In situ ergot alkaloid detection in three Balansia epichloe-infected grass species

Authors: Bacon, Charles; Hinton, Dorothy; Mitchell, Trevor; PALENCIA, EDWIN - Former ARS Employee

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/2018
Publication Date: 6/1/2018
Citation: Bacon, C.W., Hinton, D.M., Mitchell, T.R., Palencia, E. 2018. In situ ergot alkaloid detection in three Balansia epichloe-infected grass species. Journal of Applied Microbiology. 10.1111/jam.13941.
DOI: https://doi.org/10.1111/jam.13941

Interpretive Summary: The Balansia are clavicipitaceous symbiotic species associated with various species of tropical grasses. These fungi live within grasses as endophytes where they produce a variety of ergot alkaloids that are toxic to grazing livestock. Balansia epichloe is one species that is found as an endophytic weed contaminant of several grasses found growing in major forage grasses such as tall fescue. This endophytic species is found on smut grass, which is also consumed by cattle. Two other weed grasses included love grass and lace grass although we do not know their content of ergot alkaloids during the grazing year nor which grass host is toxic or if the same Balansia species infecting other grass species are identical but there are host influences. These uncertainties formed the basis for the objectives of this work. Since the taxonomy of the genus Balansia is based only on the appearance of the fungus on the leaves, we subjected these to molecular analysis using DNA bar coding. The dated procedure used to determine the accumulation of ergot alkaloids on the grasses is complex and expensive. A modern method eliminating extensive sample preparations including the lengthy fermentations, extractions, and chromatographic analyses was developed, and used to screen naturally endophytic infected pasture grass species for their ergot alkaloid content directly on the plant. This procedure is laser ablation electrospray ionization (LAESI) mass spectrometry, which is a high-throughput procedure capable of the in situ detection of trace amounts of metabolites in plant tissue. LAESI procedure was used to determine in situ chemical analyses of ergot alkaloids produced by B. epichloe symbiotic with three weed grass species, while the molecular procedure was used to determine relatedness of each species on the grass species. Results showed that production of ergot alkaloid reflected the in vitro production but that in one host no this species did not produce ergot alkaloids. Further the molecular analysis showed that this species, although morphologically identical to the two ergot alkaloid producing species, was not related and perhaps a distinct species. These results were important in establishing a reduced analysis procedure, and that there is an accumulation of ergot alkaloids in planta of weed species. Further, not all grasses infected by the same morphological species produce toxins suggesting genetic divergence from one ecotype to two distinct chemical ecological species within a host, a finding that should assist in determining the basis of endophytism by mycotoxic fungi in other more agronomically important grasses such as corn and wheat.

Technical Abstract: The Balansia are clavicipitaceous symbiotic species endophytically associated with various species of tropical grasses. Laboratory culture procedures established that grasses in tall fescue pastures are often infected with Balansia species that produced ergot alkaloids in culture. Balansia epichloe symbiotic with Sporobolus poiretii (smut grass) and two other B. epichloe–infected grasses collected from this same location were used to determine the in situ occurrence of ergot alkaloids on different grass species. The species are delineated purely morphologically and on their location on the grass host, and negative results might represent taxonomic problems. Any effects of different host species on the pattern by the same Balansia species are unknown, and negative results in culture are therefore meaningless, therefore, an in planta method is needed. Laser ablation electrospray ionization (LAESI) mass spectrometry is a high-throughput procedure capable of the in situ detection of picogram levels of metabolites in plant tissue. The grass species associated with B. epichloe included in this study were smut grass, lace grass (Eragrostis capillaries, a new host), and love grass (Eragrostis hirsuta). DNA bar coding was used to determine the relatedness of the three species on each grass regardless of their host association. These infected grasses were then analyzed for ergot alkaloids when they were showing signs of infection directly on lives with both the LAESI and on detached leaves with classical chemical examinations. The following ergot alkaloids were found, but only in the smut and love grasses: chanoclavine, ergonovine, agroclavine, and ergonovinine. The lace grass symbiotum was negative. This supported the genetic differences from the bar coding experiments, indicating that either the lace grass symbiotum is a different species or that the same species that but due to host jumping acquired genetic differences with possible host differences. Further studies on genetic divergence of such morphospecies will assist in understanding endophytism and the formation of distinct chemical ecological species.