Tolerance in maize genotypes to the green-belly stink bug: methods of screening and biochemical responses in infested plants

ABSTRACT

Identifying resistance sources using fast, sensitive, and standardized methods is key to initiating a breeding program or even a germplasm characterization. To establish the infestation density, the coexistence period, and discriminatory parameters for determining maize tolerance-type resistance to the green-belly stink bug, Diceraeus melacanthus (Hemiptera: Pentatomidae), greenhouse, and field trials were conducted using four maize genotypes (SCS154 Fortuna, SCS155 Catarina, 2B512 PW, and 2B610 PW). In both greenhouse and field trials, plants infested with a density of two adult stink bugs per plant for a coexistence period of seven days was determined to be the optimal conditions for evaluating maize genotypes tolerance. In addition, plant height and fresh weight were significantly reduced in infested plants on greenhouse trial. Conversely, D. melacanthus reduced plant height at the V5 stage and weight of thousand seeds in field trials, but plant height up to tassel, height of cob insertion, and grain yield were not affected, indicating a recovery potential of the tested genotypes. No difference in damage score was verified in the genotypes in both greenhouse and field trials. Biochemical responses of plants infested and non-infested with D. melacanthus from field trial indicated that chlorophyll and carotenoid content were reduced due to stink bug infestation. Moreover, infested plants expressed higher amounts of peroxidase and polyphenoloxidase enzymes. In field, the reduction in phenotypic and productive parameters was similar among the evaluated genotypes, and in general, the tested genotypes exhibited similar response to D. melacanthus attack.

Key words
Zea mays L.; Diceraeus melacanthus ; host plant resistance; enzymatic analysis; photosynthetic pigments

INTRODUCTION

The green-belly stink bug, Diceraeus melacanthus (Dallas) (Hemiptera: Pentatomidae), is a key pest of maize crops in the initial phase of development (Bortolotto et al. 2016). This species is widely distributed in South America, with presence in 13 states of Brazil and in Argentina, Bolivia, Colombia, Peru, Paraguay, Uruguay, and Venezuela (Panizzi 2015). Its occurrence in maize fields has increased over the past two decades, owing to the substantial adoption of no-tillage cultivation systems, second growing season maize, the perennial presence of both cultivated and wild hosts, and the escalating cultivation of Bt maize, which has led to a substantial reduction in the application of insecticides for caterpillar management (Silva et al. 2013, Sosa‐Gómez et al. 2020).

Diceraeus melacanthus inserts its stylet into the stem of a maize plant for the purpose of feeding. During this process, it injects toxic saliva, which can cause severe injuries to the plant, including a reduction in growth, the development of unproductive tillers, and the development of a rolled whorl. These injuries can result in a reduction in grain yield and, in severe and early attacks, even the death of the plant (Rosa-Gomes et al. 2011, Fernandes et al. 2020). This insect typically remains beneath crop residues during the coolest hours of the morning, increasing its activity and feeding on maize stems in the afternoon and evening hours as temperatures rise (Castilhos et al. 2021).

The most common practices adopted by growers for D. melacanthus management are seed treatment with systemic insecticides (neonicotinoids) and the spraying of registered insecticides at maize post-emergence. However, chemical control of this insect pest is often ineffective and is not sufficient to prevent extensive economic losses in many cases (Silva et al. 2021). Recent studies with D. melacanthus in maize have focused on enhancing chemical control efficacy and reducing damage to plants, by combining insecticides with bioinoculants in seed treatment (Bortolotto et al. 2022) and by using pest behavior modulators in mixtures with foliar sprays of synthetic insecticides (Morais et al. 2024).

Chemical control is the primary measure adopted by maize growers. However, the management of D. melacanthus should be approached from an integrated perspective. Plant resistance constitutes a pivotal component of integrated pest management (IPM) programs, and one of the resistance types is tolerance (plant is less damaged than susceptible ones at the same level of infestation, without interference in insect’s biology or behavior) (Baldin et al. 2019). The utilization of maize genotypes capable of withstanding the attack of D. melacanthus emerges as a promising strategy to reduce yield losses caused by this species on maize crops. Nevertheless, there is a paucity of information concerning the response of genotypes to this pest, as well as methods for the screening of tolerant genotypes.

Consequently, the present study was conducted to ascertain the infestation density and coexistence period of D. melacanthus for the purpose of screening maize genotypes; evaluate the expression of resistance of tolerance type in some maize genotypes to D. melacanthus; and assess the relationship between resistance of tolerance type and the levels of photosynthetic pigments and enzymatic activities in both infested and non-infested maize plants. The results of this study are valuable not only for breeding programs aimed at evaluating tolerance-type resistance to the green-belly stink bug, but also for guiding the selection of maize genotypes to be cultivated within an IPM framework.

