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

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

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).

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).

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).

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).

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 (P_N), stomatal conductance (g_s), intercellular carbon (C_i), 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).

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.

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