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Journal of Economic Entomology, 114(1), 2021, 377–386 doi: 10.1093/jee/toaa292 Advance Access Publication Date: 5 January 2021 Plant Resistance Research

Tolerance in Landraces to speciosa (Coleoptera: Chrysomelidae) Larvae and Its Relationship to Plant Pigments, Compatible Osmolytes, and Vigor

Eduardo Neves Costa,1,2,5, Bruno Henrique Sardinha de Souza,1,3, 1 4 Zulene Antônio Ribeiro, Durvalina Maria Mathias dos Santos, and Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 Arlindo Leal Boiça Júnior1

1Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal, Departamento de Ciências da Produção Agrícola, Universidade Estadual Paulista, 14884-900, Jaboticabal, SP, , 2Faculdade de Ciências Agrárias, Universidade Federal da Grande Dourados, 79804-970, Dourados, MS, Brazil, 3Departamento de Entomologia, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil, 4Faculdade de Ciências Agrárias e Veterinárias, Campus de Jaboticabal, Departamento de Biologia Aplicada à Agropecuária, Universidade Estadual Paulista, 14884-900, Jaboticabal, SP, Brazil, and 5Corresponding author, e-mail: [email protected]

Subject Editor: Frank Peairs

Received 30 August 2020; Editorial decision 20 November 2020

Abstract The rootworm Diabrotica speciosa (Germar) is native to and causes severe economic losses to several crops due to root feeding and disease spread. In maize (Zea mays L.), losses in production come from larval rootworm attack on plant roots resulting in plant health problems, including stalk lodging. More options for controlling this pest are needed to create well balanced, integrated pest management programs for farmers in this region. Natural sources of tolerance in maize genotypes are important for maize breeding programs, and this study investigated the expression of tolerance in several Brazilian maize landraces to D. speciosa. Plant vigor and compounds associated with plant health, including chlorophylls, carotenoids, glycine betaine, and proline were assessed for each landrace. Five landraces and one maize cultivar were selected based on their levels of antibiosis-resistance to D. speciosa that were determined in a prior screening. The percent re- duction in plant growth was used as the measure of tolerance. The landrace Azteca was classified as tolerant to D. speciosa larval feeding, displaying less reduction in plant matter despite having lower plant vigor. This landrace also had higher amounts of chlorophyl and carotenoid pigments, suggesting a positive correlation between tolerance to D. speciosa and higher contents of these photosynthetic pigments. The compatible osmolytes glycine betaine and proline do not seem to be associated with tolerance in maize landraces to D. speciosa larvae. Landrace Azteca seems promising for plant breeding, and repeated field studies are needed to confirm its suitability in maize integrated pest management.

Key words: Zea mays L., native resistance, chlorophyl, carotenoids, glycine betaine

Tolerance is characterized by the ability of a plant to withstand or Tolerance mechanisms differ from those of antibiosis and recover from injury caused by pests. Expression of toler- antixenosis. Examples of tolerance mechanisms include increased ance is determined by heritable qualities (plant genotype), allowing photosynthetic activity, compensatory regrowth, utilization of stored the tolerant genotype to withstand injury and regrow (Smith 2005). resources, and phenological changes. Conversely, other resistant From an agronomic perspective, tolerant plant cultivars and hybrids plants are characterized by the presence of morphological and/or produce more biomass and greater yield than susceptible plants chemical traits that adversely affect herbivore performance (anti- when under pest pressure. Furthermore, arthropod populations are biosis) or preference (antixenosis) (Tiffin 2000). Although tolerance not reduced when exposed to tolerant plants, as occurs for plants ex- is important in integrated pest management programs as an integral pressing antibiosis or antixenosis (Smith 2005). Therefore, tolerant determinant of economic injury levels and threshold levels of a crop, plants do not exert selection pressure on herbivore populations, thus it is less often investigated than antixenosis and antibiosis as a source they do not contribute to the emergence of resistant arthropod bio- of crop protection (Peterson et al. 2013, Cruz et al. 2016, Sperotto types (Schoonhoven et al. 2005). et al. 2018).

© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. 377 All rights reserved. For permissions, please e-mail: [email protected]. 378 Journal of Economic Entomology, 2021, Vol. 114, No. 1