MATERIALS AND METHODS

Tested insects

Green-belly stink bugs (D. melacanthus) were obtained from a colony maintained in a climate-controlled room, with a temperature set at 25 ± 2°C, relative humidity at 60 ± 10%, and a 14-hour photophase. This colony has been propagated for a minimum of 10 generations under these conditions. Soybean [Glycine max (L.) Merrill, (Fabaceae)] grains, and plants served as feeding and oviposition substrates, respectively, as described by Ribeiro et al. (2018).

The nymphs were reared in plastic containers (40 × 22 × 14 cm) with lids, featuring a central cut covered with organdy fabric to allow for ventilation. In contrast, the adults were reared in wood cages (80 × 80 × 80 cm) covered with voile-type fabric. The replacement of food and the removal of eggs occurred every two days. The eggs were placed in Gerbox cages with a portion of moist cotton. After the hatch of the nymphs, they were placed in the rearing cages in accordance with the previously described protocol.

Tested genotypes

The maize genotypes evaluated are shown in Table 1. They were chosen based on the absence of antixenosis and antibiosis to D. melacanthus, as determined in a previous study (Bueno et al. 2021).

Table 1
Maize genotypes evaluated in the tolerance assays to Diceraeus melacanthus.

Genotype	Developer	Characteristics of resistance	Proteins involved in transgenics
2B512PW	Forseed®	Bt maize	Cry1F, Cry 1A.105, Cry2Ab2 (resistance to lepidopteran insects) and CP4 EPSPS, PAT (herbicide tolerance) (Corteva 2023)
2B610PW	Forseed®	Bt maize	Cry1F, Cry 1A.105, Cry2Ab2 (resistance to lepidopteran insects) and CP4 EPSPS, PAT (herbicide tolerance) (Corteva 2023)
SCS154 Fortuna	Epagri®	Non-Bt corn, commercial material with open pollination	-
SCS155 Catarina	Epagri®	Non-Bt corn, commercial material with open pollination	-

Greenhouse trials

Determination of infestation density and coexistence period

To determine the infestation density and coexistence period to be used in the screening of tolerant genotypes, a bioassay was conducted in greenhouse conditions. For this purpose, seeds of the genotype SCS 155 Catarina were sown in 0.5-L plastic pots filled with soil, sand, manure, and substrate, in a proportion of 1:1:1:1 (v/v/v/v). The genotype SCS155 Catarina was selected for this study because it had previously demonstrated susceptibility to D. melacanthus in preliminary tests (Bueno et al. 2021).

The densities/coexistence periods that were tested included the following: no infestation; one insect per plant/one week; one insect per plant/two weeks; two insects per plant/one week and two insects per plant/two weeks. The plants were infested at the V2 growth stage (two complete expanded leaves) (Ritchie and Hanway 1966), which occurred approximately seven days after plant emergence. Prior to the infestation, insects were subjected to a 24-hour starvation period. Adult stink bugs, 5 to 10 days old and originating from the laboratory colony, were used in the trial. To prevent insect escapes, the pots were covered with organdy fabric supported by four wooden sticks and closed at the bottom with an elastic band. For each density/period combination, 10 replicates were utilized, with each plant considered a repetition, arranged under a completely randomized design.

Thirty days after plant emergence, evaluations were conducted, and the following parameters were assessed: plant height (from the base of stem to the apex of the last expanded leaf), damage score, fresh weight, and dry weight. The damage score was assigned using an injury scale (Bianco 2005) from 0 to 4, where:

• 0 = plant without injuries;

• 1 = plant with few punctures and no reduction in size;

• 2 = plant with many punctures and slightly reduced size;

• 3 = plant with whorl partially damaged, some tillering, and moderate reduction on size;

• 4 = plant with high reduction on size, tillering, and the winding of whorl.

The dry weight was measured by drying the plants in a 60°C oven until the weight remained constant.

Selection of maize genotypes tolerant to Diceraeus melacanthus

A greenhouse experiment was conducted to assess the response of maize genotypes to D. melacanthus. Maize seeds were sown in 0.5-L plastic pots and seven days after emergence (V2 phenological stage), the plants from each genotype were infested with 5–10 days old adult D. melacanthus under conditions (density of stink bugs and period of coexistence) determined based on the results from previously bioassays. The greenhouse environmental conditions were partially controlled (temperature 25 ± 2°C and 72 ± 10% relative humidity), and the plants were irrigated as needed.

