and our prior findings. The high invasive potential of the species is perhaps best underscored by transformation of P. calleryana specimens introduced from Asia into escape populations at continental scale across the United States. Our data also provided novel insight into potential issues that could be problematic for the future as P. calleryana may pose a potential threat to the economy, ecology, and native biodiversity in invaded areas.

Genetic diversity in North American Cercis Canadensis reveals an ancient population bottleneck that originated after the last glacial maximum.

Understanding of the present-day genetic diversity, population structure, and evolutionary history of tree species can inform resource management and conservation activities, including response to pressures presented by a changing climate. Cercis canadensis (Eastern Redbud) is an economically valuable understory tree species native to the United States (U.S.) that is also important for forest ecosystem and wildlife health. Here, we document and explain the population genetics and evolutionary history of this deciduous tree species across its distributed range. In this study, we used twelve microsatellite markers to investigate 691 wild-type trees sampled at 74 collection sites from 23 Eastern U.S. states. High genetic diversity and limited gene flow were revealed in wild, natural stands of C. canadensis with populations that are explained by two major genetic clusters. These findings indicate that an ancient population bottleneck occurred coinciding with the last glacial maximum (LGM) in North America. The structure in current populations likely originated from an ancient population in the eastern U.S. that survived LGM and then later diverged into two contemporary clusters. Data suggests that populations have expanded since the last glaciation event from one into several post-glacial refugia that now occupy this species' current geographic range. Our enhanced understanding benchmarks the genetic variation preserved within this species and can direct future efforts in conservation, and resource utilization of adaptively resilient populations that present the greatest genetic and structural diversity.

Microsatellite Loci Reveal Genetic Diversity of Asian Callery Pear (Pyrus calleryana) in the Species Native Range and in the North American Cultivars.

Pyrus calleryana Decne. (Callery pear) includes cultivars that in the United States are popular ornamentals in commercial and residential landscapes. Last few decades, this species has increasingly naturalized across portions of the eastern and southern US. However, the mechanisms behind this plant's spread are not well understood. The genetic relationship of present-day P.calleryana trees with their Asian P. calleryana forebears (native trees from China, Japan, and Korea) and the original specimens of US cultivars are unknown. We developed and used 18 microsatellite markers to analyze 147 Pyrus source samples and to articulate the status of genetic diversity within Asian P. calleryana and US cultivars. We hypothesized that Asian P. calleryana specimens and US cultivars would be genetically diverse and would show genetic relatedness. Our data revealed high genetic diversity, high gene flow, and presence of population structure in P. calleryana, potentially relating to the highly invasive capability of this species. Strong evidence for genetic relatedness between Asian P. calleryana specimens and US cultivars was also demonstrated. Our data suggest the source for P. calleryana that have become naturalized in US was China. These results will help understand the genetic complexity of invasive P. calleryana when developing management for escaped populations: In follow-up studies, we use the gSSRs developed here to analyze P. calleryana escape populations from across US.

Development and Characterization of 15 Novel Genomic SSRs for Viburnum farreri.

The Viburnum genus is of particular interest to horticulturalists, phylogeneticists, and biogeographers. Despite its popularity, there are few existing molecular markers to investigate genetic diversity in this large genus, which includes over 160 species. There are also few polymorphic molecular tools that can delineate closely related species within the genus. Viburnum farreri, a member of the Solenotinus subclade and one of the centers of diversity for Viburnum, was selected for DNA sequencing and development of genomic simple sequence repeats (gSSRs). In this study, 15 polymorphic gSSRs were developed and characterized for a collection of 19 V. farreri samples. Number of alleles per locus ranged from two- to- eight and nine loci had four or more alleles. Observed heterozygosity ranged from 0 to 0.84 and expected heterozygosity ranged from 0.10 to 0.80 for the 15 loci. Shannon diversity index values across these loci ranged from 0.21 to 1.62. The markers developed in this study add to the existing molecular toolkit for the genus and will be used in future studies investigating cross-transferability, genetic variation, and species and cultivar delimitation in the Viburnum genus and closely allied genera in the Adoxaceae and Caprifoliaceae.

Conventional Gel Electrophoresis and TaqMan Probes Enable Rapid Confirmation of Thousand Cankers Disease from Diagnostic Samples.

