Determining relevant traits for selecting landrace accessions of Phaseolus lunatus L. for insect resistance

Abstract

Plant-insect interactions are a determining factor for sustainable crop production. Although plants can resist or tolerate herbivorous insects to varying degrees, even with the use of pesticides, insects can reduce plant net productivity by as much as 20%, so sustainable strategies for pest control with less dependence on chemicals are needed. Selecting plants with optimal resistance and photosynthetic traits can help minimize damage and maintain productivity. Here, 27 landrace accessions of lima beans, Phaseolus lunatus L., from the Yucatan Peninsula were evaluated in the field for morphological resistance traits, photosynthetic characteristics, insect damage and seed yield. Variation was found in physical leaf traits (number, area, and dry mass of leaves; trichome density, specific leaf thickness and hardness) and in physiological traits (photosynthetic rate, stomatal conductance, intercellular carbon, water-use efficiency, and transpiration). Five accessions (JMC1325, JMC1288, JMC1339, JMC1208 and JMC1264) had the lowest index for cumulative damage with the highest seed yield, although RDA analysis uncovered two accessions (JMC1339, JMC1288) with strong positive association of seed yield and the cumulative damage index with leaf production, specific leaf area (SLA) and total leaf area. Leaf traits, including SLA and total leaf area are important drivers for optimizing seed yield. This study identified 12 important morphological and physiological leaf traits for selecting landrace accessions of P. lunatus for high yields (regardless of damage level) to achieve sustainable, environmentally safe crop production.

Introduction

In recent years, global crop productivity has been improved through the artificial selection of traits that increase yield (Lynch, 2007), but this approach has resulted in low levels of diversity, lack of expression of defense genes, and production systems that depend on high inputs of pesticides (Panda & Khush, 1995; Lynch, 2007; Chen et al., 2016). One of the main constraints on crops is insect damage to leaves; up to 20% of the net productivity in important crops can be lost despite increased pesticide use (Oerke & Dehne, 2004; Agrawal, 2011). Stout (2013) proposed an effective sustainable alternative: selecting plants with resistance and tolerance traits that reduce the impact of insect damage. The efficacy of such resistance traits in protecting a crop against herbivorous insects depends on studies that clarify their influence, their impact on herbivorous insects and their expression under different environmental conditions (Strauss & Agrawal, 1999; Stinchcombe, 2002). Some accessions with larger leaves are likely to have greater vigor and tolerance to damage by herbivorous insects (Ssekandi et al., 2016). Likewise, the thickness and hardness of leaves also have an important impact on resistance. In Fabaceae species, trichomes can also contribute to resistance against defoliating insects (Oghiakhe, Jackai & Makanjuola, 1992; Veeranna & Hussain, 1997) by hindering their movement on the plants (Tian et al., 2012; Figueiredo et al., 2013). In contrast, some plants with thinner, more fragile leaves are less preferred by insects, as in the case of Vigna radiata (L.) (Lakshminarayan, Singh & Mishra, 2008), V. mungo (Taggar & Gill, 2012), Gossypium hirsutum (Butter & Vir, 1989) and Cucumis sativus (Shibuya et al., 2009).

Legumes are the second most important group in past and current agricultural systems and for human nutrition (Blair et al., 2016). According to the FAO in 2018, beans were third in importance by planted area in Mexico, with 7.9% of the total. The Yucatan Peninsula has the greatest richness of cultivated domesticated beans in all of Mexico, and varieties have high levels of genetic diversity including those of P. lunatus (Martínez-Castillo et al., 2012), which are excellent germplasm sources for improving cultivated beans. We hypothesized that landrace accessions of broad beans (Phaseolus lunatus) have physical and physiological characteristics that confer defensive traits that may reduce the activity of herbivorous insects and optimize yields. In this context, the objective of this study was to identify accessions that were most resistant to damage caused by defoliating insects in 27 landrace accessions of lima beans (P. lunatus) by determining morphological, physiological and yield traits known to contribute to insect resistance such as dry mass of leaves, leaf area, specific leaf area (SLA), leaf thickness and hardness, and trichome density, improvement in photosynthetic capacity, and its impact on yield (Gong & Zhang, 2014).

Materials and Methods Seed sources

Seeds of 27 landraces accessions of lima beans (P. lunatus) were collected in the states of Quintana Roo, Campeche and Yucatán in the Yucatan peninsula from home gardens and rural markets. Information on the origin and genetic characterization of landraces is available in previous studies carried out by the Centro de Investigacion Cientifica de Yucatan (CICY), in Martínez-Castillo, Colunga-García & Zizumbo-Villarreal (2008) and Camacho-Pérez, Martínez-Castillo & Mijangos-Cortés (2008) (Table 1). Seeds were tested for germination, and only those lots with germination above 85% were sown.

Table 1: Origin of landrace accessions of lima bean (P. lunatus) evaluated in this study.