For each genotype, a total of six infested and six non-infested plants were utilized. Each plant was regarded as a replication that was arranged under a completely randomized design. The bioassay was examined daily, and any dead stink bugs were replaced, ensuring that there were always two stink bugs in all infested repetitions for each genotype.

Fifteen days after the removal of the insects (29 days after the emergence of the plants), the following parameters were evaluated: stem diameter between the soil and the first internode, plant height (from the stem base to the apex of the last expanded leaf), and damage score based on the Bianco (2005) scale. The above-ground fresh weight and dry weight of the plants were also measured. For the dry weight measurement, the plants were dried using an oven set at 60°C until their weight remained constant. The reductions in the aforementioned parameters induced by D. melacanthus feeding were obtained.

Field experiment

The field experiment was conducted in Chapecó, Santa Catarina state, Brazil (27°05’04”S; 52°38’12”W), during the 2017/2018 crop year, in the first (summer) growing season.

The experimental design involved the sowing of seeds on October 24, 2017, in plots measuring 20 m² (4-m width × 5-m length). The spacing between rows was kept at 0.8 m, and the average density of seeds sown per meter was 5. The experimental plots were allocated to four distinct genotypes, with each genotype assigned to an individual plot. This arrangement was implemented under a completely randomized design, ensuring that the experimental conditions were equally distributed across all plots and genotypes. The cultural treatments followed the technical recommendations for maize production in southern Brazil (Eicholz et al. 2024), except for insecticide spraying.

At the V2 stage (seven days after emergence), a pair of voile fabric cages with 1 m² (1-m length × 1-m width × 0.5-m height) were installed in each plot, with each cage covering five selected plants (1 row meter). One of the cages was infested with adults of D. melacanthus at a density of two stink bugs per plant, while the other cage was kept free of infestation. The stink bugs were confined with plants for seven days (7–14 days after emergence), a period determined according to previous results. After that period, the cages were removed, and the plants were identified with tags. To ensure the complete eradication of the infested stink bugs, a meticulous visual inspection of all plots was conducted immediately after cage removal, and the stink bugs were manually removed. Furthermore, a spray application of the insecticide thiamethoxam + lambda cyhalothrin (Engeo Pleno S; Syngenta Proteção de Cultivos LTDA., Holambra, SP, Brazil), at a dose of 250 mL.ha^-1, was utilized.

Seven days after the removal of the cages, when the plants were at the V5 stage (21 days after emergence), the height of the infested and non-infested plants was measured, and the injury score was attributed to the infested plants based on a visual scale of injuries (Bianco 2005). Furthermore, at full bloom, the height of cob insertion and the plant height from soil to tassel were measured. Finally, at physiological maturation, the grain yield per plant and the weight of one thousand seeds corrected at 13% humidity were measured, thus allowing the reduction in these parameters caused by D. melacanthus to be obtained.

Evaluation of biochemical parameters of infested and non-infested maize plants

After the removal of the cages in the field experiment, the final fully extended leaf of both infested and non-infested plants from each plot was harvested, flash frozen in liquid nitrogen, and transferred to the laboratory for maceration. Samples of macerated leaves were meticulously placed in plastic vials and transferred to the laboratory for biochemical quantification. The biochemical parameters evaluated included chlorophyll a, chlorophyll b, carotenoids, anthocyanins, peroxidase (POD), and polyphenol oxidase (PPO) content.

The extract used for POD and PPO enzymes consisted of 300 mg of fresh sample, macerated in liquid nitrogen, and homogenized with 8 mL of 0.2 M potassium phosphate buffer solution (pH 6.7). The samples were centrifuged (Hettich, 174 model Mikro 220R) (6,000 × g for 15 min at 5°C). The supernatant was removed and stored in a 175-mL amber glass. The activity of peroxidase enzyme (POD, EC 1.11.1.7) was determined according to the method described by Lima et al. (1999). The reaction consisted of 1 mL of the enzyme extract, 500 µL of H₂O₂ at 30% P.A. in 0.2 M potassium phosphate buffer (pH 6.7), and 500 µL of phenol 179 and aminoantipyrine. The tubes were subsequently placed in a water bath (30°C) for 5 min, and after this procedure, they were submerged in hot water at 85–90°C for 1 min to stop the reaction. Readings were performed at 505 nm absorbance, and POD activities were expressed in µmol H₂O₂ decomposed min^-1.g^-1.FW^-1. The polyphenol oxidase activity (PPO, EC 1.10.3.1) was analyzed according to the method by Kar and Mishra (1976) with modifications. For the reaction, 300 µL of the enzymatic extract and 1.85 mL of 0.1 M catechol were used. Subsequently, the tubes were placed in a water bath (30°C) for 30 min. The reaction was stopped in hot water (85–90°C). The absorbance was measured at 395 nm, and the results expressed in µmol catechol were transformed into min^-1.g^-1.FW^-1.