Thousand cankers disease (TCD) is caused by the fungal pathogen Geosmithia morbida and vectored by the walnut twig beetle Pityophthorus juglandis. In infected walnut and butternut (Juglans spp.) hosts and wingnut species (Pterocarya spp.) hosts, tree decline and death results in ecological disruption and economic losses. A rapid molecular detection protocol for TCD using microsatellite markers can confirm the presence of insect vector or fungal pathogen DNA, but it requires specialized expensive equipment and technical expertise. Using four different experimental approaches, capillary and conventional gel electrophoresis, and traditional polymerase chain reaction (PCR) and quantitative PCR (qPCR), we describe simplified and inexpensive processes for diagnostic confirmation of TCD. The improved and rapid detection protocols reported in this study reduce time and equipment costs associated with detection of molecular pest and pathogen DNA by (1) using conventional gel electrophoresis or TaqMan molecular probes to elucidate the detection limits for G. morbida and P. juglandis DNA and (2) identifying resources that allow visualization of positive test results for infected host plant tissue samples. Conventional gel electrophoresis and TaqMan molecular probe protocols detected presence of DNA from TCD-associated fungal and insect samples. These procedural improvements can be readily adopted by diagnostic end-users and adapted for use with other complex disease systems to enable rapid pest and pathogen detection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Odorous House Ant (Hymenoptera: Formicidae) Colony Movement in Response to Moisture, Shade, and Food Proximity.

Tapinoma sessile (Say) is a common ant throughout the United States that frequently relocates portions of its nests to form large polydomous colonies in urban areas. Despite widespread research on the control of T. sessile as a nuisance pest, relatively little work has focused on the biology of its nesting behavior and movement. We evaluated shade, moisture, and proximity to food as factors triggering colony movement in laboratory assays. Initially, T. sessile colonies moved to shaded artificial nest sites irrespective of arena moisture. Then, workers and brood were increasingly moved to moist artificial nest sites over time. Colonies moved workers and brood to near-food artificial nest sites over both 1 m and 6 m distances. Queens relocated to near-food nest sites over 1 m distances, but not 6 m distances, during the 49-d study. Results suggest that an increase either in moisture or food in proximity to a residence is likely to account for observed increases in T. sessile abundance near structures.

Habitat fragmentation influences genetic diversity and differentiation: Fine-scale population structure of Cercis canadensis (eastern redbud).

Forest fragmentation may negatively affect plants through reduced genetic diversity and increased population structure due to habitat isolation, decreased population size, and disturbance of pollen-seed dispersal mechanisms. However, in the case of tree species, effective pollen-seed dispersal, mating system, and ecological dynamics may help the species overcome the negative effect of forest fragmentation. A fine-scale population genetics study can shed light on the postfragmentation genetic diversity and structure of a species. Here, we present the genetic diversity and population structure of Cercis canadensis L. (eastern redbud) wild populations on a fine scale within fragmented areas centered around the borders of Georgia-Tennessee, USA. We hypothesized high genetic diversity among the collections of C. canadensis distributed across smaller geographical ranges. Fifteen microsatellite loci were used to genotype 172 individuals from 18 unmanaged and naturally occurring collection sites. Our results indicated presence of population structure, overall high genetic diversity (H E = 0.63, H O = 0.34), and moderate genetic differentiation (F ST = 0.14) among the collection sites. Two major genetic clusters within the smaller geographical distribution were revealed by STRUCTURE. Our data suggest that native C. canadensis populations in the fragmented area around the Georgia-Tennessee border were able to maintain high levels of genetic diversity, despite the presence of considerable spatial genetic structure. As habitat isolation may negatively affect gene flow of outcrossing species across time, consequences of habitat fragmentation should be regularly monitored for this and other forest species. This study also has important implications for habitat management efforts and future breeding programs.

Sciadopitys verticillata Resin: Volatile Components and Impact on Plant Pathogenic and Foodborne Bacteria.