Antibiosis occurs when a plant possesses traits that have adverse and causes economic losses due to larval attack on belowground effects on survival, development, fecundity, or fertility of a potential plant structures including roots and tubers of several crops, including pest arthropod, while antixenosis is characterized by negative effects maize (Viana 2010). The economic impact in agriculture and the re- on arthropod behavior, leading to delayed acceptance and possible sources expended to control this pest in Brazil have not yet been es- outright rejection of a plant as a host (Smith and Clement 2012). timated, although extensive amounts of insecticide active ingredients Some authors suggest that plants invest a lot of energy in the ex- are applied annually to different crops for control of D. speciosa pression of antibiosis (and/or antixenosis) or tolerance, and usually larvae and adults (Ávila and Santana 2011). Although it is poorly one of these strategies prevail over the other as a response to the studied, evolution of resistance of D. speciosa to insecticides has attack. (Rosenthal and Kotanen 1994, De Jong and Van Der probably occurred in South America (Cabrera Walsh et al. 2020). Meijden 2000). Attack of D. speciosa larvae has economic importance to maize, As noted above, mechanisms of tolerance often involve changes justifying the need for sustainable pest control tactics. Therefore, this in photosynthesis in injured plants including activation of dor- research aimed to evaluate tolerance in Brazilian maize landraces to mant meristems, changes in plant architecture, partitioning of feeding by D. speciosa larvae. In addition, it was investigated the photoassimilates, source-sink relationships for resource allocation, relationship between tolerance expression, plant vigor, and contents Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 and greater photosynthetic capacity (Schwachtje and Baldwin 2008, of photosynthetic pigments and compatible osmolytes, which are es- Mitchell et al. 2016). Chlorophyll plays an important role in photo- sential components of plant photosynthesis. synthesis, resulting in greater plant growth and accumulation of dry matter, as well as assisting in plant adaptation to the environment (Engel and Poggiani 1991). Carotenoids are also essential for the Materials and Methods photochemical apparatus. They are important to plant tolerance as antioxidants and phytohormone precursors, e.g., abscisic acid and Numbers of D. speciosa Larvae per Maize Plant anti-stress hormones (Howitt and Pogson 2006). Carotenoids are The experiment was performed in a greenhouse in the Department of accessory pigments in light absorption, transferring energy to the Agricultural Sciences, São Paulo State University (UNESP), School of special oxidizable chlorophyl pigment in the reaction center, since Agricultural and Veterinary Sciences, Jaboticabal, state of São Paulo, most antenna (pigment-protein) complexes contain carotenoids that Brazil, under ambient conditions of light, relative humidity, and tem- confer photo-protection to the reaction center (Blankenship 2014). perature. The climatic conditions recorded during the experimental Among osmolytes acting in plant responses to environmental period (March 2014) were: mean temperature of 24.1°C, mean relative stresses, the quaternary ammonium glycine betaine has been ex- humidity of 76.8%, and mean day length of 12 h 08 min (Unesp 2020). tensively studied (Iqbal et al. 2005, Chen and Murata 2008, Ashraf used in this research were reared according to method- 2009, Ali and Ashraf 2011), and its accumulation in maize depends ology adapted from Ávila et al. (2000). The colony of D. speciosa on plant variety (Quan et al. 2004). Glycine betaine is abundant was initiated from adults collected in common bean, Phaseolus vul- primarily in the chloroplast where it plays a vital role in protecting garis L. (Fabales: Fabaceae) (and surrounding weeds) grown in the the thylakoid membrane, contributing to maintenance of photosyn- experimental farm of UNESP, and wild insects were introduced every thetic efficiency (Genard et al. 1991) and allowing the plant to tol- two months to maintain genetic variability. erate different environmental stresses, mainly those of abiotic origin Maize seeds of cultivar ‘Cativerde 02’ were sown in 770-ml (Huang et al. 2000). Glycine betaine does not actively participate in plastic pots with an opening of 1.0 × 0.1 cm at the bottom to allow the removal of reactive oxygen species, such as superoxide radicals water drainage. The pots were filled with soil (eutrophic dusky red *- (O2 ), hydrogen peroxide (H2O2), hydroxyl radicals (OH*), and latosols; Centurion et al. 1995), organic fertilizer, and sand at 3:1:1 singlet oxygen (‘O2), which are toxic to plants in high concentra- ratio, mixed prior to use. Three seeds were sown per pot, and after tions. However, glycine betaine attenuates the harmful effects of oxi- emergence the plants were irrigated as necessary. Thinning was car- dative stress in other ways, such as activating or stabilizing enzymes ried out ~5 d after plant emergence (DAE), keeping one plant per that remove reactive oxygen species (Chen and Murata 2008). pot (replicate). The plants were infested at 10 DAE (V3 vegetative Proline is a proteinogenic amino acid with exceptional conform- stage) (Ritchie et al. 1986) with three, six, or 12 D. speciosa neonate ational rigidity, and it is essential for plant primary metabolism larvae (less than 24-h old) using a soft bristle paintbrush. The larvae (Szabados and Savouré 2010). Since the first report of proline ac- were released in the center of the pots. In total, 30 plants (replicates cumulation in perennial rye grass, Lolium perenne L. (Kemble and per treatment) were infested with D. speciosa larvae, and another 30 Macpherson 1954), several studies have demonstrated that proline plants were kept uninfested for comparison of plant biomass reduc- accumulates in plants under both biotic and abiotic stresses (Sharma tion caused by larval injury. and Dietz 2006, Szabados and Savouré 2010). Proline accumulation Eighteen days after larval infestation, plants were carefully re- may influence plant tolerance to environmental stresses by serving as moved from the pots to avoid damaging the roots. Next, the roots an antioxidant in the removal of reactive oxygen species (Matysik were immersed in a water bucket to remove greater part of soil and et al. 2002), or it can act as a molecular chaperone that protects then washed in running water. Thereafter, the height and number protein integrity and optimizes the activities of various enzymes of leaves were recorded. This methodology was used for infested (Rajendrakumar et al. 1994). and control plants. The