Antifungal rhizosphere bacteria can increase as response to the presence of saprotrophic fungi

Acknowledgments: Funding was provided by the Netherlands Organisation for Scientific Research (NWO) in the form of a personal Veni grant to A.v.d.W. This is publication number 5923 of the NIOO-KNAW Netherlands Institute of Ecology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Evaluation of a simple, non-alkaline extraction protocol to quantify soil ergosterol

Quantification of soil ergosterol is increasingly used as an estimate for soil fungal biomass. Several methods for extraction of ergosterol from soil have been published, perhaps the simplest being that described by Gong, P., Guan, X., Witter, E. [2001. A rapid method to extract ergosterol from soil by physical disruption. Appl. Soil Ecol. 17, 285–289]. This method only involves a mechanical disruption of soil mycelium by glass beads in methanol and direct HPLC analysis of the ergosterol in the filtered methanol extract. However, it has not been compared with more complex extractions that include a saponification step to liberate both free and esterified ergosterol. In this study, we have compared the Gong method with a method involving saponification for a number of sandy and clayey soils as well as for organic layers of forests. In addition, we compared both methods with respect to recovery of added ergosterol and fungal hyphae. The Gong method appeared to be as good as the method including saponification, provided that the amount of soil to be extracted was lowered with respect to the original protocol when analysing soils with an organic matter content >5%.

Mycorrhizal fungi supply nitrogen to host plants in Arctic tundra and boreal forests : 15N is the key signal

Author Posting. © NRC Research Press, 2009. This article is posted here by permission of NRC Research Press for personal use, not for redistribution. The definitive version was published in Canadian Journal of Microbiology 55 (2009): 84-94, doi:10.1139/W08-127.Symbiotic fungi’s role in providing nitrogen to host plants is well-studied in tundra at Toolik Lake, Alaska, but little-studied in the adjoining boreal forest ecosystem. Along a 570 km north–south transect from the Yukon River to the North Slope of Alaska, the 15N content was strongly reduced in ectomycorrhizal and ericoid mycorrhizal plants including Betula, Salix, Picea mariana (P. Mill.) B.S.P., Picea glauca Moench (Voss), and ericaceous plants. Compared with the 15N content of soil, the foliage of nonmycorrhizal plants (Carex and Eriophorum) was unchanged, whereas content of the ectomycorrhizal fungi was very much higher (e.g., Boletaceae, Leccinum and Cortinarius). It is hypothesized that similar processes operate in tundra and boreal forest, both nitrogen-limited ecosystems: (i) mycorrhizal fungi break down soil polymers and take up amino acids or other nitrogen compounds; (ii) mycorrhizal fungi fractionate against 15N during production of transfer compounds; (iii) host plants are accordingly depleted in 15N; and (iv) mycorrhizal fungi are enriched in 15N. Increased N availability for plant roots or decreased light availability to understory plants may have decreased N allocation to mycorrhizal partners and increased δ15N by 3‰–4‰ for southern populations of Vaccinium vitis-idaea L. and Salix. Fungal biomass, measured as ergosterol, correlated strongly with soil organic matter and attained amounts similar to those in temperate forest soils.This work was supported by the National Science Foundation (NSF OPP-0612598 and NSF DEB-0614266)

Bacterial numbers and fungal biomass (ergosterol) after 6 weeks of growth of <i>Carex arenaria</i> seedlings in quartz sand microcosms.

<p>1A: Number of bacterial colony forming units in the <i>Carex</i> rhizosphere (root-adhering sand); * indicates significant difference (p < 0.05) between microcosms with and without (control) the presence of inoculated fungi, # indicates p = 0.052 for Log-transformed data. 1B: Ergosterol concentrations. r indicates rhizophere sand (sand adhering to <i>Carex</i> roots), nr indicates sand remaining after removal of <i>Carex</i> roots. * indicates significant difference (p < 0.05) within fungal treatments between root-adhering and non-root-adhering sand. Data for both figures are the averages of 5 or 6 sand microcosms. Error bars represent standard deviation.</p

Percentage of rhizosphere bacterial isolates positive for different enzyme activities.

<p>Bacterial isolates were obtained from root-adhering soil after 6 weeks of growth of <i>Carex arenaria</i> seedlings in quartz sand microcosms. * indicates significant difference (p < 0.05) between microcosms with and without pre-inoculation of fungi. Note that experiment 1 and 2 started with different bacterial inoculums as indicated in Material & Methods. Data are the averages of three randomly selected sand microcosms. Error bars represent standard deviation. For each microcosm 40 bacterial isolates were individually screened for the different enzyme activities.</p

Pictures of the experimental set-up of sand microcosms with <i>Carex arenaria</i> (sand sedge) plants.

<p>Pictures of the experimental set-up of sand microcosms with <i>Carex arenaria</i> (sand sedge) plants.</p

Percentage of rhizosphere bacteria isolates with <i>in vitro</i> antagonistic activity against different fungi.

<p>Bacterial isolates were obtained from root-adhering soil after 6 weeks of growth of <i>Carex arenaria</i> seedlings in quartz sand microcosms. * indicates significant difference (p < 0.05) between microcosms with and without pre-inoculation of fungi, for the ANOVA test of data of the white column in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137988#pone.0137988.g003" target="_blank">Fig 3B</a> Log transformation was applied; # indicates p = 0.072. Note that experiment 1 and 2 started with different bacterial inoculums a as indicated in Material & Methods. Data are the averages of three randomly selected sand microcosms. Error bars represent standard deviation. For each microcosm 40 bacterial isolates were individually screened for in vitro antagonisms against the different fungi.</p

Schematic illustration of possible stimulation of biocontrol of soil-borne pathogenic fungi by increase of saprotrophic fungi.

<p>Organic amendments and/or other measures that stimulate growth of saprotrophic fungi can result in an increase of uptake of rhizodeposits by these fungi and, consequently, in an increase of competitive fungal pressure towards rhizosphere bacteria. As a result bacteria that are antagonistic against fungi will increase and several of these bacteria may also be antagonistic against soil-borne pathogenic fungi and form a natural barrier against fungal diseases. An advantage over introduction of antifungal biocontrol strains is that the fungus-induced stimulation occurs <i>in situ</i> with indigenous soil bacteria that are adapted to the local environmental conditions.</p