Sciadopitys verticillata (Sv) produces a white, sticky, latex-like resin with antimicrobial properties. The aims of this research were to evaluate the effects of this resin (Sv resin) on bacterial populations and to determine the impact of its primary volatile components on bioactivity. The impact of sample treatment on chemical composition of Sv resin was analyzed using Fourier transform infrared spectroscopy (FTIR) coupled with principal component analysis. The presence and concentration of volatiles in lyophilized resin were determined using gas chromatography/mass spectrometry (GC/MS). Changes in bacterial population counts due to treatment with resin or its primary volatile components were monitored. Autoclaving of the samples did not affect the FTIR spectra of Sv resin; however, lyophilization altered spectra, mainly in the CH and C=O regions. Three primary bioactive compounds that constituted >90% of volatiles (1R-α-pinene, tricyclene, and ß-pinene) were identified in Sv resin. Autoclaved resin impacted bacterial growth. The resin was stimulatory for some plant and foodborne pathogens (Pseudomonas fluorescens, P. syringae, and Xanthomonas perforans) and antimicrobial for others (Escherichia coli, Bacillus cereus, Agrobacterium tumefaciens, and Erwinia amylovora). Treatment with either 1R-α-pinene or ß-pinene reduced B. cereus population growth less than did autoclaved resin. The complex resin likely contains additional antimicrobial compounds that act synergistically to inhibit bacterial growth.

The digestive system in Zygentoma as an insect model for high cellulase activity.

The digestive system of selected phytophagous insects has been examined as a potential prospecting resource for identification of novel cellulolytic enzymes with potential industrial applications. In contrast to other model species, however, limited detailed information is available that characterizes cellulolytic activity and systems in basal hexapod groups. As part of a screening effort to identify insects with highly active cellulolytic systems, we have for the first time, identified species of Zygentoma that displayed the highest relative cellulase activity levels when compared to all other tested insect groups under the experimental conditions, including model species for cellulolytic systems such as termite and cockroach species in Rhinotermitidae (formerly Isoptera) and Cryptocercidae (formerly Blattodea). The goal of the present study was to provide a morphohistological characterization of cellulose digestion and to identify highly active cellulase enzymes present in digestive fluids of Zygentoma species. Morphohistological characterization supported no relevant differences in the digestive system of firebrat (Thermobia domestica) and the gray silverfish (Ctenolepisma longicaudata). Quantitative and qualitative cellulase assays identified the foregut as the region with the highest levels of cellulase activity in both T. domestica and C. longicaudata. However, T. domestica was found to have higher endoglucanase, xylanase and pectinase activities compared to C. longicaudata. Using nano liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS/MS) and a custom gut transcriptome we identified cellulolytic enzymes from digestive fluids of T. domestica. Among the identified enzymes we report putative endoglucanases matching to insect or arthropod enzymes and glucan endo-1,6-ß-glucosidases matching bacterial enzymes. These findings support combined activities of endogenous and symbiont-derived plant cell wall degrading enzymes in lignocellulose digestion in Zygentoma and advance our understanding of cellulose digestion in a primitive insect group.

The origin of the odorant receptor gene family in insects.

The origin of the insect odorant receptor (OR) gene family has been hypothesized to have coincided with the evolution of terrestriality in insects. Missbach et al. (2014) suggested that ORs instead evolved with an ancestral OR co-receptor (Orco) after the origin of terrestriality and the OR/Orco system is an adaptation to winged flight in insects. We investigated genomes of the Collembola, Diplura, Archaeognatha, Zygentoma, Odonata, and Ephemeroptera, and find ORs present in all insect genomes but absent from lineages predating the evolution of insects. Orco is absent only in the ancestrally wingless insect lineage Archaeognatha. Our new genome sequence of the zygentoman firebrat Thermobia domestica reveals a full OR/Orco system. We conclude that ORs evolved before winged flight, perhaps as an adaptation to terrestriality, representing a key evolutionary novelty in the ancestor of all insects, and hence a molecular synapomorphy for the Class Insecta.

Walnut Twig Beetle (Coleoptera: Curculionidae: Scolytinae) Colonization of Eastern Black Walnut Nursery Trees.