Accesion Code	Accession	Species	Collector	State	Municipality	Coordinate	Local name
1	RRS0001	Phaseolus lunatus	Roberto Ruiz	Yucatán	Izamal	–	–
2	JMC1271	Phaseolus lunatus	Jaime Martínez	Quintana Roo	Tulum	87°46′16.55″	Putsicasutsuy
3	JMC1280	Phaseolus lunatus	Jaime Martínez	Quintana Roo	Felipe C. Puerto	88°02′43″	Mulición
4	RRS0002	Phaseolus lunatus	Roberto Ruiz	Yucatán	Izamal	–	–
5	JMC1255	Phaseolus lunatus	Jaime Martínez	Campeche	Calkiní	90°03′03″	Mulición
6	JMC1304	Phaseolus lunatus	Felix Dzul Tejero	Campeche	Calkiní	90°03′03″	Mulición
7	JMC1240	Phaseolus lunatus	Jaime Martínez	Yucatán	Peto	89°24′00″	Putsicasutsuy
8	JMC1350	Phaseolus lunatus	Jaime Martínez	Campeche	Hopelchén	89°44′51.98″	X-Nuk ib
9	JMC1254	Phaseolus lunatus	Jaime Martínez	Yucatán	Tahdziú	89°30′	Putsicasutsuy
10	JMC1327	Phaseolus lunatus	Jaime Martínez	Campeche	Hecelchakán	89°58′14.48″	Mulición
11	JMC1273	Phaseolus lunatus	Jaime Martínez	Campeche	Hopelchén	89°35.57′	Putsicasutsuy
12	JMC1357	Phaseolus lunatus	Jaime Martínez	Campeche	Calkiní	89°53′90″	Mejen ib
13	JMC1345	Phaseolus lunatus	Jaime Martínez	Yucatán	Tixmehuac	89°06′31.43″	Mulición
14	JMC1270	Phaseolus lunatus	Jaime Martínez	Yucatán	Tixmehuac	89°6′56.16″	Chak ib
15	JMC1337	Phaseolus lunatus	Jaime Martínez	Campeche	Hopelchén	89°11′30″	Mulición
16	JMC1245	Phaseolus lunatus	Felix Dzul Tejero	Campeche	Calkiní	89°53′90″	Putsicasutsuy
17	JMC1208	Phaseolus lunatus	Jaime Martínez	Yucatán	Chankom	88°30′48.00″	Sac ib
18	JMC1348	Phaseolus lunatus	Jaime Martínez	Yucatán	Peto	89°24′00″	Sak X-nuk ib
19	JMC1339	Phaseolus lunatus	Jaime Martínez	Yucatán	Yaxcabá	88°49′39.69″	Sac ib
20	JMC1288	Phaseolus lunatus	Felix Dzul Tejero	Yucatán	Tekax	89°29′18″	Box ib
21	JMC1306	Phaseolus lunatus	Jaime Martínez	Yucatán	Tzucacab	89°57′35″	Mulición
22	JMC1264	Phaseolus lunatus	Jaime Martínez	Yucatán	Yaxcabá	88°49′39.69″	Chak ib
23	JMC1325	Phaseolus lunatus	Jaime Martínez	Yucatán	Tekax	89°17′16″	Mejen ib
24	JMC1297	Phaseolus lunatus	Felix Dzul Tejero	Yucatán	Tixmehuac	89°6′56.16″	Sac ib
25	JMC1313	Phaseolus lunatus	Jaime Martínez	Yucatán	Tekax	89°12′00″	Sacmejen
26	JMC1336	Phaseolus lunatus	Jaime Martínez	Yucatán	Tekax	89°17′16″	Sacmejen
27	JMC1364	Phaseolus lunatus	Jaime Martínez	Yucatán	Tekom	88°27′17″	Sac ib

DOI: 10.7717/peerj.12088/table-1

Site conditions and crop establishment

The field experiment was carried out in September, October, November and December of 2019 in the horticultural production area of the National Technological Institute of Mexico, Campus Conkal when the monthly mean temperature was 26.4 °C, the maximum was 34.7 °C, and minimum was 17.3 °C, and the monthly mean precipitation was 100.66 mm. The soil is a Leptosol, with 0.93% N, and the total contents of P, K, Ca and Mg is 2.45, 3.5, 49.38 and 2.63 g kg⁻¹, respectively. Seeds were sown directly every 60 cm in a 50-m row with a distance of 120 cm between each row. Approximately 100 plants per accession were obtained. The field was irrigated each day (7:00 to 9:00) with a drip irrigation strip system, and traditional agronomic management was applied for weed control; no pesticides or chemical fertilizers were applied. Plots were established using a split-plot experimental design with a completely random arrangement of three subplots.

Data collection

At 60 days after plant emergence (DAE), morphological and physiological leaf traits of each landrace accession were evaluated. We randomly selected five plants for each of three experimental subplots for each accession for a total of 15 plants. All leaves on each plant were counted, and area of each leaf was measured with an area meter (LI-3000C portable meter, LI-COR, Lincoln NE, USA). The dry mass of leaves was obtained by placing fresh leaves in a drying oven at 60 °C until mass was constant. The SLA (cm² g⁻¹) was calculated by dividing the leaf area (cm²) by the dry mass (g). Leaf thickness was measured with a digital micrometer (Mitutoyo model H-2780 JPN). Blade hardness (g cm⁻²) was measured with a portable penetrometer (AMS 59032 OSHA, USA). Trichomes were counted on several parts of the adaxial surface of the fully expanded youngest leaf using a stereoscope (OPTIKA ST-30FX IT) at 40 × to calculate density (no. cm⁻²) (Widstrom, McMillian & Wiseman, 1979).

An infrared gas analyzer (LI-6400 IRGA; LI-COR, Lincoln NE, USA) was used to measure gas exchange in three fully extended young leaves for each of 15 new randomly selected plants of each accession. Each leaf was measured five times for photosynthetic assimilation rate (P_N), stomatal conductance (g_s), intercellular carbon (C_i), and transpiration (E). Water-use efficiency (WUE) was then calculated as photosynthetic assimilation rate (PN) divided by transpiration rate (E). Measurements were done between 7:00 and 10:00 when flowering had started (between 45 and 60 DAE).

Leaf damage and yield

To evaluate foliar damage by chewing insects on 15 plants randomly selected per plot at 30, 45 and 60 DAE, we used the percentage damage scale of Dirzo & Domínguez (1995): (0) leaves without herbivory, (1) 1 to 5% damage, (2) 6 to 12% damage, (3) 13 to 25% damage; (4) 26 to 50% damage and (5) above 50% damage). At each sampling date, we also calculated a cumulative damage index for each accession by dividing the lowest damage value by the highest value for the accession (Sohrabi, Nooryazdan & Gharati, 2017).

For yield determinations, 15 plants were selected per accession, pods were harvested and beans removed and oven-dried at 60 °C until mass was constant. Beans were then weighted to obtain seed yield per plant (g plant⁻¹) for each accession.

Statistical analyses

We ran a one-way analysis of variance (ANOVA) to compare morphological and physiological traits, damage and seed yield among the accessions; thus, we used accession as the only factor (independent variable). When the data did not meet normality assumptions, data were transformed as follows: continuous data with the natural logarithm, discrete numbers with square root, and proportions with the arcsine of the square root. Hierarchical grouping of means tests of the Scott-Knott statistic were then applied.

These analyses were performed with the InfoStat software (Di Rienzo et al., 2016). The most important resistance and physiological tolerance traits for the accessions were identified using a principal component analysis (PCA) and the arithmetic means for each accession for a variable. The PCA was performed using correlation matrices and normalization of the model by the varimax rotation method (Dien, Beal & Berg, 2005) using SPSS version 25 (IBM, Armonk, NY, USA).

