Kimberly D. Gwinn

Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Dual biological control, of both insect pests and plant pathogens, has been reported for the fungal entomopathogens, Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) and Lecanicillium spp. (Ascomycota: Hypocreales). However, the primary mechanisms of plant disease suppression are different for these fungi. Beauveria spp. produce an array of bioactive metabolites, and have been reported to limit growth of fungal plant pathogens in vitro. In plant assays, B. bassiana has been reported to reduce diseases caused by soilborne plant pathogens, such as Pythium, Rhizoctonia, and Fusarium. Evidence has accumulated that B. bassiana can endophytically colonize a wide array of plant species, both monocots and dicots. B. bassiana also induced systemic resistance when endophytically colonized cotton seedlings were challenged with a bacterial plant pathogen on foliage. Species of Lecanicillium are known to reduce disease caused by powdery mildew as well as various rust fungi. Endophytic colonization has been reported for Lecanicillium spp., and it has been suggested that induced systemic resistance may be active against powdery mildew. However, mycoparasitism is the primary mechanism employed by Lecanicillium spp. against plant pathogens. Comparisons of Beauveria and Lecanicillium are made with Trichoderma, a fungus used for biological control of plant pathogens and insects. For T. harzianum Rifai (Ascomycota: Hypocreales), it has been shown that some fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.

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.

Mycelia and spent fermentation broth of Beauveria bassiana incorporated into synthetic diets affect mortality, growth and development of larval Helicoverpa zea (Lepidoptera: Noctuidae)

Abstract Beauveria bassiana endophytically colonises corn (Zea mays) reducing tunneling from European corn borer (Ostrinia nubilalis). Endophytic colonisation of other plants by B. bassiana has been reported, and potentially, may reduce insect feeding on these plants. We evaluated the effects on larval growth and development, and mortality of different rates of dried, ground mycelia and water-soluble metabolites from fermentation broth culture of different isolates of B. bassiana incorporated into a synthetic diet and fed to neonate bollworm, Helicoverpa zea larvae. Development was delayed, weights of larvae were lower, and mortality was high for larvae fed the highest rates (1.0 and 5.0%, w/v) of mycelia incorporated diet compared to control. Insects fed diets containing mycelia of B. bassiana isolate 11-98 had the greatest mortality. Mortality was 100% for larvae fed 5% (w/v) mycelia incorporated diet of isolate 11-98, and 61% for isolate 3-00. For insects fed low rates (0.1 to 0.5%, w/v) of mycelia incorporated diet, mortality was lower, approximately 5% for isolate 11-98, and 5 to 14% for isolate 3-00. At the 0.1% (w/v) rate of mycelia incorporated diet, development occurred at an accelerated rate, compared to fungus-free controls, indicating increased nutrition in the lowest rate fungal diet. Mortality was low for all larvae fed diets containing spent fermentation broth of B. bassiana; however, development was delayed. Insects fed the highest rate (0.5%, v/v) of spent fermentation broth-amended diet had lower pupal weights, and a greater number of days to pupation than insects fed the lowest (0.1%, v/v) rate. Insects fed the 5% (v/v) rate of spent fermentation broth of isolates 11-98 and 3-00 had the longest days to pupation.

Role of Essential Oils in Control of Rhizoctonia Damping-Off in Tomato with Bioactive Monarda Herbage

Plants in the genus Monarda produce complex essential oils that contain antifungal compounds. The objectives of this research were to identify selections of monarda that reduce Rhizoctonia damping-off of tomato, and to determine relationships between essential oil composition of 13 monarda herbages (dried and ground leaves) and disease suppression. Herbages were grouped into five chemotypes, based on essential oil composition and effective concentrations for reducing growth by 50% for Rhizoctonia solani. Replicated and repeated disease control assays were conducted with monarda herbages in greenhouse medium, with or without Rhizoctonia. Percent survival, seedling height, and stem diameter were evaluated at 8 weeks. Survival, seedling height, and stem diameter in herbage-only treatments were not different from the control (no-herbage, no-pathogen) for most herbage treatments. In the pathogen control (no-herbage + Rhizoctonia), seedling survival was 10% that of the control. In pathogen-infested media, seedling survival ranged from 65 to 80% for treatments with thymol chemotypes and 55 to 65% for carvacrol chemotypes. Effective control of Rhizoctonia damping-off was correlated with phenolic monoterpenes; herbages classified as carvacrol chemotypes effectively protected tomato seedlings from Rhizoctonia damping-off disease without phytotoxicity. This study provides evidence that monarda herbages have potential as growing media amendments for control of Rhizoctonia damping-off disease.