plants were then dried in an oven (Model Landraces are usually distinct crop genotypes that have been AS200S, QUIMIS, Diadema, SP) at 60°C for 48 h. Dry matter of the selected by humans for adaptation to particular environments, agri- aerial tissues and of the roots of plants were determined with a scale cultural practices, and customs, resulting in different shapes, sizes, (Model AS1000C, Marte, São Paulo, SP). brightness, and colors (Embrapa 2018). Landraces are considered Calculation of reduction percentages of evaluated plant param- important genetic resources for improving the genetic diversity of eters were done based on the formula proposed by Reese et al. elite germplasm in maize (Zea mays L.) (Strigens et al. 2013) (1994): X = (PC – PT)/PC*100, where X = reduction percentage; The rootworm Diabrotica speciosa (Germar) (Coleoptera: PC = value of the parameter in control plants (without larval in- Chrysomelidae) is native to South America (De La Fuente et al. 2003) festation; average of 10 plants); PT = value of the parameter in Journal of Economic Entomology, 2021, Vol. 114, No. 1 379 treatment plants (with larval infestation). The experiment was Agriculture, in the São Paulo State University (UNESP/FCAV), conducted in a completely randomized design, with 10 replicates. Jaboticabal, State of São Paulo, Brazil. A replicate was represented by one potted maize plant infested with Plants of the six maize genotypes used in the analysis of photo- different numbers of D. speciosa larvae. synthetic pigments and compatible osmolytes were grown in pots and kept in a greenhouse as previously described. The experiment was arranged in a 6 × 2 factorial scheme (6 maize genotypes × 2 Tolerance in Maize Landraces to Attack of levels of infestation [0 or 6 larvae]) for a total of 12 treatment by D. speciosa Larvae genotype combinations with 3–4 replications each. When plants The methodology used in this study for sowing, plant cultivation, were 10-d old, six D. speciosa neonate larvae were infested onto larval inoculation, and tolerance evaluation were the same described each plant in treatments receiving larvae and allowed to feed for in the previous section. This experiment was conducted twice: 18 d. After this, leaves of each maize plant (replicate) were cut with September to October and October to November 2014. The climatic scissors according to treatment regime and used either for the fresh data recorded between September to October were: mean tempera- analysis of chlorophyll and carotenoids, for the evaluation of proline ture of 24.6°C, mean relative humidity of 52.6%, and mean day by flash-freezing in liquid nitrogen, or for analysis of glycine betaine Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 length of 12h18min. From October to November, the mean tem- by oven drying (Model 320-SE, Fanem, São Paulo, SP). perature was 25°C, mean relative humidity of 60.7%, and mean day For analyses of chlorophyll a, chlorophyll b, and carotenoids length of 12 h 54 min (Unesp 2020). (carotenes + xanthophylls), three leaf discs (0.49 cm in diameter) Six maize genotypes (five landraces and one cultivar) were were collected per plant using a metal punch. Four replicates, each selected out of 19 genotypes based on their resistance levels shown consisting of three leaf disks obtained from different leaves of a in a previous study of antibiosis in the genotypes (Costa et al. maize plant were performed, except for landrace Azteca-uninfested 2018). The maize landraces were: Azteca (susceptible landrace), and the cultivar SCS 154-Fortuna-infested, where three replicates Encantilado (susceptible landrace), SCS 154-Fortuna (resistant cul- were used. The leaf discs were subsequently stored in Eppendorf tivar, developed by Epagri, Florianópolis, SC, Brazil), Palha Roxa tubes covered by aluminum foil to avoid photo-oxidation of pig- (resistant landrace), Pérola (resistant landrace), and 14 Variedades ments, and then were weighed on a precision analytical scale (Model (susceptible landrace). The optimum number of larvae for release AA-200, Denver Instrument Company, Bohemia, NY), and showed (Table 1) and age of plants at infestation were determined in pre- 0.025–0.03 g fresh weight. Next, 2 ml of acetone PA was added to vious tests. Furthermore, release of six D. speciosa larvae per maize each of the tubes and the samples were placed in an incubator shaker plant was based on the highest number of larvae from this species (Model G25, New Brunswick, NJ) for 48 h at 60 g at 4°C. Optical found in maize fields byViana and Marochi (2002). The plants were density of the filtrates was read in UV spectrophotometer (Model infested at 10 DAE (V3 vegetative stage) (Ritchie et al. 1986) with DU 640, Beckman, Fort Collins, CO) at 663, 645, 652, and 470 nm, six D. speciosa neonate larvae using a soft bristle paintbrush. A set for chlorophyll a, chlorophyll b, total chlorophyll (a + b), and carot- of plants (10 replicates/treatment) were infested with D. speciosa enoids, respectively (Arnon 1949, Lightenthaler 1987). The chloro- larvae, and another set (10 plants) were left uninfested in order to phyll and carotenoid results were expressed as µg per cm2. calculate plant biomass reduction caused by larval injury, which was Glycine betaine content in the oven dried leaves was analyzed based on the average of the 10 uninfested plants. according to the methodology of Grieve and Grattan (1983). Four replicates were performed per treatment, except for cultivar SCS 154-Fortuna-infested, landrace Palha Roxa-uninfested, and land- Plant Vigor race Pérola-infested, for which three replicates were used. The dry Control plants (uninfested) of the maize genotypes used in the tol- leaf tissue (25-mg dry matter) from each test plant was placed in erance experiment were compared to investigate if they differed in a 2-ml tube. Distilled water was added to each tube and the tubes vigor. Vigor was assessed by measuring height, number of leaves, were shaken in an incubator shaker for 4 h at 25°C. Next, the and dry matter of aerial tissues and of roots, using 10 replicates in a tubes were centrifuged at 10,000 g for 10 min at 25°C. After cen- completely randomized design. trifugation, the supernatant was collected to obtain the aqueous extract. Tubes were then filled with 250 µl aqueous extract and Contents of Photosynthetic Pigments and 250 µl H2SO4 2N, and this solution was cooled in an ice bath for Compatible Osmolytes 60 min at 0°C in a refrigerator. Next, 200 µl of cooled KI-I2 re- The analyses described below were performed in the Laboratory agent (iodine potassium iodide solution) was added, and the tubes of Plant Physiology of the Department of Biology Applied to were kept in an ice bath for 16 h at 0°C in a refrigerator. After