Thousand cankers disease, caused by the invasive bark beetle Pityophthorus juglandis Blackman and an associated fungal pathogen Geosmithia morbida M.Kolarík, E. Freeland, C. Utley, N. Tisserat, currently threatens the health of eastern black walnut (Juglans nigra L.) in North America. Both the beetle and pathogen have expanded beyond their native range via transport of infested walnut wood. Geosmithia morbida can develop in seedlings following inoculation, but the ability of P. juglandis to colonize young, small diameter trees has not been investigated. This study assessed the beetle's colonization behavior on J. nigra nursery trees. Beetles were caged directly onto the stems of walnut seedlings from five nursery sources representing a range of basal stem diameter classes. Seedlings were also exposed to P. juglandis in a limited choice, field-based experiment comparing pheromone-baited and unbaited stems. When beetles were caged directly onto stems, they probed and attempted to colonize seedlings across the range of diameters and across sources tested, including stems as small as 0.5 cm in diameter. In the field experiment, beetles only attempted to colonize seedlings that were baited with a pheromone lure and appeared to prefer (though not statistically significant) the larger diameter trees. Despite several successful penetrations into the phloem, there was no evidence of successful progeny development within the young trees in either experiment. Further investigation is recommended to better elucidate the risk nursery stock poses as a pathway for thousand cankers disease causal organisms.

Trap Style, Bait, and Height Deployments in Black Walnut Tree Canopies Help Inform Monitoring Strategies for Bark and Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae).

Knowledge about which bark and ambrosia beetle species are active and at what heights in black walnut canopies is not well understood. Neither is the role of these beetles in spreading Thousand Cankers Disease. To assist with future planned research, which will assess the extent to which these beetle species are associated with Geosmithia morbida Kolarík, Freeland, Utley, and Tisserat (Ascomycota: Hypocreales: Bionectriaceae), experiments were undertaken to monitor bark and ambrosia beetles in urban landscapes and parks in Tennessee between 2011 and 2013. Within mature walnut tree canopies, sticky panel, modified soda bottle, and Lindgren traps were deployed at different heights, with and without ethanol as an attractant and with and without walnut stem sections, or in situ limbs that had been girdled or injection with ethanol to simulate stressed tree tissues. Bark and ambrosia beetle species (Coleoptera: Curculionidae: Scolytinae) collected in greatest abundance included Ambrosiodmus rubricollis (Eichhoff), Ambrosiophilus atratus (Eichhoff), Cnestus mutilatus (Blandford), Dryoxylon onoharaense (Murayama), Euwallacea validus (Eichhoff), Monarthrum fasciatum (Say), Monarthrum mali (Fitch), Xyleborinus saxesenii (Ratzeburg), Xyleborus affinis Eichhoff, Xyleborus ferrugineus (Fabricius), Xylosandrus crassiusculus (Motschulsky), and Xylosandrus germanus (Blandford). C. mutilatus, X. saxesenii, and X. crassiusculus were more active higher in trees than most other species and were strongly attracted to ethanol via all means of lure deployment. C. mutilatus, which were captured from April through October and increased in abundance across the 3-yr study, were most abundant in late May with a second activity period in late August.

Expression of an endoglucanase from Tribolium castaneum (TcEG1) in Saccharomyces cerevisiae.

Insects are a largely unexploited resource in prospecting for novel cellulolytic enzymes to improve the production of ethanol fuel from lignocellulosic biomass. The cost of lignocellulosic ethanol production is expected to decrease by the combination of cellulose degradation (saccharification) and fermentation of the resulting glucose to ethanol in a single process, catalyzed by the yeast Saccharomyces cerevisiae transformed to express efficient cellulases. While S. cerevisiae is an established heterologous expression system, there are no available data on the functional expression of insect cellulolytic enzymes for this species. To address this knowledge gap, S. cerevisiae was transformed to express the full-length cDNA encoding an endoglucanase from the red flour beetle, Tribolium castaneum (TcEG1), and evaluated the activity of the transgenic product (rTcEG1). Expression of the TcEG1 cDNA in S. cerevisiae was under control of the strong glyceraldehyde-3 phosphate dehydrogenase promoter. Cultured transformed yeast secreted rTcEG1 protein as a functional ß-1,4-endoglucanase, which allowed transformants to survive on selective media containing cellulose as the only available carbon source. Evaluation of substrate specificity for secreted rTcEG1 demonstrated endoglucanase activity, although some activity was also detected against complex cellulose substrates. Potentially relevant to uses in biofuel production rTcEG1 activity increased with pH conditions, with the highest activity detected at pH 12. Our results demonstrate the potential for functional production of an insect cellulase in S. cerevisiae and confirm the stability of rTcEG1 activity in strong alkaline environments.

Identification, cloning, and expression of a GHF9 cellulase from Tribolium castaneum (Coleoptera: Tenebrionidae).