The photosynthetic pigment contents were determined using the method proposed by Sims and Gamon (2002). The samples were read in a spectrophotometer, for chlorophyll a, at 663 nm; for chlorophyll b, at 647 nm; for anthocyanins, at 537 nm; and for carotenoids, at 470 nm. All steps were performed in a dark environment.

Statistical analyses

All bioassays and field trials were conducted in a completely randomized design. A 4 × 2 factorial analysis of variance (ANOVA) was conducted to compare the main effects of genotype and infestation, as well as the interaction effect between them, on the variables of phenotype, productivity, and biochemistry.

For analysis, the Shapiro-Wilk’s test was initially employed to verify the normal distribution of residues of the obtained data, and the Bartlett’s test was used to verify the homogeneity of variance. When a significant effect was obtained, the averages were compared by means of the Tukey’s test (p < 0.05). The damage score was submitted to the F test (p < 0.05). All statistical analyses were performed using the software R, version 4.1.0 (R Core Team 2021).

RESULTS

Determination of infestation density and coexistence period of Diceraeus melacanthus

The results of a greenhouse trial revealed significant effects of infestation density and coexistence period of green-belly stink bug on the damage rating and parameters of development of potted maize plants (Table 2). The level of infestation of one stink bug per one week (7–14 days after emergence) resulted in plant damage, as measured by a damage rating scale, plant height, and fresh and dry weight, that was comparable to that observed in the control (not infested) (Table 2). Conversely, alternative conditions involving different stink bug population densities (one stink bug per two weeks, two stink bugs per one week, and two stink bugs per two weeks) resulted in substantial plant height and fresh weight reductions, as well as a heightened plant damage, as measured by the damage scale (Table 2). However, the impact on dry weight was observed to be influenced solely by the extended coexistence period and increased infestation density.

Table 2
Effects of different levels of infestation densities^{<tfn href="tfn02">1</tfn>} and coexistence periods^{<tfn href="tfn03">2</tfn>} of Diceraeus melacanthus adults on the damage rating and parameters of initial development of potted maize plants in a greenhouse trial.

Treatments	Damage score (scale 0–4)*	High (cm)*	Fresh weight (g)*	Dry weight (g)*
No infestation	0.00 ± 0.00 b	91.88 ± 2.38 a	49.92 ± 4.45 a	5.32 ± 0.40 a
1 insect/1 week	0.44 ± 0.18 ab	77.78 ± 3.09 ab	45.89 ± 6.91 abc	4.76 ± 0.49 ab
1 insect/2 weeks	1.33 ± 0.33 a	69.83 ± 3.73 b	37.30 ± 2.65 bc	4.17 ± 0.28 ab
2 insects/1 week	1.56 ± 0.47 a	61.27 ± 7.37 bc	29.19 ± 4.68 c	3.78 ± 0.66 ab
2 insects/2 weeks	1.33 ± 0.24 a	45.33 ± 5.59 c	25.27 ± 5.07 c	2.81 ± 0.66 b
F	30.444	13.309	4.5348	3.4745
df	4, 40	4, 40	4, 40	4, 40
p-value	0.0014	< 0.0001	0.0041	0.0158

• ^*
  Means followed by different letters in a column indicate a significant difference between the treatments by the Tukey’s test (p < 0.05);
• ¹
  infestation density of 0 (control), one, or two adult stink bugs per potted plant;
• ²
  coexistence period was considered after seven days of plant emergence, i.e., one week = seven to 14 days after plant emergence; two weeks = seven to 21 days after plant emergence.

In view of these findings and in consideration of the necessity for expeditious and sensitive screening methods to identify genotypes exhibiting tolerance to D. melacanthus, we determined that the subsequent trials would be conducted using a level of infestation of two stink bugs per plant and a coexistence period of one week (7–14 days after emergence). The objective of the subsequent trials was to identify tolerant genotypes.

Tolerance of maize genotypes to Diceraeus melacanthus: greenhouse trial

Regardless of the genotype, the maize infestation with a density of two stink bugs per plant and with a coexistence period of one week (7–14 days after emergence) resulted in a reduction of plant height, and fresh weight and caused a significantly higher damage score in the infested genotypes (Table 3). However, this level of infestation did not lead to a significant difference in stem diameter and dry weight between the infested and non-infested plants at the endpoint of the bioassay (Table 3). The interaction between maize genotype and infestation was not significant for any of these variables (Table 3).