Table 1. Percent reduction (%, mean ± SE) in height, number of leaves, and aerial tissues and roots dry matter of maize plants infested with different numbers of Diabrotica speciosa larvae

Dry matter (g)

Treatment Height (cm) (n = 90) Leaves (n = 90) Aerial tissues (n = 90) Roots (n = 90)

Three larvae 6.2 ± 1.4c 3.7 ± 1.2ab 0.8 ± 0.6b 9.4 ± 3.3c Six larvae 15.9 ± 2.5b 2.1 ± 1.0b 5.3 ± 2.3a 17.0 ± 3.3b Twelve larvae 25.2 ± 2.4a 5.7 ± 1.3a 4.9 ± 2.1a 27.0 ± 3.4a

Means followed by the same letter in columns are not significantly different by Tukey’s (height) or Kruskal–Wallis’ tests α( = 0.05). Reduction percentage formula: X = (PC − PT)/PC*100, where X = reduction percentage; PC = value of the parameter in control plants (without larval infestation, average of 10 plants); PT = value of the parameter in treatments (with D. speciosa larval infestation). 380 Journal of Economic Entomology, 2021, Vol. 114, No. 1 the extraction process was completed, the tubes were centrifuged Results (Avanti-J25, Beckman, Fort Collins, CO) at 10,000 g for 15 min, Numbers of D. speciosa Larvae per Maize Plant and the supernatant discarded. The precipitate was washed twice A greater reduction in plant height (F = 19.07; df = 2, 87; P < 0.0001) with 2 ml of H2SO4 (1N), and centrifuged at 10,000 g for 5 min at 0°C. The precipitate was dissolved in 3 ml 1, 2-dichloroethane was observed when maize plants were infested with 12 D. speciosa by shaking vigorously. Two hours later, the samples were read in larvae compared to six and three larvae (Table 1). Similarly, maize a spectrophotometer at 365 nm (Grieve and Grattan 1983) for plants showed a greater reduction in plant height when infested with glycine betaine content. The standard curve for glycine betaine six than three D. speciosa larvae. Maize plants exhibited a greater reduction in number of leaves (50–200 μg/ml) was prepared in 1-N H SO . The values were ex- 2 4 (H = 6.17; df = 2, 87; P < 0.0001) upon attack of 12 D. speciosa pressed as µg glycine betaine per gram of dry mass. Determination of proline content was carried out following Bates larvae compared to an infestation with six larvae; however, a similar et al. (1973). For each of the three replicates, a 0.5 g sample of the reduction in number of leaves was registered when infesting maize thawed leaf sample collected previously was macerated in liquid ni- plants with three and 12 D. speciosa larvae (Table 1). trogen with pestle and mortar. About 10 ml of 3% sulphosalicylic Reduction in aerial tissues dry matter was greater (H = 11.33; Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 acid was added to the macerated leaf material, and the extract was df = 2, 87; P = 0.0035) in maize plants infested with 12 and 6 filtered twice to remove the large leaf material. Next, 1 ml of the ex- D. speciosa larvae compared with an attack of three larvae (Table 1). tract was transferred into an assay tube, 1-ml acetic acid and 1-ml With respect to roots dry matter, a greater reduction (H = 15.59; ninhydrin solution were added, and the samples were homogenized df = 2, 87; P < 0.0001) was noted in maize plants infested with 12 in a tube shaker (AT 56A, Phoenix, Rochester, NY). The solutions D. speciosa larvae compared to six or three larvae. In addition, a were subjected to a thermostatic bath at 100°C for 1 h. The assay greater reduction in roots dry matter was found in maize plants in- tubes containing the solutions were stored in ice until cool, and then fested with six than three D. speciosa larvae (Table 1). 2 ml of toluene were added and the tubes were shaken again. Proline content was determined using a spectrophotometer at 520 nm. The Tolerance in Maize Landraces to Attack of correction factor was determined using 2-ml toluene as the standard D. speciosa Larvae solution. The results were expressed as µmol proline per gram of leaf tissue. In the first assay, there were no differences in percentage decrease in plant height (H = 4.75; df = 5, 53; P = 0.4459), number of leaves (H = 8.13; df = 5, 53; P = 0.1490), and aerial tissues dry matter Statistical Analysis (H = 4.57; df = 5, 53; P = 0.4701). Genotypes differed in their toler- Data were checked for normality of residuals (Shapiro and Wilk ance as defined as the percentage of the reduction of root dry matter 1965) and homogeneity of variances (Levene 1960). Percentage re- (H = 13.25; df = 5, 53; P = 0.0211). Plants of the landrace Azteca duction data were not normally distributed and were not normalized had a lower percent decrease in root dry matter than cultivar SCS by any transformation (except reduction in plant height in the test of 154-Fortuna, whose plants showed percent decrease seven-fold number of larvae); therefore, they were analyzed by a nonparametric greater than that of landrace Azteca (Table 2). test (Kruskal and Wallis 1952) (α = 0.05), followed by multiple In the second assay, there was no differences regarding the per- comparison of means (Dunn 1964). Data for chemical analyses dis- cent reductions in plant height (H = 9.11; df = 5, 50; P = 0.1046) played normality of residuals and homogeneity of variances, and and number of leaves (H = 8.09; df = 5, 50; P = 0.1516). However, were analyzed by two-way analysis of variance for the main effects landrace Azteca showed a lower percent decrease in aerial tissues dry of genotype (G), infestation (I), and genotype by infestation (G × matter (H = 11.32; df = 5, 50; P = 0.0453) compared with cultivar I) interaction. Some parameters of control plants used to compare SCS 154-Fortuna. The latter genotype displayed a percent decrease plant vigor diverged for normality of residuals and homogeneity of of aerial tissues dry matter six-fold greater than that of landrace variances. Data that did not meet these statistical requirements were: Azteca (Table 2). Considering the percent decrease of root dry assay 1 = dry matter of aerial tissues and dry matter of roots, which matter, landrace Encantilado showed lower reduction than cultivar were log(x)- and squareroot (x + 0.5)-transformed, respectively, ac- SCS 154-Fortuna (H = 14.67; df = 5, 50; P = 0.0119), which had ca. cording to an analysis of transformation (Box and Cox 1964); and sixfold greater percent decrease than that of landrace Encantilado. assay 2 = height and dry matter of roots, which were (x)2- and log(x)- A significant positive correlation was found between percent transformed, respectively, according to Box-Cox test. These data decrease in aerial tissues and root dry matter for cultivar SCS were subjected to one-way analysis of variance. When significant 154-Fortuna in the second assay (Fig. 1d), whereas no significant differences were found, means were separated by Tukey’s honestly correlation was observed between these plant growth parameters significant difference test (Tukey 1949; α = 0.05), except dry matter for landrace Azteca in either assay (Fig. 1a and b). Significant posi- of roots in assay 2, where means were separated using another tive correlations were also observed for landraces Encantilado parametric test (Duncan 1955; α = 0.05). Assays 1 and 2 were ana- (r = 0.9014; P = 0.0004) and Pérola (r = 0.8846; P = 0.0081) in lyzed separately. Statistical analysis was performed in SAS 9.0 (SAS assay 1 and for landrace 14 Variedades in assay 2 (r = 0.7500; Institute 2003). As reduction percentages of aerial tissues and root P = 0.0321) (data not shown). In addition, no significant correlations dry matter did not meet the statistical assumptions, a nonparametric were recorded for the cultivar SCS 154-Fortuna (Fig. 1c) and the correlation test (Spearman 1904) (α = 0.05) was applied to investi- landraces Palha Roxa (r = 0.2258; P = 0.6670) and 14 Variedades gate possible correlations between those parameters. The correlation (r = 0.5069; P = 0.1347) in assay 1, nor for the landraces Encantilado analysis was performed in the Free Statistics Software (version 1.2.1) (r = 0.5313; P = 0.1755), Palha Roxa, (r = 0.5608; P = 0.1482), and (Wessa 2020). Pérola (r = 0.4186; P = 0.3020) in assay 2 (data not shown). Journal of Economic Entomology, 2021, Vol. 114, No. 1 381