To determine possible associations between variables, we subjected all data for physical and physiological traits, seed yield and cumulative damage index for each of the 27 accessions using a redundancy analysis (RDA) (Legendre & Legendre, 1998). The RDA was chosen over a canonical correspondence analysis due to the length of the gradient for the variables (Ter Braak & Smilauer, 2002). Gradient length was calculated using a detrended correspondence analysis (DCA) (Hill, 1979). The significance of the damage and yield index on the ordering of morphological and physiological variables, was analyzed using a Monte Carlo random permutation test (499 permutations, p < 0.05) using Canoco 4.5 (Ter Braak & Smilauer, 2002).

Results Physical traits of resistance

Significant differences in the physical traits were found among the accessions (Scott-Knott p < 0.005). The accessions with the most leaves were JMC1339, JMC1306, JMC1364, JMC1208, JMC1264, JMC1313, JMC1336, JMC1288 and JMC1348 had the fewest (Fig. 1A). JMC1339 had the largest leaf area, JMC1255 and JMC1348 the smallest (Fig. 1B). JMC1255, RRS0002, JMC1280 JMC1348 and JMC1304 had the highest leaf dry mass; JMC1339 had the lowest (Fig. 1C). JMC1255 had the thickest leaves, and JMC1325 accession had the thinnest (Fig. 1D). JMC1339 had the highest SLA, and JMC1255, JMC1348, RRS002, JMC1280, JMC1304 and JMC1357 had the lowest (Fig. 1E). The highest trichome densities were found on JMC1306 and JMC1280, the lowest on JMC1208 (Fig. 1F). JMC1264, JMC1325 and JMC1313 had the hardest leaves, and the rest had softer leaves (Fig. 1G).

Figure 1: Means (±SD) at 60 days after emergence for leaf resistance traits of 27 Phaseolus lunatus landrace accessions from southeastern Mexico. Different letters above histobars denote a significant difference among accessions (Scott-Knott, p [less than] 0.05). (A) Number of leaves, (B) leaf area, (C) dry mass of leaves, (D) thickness, (E) specific leaf area, (F) number of trichomes, (G) hardness. Accession codes on x-axis: 1 = RRS0001, 2 = JMC1271, 3 = JMC1280, 4 = RRS0002, 5 = JMC1255, 6 = JMC1304, 7 = JMC1240, 8 = JMC1350, 9 = JMC1254, 10 = JMC1327, 11 = JMC1273, 12 = JMC1357, 13 = JMC1345, 14 = JMC1270, 15 = JMC1337, 16 = JMC1245, 17 = JMC1208, 18 = JMC1348, 19 = JMC1339, 20 = JMC1288, 21 = JMC1306, 22 = JMC1264, 23 = JMC1325, 24 = JMC1297, 25 = JMC1313, 26 = JMC1336, 27 = JMC1364. Days after emergence (DAE), specific leaf area (SLA). DOI: 10.7717/peerj.12088/fig-1

Physiological traits

The physiological variables also differed significantly among the accessions (Scott-Knott p < 0.005). Accessions JMC1273, JMC1325, JMC1264, RRS0002, JMC1245, JMC1288, JMC1313, JMC1304, JMC1280, JMC1357, JMC1270 and JMC1350 had the highest photosynthetic rate (P_N), JMC1336 and JMC1339 the lowest (Fig. 2A). WUE was highest in JMC1337, JMC1245, and JMC1270, and lowest in JMC1336 (Fig. 2B). JMC1364 had the highest g_s, for overall (Fig. 2C). Transpiration was highest in JMC1273 and JMC1364 (Fig. 2D). JMC1336 and JMC1364 had the highest C_i for overall (Fig. 2E).

Figure 2: Means (±SD) at 60 days after emergence for physiological tolerance traits of 27 Phaseolus lunatus landraces accessions from southeastern Mexico. Different letters above histobars denote a significant difference among accessions at 60 DAE (Scott-Knott, p [less than] 0.05). (A) Photosynthesis assimilation rate (PN), (B) water-use efficiency (WUE), (C) stomatal conductance (gs), (D) transpiration (E), (E) intercellular carbon (Ci). Accession codes on x-axis: 1 = RRS0001, 2 = JMC1271, 3 = JMC1280,4 = RRS0002, 5 = JMC1255, 6 = JMC1304, 7 = JMC1240, 8 = JMC1350, 9 = JMC1254, 10 = JMC1327, 11 = JMC1273, 12 = JMC1357, 13 = JMC1345, 14 = JMC1270, 15 = JMC1337, 16 = JMC1245, 17 = JMC1208, 18 = JMC1348, 19 = JMC1339, 20 = JMC1288, 21 = JMC1306, 22 = JMC1264, 23 = JMC1325, 24 = JMC1297, 25 = JMC1313, 26 = JMC1336, 27 = JMC1364. DOI: 10.7717/peerj.12088/fig-2

Leaf damage and yield

The percentage of leaf damage differed significantly among all accessions (Scott-Knott p < 0.005) and at DAE (Scott-Knott p < 0.005). The highest percentages of damage were found at 45 DAE, followed by 60 and 30 DAE. At 30 DAE, accessions JMC1271, JMC1255 and RRS0001 had the highest percentages of damage, and JMC1325, JMC1336, JMC1297, JMC1313, JMC1288, JMC1270, JMC1364, JMC1348, JMC1337, JMC1245, JMC1339, JMC1306 and JMC1264 had the lowest (Fig. 3A). For the damage index, RRS0001, JMC1271 and JMC1255 had the highest values; JMC1297, JMC1325, JMC1306, JMC1273 and JMC1264 had the lowest (Table 2). At 45 DAE, JMC1273 had the highest percentage of foliar damage; JMC1306, JMC1264 and JMC1357 had the lowest (Fig. 3A). The damage index at 45 DAE showed that JMC1255, JMC1273, JMC1280 and JMC1339 had the greatest damage; JMC1306, JMC1264, JMC1325 and RRS0002 had the lowest (Table 2). At day 60 DAE, the highest percentages of leaf damage were on JMC1348, JMC1336, JMC1339, JMC1288, JMC1327, the lowest were on JMC1297, JMC1357, JMC1306, JMC1304, JMC1255 and JMC1325 (Fig. 3C). In addition, the damage index was notably higher for JMC1336 than for JMC1348, JMC1339, JMC1288, JMC1306, JMC1264, JMC1325, JMC1297 and JMC1313, which had the lowest values (Table 2). Seed yields also differed significantly among accessions (Scott-Knott p < 0.005), the accessions JMC1325 and JMC1348 had the highest yield (Table 2).