Table 3
Effects of Diceraeus melacanthus infestation<tfn href="tfn04">1</tfn> on the damage rating and initial development of different maize genotypes in a greenhouse trial<tfn href="tfn06">*</tfn>.

Genotypes	Non-infested	Infested	Mean	R%
Diameter (cm)
SCS155 Catarina	1.38 ± 0.05	1.43 ± 0.06	1.41 a	-3.94 b
SCS154 Fortuna	1.33 ± 0.06	1.35 ± 0.08	1.34 ab	-1.88 ab
2B512PW	1.32 ± 0.03	1.28 ± 0.07	1.30 ab	2.51 ab
2B610PW	1.33 ± 0.05	1.18 ± 0.02	1.26 b	10.88 a
Means	1.34 A	1.31 A
p (Genotype)	0.0475
p (Infestation)	0.4198
p (Interaction genotype × infestation)	0.2862
High (cm)
SCS155 Catarina	82.00 ± 2.16	78.67 ± 3.37	80.33 ns	3.73 ns
SCS154 Fortuna	83.00 ± 1.34	78.33 ± 1.31	80.66	5,48
2B512PW	87.50 ± 1.23	79.00 ± 0.86	83.25	9.62
2B610PW	84.17 ± 1.76	77.83 ± 1.96	81.00	7.32
Means	84.17 A	78.46 B
p (Genotype)	0.4161
p (Infestation)	0.0001
p (Interaction genotype × infestation)	0.5623
Damage score (scale 0–4)
SCS155 Catarina	0.00 ± 0.00	0.67 ± 0.21	0.33 ns	-
SCS154 Fortuna	0.00 ± 0.00	1.00 ± 0.00	0.50	-
2B512PW	0.00 ± 0.00	1.00 ± 0.00	0.50	-
2B610PW	0.00 ± 0.00	0.50 ± 0.22	0.25	-
Means	0.00 B	0.79 A
p (Genotype)	0.0620
p (Infestation)	< 0.0001
p (Interaction genotype × infestation)	0.0620
Fresh weight (g)
SCS155 Catarina	33.60 ± 2.26	34.82 ± 1.13	34.21 ns	-5,64 b
SCS154 Fortuna	34.78 ± 1.28	33.94 ± 1.68	34.36	1,61 b
2B512PW	33.51 ± 1.89	28.34 ± 1.70	30.92	13.87 a
2B610PW	34.57 ± 1.09	29.71 ± 1.54	32.14	13.63 a
Means	34.11 A	31.70 B
p (Genotype)	0.1118
p (Infestation)	0.0411
p (Interaction genotype × infestation)	0.1507
Dry weight (g)
SCS155 Catarina	3.58 ± 0.22	4.03 ± 0.17	3.81 ns	-14.38 b
SCS154 Fortuna	3.39 ± 0.17	3.49 ± 0.31	3.44	-4.54 ab
2B512PW	3.78 ± 0.23	3.26 ± 0.25	3.52	12.14 a
2B610PW	3.74 ± 0.19	3.40 ± 0.29	3.57	8.06 a
Means	3.62 A	3.54 A
p (Genotype)	0.4423
p (Infestation)	0.6370
p (Interaction genotype × infestation)	0.1694

• ¹
  In this trial, it was used the level of infestation of two stink bugs per plant and the coexistence period of one week (7–14 days after emergence) based on previous results; R%: reduction (%) = 100 - (100 × I / N.I.), where: I = value obtained on infested plants, and N.I. = value obtained on non-infested plants;
• ^ns
  not significant difference;
• ^*
  means (± standard error) in the column of each assessed parameter followed by different lowercase letters or means in a row followed by different uppercase letters are significantly different (p ≤ 0.05; analysis of variance and Tukey’s test).

A comparison of the genotypes was conducted in an independent manner. The results indicated that SCS155 Catarina exhibited a larger steam diameter compared to 2B610PW, without significant differences being observed between the genotypes of SCS154 Fortuna and 2B512PW (Table 3). Furthermore, the evaluated genotypes did not demonstrate significant differences in terms of plant height, damage score, fresh weight, and dry weight (Table 3).

A consideration of the reduction rate for the evaluated parameters revealed that the reduction in stem diameter was lower in SCS155 Catarina in comparison to 2B610PW. The hybrids 2B512PW and 2B610PW exhibited a higher reduction in fresh weight and dry weight, while the reduction in plant height was similar for all genotypes (Table 3).