Table 2. Percent reduction (%, mean ± SE) in height, number of leaves, and aerial tissues and roots dry matter of maize genotypes sub- jected to larval attack of Diabrotica speciosa

Dry matter (g)

Genotype Height (cm) Leaves Aerial tissues Roots

Assay 1 (n = 59) (n = 59) (n = 59) (n = 59) Azteca 5.7 ± 3.0a 1.6 ± 1.6a 10.9 ± 5.6a 3.7 ± 1.9b Encantilado 6.4 ± 4.2a 2.9 ± 1.2a 12.8 ± 6.4a 26.2 ± 7.1ab SCS 154-Fortuna 10.4 ± 3.7a 5.1 ± 1.2a 15.2 ± 5.5a 28.0 ± 6.3a Palha Roxa 2.3 ± 1.1a 3.8 ± 1.7a 5.1 ± 3.3a 6.7 ± 4.7ab Pérola 5.7 ± 2.6a 6.1 ± 2.0a 6.1 ± 2.6a 12.7 ± 5.9ab 14 Variedades 7.1 ± 2.6a 2.7 ± 1.7a 16.1 ± 5.3a 16.5 ± 6.1ab Assay 2 (n = 56) (n = 56) (n = 56) (n = 56)

Azteca 11.8 ± 3.1a 6.8 ± 2.3a 4.4 ± 3.0b 12.6 ± 6.9ab Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 Encantilado 3.9 ± 1.7a 8.3 ± 2.6a 6.0 ± 3.1ab 6.7 ± 3.4b SCS 154-Fortuna 20.2 ± 7.2a 3.0 ± 2.0a 30.3 ± 9.6a 39.5 ± 9.5a Palha Roxa 7.2 ± 2.8a 3.3 ± 1.6a 11.5 ± 4.1ab 19.5 ± 7.3ab Pérola 14.6 ± 2.6a 5.7 ± 3.1a 10.8 ± 4.7ab 27.0 ± 5.0ab 14 Variedades 8.1 ± 3.5a 1.5 ± 1.0a 3.3 ± 2.3ab 10.1 ± 5.8ab

Means followed by the same letter in columns are not significantly different by Kruskal–Wallis’s test α( = 0.05). Reduction percentage formula: X = (PC − PT)/ PC*100, where X = reduction percentage; PC = value of the parameter in control plants (without larval infestation, average of 10 plants); PT = value of the par- ameter in treatments (with D. speciosa larval infestation).

Fig. 1. Spearman’s correlation analysis between reduction percentage in aerial tissues dry matter and roots dry matter in maize genotypes comparing infested with uninfested plants by Diabrotica speciosa larvae. The same experiment was performed twice (assays 1 and 2). This type of correlation analysis is indicated for non-normal data. NS Non significant; ** Significant at 1% probability;n = sample size. 382 Journal of Economic Entomology, 2021, Vol. 114, No. 1

Plant Vigor Contents of Photosynthetic Pigments and In the first assay, maize genotypes showed a similar plant height Compatible Osmolytes (F = 1.65; df = 5, 52; P = 0.1640). Conversely, differences were found Significant differences among genotypes in chlorophyll a contents in numbers of leaves (F = 2.67; df = 5, 52; P = 0.0320), aerial tissues were found (F = 5.00; df = 5, 34; P = 0.0015), whereas no differences dry matter (F = 3.40; df = 5, 52; P = 0.0099), and root dry matter were observed for infestation (I) (F = 0.25; df = 1, 34; P = 0.6203) (F = 2.55; df = 5, 52; P = 0.0387) among maize genotypes (Table 3). or the G × I interaction (F = 1.20; df = 5, 34; P = 0.3318) (Table 4). Landrace Encantilado showed lower numbers of leaves than cul- Higher chlorophyll a content was observed in leaves of landrace tivar SCS 154-Fortuna, but these genotypes did not differ from the Azteca, differing from that of landraces Palha Roxa, Pérola, and 14 others. On the other hand, landrace Azteca displayed lower aerial Variedades. The content of chlorophyll a in leaves of landrace Azteca tissues dry matter than cultivar SCS 154-Fortuna and landrace 14 was 1.87-fold higher than the content in landrace Pérola. Variedades. Landrace Azteca also exhibited lower root dry matter With regards to chlorophyll b, significant differences were ob- than all maize genotypes. served for genotype (F = 5.04; df = 5, 34; P = 0.0015), but not for In the second assay, significant differences among genotypes were infestation (F = 0.15; df = 1, 34; P = 0.7049) or the G × I interaction observed in plant height (F = 7.71; df = 5, 52; P < 0.0001), aerial tis- (F = 1.31; df = 5, 34; P = 0.2859) (Table 4). Greater chlorophyll b Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 sues dry matter (F = 4.31; df = 5, 51; P = 0.0024), and root dry matter content was found in leaves of landrace Azteca, differing from the (F = 2.41; df = 5, 51; P = 0.0487) (Table 3). Plants of landrace Pérola contents of landraces Palha Roxa, Pérola, and 14 Variedades. The showed greater height than plants of the other genotypes. Landrace content of chlorophyll b in leaves of landrace Azteca was 1.81-fold Azteca displayed lower aerial tissues dry matter than cultivar SCS higher than those of the landraces Palha Roxa and Pérola. 154-Fortuna and landraces Palha Roxa and Pérola. Landrace Azteca Concerning total chlorophyll (a + b) contents, differences were also had one of the lowest values for root dry matter, not differing only reported for the effects of genotype (F = 5.02; df = 5, 34; from that of landrace 14 Variedades, which showed the lowest mean P = 0.0015). No significant effect of infestation were found for total of root dry matter and differed from all other genotypes. However, chlorophyll contents (F = 0.21; df = 1, 34; P = 0.6484) or the G × no differences were observed in number of leaves among maize I interaction (F = 1.23; df = 5, 34; P = 0.3169) (Table 4). The highest genotypes (F = 1.94; df = 5, 52; P = 0.1037). total chlorophyll content was observed in leaves of landrace Azteca,

