Endophytic Relationship Research Articles

Association Between Fusarium spp. on Spartina alterniflora and Dieback Sites in Connecticut and Massachusetts

Sudden vegetation dieback (SVD) is defined as the loss and lack of recovery of smooth cordgrass (Spartina alterniflora) in salt marshes. A new species of a moderately pathogenic fungus called Fusarium palustre is consistently found in SVD sites, but greenhouse tests revealed that it is not capable of causing mortality of healthy plants. Similarly, root-knot nematodes (Meloidogyne spartinae) are also found in SVD sites, but their incidence in marshes affected by SVD is not known. To understand more about the ecology of F. palustre and M. spartinae, salt marshes along Connecticut's Long Island Sound and Massachusetts' Cape Cod that exhibited SVD and those that did not, were visited during the summers of 2007, 2008, and 2009. Belowground and aboveground tissues of smooth cordgrass plants from 18 marshes were removed, washed, and assayed for Fusarium spp. to determine if patterns between the incidence of the different species of Fusarium, their virulence on S. alterniflora, root-knot nematodes (M. spartinae), and the health of the marsh could be revealed. There were significantly more colonies of Fusarium growing from plants in SVD sites (6.1%) than in healthy marshes where no SVD was present (<1.0%). The incidence of Fusarium spp. from plants at the perimeter of the SVD site was not statistically different from asymptomatic plants 10-20 m from the SVD edge. The majority of isolates could be assigned to one of two species, F. palustre or another slightly pathogenic group called Fusarium cf. incarnatum (88% in 2007, 62% in 2008, and 96% in 2009). The ratio of F. palustre to F. cf. incarnatum was 6.7, 2.7, or 2.1 for 2007, 2008, or 2009, respectively. Greenhouse tests on healthy S. alterniflora revealed that isolates of F. palustre were more virulent than F. cf. incarnatum, regardless of whether they were recovered from plants in healthy marshes or in SVD sites. Root-knot nematodes were found sporadically and could not be associated with SVD. Factorial greenhouse experiments did not demonstrate any interaction between F. palustre and M. spartinae providing no experimental evidence that combining Fusarium and root-knot nematodes could cause mortality. The presence of Fusarium on S. alterniflora in healthy marshes also suggests an endophytic relationship that may subsequently function in the breakdown of tissue when plants are compromised.

Prevalence of an intraspecific Neotyphodium hybrid in natural populations of stout wood reed (Cinna arundinacea L.) from eastern North America

Members of genus Neotyphodium are asexual derivatives of sexual Epichloë species and maintain endophytic relationships with many cool-season grasses. Most Neotyphodium species analyzed so far are interspecific hybrids with combined or partial genomes of two or three ancestral species. In this study we characterized Neotyphodium isolates from Cinna arundinacea, a perennial cool-season grass from eastern North America. A total of 23 isolates grouping into two distinct morphotypes were obtained from five local populations of C. arundinacea. PCR amplification and cloning of translation-elongation factor 1-α (tefA) and β-tubulin (tubB) genes of 10 isolates comprising both morphotypes (two isolates per location) revealed that all 10 contain two copies of tefA and tubB genes. Surprisingly phylogenetic analysis of mainly non-coding sequence from these genes revealed that both copies in each isolate were inherited from Epichloë typhina ancestors, indicating that the C. arundinacea endophytes arose through intraspecific hybridization between two E. typhina progenitors with extant relatives infecting hosts Poa nemoralis and Poa pratensis. Furthermore the tefA sequences were identical between isolates, as were tubB sequences, despite obvious morphological differences. Profiling of alkaloid biosynthetic genes from these isolates indicated the presence of the peramine biosynthetic gene (perA) and the absence of genes required for biosynthesis of lolines, indole-diterpenes and ergot alkaloids. Thus this endophyte is potentially capable of producing peramine in planta and providing protection to its host from insect pests. The absence of genes for indole-diterpenes and ergot alkaloid biosynthesis makes this endophyte a candidate for agricultural applications. Based on our phylogenetic analysis, alkaloid profiling and description of morphological characteristics, we propose the name Neotyphodium schardlii for these isolates from C. arundinacea, a new member of genus Neotyphodium and the first described to have arisen through intraspecific hybridization.

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species ( Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.

