DataSheet_1_Aboveground herbivory does not affect mycorrhiza-dependent nitrogen acquisition from soil but inhibits mycorrhizal network-mediated nitrogen interplant transfer in maize.pdf

Arbuscular mycorrhizal fungi (AMF) are considered biofertilizers for sustainable agriculture due to their ability to facilitate plant uptake of important mineral elements, such as nitrogen (N). However, plant mycorrhiza-dependent N uptake and interplant transfer may be highly context-dependent, and whether it is affected by aboveground herbivory remains largely unknown. Here, we used 15N labeling and tracking to examine the effect of aboveground insect herbivory by Spodoptera frugiperda on mycorrhiza-dependent N uptake in maize (Zea mays L.). To minimize consumption differences and 15N loss due to insect chewing, insect herbivory was simulated by mechanical wounding and oral secretion of S. frugiperda larvae. Inoculation with Rhizophagus irregularis (Rir) significantly improved maize growth, and N/P uptake. The 15N labeling experiment showed that maize plants absorbed N from soils via the extraradical mycelium of mycorrhizal fungi and from neighboring plants transferred by common mycorrhizal networks (CMNs). Simulated aboveground leaf herbivory did not affect mycorrhiza-mediated N acquisition from soil. However, CMN-mediated N transfer from neighboring plants was blocked by leaf simulated herbivory. Our findings suggest that aboveground herbivory inhibits CMN-mediated N transfer between plants but does not affect N acquisition from soil solutions via extraradical mycorrhizal mycelium.</p

Dietary bergapten negatively affects cowpea bruchid development and fecundity.

<p>(A) Developmental time (days, mean±SE) of bruchids when fed bergapten at doses ranging from 0 to 800 ppm as shown. Data were analyzed using a one-way ANOVA (<i>F</i>_{6, 325} = 179.6, <i>P</i><0.001). Tukey's multiple range test was used to compare the difference between treatments. Means followed by different letters indicate significant difference between treatments (Tukey test: <i>P</i><0.05). Developmental time is defined as the time period from egg laying to adult emergence. (B) Use of acetone as bergapten solvent has no effect on cowpea bruchid development. Acetone (10%), equivalent of the amount used in making 1,000 ppm bergapten, was mixed with cowpea flour for making artificial seeds, followed by lyophilization. Developmental time data was analyzed by independent <i>t</i>-test. Means followed by the same letter are not significantly different (<i>t</i>_{1, 59}  = 1.682, <i>P</i> = 0.098). (C) Fecundity (eggs per female, mean±SE) when fed to diet containing 250 ppm bergapten. Different letters indicate significant difference between treatments (Independent <i>t</i> test: <i>t</i>_{1, 4}  = 8.733, <i>P</i> = 0.001).</p

Cowpea bruchid midgut genes coregulated by bergapten and scN.

a<p>: Numbers shaded gray indicate genes down-regulated by two-fold or more (P≤0.05) in response to dietary bergapten or scN, Unshaded are up-regulated, and underlined indicate the genes are induced or repressed by both bergapten or scN.</p

Summary of sequence annotation of bergapten-response genes from cowpea bruchid midgut based on (A) biological function, (B) molecular function and (C) KEGG pathway analyses.

<p>The 4^th instar larvae reared on artificial diet containing 250 ppm bergapten and control diet, respectively, were removed, their midguts were dissected and total RNA was extracted, followed by microarray hybridization. The BLAST2GO software was used for BlastX search (<i>E</i>-value cutoff, 10⁻⁶) and KEGG pathway mapping of bergapten-responsive genes. Shown are KEGG pathways with at least three genes mapped.</p

scN potentiates the anti-insect effect of bergapten.

<p>Developmental time (days, mean±SE) of bruchids when fed the control diet and diet containing 1,000 ppm scN, 250 ppm bergapten, or 1000 ppm scN + 250 ppm bergapten, respectively, was analyzed by one-way ANOVA (<i>F</i>_{3, 128} = 68.1, <i>P</i><0.001). Tukey's multiple range test was used to compare the difference between treatments. Means followed by different letters indicate significant difference between treatments (Tukey test: <i>P</i><0.05). Developmental time is defined as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041877#pone-0041877-g001" target="_blank">Fig. 1</a>.</p

scN attenuates transcriptional changes induced by bergapten.

<p>Selected bergapten- and scN-coregulated genes involved in polysaccharide or protein degradation (<i>CmGH5</i>, <i>CmCatL</i>s, <i>CmCatB</i>), detoxification (<i>CmCYP6G1</i>s, <i>CmGST</i>, <i>CmPOD</i>), defense (<i>CmDrsL1-1</i>), development (<i>CmJHE7</i>) and transport (<i>CmSUT1</i>) were subjected to qPT-PCR analyses. Total RNA was extracted from midgut of the 4^th instar larvae feeding on artificial diet containing 1,000 ppm scN, 250 ppm bergapten or 1,000 ppm scN + 250 ppm bergapten, respectively. Insects feeding on diet without bergapten or scN served as the control. Reverse transcription and qRT-PCR reactions were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041877#s2" target="_blank">Material and Methods</a>. Transcript fold induction derived from qRT-PCR is shown as bar graphs. The lower panel shows microarray results of the corresponding genes. “+”, “−”: up- or down-regulation when subjected to scN or bergapten treatment. <i>CmGH5</i>, Glycoside hydrolase; <i>CmCatLa</i> and <i>CmCatLb</i>, cathepsin L-like proteases; <i>CmCatB</i>, cathepsin B-like protease; <i>CmCYP6G1-2</i> and <i>CmCYP6G1-3</i>, Cytochrome P450s; <i>CmGST</i>, Glutathione S-transferase; <i>CmDrsL1-1</i>, Drosomycin-like I; <i>CmJHE7</i>, Juvenile hormone esterase; <i>CmSUT1</i>, Sugar transporter 1; <i>CmPOD</i>, Peroxidase precursor.</p