Tolerance of maize genotypes to Diceraeus melacanthus in field conditions

In field conditions, the damage score of D. melacanthus on the maize genotypes ranged from 1.68 to 2.04, and no significant difference was observed among them considering the same infestation density and coexistence period (Table 4). For all variables assessed, the interaction between maize genotype and infestation was not significant either (Table 5). The infestation of maize plants reduced plant height at V5 stage and weight of thousand seeds (Table 5). However, the level of infestation and the coexistence period did not alter the height of cob insertion, plant height, and grain yield at physiological maturation (Table 5).

Table 4
Damage score (± standard error) of Diceraeus melacanthus for different maize genotypes in a field trial.

Genotypes	Damage score
SCS155 Catarina	1.68 ± 0.21 ns
SCS154 Fortuna	2.00 ± 0.08
2B512PW	1.96 ± 0.26
2B610PW	2.04 ± 0.23

• ^ns
  not significant difference by F Test (F = 0.769, df = 3, 32; p = 0.5199)

Table 5
Effects of Diceraeus melacanthus infestation^{<tfn href="tfn08">1</tfn>} on parameters of initial development and yield components of different maize genotypes in a field trial.

Genotypes	Non-infested	Infested	Mean	R%
Plant height at V5 (cm)
SCS155 Catarina	55.4 ± 2.2	38.8 ± 2.8	47.08 ab	29.89 ns
SCS154 Fortuna	48.3 ± 2.1	33.7 ± 1.6	40.97 b	29.42
2B512PW	54.4 ± 3.1	38.0 ± 2.6	46.21 ab	29.61
2B610PW	58.8 ± 3.5	38.9 ± 1.8	48.85 a	33.48
Means	54.22 A	37.34 B
p (Genotype)	0.0199
p (Infestation)	< 0.0001
p (Interaction genotype × infestation)	0.7388
Cob insertion height (cm)
SCS155 Catarina	131.8 ± 5.0	132.3 ± 2.7	132.05 a	-1.03 ns
SCS154 Fortuna	120.5 ± 5.8	113.7 ± 6.0	117.10 b	5.65
2B512PW	113. ± 5.2	112.0 ± 3.1	112.54 b	0.27
2B610PW	123.7 ± 3.3	134.4 ± 3.7	129.04 a	-8.68
Means	122.27 A	123.09 A
p (Genotype)	0.0001
p (Infestation)	0.7788
p (Interaction genotype × infestation)	0.2219
Plant height (tassel)
SCS155 Catarina	243.0 ± 4.7	231.4 ± 3.6	237.19 a	4.56 ns
SCS154 Fortuna	220.6 ± 7.2	210.1 ± 6.7	215.32 b	4.76
2B512PW	213.9 ± 3.9	207.1 ± 4.8	210.48 b	3.11
2B610PW	221.0 ± 3.1	224.7 ± 5.6	222.88 b	-1.69
Means	224.62 A	218.31 A
p (Genotype)	< 0.0001
p (Infestation)	0.0627
p (Interaction genotype × infestation)	0.3502
Grain Yield (g/cob)
SCS155 Catarina	142.8 ± 13.7	141.2 ± 13.3	142.01 b	1.09 ns
SCS154 Fortuna	169 ± 25.5	144.1 ± 10.9	156.54 ab	7.25
2B512PW	162.5 ± 9.1	146.6 ± 17.6	154.53 ab	7.61
2B610PW	200.8 ± 12.9	179.8 ± 10.2	190.34 a	8.77
Means	168.78 A	152.93 A
p (Genotype)	0.0087
p (Infestation)	0.1104
p (Interaction genotype × infestation)	0.8406
Weight of thousand seeds (g)
SCS155 Catarina	370.8 ± 11,9	345.8 ± 22.2	358.28 a	6.02 ns
SCS154 Fortuna	356.0 ± 11.9	329.1 ± 23.6	342.54 a	6.67
2B512PW	312.9 ± 10.8	288.1 ± 8.0	300.52 b	7.58
2B610PW	302.8 ± 5.5	300.4 ± 5.3	301.61 b	0.77
Means	335.63 A	315.85 B
p (Genotype)	< 0.0001
p (Infestation)	0.0352
p (Interaction genotype × infestation)	0.7430

• ¹
  In this trial, it was used the level of infestation of two stinks bug per plant and the coexistence period of one week (7–14 days after emergence) based on a previous bioassay; R%: reduction (%) = 100 - (100 × I / N.I.), where: I = value obtained on infested plants and N.I. = value obtained on non-infested plants;
• ^ns
  ns: not significant difference;
• * means (± standard error) in the column of each assessed parameter followed by different lowercase letters or means in a row followed by different uppercase letters are significantly different (p ≤ 0.05; analysis of variance and Tukey’s test).