Figure 3: Mean (±SD) leaf damage caused by herbivorous insects at three ages of 27 Phaseolus lunatus landrace accessions from southeastern Mexico. Different letters above histobars denote a significant difference among accessions at (A) 30, (B) 45 and (C) 60 days after emergence (DAE) (Scott-Knott, p [less than] 0.05). DOI: 10.7717/peerj.12088/fig-3

Table 2: Estimates of cumulative damage index at three growth ages (30, 45 and 60 days after emergence; DAE) and of seed yield at 60 DAE for lima bean (P. lunatus). The index was calculated by dividing the lowest recorded number by the highest recorded number for each accession, at 30 45 y 60 DAE (days after emergence). For seed yield, different letters within a column denote a significant difference among accessions within the each DAE (Scott-Knott, p < 0.05). Means (±SD).

Accession code	Accessions	Damage index 30 DAE	Damage index 45 DAE	Damage index 60 DAE	Seed yield(g plant−1)
1	RRS0001	0.14	0.24	0.14	53.6 ± 0.62	c
2	JMC1271	0.11	0.03	0.11	49.7 ± 0.93	c
3	JMC1280	0.03	0.38	0.09	53.6 ± 0.62	c
4	RRS0002	0.02	0.02	0.21	49.4 ± 0.74	d
5	JMC1255	0.14	0.42	0.04	38.5 ± 0.61	d
6	JMC1304	0.07	0.07	0.18	33.0 ± 0.75	e
7	JMC1240	0.06	0.11	0.23	28.1 ± 0.92	e
8	JMC1350	0.06	0.04	0.24	34.5 ± 0.77	e
9	JMC1254	0.03	0.09	0.11	31.3 ± 0.92	e
10	JMC1327	0.04	0.03	0.21	34.9 ± 0.74	e
11	JMC1273	0.01	0.42	0.21	58.8 ± 1.69	b
12	JMC1357	0.02	0.11	0.03	35.0 ± 0.74	e
13	JMC1345	0.03	0.14	0.03	32.8 ± 0.61	e
14	JMC1270	0.03	0.22	0.23	29.0 ± 0.92	e
15	JMC1337	0.03	0.08	0.25	38.4 ± 0.92	d
16	JMC1245	0.03	0.08	0.07	30.8 ± 0.84	e
17	JMC1208	0.03	0.16	0.03	52.4 ± 0.01	b
18	JMC1348	0.05	0.05	0.18	66.6 ± 0.92	a
19	JMC1339	0.02	0.33	0.02	54.9 ± 0.92	c
20	JMC1288	0.09	0.34	0.02	55.9 ± 2.24	c
21	JMC1306	0.01	0.00	0.03	42.4 ± 0.43	d
22	JMC1264	0.01	0.00	0.02	51.9 ± 0.92	c
23	JMC1325	0.00	0.02	0.02	67.0 ± 0.92	a
24	JMC1297	0.00	0.02	0.06	30.8 ± 0.92	e
25	JMC1313	0.02	0.20	0.03	39.5 ± 0.92	d
26	JMC1336	0.00	0.04	0.59	42.1 ± 0.74	d
27	JMC1364	0.03	0.10	0.27	42.9 ± 0.61	d

DOI: 10.7717/peerj.12088/table-2

Variation in resistance and physiological traits

In the PCA for the 12 resistance and gas-exchange traits evaluated, five main components were significant with values >1. These components together explained 86.72% of the variation. PC1 explained 32.45% of the total variation in the original data, PC2 18.32%, PC3 15.20%, PC4 11.02% and PC5 explained 9.72% (Fig. 4, Table 3). PC1 consisted of leaf area, SLA, number of leaves and dry mass; PC2 consisted only of P_N, PC3 of WUE, PC4 of g_s and C_i, and PC5 was formed solely by trichome density (Fig. 4, Table 3).

Figure 4: Biplot of principal component analysis of 27 Phaseolus lunatus landrace accessions from southeastern Mexico based on 12 morphological and physiological leaf traits. (A) PC1 and PC2, (B) PC2 and PC3. Morphological traits: trichomes (Tr), thickness (Th), hardness (Ha), dry mass of leaves (Dm). Physiological traits: photosynthesis assimilation rate (PN), water-use efficiency (WUE), stomatal conductance (gs), transpiration (E), intercellular carbon (Ci). DOI: 10.7717/peerj.12088/fig-4

Table 3: Variance explained in PCA by five principal components derived from 12 leaf traits of lima bean (P. lunatus) and contributions of the original variables to each component. Specific leaf area (SLA), photosynthesis assimilation rate (PN), stomatal conductance (gs), intercellular carbon (Ci), water-use efficiency (WUE), transpiration (E), days after emergence (DAE).

	Principal component axes
Axes	PC1	PC2	PC3	PC4	PC5
Eigenvalue	3.895	2.199	1.825	1.323	1.167
Explained proportion of variation (%)	32.456	18.322	15.206	11.026	9.722
Cumulative proportion of variation (%)	32.456	50.778	65.984	77.010	86.732
Trait	Correlationmatrix
Leaf area (cm2)	0.974	−0.053	0.093	0.033	−0.056
Dry mass (g)	−0.776	−0.011	0.267	−0.221	−0.042
SLA (cm2 g−1)	0.867	−0.085	0.161	−0.06	−0.036
Trichomes (cm2)	−0.072	−0.023	0.023	−0.078	0.961
Hardness (g cm−2)	0.454	0.656	−0.129	−0.18	0.182
Thickness (mm)	−0.389	−0.162	0.62	−0.123	−0.486
Number of leaves	0.914	0.092	0.162	0.025	0.086
PN [µmol (CO2) m−2 s−1]	−0.283	0.911	−0.14	−0.042	−0.097
gs [mol (H2O) m−2 s−1]	0.207	0.184	0.248	0.846	0.146
Ci [µmol (CO2) mol−1]	−0.091	−0.291	−0.044	0.87	−0.24
WUE [µmol (CO2) mmol (H2O)−1]	−0.333	0.248	−0.838	−0.157	−0.107
E [mmol (CO2) mol (H2O)−1]	0.058	0.612	0.667	0.349	0.017

DOI: 10.7717/peerj.12088/table-3

Association of resistance traits and physiology to performance

The RDA showed a reduced separation of the morphological and physiological variables (eigenvalues axis 1 < 0.1; cumulative variance 99.1%), although the axes were marginally significant (axis 1: F = 2.1, p = 0.054; all axes: F = 1.06, p = 0.056) (Fig. 5). In addition, the damage index was significantly higher (Monte Carlo test, F = 2.04, p = 0.02) in accessions JMC1339 and JMC1288, which had the most leaves and greatest SLA and foliar area. Although the difference in seed yield was not significant, it did tend to be higher in these accessions (Fig. 5). In contrast, JMC1270, JMC1245 and JMC1254 had the least damage, but the lowest seed yield. In addition, they had the highest values of foliar biomass, for gas-exchange variables (P_N, E, g_s, WUE and C_i) and defense traits (hardness, thickness, and trichome density).

