Abstract
The amounts and types of epicuticular waxes on onion (Allium cepa) leaves affect feeding damage by onion thrips (Thrips tabaci), a serious insect pest of onion. This study used gas chromatography mass spectrometry (GCMS) to measure amounts of epicuticular waxes on foliage of two plants from each of 50 plant introductions (PI) and inbred lines with high (waxy) and low (glossy) amounts of wax. Wax amounts on leaves of the same plants were measured twice (once in the greenhouse and once after moving plants outside) and were significantly (P < 0.01) correlated; however, wax amounts on leaves of plants grown in the greenhouse were approximately twice that of the same plants grown outside. Hentriacontanone-16 (H16) was the predominant wax on leaves of all PIs except PI 289689, and amounts of H16 were significantly correlated with amounts of fatty alcohols and total wax. Five plants from 17 of the PIs were grown in the greenhouse, wax amounts measured using GCMS, and results were significantly correlated with earlier evaluations. Results indicate that measurements of waxes on onion foliage should occur under protected conditions to better characterize phenotypic variation, and selection of higher amounts of waxes other than H16 may be effective toward the development onions suffering less thrips damage.
Epicuticular waxes accumulate on aerial surfaces of terrestrial plants to prevent water loss and offer production benefits such as disease resistance and tolerance to herbicides (Baker, 1982). Epicuticular waxes are made of several classes of lipids, including fatty acids and alcohols, ketones, aldehydes, and alkanes (Eigenbrode and Espelie, 1995). The composition of lipids that form epicuticular waxes differs among plant species (Baker, 1982; Eigenbrode and Espelie, 1995). Khosa et al. (2020) reported that the primary waxes on onion (Allium cepa) foliage are the fatty alcohols heptadecanol-1 (Hept1), hexacosanol-1 (Hex1), octacosanol-1 (Oct1), and triacontanol-1 (Tri1); alkanes heptacosane (Hept), nonacosane (Non), and hentriacontane (Hent); ether 1-ethenyloxyoctadecane (Octd); and ketone hentriacontanone-16 (H16).
Onion foliage can be visually classified based on relative amounts of epicuticular waxes. Leaves of wild-type onion have a dark blue-green color, accumulate relatively high amounts of leaf waxes, and are referred to as “waxy.” Onions with leaves that are light green in color accumulate significantly less total wax relative to waxy onions and are referred to as “glossy.” There exist foliage types that are visually intermediate between the glossy and waxy phenotypes and are referred to as “semi-glossy.” Previous studies have shown that the total amount of wax and the relative proportion of H16 to total wax are associated with visual leaf appearance; onions with lower amounts of total wax and/or lower proportions of H16 relative to total wax exhibit the glossy or semi-glossy phenotype, whereas onions with larger amounts of total wax and a higher proportion of H16 appear waxy (Damon et al., 2014; Munaiz et al., 2020).
Onion thrips (Thrips tabaci) is a serious insect pest of onion causing significant losses to both the bulb and seed crops (Diaz-Montano et al., 2010; Fournier et al., 1995; Jones et al., 1934). Feeding by thrips larvae causes the greatest leaf damage (Coudriet et al., 1979), and thrips can transmit Iris yellow spot virus, causing further losses (Gent et al., 2004; Pozzer et al., 1999). Control of thrips is difficult because of high reproductive capacity, commonly occurring host plants, and overlapping generations. The insect tends to accumulate between the narrow leaves of onion where sprays may not reach (Jones et al., 1934; Lall and Singh, 1968). To control thrips, growers spray insecticides many times over the growing season at significant cost and may contribute to development of insecticide resistances (Allen et al., 2005; Herron et al., 2008; Shelton et al., 2003, 2006). Development of onions that suffer less thrips damage would allow growers to reduce the numbers of sprays and potentially delay development of insecticide resistance.