Table 3. Height, number of leaves, aerial tissues and roots dry matter (mean ± standard error) of control plants (uninfested) of maize geno- types for plant vigor evaluation

Dry matter (g)

Genotype Height Leaves Aerial tissues Roots

Assay 1 (n = 58) (n = 58) (n = 58) (n = 58) Azteca 58.4 ± 2.5a 6.0 ± 0.3ab 2.1 ± 0.1b 1.1 ± 0.1b Encantilado 64.1 ± 3.8a 5.4 ± 0.1b 2.6 ± 0.2ab 1.7 ± 0.3a SCS 154-Fortuna 70.2 ± 4.0a 6.5 ± 0.2a 3.4 ± 0.3a 2.3 ± 0.3a Palha Roxa 65.0 ± 2.7a 6.2 ± 0.1ab 3.0 ± 0.3ab 1.9 ± 0.1a Pérola 63.4 ± 3.0a 5.7 ± 0.2ab 2.4 ± 0.2ab 1.6 ± 0.2a 14 Variedades 69.7 ± 3.9a 6.1 ± 0.2ab 3.3 ± 0.3a 1.9 ± 0.2a Assay 2 (n = 57) (n = 57) (n = 57) (n = 57) Azteca 76.2 ± 5.8b 6.5 ± 0.2a 2.8 ± 0.1b 1.6 ± 0.1ab Encantilado 82.7 ± 8.4b 6.5 ± 0.4a 3.2 ± 0.2ab 1.8 ± 0.2a SCS 154-Fortuna 75.9 ± 4.1b 5.9 ± 0.1a 4.1 ± 0.3a 1.9 ± 0.1a Palha Roxa 84.4 ± 3.7b 6.6 ± 0.2a 4.1 ± 0.4a 1.9 ± 0.3a Pérola 103.5 ± 1.9a 7.0 ± 0.2a 4.2 ± 0.2a 1.8 ± 0.0a 14 Variedades 81.8 ± 2.6b 6.5 ± 0.2a 3.6 ± 0.2ab 1.2 ± 0.0b

Means followed by the same letter in columns are not significantly different by Tukey’s or Duncan’s tests (root dry matter in Assay 2), α( = 0.05).

Table 4. Leaf contents (mean ± SE) of chlorophyll and carotenoids (carotene + xanthophyll) expressed as µg/cm2 in maize genotypes

Chlorophyll a Chlorophyll b Total Chlorophyll (a + b) Carotenoidsa

Genotype (n = 46) (n = 46) (n = 46) (n = 46)

Azteca 8.1 ± 0.4a 3.2 ± 0.1a 11.3 ± 0.6a 7.5 ± 0.3a Encantilado 6.1 ± 0.7ab 2.4 ± 0.3ab 8.5 ± 1.1ab 5.5 ± 0.7ab SCS 154-Fortuna 5.6 ± 1.0ab 2.2 ± 0.4ab 7.9 ± 1.4ab 5.2 ± 0.9ab Palha Roxa 4.4 ± 0.4b 1.7 ± 0.1b 6.2 ± 0.6b 4.1 ± 0.4b Pérola 4.4 ± 0.3b 1.7 ± 0.1b 6.1 ± 0.4b 4.0 ± 0.2b 14 Variedades 4.9 ± 0.4b 2.0 ± 0.1b 6.9 ± 0.6b 4.4 ± 0.4b