Figure 5: Redundancy analysis showing the ordination of morphological and physiological leaf traits associated with the cumulativedamage index and seed yield of 27 landraces accessions of Phaseolus lunatus from southeastern Mexico. Accessions: 1 = RRS0001, 2 = JMC1271, 3 = JMC1280,4 = RRS0002, 5 = JMC1255, 6 = JMC1304, 7 = JMC1240, 8 = JMC1350, 9 = JMC1254, 10 = JMC1327, 11 = JMC1273, 12 = JMC1357, 13 = JMC1345, 14 = JMC1270, 15 = JMC1337, 16 = JMC1245, 17 = JMC1208, 18 = JMC1348, 19 = JMC1339, 20 = JMC1288, 21 = JMC1306, 22 = JMC1264, 23 = JMC1325, 24 = JMC1297, 25 = JMC1313, 26 = JMC1336, 27 = JMC1364. Morphological traits: trichomes (Tr), thickness (Th), hardness (Ha), dry mass of leaves (Dm). Physiological traits: photosynthesis assimilation rate (PN), water-use efficiency (WUE), stomatal conductance (gs), transpiration (E), intercellular carbon (Ci). DOI: 10.7717/peerj.12088/fig-5

Discussion

The data showed a wide variation in the evaluated foliar characteristics, in line with the high diversity among the evaluated landrace accessions found by Ballesteros (1999) and Martínez-Castillo et al. (2004) for P. lunatus in the Yucatan Peninsula. In the search for pest resistance among select landrace accessions, genetic variation is a key element because the wider genetic pool increases the likelihood of finding highly resistant populations as found for Vigna umbellata resistant to Callosobruchus chinensis (L.) (Somta et al., 2008), P. vulgaris resistant to Callosobruchus chinensis (Ku-Hwan et al., 2002), and Pisum fulvum resistant to Bruchus pisorum (Clement, Hardie & Elberson, 2002). The effectiveness of species belonging to Fabaceae in resisting damage caused by pest insects is likely a function of multiple defense mechanisms ranging from morphological characteristics to physiological adaptations (Bonte et al., 2010).

When evaluating the resistance traits independently, we found distinct differences among the accessions as reported for other traits of native and cultured materials, thus allowing selection of populations with desirable defense characteristics (Maag et al., 2015; Moya-Raygoza, 2016; Dos Santos et al., 2020). Although many studies have evaluated physical resistance traits, chemical defenses, and biological interaction networks in agricultural production systems, these factors can also contribute to germplasm selection and crop improvement (Chen et al., 2008; Mitchell et al., 2016). Some accessions with larger leaves are likely to have greater vigor and tolerance to damage by herbivorous insects (Ssekandi et al., 2016). Likewise, the thickness and hardness of leaves also have an important impact on resistance; as leaf thickness increases, some sucking insect larvae spend less time on the leaf blade (Rao, 2002; Saheb et al., 2018). To produce large, thick leaves, the plant must also have a greater photosynthetic capacity to generate the necessary photoassimilates. Furthermore, stomatal conductance and transpiration rates play a fundamental role in the WUE of plants with large leaves such as those of P. lunatus. Here, the accessions with outstanding physiological traits such photosynthetic assimilation also had outstanding morphological characteristics for resisting insect damage. Plants with harder leaves require greater effort by leaf-eating insects (Schofeld et al., 2011). Accession JMC1325 in our study conditions produced the hardest and thinnest leaves, so it is a good candidate for further studies on resistance to foliar damage since some plants with thinner leaves are less preferred by insects, as in the case of Vigna radiata (L.) (Lakshminarayan, Singh & Mishra, 2008), V. mungo (Taggar & Gill, 2012), Gossypium hirsutum (Butter & Vir, 1989) and Cucumis sativus (Shibuya et al., 2009). Leaf hardness has been positively associated with the nutritional quality of the leaf; thus, the insects may be able to evaluate and select their food (Larcher, 2006). Therefore, the harder leaves of some accessions might not always be defensive traits, but rather provide a better food source for certain insects, making the plant more susceptible to defoliation (Schädler et al., 2007; Caldwell, Read & Sanson, 2016). In addition, we found that for another widely studied morphological trait, trichome density, accession JMC1306 has a high density. This trait and its contribution to plant defense is difficult to generalize among plant species (Dos Santos et al., 2020). For example, in Fabaceae species, trichomes can contribute to resistance against defoliating insects (Oghiakhe, Jackai & Makanjuola, 1992; Veeranna & Hussain, 1997) by hindering pest movement on the plants (Tian et al., 2012; Figueiredo et al., 2013).

Physiological traits also varied among accessions. Groups of accessions with high values for a particular trait were found, for example, for P_N with 13 accessions; for E, C_i and WUE with two; and for g_s with only one variable. For P_N and E, the evaluated accessions had considerably high levels (P_N = 24 P_N [µmol (CO₂) m⁻² s⁻¹] and E = 11 [mmol (H₂O) m⁻² s⁻¹] (Ribeiro et al. (2004), since values ranged from 25 to 29 [µmol (CO₂) m⁻² s⁻¹] for P_N and 8 to 9 [mmol (H₂O) m⁻² s⁻¹] for E. Thus, accessions with higher P_N seem to have a greater carboxylation capacity in the environmental conditions of the region, and in the case of accessions with high E values, a greater release of water molecules as result of large stomatal openings (Meneses-Lazo et al., 2018). In addition, in the case of g_s, most accessions have low values, which could be interpreted as an indicator of drought tolerance (Khazaei et al., 2019), although sometimes the ability to regulate g_s can be a better strategy than having low values, as found for Phaseolus vulgaris L. (Rosales et al., 2012). For C_i, we only found two accessions at the highest levels, which could be the result of a differential photosynthetic adjustment between accessions, and in the ability to regulate mitochondrial respiration such as its photorespiration, impacting the release of CO₂ (Lawlor & Cornic, 2002). For WUE, accessions JMC1337, JMC1245, and JMC1270 had higher values than the rest and large leaves, a desirable combination of traits for insect resistance.