As indicated in Table 5, a statistically significant variation in the evaluated parameters was observed in the tested genotypes, but without significant interaction. At the V5 stage, the plant height of 2B610PW surpassed that of SCS154 Fortuna, but after tasseling, SCS155 Catarina exhibited a greater height in comparison to the other genotypes under consideration. Furthermore, SCS155 Catarina and 2B610PW exhibited a greater cob insertion height compared to SCS154 Fortuna and 2B512PW. The 2B610PW genotype demonstrated higher grain yield, while SCS155 Catarina exhibited lower yield. Intermediate yield was observed in SCS154 Fortuna and 2B512PW. As for weight of one thousand seeds, higher values were obtained in the open pollination genotypes SCS154 Fortuna and SCS155 Catarina in comparison to the Bt corn hybrids 2B610PW and 2B512PW (Table 5). The reduction in plant height at V5, cob insertion height, plant height at tasseling, grain yield, and weight of thousand seeds caused by D. melacanthus was similar in all evaluated genotypes (Table 5).

Biochemical parameters of infested and non-infested maize plants

The contents of chlorophyll a, chlorophyll b, and carotenoids were lower in infested plants in comparison to non-infested ones (Table 6). For these three photosynthetic pigments, the genotype 2B512PW showed higher expression than SCS155 Catarina, while SCS155 Fortuna and 2B610PW presented an intermediate level, with no significant difference from any genotype. However, the infestation and genotypes exhibited no significant effect on anthocyanin content (Table 6).

Table 6
Biochemical parameters of maize plants infested and non-infested with Diceraeus melacanthus adults<tfn href="tfn10">*</tfn>.

Genotypes	Non-infested	Infested	Mean	R%
Chlorophyll a (µg·g-1)
SCS155 Catarina	32.92 ± 3.13	22.61 ± 0.82	27.76 b	-3.94 b
SCS154 Fortuna	34.10 ± 0.47	28.06 ± 1.39	31.08 ab	-1.88 ab
2B512PW	37.13 ± 3.04	35.60 ± 3.22	36.36 a	2.51 ab
2B610PW	35.50 ± 1.60	26.38 ± 1.59	30.94 ab	10.88 a
Means	34.91 A	28.16 B
p (Genotype)	0.003859
p (Infestation)	0.0001
p (Interaction genotype × infestation)	0.1978
Chlorophyll b (µg·g-1)
SCS155 Catarina	12.72 ± 1.62	8.51 ± 0.45	10.62 b	3.73 ns
SCS154 Fortuna	14.06 ± 0.89	11.56 ± 0.72	12.81 ab	5,48
2B512PW	14.67 ± 1.70	14.45 ± 1.37	14.56 a	9.62
2B610PW	14.41 ± 2.12	9.46 ± 0.81	11.93 ab	7.32
Means	13.96 A	10.99 B
p (Genotype)	0.0399
p (Infestation)	0.0033
p (Interaction genotype × infestation)	0.3052
Anthocyanins (µg·g-1)
SCS155 Catarina	3.71 ± 0.57	3.86 ± 0.30	3.78 ns	-
SCS154 Fortuna	3.97 ± 0.22	3.18 ± 0.76	3.57	-
2B512PW	2.35 ± 0.30	3.05 ± 0.58	2.70	-
2B610PW	1.38 ± 0.38	3.27 ± 0.91	2.33	-
Means	3.34 A	2.85 A
p (Genotype)	0.0683
p (Infestation)	0.1619
p (Interaction genotype × infestation)	0.1831
Carotenoids (µg·g-1)
SCS155 Catarina	11.60 ± 0.55	7.86 ± 0.38	9,73 b	-5,64 b
SCS154 Fortuna	12.40 ± 0.41	9.31 ± 0.40	10.86 ab	1,61 b
2B512PW	12.35 ± 1.12	11.22 ± 0.88	11.78 a	13.87 a
2B610PW	12.51 ± 0.32	9.08 ± 0.89	10.79 ab	13.63 a
Means	12.22 A	9.37 B
p (Genotype)	0.0433
p (Infestation)	< 0.0001
p (Interaction genotype × infestation)	0.2359
Peroxidase (µmol H 2 O 2 min -1 g -1 FW -1 )
SCS155 Catarina	0.31 ± 0.04 Ab	0.34 ± 0.02 Ab		0.325
SCS154 Fortuna	0.41 ± 0.02 Aa	0.45 ± 0.03 Aa		0.43
2B512PW	0.27 ± 0.02 Bb	0.39 ± 0.02 Aab		0.33
2B610PW	0.25 ± 0.02 Bb	0.47 ± 0.01 Aa		0.36
Means	0.31	0.41
p (Genotype)	0.0003
p (Infestation)	< 0.0001
p (Interaction genotype × infestation)	0.0010
Polyphenol oxidase (µmol catecol min -1 g -1 FW -1 )
SCS155 Catarina	2,751.66 ± 78.46 Bb	3,081.28 ± 46.74 Aa		2,916.47
SCS154 Fortuna	3,046.70 ± 67.08 Aa	3,122.69 ± 100.00 Aa		3,084.69
2B512PW	2,438.39 ± 94.72 Bc	3,059.38 ± 57.41 Aa		2,748.88
2B610PW	2,155.09 ± 38.63 Bd	3,296.18 ± 50.18 Aa		2,725.63
Means	2,597.96	3,139.88
p (Genotype)	< 0.0001
p (Infestation)	< 0.0001
p (Interaction genotype × infestation)	<0.0001