Means followed by the same letter in columns are not significantly different by Tukey’s test α( = 0.05). aCarotene + xanthophyll. Journal of Economic Entomology, 2021, Vol. 114, No. 1 383 which differed from that of the landraces Palha Roxa, Pérola, and 14 It is important to note that differences in larval feeding on roots Variedades. The content of total chlorophyll in the landrace Azteca of the maize landraces were expected because of the different levels was 1.84-fold higher than the content in landrace Pérola. of antibiosis they possess (Costa et al. 2018). Antibiosis is a resist- Significant differences were found for carotenoid contents among ance category whose effects are commonly difficult to distinguish genotypes (F = 5.35; df = 5, 34; P = 0.0010), whereas no differences from those of antixenosis (Smith 2005, Stout 2013). Therefore, it were observed for the effects of infestation (F = 0.26; df = 1, 34; is possible that roots of the susceptible genotypes were more pre- P = 0.6147) or the G × I interaction (F = 1.19; df = 5, 34; P = 0.3366) ferred by larvae than roots of the resistant genotypes, or the latter (Table 4). Higher carotenoid contents were observed in leaves of genotypes may have affected rootworm biology, leading to reduced landrace Azteca than in the landraces Palha Roxa, Pérola, and 14 root injury. However, landrace Azteca was not negatively affected Variedades. Carotenoid content in Azteca was 1.83-fold higher than probably due to root recovery. Compensatory growth was observed that observed in Pérola. in roots of the landraces Azteca and Encantilado, with new tissues Contents of glycine betaine were not affected by genotype (F = 1.87; emerging from rootworm-injured areas (data not shown). df = 5, 32; P = 0.1270), infestation (F = 1.73; df = 1, 32; P = 0.1984), or Maize tolerance to the western corn rootworm Diabrotica by their interaction (F = 1.39; df = 5, 34; P = 0.2538) (data not shown). virgifera virgifera LeConte (Coleoptera: Chrysomelidae), the major Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 Glycine betaine contents ranged from 2.08 to 3.38 µg g-1 dry matter rootworm maize pest in the United States, is a result of greater root among maize genotypes. Also, contents of proline did not differ among volume compared with those of susceptible genotypes (Painter 1968, maize genotypes (F = 1.32; df = 5, 24; P = 0.2875), larval infestation Smith 2005). However, this was not observed in our study when (F = 0.00; df = 1, 24; P = 0.9534), or the G × I interaction (F = 1.94; evaluating tolerance in landrace Azteca to D. speciosa; our assess- df = 5, 24; P = 0.1251) (data not shown). Proline contents varied from ments of plant vigor showed this tolerant landrace maize exhibited 0.04 to 0.14 µmol/g fresh matter. lower root dry matter compared with that of the other genotypes, es- pecially in the first assay (Table 2). Furthermore, the susceptible cul- tivar SCS 154-Fortuna displayed one of the greatest root dry matter. Therefore, root size or volume was not associated with tolerance in Discussion the maize landraces to D. speciosa larvae in the current study. Tolerance seems to be the least-investigated type of plant de- Price (1991) stated that plant vigor can provide tolerance to fense. A survey of more than 200 reports on host plant resistance herbivory in several plant species. In accordance with this, Allsop to arthropod pests in vegetable crops indicated that tolerance was and Cox (2002) studied sugarcane tolerance to the root-feeding pest involved in as little as 10% cases, whereas the remaining 90% of Lepidiota stigma (F.) (Coleoptera: Scarabaeidae), and observed that studies were related to antixenosis and antibiosis (Stoner 1992). clonal variation in tolerance correlated with increased plant vigor. Tolerance has been documented to occur in at least 13 crop species However, we did not observe this trend in our research, since tol- (Eigenbrode and Clement 1999), and its lower reported frequency erant landrace Azteca showed low aerial tissues dry matter in both in studies relative to the other two categories of resistance may re- assays, suggesting weaker plant vigor when compared to the other flect low attention paid to it (Schoonhoven et al. 2005, Peterson studied landraces (Table 2). Conversely, the susceptible cultivar SCS et al. 2013, Sperotto et al. 2018). In this context, we selected maize 154-Fortuna had greater aerial tissues dry matter in both assays, in landraces varying in antibiosis levels against D. speciosa (Costa addition to showing greater numbers of leaves in the first assay, sug- et al. 2018), and evaluated the tolerance levels to this important gesting increased plant vigor. According to Gonçalves-Alvim et al. maize pest. (2001), vigor is defined as any plant or plant module that grows Overall, landrace Azteca was the most tolerant to D. speciosa rapidly and reaches a larger size in relation to the mean growth rate. larval injury, and it is noteworthy to mention that this landrace was With regards to maize leaf pigments, leaves of landrace Azteca the most susceptible in a previous screening for antibiosis (Costa displayed higher contents of chlorophyll a, chlorophyll b, total et al. 2018). In this antibiosis study, insects reared on landrace chlorophylls (a + b), and carotenoids (Table 3). There was a signifi- Azteca displayed shorter -to-adult period, greater survival, cant correlation (negative) between high contents of photosynthetic higher number of viable eggs, and higher percent viable eggs. Results pigments and low percent reductions in plants dry matter for land- obtained in Assay 1 herein showed that this maize landrace had the race Azteca. Chlorophylls are closely involved in all major aspects lowest percent reduction in root dry matter, although no significant of photosynthesis: capture and transfer of light, and conversion of differences were found for percentage reductions in dry matter of energy. Therefore, the final plant biomass production largely de- aerial tissues, height, and numbers of leaves (Table 1). In Assay 2, pends on the efficiency with which leaves convert radiant energy into landrace Azteca stood out for possessing the lowest percent decrease chemical energy through photosynthesis (Assis and Mendes 1989). in dry matter of aerial tissues, despite showing percent decrease in Low efficiency of solar energy conversion may be due to chloro- root dry matter similar to that of other landraces (Table 1). In add- phyll antenna size (Melis 2009). This author suggests that generation ition, there was no significant correlation between percent decrease of truncated light-harvesting chlorophyll antenna size (tla) strains in dry matter of aerial tissues and percent decrease in root dry matter in plants and other photosynthesizing organisms may help mitigate for landrace Azteca. On the other hand, cultivar SCS 154-Fortuna excess sunlight absorption and the resultant wasteful dissipation of showed a positive significant correlation between these variables in excitation energy, maximizing solar-to-product energy conversion the second assay, indicating reduction in dry matter of aerial tissues efficiency and photosynthetic yield in high-density mass cultiva- resulted from rootworm feeding on the roots (Fig. 1). Differences in tion. Therefore, a plausible explanation for the paradoxical rela- results across assays may be associated with occurrence of longer tionship shown by landrace Azteca, with low plant dry matter and sunlight period in the second assay, favoring plant growth and re- high chlorophyll content, may be related to chlorophyll antenna size. covery from insect herbivory. In summary, landrace Azteca differed Thus, higher contents of chlorophylls and carotenoids did not render in both assays from the cultivar SCS 154-Fortuna, which was the landrace Azteca more productive when growing in the absence of most susceptible, suggesting that growth of landrace Azteca was not larvae, but possibly these greater contents assisted the plants in re- negatively affected by larval attack. covering from larval injury. This may have occurred mainly due to 384 Journal of Economic Entomology, 2021, Vol. 114, No. 1 greater content of carotenoids, which also serve roles as antioxidants in insect growth rates, fertility, fecundity or longevity, and increased and hormone precursors, e.g., abscisic acid and anti-stress hormones insect mortality rates (Schoonhoven et al. 2005, Smith 2005). (Howitt and Pogson, 2006), which can help plants to withstand in- Therefore, it is possible to consider that landrace Azteca represents sect herbivory. Of course there are other compounds that may have a classic example of tolerance due to the observed lower reductions influenced tolerance in the landraces. Maize plant growth/develop- in plant growth after larval feeding and the absence of detrimental ment processes need several enzymes that might differ by plant geno- effects on the growth and survival of infesting D. speciosa larvae. types, causing differences in plant dry matter (Causse et al. 1995). On the other hand, the genotypes Palha Roxa, Pérola, and SCS Different factors can contribute to maize antibiosis (or 154-Fortuna were selected in that antibiosis test for showing resist- antixenosis) and tolerance to Diabrotica species. Xie et al. (1990) ance. Nevertheless, in the current research we observed that these studied the role of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one genotypes were not tolerant to D. speciosa larval feeding, showing (DIMBOA) in maize resistance to D. v. virgifera by comparing a line higher percentage reductions in plant aerial tissues dry matter than with high DIMBOA content in roots (ITR 3872) with a line with low the other genotypes in the second assay. Thus, we suggest that mech- DIMBOA content (NTR-2 Ger. 4042), and observed that the high anisms involved in maize landrace tolerance to D. speciosa are likely