For all traits described, both resistance and physiological, we highlighted those with the most appropriate values for optimal performance, depending on the type of trait, but an accession should not be selected on the basis of one variable (Capblancq et al., 2018; Vangestel et al., 2018). In this regard, seven accessions—JMC1339, JMC1288, JMC1264, JMC1325, JMC1208 and JMC1313—had the lowest damage indices at 60 DAE. On the basis of leaf damage, they could have a better resistance throughout their ontogeny (Nzungize et al., 2012), but when we also consider higher seed yield, we found that five accessions—JMC1325, JMC1288, JMC1339, JMC1208 and JMC1264—were the best performers. These accessions had the highest yields (67.0 ± 0.92 to 51.9 ± 0.92 g plant⁻¹) and lowest damage indices (0.02 to 0.03). However, when we analyzed with the RDA all accessions simultaneously with the morphological and physiological traits and cumulative damage index during the experiment, we found that only JMC1339 and JMC1288 maintained an optimum seed yield (54.9 ± 0.92 to 55.9 ± 2.24 g plant⁻¹), despite having a high cumulative damage index. Interestingly, we found a strong positive association of leaf production, SLA and leaf area with the cumulative damage index. Overall, this finding may not appear to be very surprising since some plants can maintain high yields while being more susceptible to damage by pests (Lale & Kolo, 1998; Kimiti, Odee & Vanlauwe, 2009; Keneni, Bekele & Imtiaz, 2011; Kiptoo et al., 2016), perhaps because plants that allocate more resources to defense will have less to allocate toward growth or reproduction (Gong & Zhang, 2014). However, we emphasize the association of a higher cumulative damage index with greater leaf production, SLA and leaf area because SLA has been suggested as critical driver of variation in resource availability above ground (Poorter et al., 2012), which might help lima bean compensate for the resource limitation caused by the lost leaf area or even maximize light capture area (high SLA) (Evans & Poorter, 2001; Freschet, Swart & Cornelissen, 2015) through unfolding its leaves in such a way as to avoid leaf overlap (Santiago & Wright, 2007).

Our main results revealed positive correlations between foliar resistance traits and herbivorous insect damage levels but a negative correlation between these traits and yield. Nevertheless, we measured only 45 individuals for each of 27 accessions in only one environment, and the genetic diversity among the accessions has not been assessed. Although it is difficult to generalize the strength of the resistance traits and their possible correlations with insect damage and bean yield, our study highlights the importance of morphological traits such as greater leaf production, leaf area and SLA in relation to increased plant productivity (McNickle & Evans, 2018) through the capture of more light energy and efficient use of available resources in plants (Maschinski & Whitham, 1989).

Conclusions

Our measurements and comparisons of resistance, physiological and yield traits in landrace accessions of P. lunatus in the Yucatan Peninsula highlights the great diversity in germplasm resources. Five accessions, JMC1325, JMC1288, JMC1339, JMC1208 and JMC1264, performed the best in the field in terms of seed yield and lowest cumulative damage index, even though two, JMC1339 and JMC1288, had the greatest damage. Our results found a positive correlation between high values for “resistance traits” and actual resistance to herbivorous insect damages, but a negative correlation between these traits and yield. Our study identifies important morphological (number of leaves, leaf area, and dry mass of leaves; trichome density, specific leaf thickness and hardness) and physiological traits (photosynthetic rate, stomatal conductance, intercellular carbon, water-use efficiency and transpiration) for selecting lima bean accessions belonging to landrace accessions with high yields (regardless of the damage they may suffer) when no agrochemicals are used, despite the limitations of our study. This is the first step toward identifying resistant lines of lima beans for sustainable, safe production in the Yucatan Peninsula.

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Roberto Rafael Ruiz-Santiago conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Horacio Salomón Ballina-Gómez conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Esau Ruiz-Sánchez conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Jaime Martínez-Castillo, René Garruña-Hernández and Rubén Humberto Andueza-Noh performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Data Availability

The following information was supplied regarding data availability:

The raw measurements are available in the Supplementary File.

Funding

This work was supported by CONACYT with the doctoral scholarship to Roberto Rafael Ruiz-Santiago (No. 845968), CONACYT project (no. YUC-2018-03-01-119959) and Tecnológico Nacional de México (TECNM) project (no. 8983.20-P): “Selección de variedades locales con características morfológicas y de defensa en contra de insectos herbívoros en el cultivo de Phaseolus lunatus L.”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

Agrawal AA. 2011. Current trends in the evolutionary ecology of plant defence. Functional Ecology 25:420-432

Ballesteros GA. 1999. Ballesteros GA 1999. Contribuciones al conocimiento del frijol Lima (Phaseolus lunatus L.) en América Tropical. In: Colegio De Posgraduados. Montecillos: Estado De México.

Blair MW, Wu X, Bhandari D, Zhang X, Hao J. 2016. Role of legumes for and as horticultural crops in sustainable agriculture. In: Nandwani D. Organic Farming for Sustainable Agriculture. Sustainable Development and Biodiversity.