The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-ß-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-ß-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.

Characterization of cellulolytic activity from digestive fluids of Dissosteira carolina (Orthoptera: Acrididae).

Previous screening of head-derived and gut fluid extracts of Carolina grasshoppers, Dissosteira carolina (L.) revealed relatively high activity against cellulase substrates when compared to other insect groups. In this work we report on the characterization and identification of enzymes involved in cellulolytic activity in digestive fluids of D. carolina. In zymograms using carboxymethylcellulose (CMC) as substrate, we detected four distinct cellulolytic protein bands in D. carolina gut fluids, common to all developmental stages. These cellulolytic enzymes were localized to foregut and midgut regions of the D. carolina digestive tract. Cellulases were purified from D. carolina head and gut fluid extracts by liquid chromatography to obtain N-terminal amino acid sequence tags. Database searches with sequence tags from head fluids indicated high similarity with invertebrate, bacterial and plant beta1,4-endoglucanases, while no homologues were identified for the gut-derived protein. Our data demonstrate the presence of cellulolytic activity in the digestive system of D. carolina and suggest that cellulases of endogenous origin are present in this organism. Considering that this grasshopper species is a pest of grasses, including switchgrass that has been suggested bioethanol feedstock, characterization of insect cellulolytic systems may aid in developing applications for plant biomass biodegradation for biofuel production.

Prospecting for cellulolytic activity in insect digestive fluids.

Efficient cellulolytic enzymes are needed to degrade recalcitrant plant biomass during ethanol purification and make lignocellulosic biofuels a cost-effective alternative to fossil fuels. Despite the large number of insect species that feed on lignocellulosic material, limited availability of quantitative studies comparing cellulase activity among insect taxa constrains identification of candidate species for more targeted identification of effective cellulolytic systems. We describe quantitative determinations of the cellulolytic activity in gut or head-derived fluids from 68 phytophagous or xylophagous insect species belonging to eight different taxonomic orders. Enzymatic activity was determined for two different substrates, carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), approximating endo-beta-1,4-glucanase and complete cellulolytic activity, respectively. Highest CMC gut fluid activities were found in Dictyoptera, Coleoptera, Isoptera, and Orthoptera, while highest MCC gut fluid activities were found in Coleoptera, Hymenoptera, Lepidoptera, and Orthoptera. In most cases, gut fluid activities were greater with CMC compared to MCC substrate, except in Diptera, Hymenoptera, and Lepidoptera. In contrast, cellulolytic activity levels in most head fluids were greater on the MCC substrate. Our data suggests that a phylogenetic relationship may exist for the origin of cellulolytic enzymes in insects, and that cellulase activity levels correlate with taxonomic classification, probably reflecting differences in plant host or feeding strategies.

Emission of volatile chemicals from flowering dogwood (cornus Florida L.) flowers.

Reproduction of flowering dogwood trees occurs via obligate out-crossing, and U.S. native bees have been suggested to be primary pollinators of this ecologically and economically important deciduous tree. Whether floral volatiles play a role in reproduction of the dogwood remains unclear. Objectives of this study were to identify principal volatile chemicals emitted from dogwood flowers and to assess a temporal volatile emission profile and volatile consistency across four cultivars. Inflorescences with intact bracts and 5 cm flower pedicel were removed from dogwood trees and subjected to headspace volatile collection. Six principal volatile compounds were detected from the flowers of the cultivar 'World's Fair' with 3-formylpyridine as the most abundant constituent. Subsequent headspace analyses performed using inflorescences without bracts or floral pedicels alone indicated that 3-formylpyridine, E-beta-ocimene, S-linalool, and ketoisophorone were mainly emitted from inflorescences. Experiments were also conducted to determine whether volatile emissions differed across time and between different cultivars of flowering dogwood. When volatile emission was analyzed for 48 h using 12 h light/dark cycles, the emission of several volatile compounds displayed diurnal patterns. Finally, whereas florets in inflorescences of four different dogwood cultivars emitted similar levels of the six principal floral volatile chemicals, 'Cherokee Brave' flowers alone yielded 4-methoxybenzaldehyde and germacrene-D. The implications of the findings of this study to dogwood breeding programs are discussed.

Beauveria bassiana: endophytic colonization and plant disease control.

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.