Onion plants with glossy foliage suffer significantly less thrips damage relative to waxy plants (Boateng et al., 2014; Cramer et al., 2014; Damon et al., 2014; Diaz-Montano et al., 2010, 2012; Jones et al., 1934); however, glossy onions are not commercially viable because the low amounts of leaf waxes are associated with susceptibility to powdery mildew and herbicides (Baker, 1982; Mohan and Molenaar, 2005). Onions with semi-glossy foliage suffer less thrips damage relative to waxy foliage and have the benefit of accumulating more wax than plants with glossy foliage (Damon et al., 2014; Diaz-Montano et al., 2010; Munaiz et al., 2020). The semi-glossy inbred ‘B5351’ suffers significantly less thrips damage compared with waxy plants (Damon et al., 2014) and the semi-glossy phenotype is conditioned by quantitative trait loci (QTL) on chromosomes 2 and 5. The QTL on chromosome 2 reduces amounts of fatty alcohols Oct1 and Tri1; the QTL on chromosome 5 reduces amounts of H16 and maps with the visual phenotype (Damon and Havey, 2014). Therefore, leaves of B5351 accumulate significantly lower amounts of both fatty alcohols and H16 resulting in significantly less total wax relative to waxy plants.
In this study, GCMS was used to measure natural variation for the amounts and types of epicuticular waxes on leaves of diverse onion accessions, with the long-term objective of developing onions with unique epicuticular-wax profiles and potentially reduce losses by onion thrips.
Materials and Methods
Plant materials.
Plant introductions (PIs) 124525, 164807, 165498, 168962, 168966, 171475, 171477, 172701, 172702, 172703, 172704, 174018, 248754, 249899, 251325, 251509, 255557, 258956, 264320, 264321, 264325, 264631, 264648, 269305, 269306, 271039, 273211, 274780, 287540, 288072, 288270, 288272, 288902, 288903, 288908, 288909, 289689, 289690, 293756, 321384, 321385, 342943, 343048, 344392, 370338, 430371, 433312, 433330, 433331, and 433332 of onion were selected from the U.S. Department of Agriculture germplasm collection based on bloom (visual assessment of leaf waxiness) scores ≤5 on a 1 (low) to 10 (high) scale as reported on the Germplasm Resource Information Network (https://www.ars-grin.gov/). Seed was planted in a soilless-mix (Promix, Sun-Gro Horticulture, Agawam, MA) in February in a greenhouse on the University of Wisconsin (UW)–Madison campus together with the glossy inbred B9885 and waxy doubled haploid (DH) 2107 (Damon et al., 2014). After 4 weeks, plants were transplanted into pots with the soil-mix and pots were randomly arranged on benches in a greenhouse with supplemental lighting for 14 h at 25 °C days and 20 °C nights. Plants were watered daily and fertilized once per week with 20N–8.7P–16.6K (Peter’s Professional 20–10–20, Everris, Dublin, OH). Plants were inspected weekly for thrips and no insecticide sprays were required.
Ten weeks after seed sowing, plants had ≈6 true leaves when two adjacent leaf segments were collected from the middle region of the youngest fully extended leaf (generally the fifth true leaf) from each of two randomly selected plants from each PI. The fresh weight of each leaf piece was measured, and the piece was placed into a 16 × 100 mm glass tube (Thermo-Fisher Scientific, Waltham, MA). Docosane (Sigma-Aldrich, St. Louis, MO) dissolved in high-performance liquid chromatography (HPLC)-grade chloroform (Sigma-Aldrich) was added onto the surface of each leaf piece at 100 μg·g−1 fresh weight. Immediately after the first sampling, pots containing the plants were moved outside and randomly arranged in an open frame adjacent to the greenhouses, covered with black mesh for 2 d, and then grown under natural environmental conditions. Plants were watered only if soil was dry, otherwise were rain fed, and fertilized with 20N–8.7P–16.6K once per week. Plants were inspected for thrips as described earlier, and no insecticide sprays were required. Four weeks later, the plants had ≈10 leaves when two leaf segments were collected from the middle region of the youngest, fully extended leaf from each plant, fresh weights measured, and docosane added as described earlier. For both samplings, leaf pieces were submerged in HPLC grade chloroform and after 1 min, the leaf piece was removed and discarded. Preparation for GCMS and run conditions are described subsequently.
Five plants from each of 17 PIs (164807, 168962, 172702, 248754, 249899, 258956, 264320, 264321, 264325, 271039, 273211, 274780, 288272, 289689, 293756, 321385, and 343048) were selected based on visually semi-glossy foliage and diversity for wax profiles. Plants of these PIs, waxy DH2107, and glossy B9885 were grown in a greenhouse as described above. Ten weeks after sowing, two leaf pieces from each of five plants from each PI, glossy B9885, and waxy DH2107 were sampled for GCMS and docosane added based on fresh weights of leaf pieces as described above.