DIMBOA line (but not the low DIMBOA line) adversely affected not associated with mechanisms that are related to the expression of Downloaded from https://academic.oup.com/jee/article/114/1/377/6063497 by guest on 28 September 2021 the number of emerged adults, adult weight, and adult head-capsule either antibiosis or antixenosis. width. In addition, ITR 3872 (high DIMBOA line) displayed signifi- Results of this work also revealed that the quaternary ammo- cantly less damage than NTR-2 Ger. 4042 (low DIMBOA line) con- nium glycine betaine and the amino acid proline are likely not sidering most plant parameters evaluated, such as plant height, stem associated with maize tolerance to D. speciosa larvae. This was con- thickness, plant fresh weight, root fresh weight, plant dry weight, cluded because there was no accumulation of these compounds after and root dry weight. Costa et al. (2018) mentioned some morpho- insect injury, which would otherwise characterize them as compat- logical barriers that may be associated with maize resistance to ible osmolytes participating in osmotic adjustment. In the literature, D. speciosa, e.g., lignin, fibers, and cellulose. there are references addressing the role of glycine betaine and proline Commonly these secondary compounds possess toxic and/or in tolerance of maize plants under different abiotic stresses. Glycine antinutritional effects on insects, and are not directly associated with betaine has been reported to assist plants in tolerating cold (Farooq plant tolerance. Conversely, tolerance to Diabrotica larval herbivory et al. 2008), drought, and salinity (Chen and Murata 2008). For seems to be associated with traits such as rapid plant regrowth and proline, there are reports on its function in mitigating the effects of phytohormones. Bohn et al. (2018) affirmed that tolerant geno- water stress (Raymond and Smirnoff 2002), promoting heat toler- types generally possess large root systems and superior root re- ance (Li et al. 2013), benefiting plant yield (Špoljarević et al. 2011), growth after root damage by D. v. virgifera. Qu et al. (2016) showed and playing a role in regulation of protein synthesis (Wang et al. that D. v. virgifera feeding induced specific patterns of lateral root 2014). Therefore, most of the studies about the effects of proline or growth, which are related with shift in auxin biosynthesis from glycine betaine in plant tolerance focused abiotic stress. indole-3-pyruvic acid to indole-3-acetonitrile. However, in some The few studies currently available on the effects of these cases it seems that the same compound is related with both antibiosis compounds in insect herbivory show that their accumulation (or antixenosis) and tolerance. For example, phytohormones associ- is beneficial to herbivores. Zuñiga and Corcuera (1987) stated ated with plant growth are thought to participate in plant defense, that glycine betaine application to detached shoots of barley in- such as auxin, gibberillic acid, and cytokinin (Bari and Jones 2009, creased population growth of Schizaphis graminum (Rondani) Dervinis et al. 2010). (Hemiptera: Aphididae), and therefore, glycine betaine accumula- Based on the results found in our study, in which larval feeding ex- tion in water-stressed barley plants may benefit insect herbivores. erted no influence on chlorophyll or carotenoid content, we inferred Haglund (1980) reported that grasshoppers detect and preferen- that there was no increase in the concentrations of these photosyn- tially feed on grasses treated with the amino acids proline and thesizing pigments in response to rootworm attack. In other words, valine, and this may contribute to acridid population outbreaks the higher chlorophyll and carotenoid content in leaves of the land- on drought-stressed plants. race Azteca were constitutive and did not increase after D. speciosa In conclusion, our investigation suggests that tolerance expressed larval attack. This observation is in accordance with Tiffin (2000), by landrace maize Azteca is associated with higher leaf contents who stated that photosynthetic activity is not induced as a response of chlorophylls and carotenoids, and these do not seem to be en- to the feeding of all and does not always lead to com- hanced upon rootworm herbivory, but are constitutively present. pensatory growth, probably because the plants need to use their re- Thus, knowledge acquired from this research can serve as an im- sources for the production of defensive compounds. However, this is portant tool in plant breeding programs aimed at the development not the rule for all insects and plants species. For some plant species, of maize cultivars and hybrids with tolerance traits to D. speciosa herbivory has resulted in increased photosynthetic rates (Strauss and larvae. However, repeated field tests are still needed to obtain more Agrawal 1999, Retuerto et al. 2004). Information on compensatory realistic estimates of plant yield after natural rootworm infestation. growth in maize as a response to herbivory is scarce in the literature. This study also revealed that glycine betaine and proline are com- Riedell and Reese (1999) found that maize root systems injured by pounds that cannot be considered reliable proxies for phenotyping D. v. virgifera larvae had accelerated adventitious root axis growth maize plant tolerance to D. speciosa larvae. It is also important to and development in the nodes situated immediately above damaged note that when selecting genotypes for evaluation of resistance, a nodes, especially under moderate root feeding injury. genotype not possessing antixenosis and/or antibiosis may express In a previous antibiosis test conducted by our research group tolerance to a same insect species. Therefore, to prevent underesti- (Costa et al. 2018), Azteca was the most susceptible landrace maize mation of the usefulness of maize genotypes in integrated pest man- to D. speciosa among 19 genotypes (17 landraces and 2 cultivars) agement, tolerance should also be evaluated when investigating host screened. Effects of antibiosis-resistance are manifested by reductions plant resistance. Journal of Economic Entomology, 2021, Vol. 114, No. 1 385

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