Soybean Cyst Nematodes Influence Aboveground Plant Volatile Signals Prior to Symptom Development

Abstract

Soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive soybean pests worldwide. Unlike many diseases, SCN doesn't show above ground evidence of disease until several weeks after infestation. Knowledge of Volatile Organic Compounds (VOCs) related to pests and pathogens of foliar tissue is extensive, however, information related to above ground VOCs in response to root damage is lacking. In temporal studies, gas chromatography-mass spectrometry analysis of VOCs from the foliar tissues of SCN infested plants yielded 107 VOCs, referred to as Common Plant Volatiles (CPVs), 33 with confirmed identities. Plants showed no significant stunting until 10 days after infestation. Total CPVs increased over time and were significantly higher from SCN infested plants compared to mock infested plants post 7 days after infestation (DAI). Hierarchical clustering analysis of expression ratios (SCN: Mock) across all time points revealed 5 groups, with the largest group containing VOCs elevated in response to SCN infestation. Linear projection of Principal Component Analysis clearly separated SCN infested from mock infested plants at time points 5, 7, 10 and 14 DAI. Elevated Styrene (CPV11), D-Limonene (CPV32), Tetradecane (CPV65), 2,6-Di-T-butyl-4-methylene-2,5-cyclohexadiene-1-one (CPV74), Butylated Hydroxytoluene (CPV76) and suppressed Ethylhexyl benzoate (CPV87) levels, were associated with SCN infestation prior to stunting. Our findings demonstrate that SCN infestation elevates the release of certain VOCs from foliage and that some are evident prior to symptom development. VOCs associated with SCN infestations prior to symptom development may be valuable for innovative diagnostic approaches.

Keywords: GC-MS; VOCs; early disease detection; soybean; soybean cyst nematode.

Figure 1

Schematic diagram of volatile collection system for GC-MS analysis. Air entering the chamber was scrubbed using activated charcoal. Volatiles were pulled from the chambers via a vacuum pump and collected by HayeSepQ resin for GC-MS analysis. To ensure only foliar volatiles were collected a parafilm barrier was placed at the base of each soybean plant.

Figure 2

Experimental data processing and analysis pathway for the VOC peak alignment with linear regression approach.

Figure 3

Symptom progression of soybean seedlings infested with SCN. (A) Height measurements of SCN infested and mock infested plants at 1, 3, 5, 7, 10 and 14 days after infestation (DAI). (B) Image of SCN infested plant (right) and mock infested plant (left) at 14 DAI. The plant height was measured from the soil line to the top leaf. The data shown represents an average ± SE plant height and significant differences based on Student's t-test (*P ≤ 0.05, ***P < 0.01, n = 10).

Figure 4

Total volatile emission from all 107 CPVs over time for SCN and mock treated plants. Graph includes linear regression slopes for both mock and SCN infested plants and goodness of fit (R²). The data indicates average ± SE total VOC concentration and significant differences based on Student's t-test (*P ≤ 0.05, **P < 0.01, n = 10).

Figure 5

Clustering analysis of 107 CPVs in response to SCN infestation. The heat map was arranged according to CPV log² fold change (Infestation/Mock). Five groups were identified, arranged from left to right as elevated, suppressed, no change, and oscillating expression Infested/mock ratios, respectively. Red, blue, and white indicate elevated, suppressed, and no change in expression ratios of volatile emissions. The six rows illustrate the expression patterns of CPVs during the time points 1, 3, 5, 7, 10, and 14 DAI. * indicates statistically significant Infested/Mock ratio (permutation test P < 0.05).

Figure 6

Linear projection of principal component analysis (PCA) of identified and significant CPVs for all 6 time points. Explained variance was 60% with a cumulative variance of 0.602 and component variance of 0.195. PCA was based on 18 CPVs over 1, 3, 5, 7, 10, and 14 DAI. Mock time points are assigned blue circles and SCN time points are assigned red circles.

Figure 7

Temporal expression patterns of identified CPVs with significant differences in expression between SCN infested and mock treatments. Time points were 1, 3, 5, 7, 10, and 14 DAI. Red blocking indicates statistically significant emission and blue blocking indicates significant suppression of VOCs by infested plants (permutation test P < 0.05; n = 20). BHT-quinone methide and BHT abbreviations for 2,6-Di-T-butyl-4-methylene-2,5-cyclohexadiene-1-one and Butylated Hydroxytoluene, respectively. Images show representative mock (left) and SCN infested (right) plants at different timepoints.