Preparation of leaf samples for GCMS.
The tubes containing waxes dissolved in chloroform were allowed to dry in a fume hood for ≈10 to 14 d. As soon as the chloroform in a tube had evaporated, 250 μL of anhydrous HPLC-grade chloroform (Sigma-Aldrich) was added, the tube was tightly capped and gently rotated to remove dried deposits from sides of the tubes. Once all samples had anhydrous chloroform, 300 μL of acetonitrile (Fisher Scientific, Waltham MA) and 105 μL of N,O-Bis(trimethylsilyl)trifluoroacetamide (Sigma-Aldrich) were added to each tube. The tubes were tightly capped, derivatized at 80 °C for 30 min, and placed in a shaker at 100 rpm for 5 min. Contents were then filtered through 13 mm diameter, 0.22 μm pore size, PVDF-sterile filters (Fisher Scientific) into GCMS vials. Samples were injected into a column (SH-Rxi-5il MS; Shimadzu, Kyoto, Japan) on a GCMS (QP2010 SE, Shimadzu) with a Hamilton syringe using a split injection method. The initial column oven temperature was 150 °C, and the injection temperature was 250 °C. Samples were run on a cycle of 150 °C for 10 min, then a ramp up of temperature to 300 °C over 15 min and held at 300 °C for 10 min.
Data analysis.
Post-run GCMS analyses (Shimadzu) were used to determine peak areas and identities of waxes as described by Khosa et al. (2020). Peak areas were adjusted by dividing the peak areas for docosane and therefore represent peak areas per gram leaf fresh weight. Adjusted peak areas were averaged when two samples were taken from the same leaf. Pearson correlations and analyses of variance (ANOVAs) were calculated using RStudio (R Foundation for Statistical Computing, Vienna, Austria). For multiple measurements of waxes on leaves of the same plants, two-way repeated-measures ANOVAs (accessions and samplings as sources of variation) were calculated and Tukey’s multiple comparisons test was used to compare means at P = 0.05.
Results and Discussion
Variation for epicuticular waxes among onion PIs.
GCMS analyses of epicuticular waxes from 50 onion PIs listed in GRIN as having relatively low visual waxiness (bloom) revealed that leaves of all but one of the PIs accumulated Hept1, Hex1, Oct1, Tri1, Octd, Non, Hent, and H16 (Supplemental Table 1). Peak areas for the alkane Hept were absent or below the detection threshold. The sole exception was PI 289689 for which plants accumulated no waxes with greater than 28 carbons as previously reported (Havey et al., 2021). H16 was the most prevalent wax on the foliage of all PIs except 289689 and amounts of H16 and total wax were significantly (P < 0.001) correlated in both the greenhouse (0.977) and outside (0.962). Amounts of total wax on plants grown in the greenhouse and then outside were significantly (P < 0.01) correlated at 0.663; however, outside-grown plants accumulated on average 55% less total wax than the same plants grown in a greenhouse (regression coefficient of 0.45 in Fig. 1). Over years, we have noticed that the glossy and semi-glossy phenotypes are visually easier to score in field plots and that plants grown in the greenhouse tend to become waxier over time, making visual phenotypes less distinctive. This indicates that glossy and semi-glossy plants may have the same relative proportions of individual waxes but may be slower accumulators of epicuticular waxes relative to waxier phenotypes and slower to replace leaf waxes lost due to environmental factors such as rain or wind.
For all waxes except Hent, there were highly significant (P < 0.001) differences among accessions and between the two repeated measures in time (Table 2), as expected given the phenotypic differences of glossy B9985 and waxy DH2107 vs. the PIs and the overall lower wax amounts on leaves of plants grown outside. The significant interactions between accession and sampling for individual waxes and total wax (Table 2) is consistent with different amounts of waxes under greenhouse and outside conditions. These results indicate that waxes on onion foliage should be measured under protected conditions to better characterize phenotypic variation.
