Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: an additional branch of the soil nitrogen cycle

Abstract

The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with (15)N-labeled nitrogen, and we tracked the incorporation of (15)N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.

FIG 1

Percentages of plant nitrogen derived from ¹⁵N-injected wax moth larvae by five species of the endophytic, insect-pathogenic fungus Metarhizium (M. robertsii, M. acridum, M. guizhouense, M. brunneum, and M. flavoviride). Four plant species were used, wheat (Triticum aestivum), switchgrass (Panicum virgatum), soybean (Glycine max), and haricot bean (Phaseolus vulgaris). Results are means of three separate trials done in duplicate. Solid circles and open circles represent treatments with and without wax moth larvae, respectively. Amounts of insect-derived nitrogen in leaves were determined by NOI-5 emission spectrophotometer. Results were analyzed using ANOVA. Standard deviations not shown are less than 1% of the means.

FIG 2

Percentages of plant nitrogen derived from ¹⁵N-injected wax moth larvae by the endophytic, insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Two plant species were used, switchgrass (Panicum virgatum) and haricot bean (Phaseolus vulgaris). Results are means of three separate trials done in duplicate. Solid circles and open circles represent treatments with and without wax moth larvae, respectively. Amounts of insect-derived nitrogen in leaves were determined by NOI-5 emission spectrophotometer. Results were analyzed using ANOVA. Standard deviations not shown are less than 1% of the means.

FIG 3

Time course of fungal propagules found per gram of soil after placement of infected insects. Soil was sampled within 0.5 cm from plant roots at a depth of 2 cm. Soil was plated on selective medium, and CFU were counted. (A) CFU of Metarhizium species per gram of soil. Open circles, M. brunneum; black circles, M. robertsii; gray circles, M. flavoviride; black squares, M. acridum; open squares, M. guizhouense. (B) CFU of Beauveria bassiana and Lecanicillium lecanii per gram of soil. Open circles, B. bassiana; closed circles, L. lecanii. Standard deviations of the means are shown. n = 5 for each time point.

FIG 4

Confocal micrographs obtained after plants had grown in soil microcosms with wax moth larvae and Metarhizium expressing GFP for 7 days. GFP micrographs of EIPF associating with plant roots at 7 days are overlaid over bright-field images. Magnification, ×400. Bars represent 10 μm. (A) Metarhizium guizhouense. (B) Metarhizium robertsii. (C) Beauveria bassiana. (D) Lecanicillium lecanii.

FIG 5

Percentages of plant nitrogen derived from ¹⁵N-injected wax moth larvae by the endophytic, insect-pathogenic fungus M. robertsii under field conditions. Three dominant grass species were present in the field site, timothy (Phleum pratense), smooth bromegrass (Bromus inermis), and orchard grass (Dactylis spp.). Open circles, ¹⁵N-labeled, M. robertsii-infected insects under 30-μm mesh; black squares, ¹⁵N-labeled, M. robertsii-infected insects under 1-μm mesh; closed circles, ¹⁵N-labeled, uninfected insects under 1-μm mesh; gray circles, ¹⁵N-labeled, uninfected insects under 30-μm mesh; white squares, grass-only controls. Results are means of two trials done in duplicate. Standard deviations are less than 1% of the means.

FIG 6

Plant growth measurements taken every 4 days for 16 days. Three plant species were used in order to ascertain overall plant health under 3 separate growth conditions. Closed circles, plants grown in the presence of Metarhizium-infected insects; open circles, plants grown in the presence of Mertarhizium only; gray circles, plants grown in the presence of insects alone. Whole-plant and root weights of the indicated plants are shown. Standard deviations are less than 1% of the mean.