Endophyte Isolate and Host Grass Effects on Chaetocnema pulicaria (Coleoptera: Chrysomelidae) Feeding

ABSTRACT Endophytic fungi belonging to the genus Neotyphodium, confer resistance to infected host grasses against insect pests. The effect of host species, and endophtye species and strain, on feeding and survival of the corn flea beetle, Chaetocnema pulicaria Melsheimer (Coleoptera: Chrysomelidae) was investigated. The grass—endophyte associations included natural and artificially derived associations producing varying arrays of common endophyte-related alkaloids or alkaloid groups, peramine, lolitrem B, ergovaline, and the lolines. Preference and nonpreference tests showed that C. pulicaria feeding and survival were reduced by infection of tall fescue with the wild-type strain of N. coenophialum, the likely mechanism being antixenosis rather than antibiosis. In the preference tests, endophyte and host species effects were observed. Of the 10 different Neotyphodium strains tested in artificially derived tall fescue associations, eight strongly deterred feeding by C. pulicaria, whereas the remaining two strains had little or no effect on feeding. Infection of tall fescue with another fungal symbiont, p-endophyte, had no effect. Perennial ryegrass, Lolium perenne L., infected with six strains of endophyte, was moderately resistant to C. pulicaria compared with endophyte-free grass, but four additional strains were relatively inactive. Six Neotyphodium—mendow fescue, Festuca pratensis Huds., associations, including the wild-type N. uncinatum—mendow fescue combination, were resistant, whereas three associations were not effective. Loline alkaloids seemed to play a role in antixenosis to C. pulicaria. Effects not attributable to the lolines or any other of the alkaloids examined also were observed. This phenomenon also has been reported in tests with other insects, and indicates the presence of additional insect-active factors.

Fungal mutualists enhance growth and phytochemical content in Echinacea purpurea

An emerging paradigm in sustainable biotechnique is the use of mutualists to enhance plant growth and secondary metabolism. Our objective was to determine impact of two groups of fungal mutualists on growth and phytochemistry of Echinacea purpurea. Growth, development, and phytochemical concentration were measured in greenhouse-grown 12-week-old plants colonized by arbuscular mycorrhizal fungi (AMF) (Rhizophagus intraradices and Gigaspora margarita) or the endophytic entomopathogen, Beauveria bassiana. In one experiment, all measured growth parameters were increased in mycorrhizal plants. Biomass of AMF-colonized plants was over 13-fold greater than non-mycorrhizal controls receiving the same levels of phosphorous, and over 4-fold greater than non-mycorrhizal controls given additional phosphorous. Endophytic colonization by B. bassiana had minor effects on growth. Colonization by AMF and B. bassiana alone or in combination altered concentrations of phytochemicals (pigments, polyphenolics, alkylamides, and terpenes). Mycorrhizal plants produced up to 4.6-fold higher concentration of polyphenolics. Specific alkylamides increased 1.7 fold in plants colonized only with B. bassiana and up to a 2.4-fold increase in plants colonized by both mutualists. Changes in other phytochemical classes were related to differences in plant size induced by AMF. Phytochemical content (concentration × biomass) was increased up to 30-fold in mycorrhizal plants. Phytochemical relationships to plant biomass were confirmed in a second experiment in which non-mycorrhizal plants were fertilized to produce biomass equivalent to that of mycorrhizal plants. Based on this study, mycorrhizal colonization of E. purpurea enhances phytochemical content; this has major implications for the natural product industries and growers of E. purpurea.