Antibiosis, mycoparasitism, and colonization success for endophytic Trichoderma isolates with biological control potential in Theobroma cacao

Theobroma cacao (cacao) suffers severe yield losses in many major production areas due to fungus-induced diseases. Cacao supports a complex endophytic microbial community that offers candidates for biocontrol of cacao diseases. Endophytic isolates of Trichoderma species were isolated from the live sapwood of trunks of Theobroma species, pods of Theobroma species, and a liana ( Banisteriopsis caapi). Fifteen isolates of Trichoderma, potentially representing seven species, were selected for characterization of the influence of seedling inoculation on the establishment of endophytic growth in cacao seedlings. An isolate of Colletotrichum gloeosporioides was also included. The isolates studied in vitro varied in their abilities to produce metabolites inhibitory to Moniliophthora roreri and in their abilities to parasitize M. roreri cultures. The five inoculation methods used were: (1) inoculation of germinating seed on agar plates; (2) plate inoculation followed by planting in sterile soil; (3) planting sterile seed in pre-inoculated soil; (4) inoculation of emerged seedlings at the soil surface; and (5) inoculation of emerged seedlings between the cotyledon and stem. All the isolates studied were able to colonize Theobroma cacao seedlings, but isolates DIS 110a ( Trichoderma cf. harzianum), DIS 219b ( T. hamatum), DIS 219f ( T. harzianum), and TA ( T. asperellum) were the most efficient across inoculation methods. These same isolates also caused moderate to severe discoloration of roots of cacao seedlings germinated on water agar plates. Isolates DIS 173a ( T. spirale), DIS 185c ( T. stromaticum), and Col ( Colletotrichum gloeosporioides) were inefficient colonizers of cacao. Most of the isolates studied were able to establish an endophytic relationship with cacao by colonizing the above ground portions of the cacao seedling, and exploitation of this characteristic could lead to the development of novel biocontrol strategies for control of cacao diseases.

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana ( Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana ( Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.

Field performance of maize grown from Fusarium verticillioides-inoculated seed

Fusarium verticillioides is an important fungus occupying dual roles in the maize plant. The fungus functions as an endophyte, a fungal/host interaction beneficial to the growth of some plants. At other times, the fungus may function as a mycotoxin producing pathogen. The advantages and/or disadvantages of the endophytic relationship must be established in order to target appropriate sites for controlling diseases and mycotoxins in maize. One possibility could be to ensure seed maize is fungal free prior to planting. Reciprocal inoculations were made with two fungal isolates on seed of two maize genotypes. Yield was measured at harvest by ear and seed characters and vegetative growth at one-month intervals for plant survival, height, weight and stem diameter. Yield and vegetative growth differed among mature plants only once based on seed inoculation status. In 1998, plant weight was reduced and seed weight per ear was increased for the dent maize, GT-MAS: gk, grown from F. verticillioides RRC 374-inoculated seed compared to other seed treatments. Most vegetative characters were reduced at the first collection for Silver Queen plants grown from F. verticillioides-inoculated seed in 1997 and 1999, but not in 1998. However, no significant differences occurred among mature Silver Queen plants during any of the three growing seasons. In conclusion, yield and vegetative growth of mature maize plants grown from F. verticillioides-inoculated seed were equal to or greater than plants grown from non-inoculated seed under south Georgia field conditions during 1997, 1998, and 1999.

Seasonal synchrony of a green algal endophyte, Acrosiphonia (Codiolales), with its red algal hosts, Mastocarpus and Mazzaella (Gigartinales)

A.V. Sussmann, R. Scrosati and R.E. DeWreede. 2005. Seasonal synchrony of a green algal endophyte, Acrosiphonia (Codiolales), with its red algal hosts, Mastocarpus and Mazzaella (Gigartinales). Phycologia 44: 129–132.‘Codiolum’ and ‘chlorochytrium’ (both unicellular sporophyte phases of the filamentous green alga Acrosiphonia) are endophytic in the red algal crust ‘petrocelis’ (sporophyte of Mastocarpus papillatus) and in the bladed red alga Mazzaella splendens, respectively, in southern British Columbia, Canada. This study is the first to investigate experimentally the synchrony of host and endophyte seasonal abundance for any host–endophyte system. A correlation study of time series data on abundance indicated that in 1997 and 1998 seasonal synchrony occurred for both host–endophyte associations; this was predicted for interactions where the host is highly seasonal and the host–endophyte relationship is highly specific. We suggest that Acrosiphonia's sporophyte phase may have evolved to synchronize colonization with increasing host abundance, and synchronize reproductive phenology and host exit (as zoospores) with decreasing host abundance.