Bonte D, De Roissart A, Vandegehuchte ML, Ballhorn DJ, Van Leeuwen T, De la Peña E. 2010. Local adaptation of aboveground herbivores towards plant phenotypes induced by soil biota. PLOS ONE 5:11174

Butter NS, Vir BK. 1989. Morphological basis of resistance in cotton to the whitefly Bemisia tabaci. Phytoparasitica 17:251-561

Caldwell E, Read J, Sanson GD. 2016. Which leaf mechanical traits correlate with insect herbivory among feeding guilds? Annals of Botany 117:349-361

Camacho-Pérez L, Martínez-Castillo J, Mijangos-Cortés JO. 2008. Genetic structure of Lima bean (Phaseolus lunatus L.) landraces grown in the Mayan area. Genetic Resources and Crop Evolution 65:229-241

Capblancq T, Luu K, Blum MGB, Bazin E. 2018. Evaluation of redundancy analysis to identify signatures of local adaptation. Molecular Ecology Resources 18:1223-1233

Chen M, Huang Y, Liu G, Qin F, Yang S, Xu X. 2016. Effects of enhanced UV-B radiation on morphology, physiology, biomass, leaf anatomy and ultrastructure in male and female mulberry (Morus alba) saplings. Environmental and Experimental Botany 129:85-93

Chen Y, Schmelz EA, Wäckers F, Ruberson JR. 2008. Cotton plant, Gossypium hirsutum L. defense in response to nitrogen fertilization. Journal of Chemical Ecology 34:1553-1564

Clement SL, Hardie DC, Elberson LR. 2002. Variation among accessions of Pisum fulvum for resistance to pea weevil. Crop Science 42:2167-2173

Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW. 2016. Grupo InfoStat. Universidad Nacional De Córdoba. Argentina.

Dien J, Beal DJ, Berg P. 2005. Optimizing principal components analysis of event-related potential analysis: matrix type, factor loading weighting, extraction, and rotations. Clinical Neurophysiology 116:1808-1825

Dirzo R, Domínguez CA. 1995. Plant-herbivore interactions in Mesoamerican tropical dry forest. In: Bullock S, Mooney H, Medina E, eds. Seasonally dry tropical forest. Cambridge: Cambridge University Press. 305-325

Dos Santos LFC, Ruiz-Sánchez E, Andueza-Noh RH, Garruña Hernández R, Latournerie-Moreno L, Mijangos-Cortés JO. 2020. Leaf damage by Spodoptera frugiperda J. E. Smith (Lepidoptera: Noctuidae) and its relation to leaf morphological traits in maize landraces and commercial cultivars. Journal of Plant Diseases and Protection 127:103-109

Evans JR, Poorter H. 2001. Photosynthetic acclimation of plants to growth irradiance: the relative importance of specific leaf area and nitrogen partitioning in maximizing carbon gain. Plant, Cell & Environment 24:755-767

FAO. 2018. World food and agriculture –Statistical pocketbook. data

Figueiredo AST, Resende JTV, Morales RGF, Gonçalves APS, Da Silva PR. 2013. The role of glandular and non-glandular trichomes in the negative interactions between strawberry cultivars and spider mite. Arthropod-Plant Interactions 7:53-58

Freschet GT, Swart EM, Cornelissen JHC. 2015. Integrated plant phenotypic responses to contrasting above- and below-ground resources: key roles of specific leaf area and root mass fraction. New Phytologist 206:1247-1260

Gong B, Zhang G. 2014. Interactions between plants and herbivores: a review of plant defense. Ecological Society of China 34:325-336

Hill MO. 1979. DECORANA-A FORTRAN program for detrended correspondence analysis and reciprocal averaging. Ithaca: Cornell University. 1-52

Keneni G, Bekele E, Imtiaz M. 2011. Breeding chickpea (Cicer arietinum [Fabaceae]) for better seed quality inadvertently increased susceptibility to adzuki bean beetle (Callosobruchus chinensis [Coleoptera: Bruchidae]) International Journal of Tropical Insect Science 31:249-261

Khazaei H, Wach D, Pecio A, Vandenberg A, Stoddard FL. 2019. Genetic analysis of photosynthesis-related traits in faba bean (Vicia faba L.) for crop improvement. Plant Breeding 138:761-769

Kimiti JM, Odee DW, Vanlauwe B. 2009. Area under grain legumes cultivation and problems faced by smallholder farmers in legume production in the semi-arid eastern Kenya. Journal of Sustainable Development in Africa 11:305-315

Kiptoo GJ, Kinyua M, Kiplagat O, Wanjala FME, Kiptoo JJ, Cheboi JJ, Kimno SK, Rotich G, Ngurwe JK. 2016. Evaluation of common bean (Phaseolus vulgaris L.) varieties for resistance to bean stem Maggot (Ophiomyia spp.) in Kenya. American Journal of Experimental Agriculture 12:1-7

Ku-Hwan L, Seung-Keun J, Sang IIP, Beom-Heon S, Yong-Gu C, Hong-Sig K. 2002. Agronomic characteristics of resistant mungbean to Callosbruchus chinensis. Journal of Agricultural Science 19:41-52

Lakshminarayan S, Singh PS, Mishra DS. 2008. Relationship between whitefly population, YMV disease and morphological parameters of green gram germplasm. Environment and Ecology 26:978-982

Lale NES, Kolo AA. 1998. Susceptibility of eight genetically improved local cultivars of cowpea to Callosobruchus maculatus F. (Coleoptera: Bruchidae) in Nigeria. International Journal of Pest Management 44:25-27

Larcher W. 2006. Ecofisiologia vegetal. São Carlo: RiMa. 525-550

Lawlor DW, Cornic G. 2002. Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants. Plant, Cell and Environment 25:275-294

Legendre P, Legendre L. 1998. Numerical ecology. Amsterdam: Elsevier. 380

Lynch JP. 2007. Roots of the second green revolution. Australian Journal of Botany 55:493-512

Maag D, Erb M, Bernal JS, Wolfender JL, Turlings TCJ, Glauser G. 2015. Maize domestication and anti-herbivore defences: leaf-specifc dynamics during early ontogeny of maize and its wild ancestors. PLOS ONE 8:e0135722

Martínez-Castillo J, Colunga-García MP, Zizumbo-Villarreal D. 2008. Genetic erosion and in situ conservation of Lima bean (Phaseolus lunatus L.) landraces in its Mesoamerican diversity center. Genetic Resources and Crop Evolution 55:1065-1077

Martínez-Castillo J, Camacho-Perez L, Coello-Coello J, Andueza-Noh R. 2012. Wholesale replacement of Lima bean (Phaseolus lunatus L.) landraces over the last 30 years in northeastern Campeche, Mexico. Genetic Resources and Crop Evolution 59:191-204

Martínez-Castillo J, Zizumbo-Villarreal D, Perales-Rivera H, Colunga-García M. 2004. Intraspecific diversity and morpho-phenological variation in Phaseolus lunatus L. from the Yucatan Peninsula, Mexico. Economic Botany 58:354-380

Maschinski J, Whitham TG. 1989. The continuum of plant responses to herbivory: the influence of plant association, nutrient availability, and timing. The American Naturalist 134:1-19