We calculated correlations among amounts of individual waxes measured on the same plants grown in the greenhouse and outside and observed significant (P < 0.05) correlations between the two environments for Non, Hex1, Octd, Oct1, Hept1, H16, Tri1, and total wax (Table 2). The correlation for Hent was not significant. Amounts of the individual fatty alcohols (Hept1, Hex1, Oct1, and Tri1) were significantly (P < 0.001) correlated across the two environments, and amounts of the ketone H16 were significantly correlated with amounts of the individual fatty alcohols (P < 0.001) and Octd (P < 0.05). Amounts of the alkanes Non and Hent were significantly correlated (P < 0.001) with each other but not correlated with amounts of any of the other waxes (Table 1). These correlations are consistent with the production of leaf waxes in plants that begins with fatty acids with 26 to 32 carbons as substrates for the acyl-reduction pathway to produce fatty alcohols with even numbers of carbons, and the decarbonylation pathway to produce alkanes and ketones with odd number of carbons (Millar et al., 1999). Overall, the amounts of alkanes were relatively small compared with the ketone H16, indicating that production of alkanes may be reduced in favor of more H16. This would suggest that it may be possible to select onions with higher amounts of fatty alcohols vs. alkanes/ketone because these classes of waxes are produced by two biochemical pathways.
Pearson correlations (above diagonal) and levels of significancez (below diagonal) among amounts of individual waxes on onion plants from 50 plant introductions with glossy and waxy controls sampled once in the greenhouse and again after moving outside.
Levels of significance from analyses of variance for repeated measures of amounts of individual waxes and total wax on onion plants from plant introductions with glossy and waxy controls (Acc) sampled once in the greenhouse and then outside with interaction.
Epicuticular wax profiles for a subset of semi-glossy 17 PIs.
Amounts of individual waxes and total wax (Supplemental Table 2) were significantly different (P < 0.001) among the 17 PIs, waxy DH2107, and glossy B9885. Even though plants from all 17 of the PIs were visually nonwaxy in the greenhouse, some of the PIs had individual plants that were not significantly different from waxy DH2107 for total wax and some individual waxes (Table 3). This was also apparent from boxplots of total wax which show that glossy or semi-glossy accessions can overlap with waxy DH2107 (Fig. 2). These results are consistent with Munaiz et al. (2020), who identified specific semi-glossy selections with the same amount of total wax as waxy plants. Glossy B9885 had significantly (P < 0.05) less H16 than waxy DH2107 in agreement with Damon et al. (2014); however, this glossy inbred was not different for amounts of most of the other waxes. PI 289689 had significantly more of Hex and Hent than waxy DH2107 and was not significantly different amount of Oct1 relative to DH2107.
Mean peak areas adjusted to gram leaf fresh weight for individual epicuticular waxes and total wax for 17 plant introductions of onion, waxy DH2107, and glossy B9885 grown in a greenhouse. Accessions (Acc) are sorted by amount of total wax and waxes are listed in columns from highest to lowest amounts on waxy DH2107. Means followed by the same letter were not significantly different at P = 0.05 using Tukey’s multiple comparisons test.
Conclusions
Previous studies reported that onions with lighter green foliage suffer less feeding damage from onion thrips (Diaz-Montano et al., 2010; Jones et al., 1934), and this lighter green leaf color is associated with lower amounts of H16 or total wax, or higher amounts of other waxes (Damon et al., 2014; Munaiz et al., 2020). Our results indicate that amounts of waxes on onion foliage should be measured under protected conditions to better reveal phenotypic variation. We previously reported that multiple sampling of leaves on the same plant over time and environments may not be necessary (Khosa et al., 2020), and resources may be better used to sample greater numbers of plants. Reducing the amount of H16 while maximizing amounts of fatty alcohols and/or alkanes may produce onion populations with semi-glossy foliage that accumulate enough total epicuticular wax to perform well in field production. These plants with unique epicuticular-wax profiles could then be evaluated for thrips damage under environmentally controlled and field conditions.
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Mean peak areas per gram leaf fresh weight for eight epicuticular waxes and total wax on foliage of two onion plants from each of 50 plant introductions (PI; six-digit numbers) and control inbreds (glossy B9885 and waxy DH2107) sampled in the greenhouse (GH) and then again after moving outside (OUT).
Mean peak areas per gram leaf fresh weight for individual epicuticular waxes and total wax on foliage of five plants from 17 plant introductions (PI; six-digit numbers) and control inbreds (glossy B9885 and waxy DH2107) of onion grown in a greenhouse. Values are peak areas adjusted to leaf fresh weights.