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

Field and greenhouse studies were conducted to determine the proclivity of Bacillus thuringiensis (Bt) (Berliner)‐transgenic corn (Zea mays L.) to form an endophytic relationship with Beauveria bassiana, and to evaluate the corn for possible plant pathological effects associated with this relationship. Beauveria bassiana (Balsamo) Vuillemin was applied as a granular formulation to two separate lines of corn, expressing Bt events MON802 and MON810, and their corresponding isolines. There were no significant differences in levels of endophytism between transgenic events or their near‐isolines. In greenhouse studies, B. bassiana was applied as a liquid seed treatment to Bt transgenic corn hybrids Pioneer 34R06 (event MON810) and Ciba Max 454 (event 176) and their near isolines at a rate of 2 × 1010 conidia per ml. There were no significant differences in seed germination or presence of root pathogens in transgenic or isoline seeds soaked in a B. bassiana suspension. The same lines of corn were used in field experiments with treatments of seeds soaked in a suspension of B. bassiana, a foliar application of a granular formulation of B. bassiana, and corresponding untreated checks. Plants were sampled throughout the growing season and evaluated for growth of individual plant components, including sheaths, leaves, stem, husk, ear, plant leaf‐to‐stem ratio and overall plant growth. There were no significant differences in overall plant growth between the B. bassiana treatments or in the growth of each plant component. The results of this study indicate that B. bassiana readily forms an endophytic relationship with transgenic and non‐transgenic corn and causes no plant pathology.

Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana.

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.

Persistent, systemic, asymptomatic infections of Albugo candida, an oomycete parasite, detected in three wild crucifer species

Field observations and published data suggested that the oomycete parasite Albugo candida (Pers.) Kuntze could exist as latent, asymptomatic, systemic infections. The wild crucifer species Lepidium campestre (L.) R.Br., Arabis lyrata L., and Erysimum menziesii ssp. eurekense R.A. Price were chosen as models to test this hypothesis. Detection of the parasite in asymptomatic host tissue was based on polymerase chain reaction amplification of ribosomal DNA internal transcribed spacer region I of A. candida using primers designed to discriminate between plant and parasite DNA template. Albugo candida was detected in high proportions of asymptomatic field plants (vegetative and reproductive), greenhouse grown vegetative rosettes, and laboratory germinated seedlings of each species. For example, A. candida appeared to be systemic in all 20 field plants and 10 inoculated rosettes of L. campestre. Albugo candida was present in uninoculated rosettes and seedlings (up to 98%) suggesting seed borne transmission of the pathogen. Although the parameters controlling symptom development are still unclear, presence of the pathogen appeared not to be sufficient for symptom expression. Persistent, asymptomatic, systemic infections that are vertically transmitted through seed suggest that A. candida may possibly form endophytic relationships with at least some of its crucifer hosts.Key words: fungal endophyte, latent infection, PCR detection, rDNA ITS I.

Endophytic relationship between an entomopathogenic fungus Beauveria bassiana and corn (zea mays)

Fungi are known to colonize plants, usually as phytopathogens or symbionts. This is the first report of the entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) penetrating and colonizing a plant in a manner similar to when it invades a typical insect host Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae). Electron microscopy was used to document this fungus penetrating the leaves of corn (Zea mays) and establishing an endophytic relationship within the plant.Several studies have shown B. bassiana to adhere to insect cuticle and penetrate with or without forming appressorial penetration structures. Investigations have concluded that the young, germinating hyphae may produce sequentially a proteinase followed by chitinase to dissolve the cuticular envelope of target insects. Within three days after inoculation, viable B. bassiana conidia germinated and formed vegetative mycelia which grew randomly over the corn leaf surface (Fig. 1). Often a germ tube is formed from a conidium and elongates only a short distance before terminating its growth and penetrating the leaf surface (Figs. 2 and 3).

Geographic Survey of Fungal Endophyte Community Composition in Leaves of Coastal Redwood

This geographic survey of the leaf fungal endophytes in Sequoia sempervirens (coastal redwood) is the initial stage in a larger study to explore their symbiotic relationships. Leaves from six sites along a north to south transect, representing the range of Sequoia sempervirens from southern Oregon to the central coast of California, were sampled for endophytes within a 31-day period. Of the 16 species identified, one endophyte Pleuroplaconema sp. was isolated most frequently from all sites, being detected in 55.4%-74.6% of leaves. The lowest percentage of Pleuroplaconema sp. at the southernmost site also had the lowest total infection frequency with 18.6% of leaves being uninfected. Redwood at all other sites had between 1–4% frequency of uninfected leaves. Other species with consistent presence in the redwood populations were: Cryptosporiopsis abietina, Phomopsis occulta, Hypoxylon bipapillatum, Gelatinosporium sp., Nodulisporium sp., Geniculosporium sp, Aureobasidium sp., and Phlyctema sp. The relatively constant endophyte composition in Sequoia sempervirens leaves along this north to south transect provides a basis to explore the mutualistic, pathogenic and saprobic nature of these endophytic relationships.