McNickle GG, Evans WD. 2018. Toleration games: compensatory growth by plants in response to enemy attack is an evolutionarily stable strategy. AoB Plants 10:1-14

Meneses-Lazo RE, Garruña Hernández R, Latournerie-Moreno L, Andrade-Torres JL, Pérez-Gutiérrez A. 2018. Phenological and physiological characterization of experimental habanero pepper varieties with high agronomic potential. Revista Fitotecnia Mexicana 41:67-74

Mitchell C, Brennan RM, Graham J, Karley AJ. 2016. Plant defense against herbivorous pests: exploiting resistance and tolerance traits for sustainable crop protection. Frontiers in Plant Science 7:1132

Moya-Raygoza G. 2016. Early development of leaf trichomes is associated with decreased damage in teosinte, compared with maize, by Spodoptera frugiperda (Lepidoptera: Noctuidae) Annals of the Entomological Society of America 109:737-743

Nzungize JR, Lyumugabe F, Busogoro JP, Baudoin J-P. 2012. Pythium root rot of common bean: biology and control methods. A review. Biotechnology. Agronomy, Society and Environment 16:405-413

Oerke EC, Dehne HW. 2004. Safeguarding production-losses in major crops and the role of crop protection. Crop Protection 23:275-285

Oghiakhe LEN, Jackai L, Makanjuola WA. 1992. A rapid visual field screening technique for resistance of cowpea (Vigna unguiculata) to the legume pod borer, Maruca testulalis (Lepidoptera: Pyralidae) Bulletin of Entomological Research 82:507-512

Panda N, Khush GS. 1995. Host plant resistance to insects. Biddles, Guildford: CAB International in Association with International Rice Research Institute.

Poorter H, Niklas KJ, Reich PB, Oleksyn J, Poot P, Mommer L. 2012. Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control. New Phytologist 193:30-50

Rao RVS. 2002. Method of screening for resistance to leaf miner, Aproaerema modicella (Deventer) under greenhouse conditions. Annals of Plant Protection Sciences 10:38-41

Ribeiro RV, Dos Santos MG, Souza GM, Machado EC, De Oliveira RF, Angelocci LR, Pimentel C. 2004. Environmental effects on photosynthetic capacity of bean genotypes. Pesquisa AgropecuáRia Brasileira 39:615-623

Rosales MA, Ocampo E, Rodríguez-Valentín R, Olvera-Carrillo Y, Acosta-Gallegos J, Covarrubias AA. 2012. Physiological analysis of common bean (Phaseolus vulgaris L.) cultivars uncovers characteristics related to terminal drought resistance. Plant Physiology and Biochemistry 56:24-34

Saheb YP, Hari-Prasad KV, Swarajya KL, Rani JS. 2018. Mechanisms of resistance in groundnut genotypes against leaf bud borer, Anarsia ephippias (Meyrick) Journal of Entomology and Zoology Studies 6:694-705

Santiago LS, Wright SJ. 2007. Leaf functional traits of tropical forest plants in relation to growth form. Functional Ecology 21:19-27

Schädler M, Roeder M, Brandl R, Matthies D. 2007. Interacting effects of elevated CO2, nutrient availability and plant species on a generalist invertebrate herbivore. Global Change Biology 13:1005-1015

Schofeld RMS, Emmett KD, Niedbala JC, Nesson MH. 2011. Leafcutter ants with worn mandibles cut half as fast, spend twice the energy, and tend to carry instead of cut. Behavioral Ecology and Sociobiology 65:969-982

Shibuya T, Hirai N, Sakamoto Y, Komuro J. 2009. Effects of morphological characteristics of Cucumis sativus seedlings grown at different vapor pressure deficits on initial colonization of Bemisia tabaci (Hemiptera: Aleyrodidae) Journal of Economic Entomology 102:2265-2267

Sohrabi F, Nooryazdan HR, Gharati. 2017. plant resistance to the moth Tuta absoluta (Meyrick) (Lepidoptera:Gelechiidae) in tomato cultivars. Neotropical Entomology 46:203-209

Somta P, Kaga A, Tomooka N, Isemura T, Vaughan DA, Srivines P. 2008. Mapping of quantitative trait loci for a new source of resistance to bruchids in the wild species Vigna nepalens Tateishi & Maxted (Vigna subgenus Ceratotropis) Theoretical and Applied Genetics 117:621-628

Ssekandi JW, Mulumba J, Colangelo P, Nankya R, Fadda C, Karungi J, Otim M, De Santis P, Jarvis DI. 2016. The use of common bean (Phaseolus vulgaris) traditional varieties and their mixtures with commercial varieties to manage bean fly (Ophiomyia spp.) infestations in Uganda. Journal of Pest Science 89:45-57

Stinchcombe J. 2002. Can tolerance traits impose selection on herbivores? Evolutionary Ecology 16:595-602

Stout MJ. 2013. Re-evaluating the conceptual framework for applied research on host-plant resistance. Insect Science 20:263-272

Strauss SY, Agrawal AA. 1999. The ecology and evolution of plant tolerance to herbivory. Trends in Ecology & Evolution 14:179-185

Taggar GK, Gill RS. 2012. Preference of whitefly, Bemisia tabaci, towards black gram genotypes: role of morphological leaf characteristics. Phytoparasitica 40:461-474

Ter Braak CJF, Smilauer P. 2002. CANOCO reference manual and canodraw for windows user’guide: software for canonical community ordination (version 4.5). Ithaca: Microcomputer Power. 500

Tian D, Tooker J, Peiffer M, Chung SH, Felton GW. 2012. Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum) Planta 236:1053-1066

Vangestel C, Andrew JE, Wegrzyn JL, St. Clair JB, Neale DB. 2018. Linking phenotype, genotype and environment to unravel genetic components underlying cold hardiness in coastal Douglas-fir (Pseudotsuga Menziesii Var. Menziesii) Tree Genetics & Genomes 14:10

Veeranna R, Hussain M. 1997. Trichomes as physical barriers for cowpea podborer, Maruca testulalis (Geyer) (Lepidoptera: Pyralidae) Insect Environment 3:1-15

Widstrom NW, McMillian WW, Wiseman BR. 1979. Ovipositional preference of the corn earworm and the development of trichomes on two exotic corn selections. Environmental Entomology 8:833-839