Corn Flea Beetle (Chaetocnema Pulicaria) Responses to Natural Endophytes of Tall Fescue, Meadow Fescue, and Perennial Ryegrass

Adult corn flea beetle (Chaetocnema pulicaria) were less numerous on plots containing tall fescue (Festuca arundinacea) infected with the endophytic fungus Neotyphodium coenophialum than on endophyte-free tall fescue plots (Kirfman et al. 1986). The experiments described in this paper were aimed to ascertain whether the endophyte-mediated resistance to corn flea beetle was a consequence of antibiosis or feeding deterrence, and whether grass-endophyte associations other than tall fescue-N. coenophialum were also resistant.

Development and characterization of simple sequence repeats for Bipolaris sorokiniana and cross transferability to related species

Simple sequence repeats (SSR) markers were developed from a small insert genomic library for Bipolaris sorokiniana, a mitosporic fungal pathogen that causes spot blotch and root rot in switchgrass. About 59% of sequenced clones (n = 384) harbored SSR motifs. After eliminating redundant sequences, 196 SSR loci were identified, of which 84.7% were dinucleotide repeats and 9.7% and 5.6% were tri- and tetra-nucleotide repeats, respectively. Primer pairs were designed for 105 loci and 85 successfully amplified loci. Sixteen polymorphic loci were characterized with 15 B. sorokiniana isolates obtained from infected switchgrass plant materials collected from five states in USA. These loci successfully cross-amplified isolates from at least one related species, including Bipolaris oryzae, Bipolaris spicifera and Bipolaris victoriae, that causes leaf spot on switchgrass. Haploid gene diversity per locus across all isolates studied varied 0.633–0.861. Principal component analysis of SSR data clustered isolates according to their respective species. These SSR markers will be a valuable tool for genetic variability and population studies of B. sorokiniana and related species that are pathogenic on switchgrass and other host plants. In addition, these markers are potential diagnostic tools for species in the genus Bipolaris.

First Report of Cercospora apii s. lat. Causing a Leaf Spot on Cardinal Flower (Lobelia cardinalis) in the United States

Cardinal flower (Lobelia cardinalis L.: Campanulaceae) is a wildflower common throughout much of the eastern United States and grows in a variety of habitats. It is used as an ornamental plant because its bright red flowers appear in the fall and attract hummingbirds. The foliage is generally free of disease when the plant is not flowering, although a few leaf and stem lesions may be present during the growing season. In August/September 2016 and 2017, plants established in eastern Tennessee developed numerous spots on stems and leaves. The round- to lenticular-shaped lesions had dry, white, papery necrotic centers surrounded by reddish-purple host tissue. There were no signs of a pathogen. Leaves were washed in running water for 5 min, surface disinfested with 1.05% NaClO for 15 min, and rinsed three times in sterile water. Lesions and some green tissue from leaves were transferred to half-strength potato dextrose agar (PDA) augmented with 10 mg rifampicin/liter, and incubated in the dark. Mycelium was i...

Bioactive Natural Products in Plant Disease Control

Abstract Increasing sustainability of agricultural production systems increases availability of safe and nutritious food thus addressing the grand agricultural challenge of food security. Sustainability can be incorporated into plant production by expanded use of bioactive natural products for plant protection. In both ancient and modern agriculture compounds derived from plants and microorganisms have been used as biopesticides to reduce impact of disease in plant production. This review will focus on the use of microbial or botanical natural products that act as antimicrobial agents and/or as natural inducers of the host defense system. Commercial products based on extracts of plants (particularly neem, citrus, and Giant Knotweed), proteins from plant pathogens, and mixes of oils derived from herbs and seeds are available for producers. Some products act directly on the fungus, and others stimulate the innate immunity system of the host plant. Antifungal activity of extracts and essential oils is an active area of research, and much discovery-based research is conducted to determine novel chemical combinations or to discover value-added roles for waste streams in plant-based industries. Chemical basis of antifungal activity is not always determined, but active fractions typically include phenolics, flavones, quinones, tannins, terpenes, essential oils, alkaloids, and saponins. Impact of these compounds on fungal growth will be discussed, and available literature on use of these compounds in plant protection formulations will be addressed. Application of basic knowledge to develop innovative control strategies (e.g., biopesticides that turn on plant resistance, plants engineered to overproduce bioactive natural products or produce antimicrobial proteins) will also be discussed.