• ^*
  Means (± standard error) in the column of each assessed parameter followed by different lowercase letters or means in a row followed by different uppercase letters are significantly different (p ≤ 0.05; analysis of variance and Tukey’s test).

The interaction between genotypes and infestation was found to be significant for POD and PPO enzymes (Table 6). POD content was found to be significantly higher in infested plants from the hybrids 2B512PW and 2B610PW, while no difference was observed for SCS155 Catarina and SCS155 Fortuna. Conversely, an increase in PPO expression was observed in infested plants from the genotypes SCS155 Catarina, 2B512PW, and 2B610PW (Table 6). In non-infested plants, POD and PPO levels were higher in SCS154 Fortuna, while no significant differences in PPO levels were detected among the genotypes in infested plants. Conversely, SCS155 Catarina exhibited the lowest levels of POD under infestation (Table 6).

DISCUSSION

The initial step in the successful implementation of a breeding program aimed at obtaining resistant maize lines or even the characterization of a wide germplasm is the identification of sources of resistance by means of fast, sensitive, and standardized methods. In this study, we verified that the height from infested plants significantly differed from non-infested ones in all genotypes evaluated in the greenhouse, and consequently, we highlight that it is an interesting parameter to be assessed in studies of this nature. Conventional and transgenic genotypes may respond differently to D. melacanthus attack, and conventional maize exhibits a greater reduction in height in comparison to transgenic ones (Crosariol Netto et al. 2015). However, this tendency was not verified in the greenhouse experiment in which no difference among genotypes was obtained.

In the greenhouse experiment, the fresh weight of infested plants was lower, but no difference was observed in the dry weight. This can be explained by the fact that, in general, plants have an allometric relationship between leaf dry weight and fresh weight (Huang et al. 2019). The reduction in fresh weight and dry weight was more pronounced in the hybrids 2B512PW and 2B610PW. The dry weight is regarded as a more suitable and reliable parameter for assessing plant performance (Huang et al. 2017), and it remained unaltered by the D. melacanthus infestation in the maize varieties SCS155 Catarina and SCS154 Fortuna, suggesting that the main initial impact of this stink bug on maize plants was predominantly a reduction in plant height. In contrast, Fernandes et al. (2020) observed a significant reduction in dry weight of maize plants infested with D. melacanthus under greenhouse conditions. However, in their study, the plants were subjected to a longer coexistence period (14 days).

In the field trial, plant height at the V5 stage was the most damaged parameter, with an overall reduction of approximately 30%. However, at full bloom, the height of cob insertion and the height of plants up to tassel did not differ significantly between infested and non-infested plants of all genotypes, indicating that maize plants attacked by D. melacanthus have a recovery capacity depending on the intensity of the damage. As previously documented by Bianco (2016), maize exhibits a recovery potential of 100 and 90% when subjected to damage scores 2 and 3, respectively, particularly in the absence of drought and supplemental nitrogen fertilization. Our findings are consistent with those reported by Bridi et al. (2016), who did not observe a reduction in plant height at the tasseling stage in maize plants infested with D. melacanthus either.

The plant yield from infested and non-infested plants did not differ significantly in the field trial. According to the findings of Bianco (2016), maize plants can tolerate damage scores 1 and 2 without significantly reducing its productivity, which is in line with the results of the present study. The average damage score observed for the genotypes approximated 2. Crosariol Netto et al. (2015) did not find significant discrepancies in attack symptoms between the different genotypes when evaluating the damage of D. melacanthus on five maize hybrids either. An average reduction of 21.07% in grain yield of several maize cultivars infested with D. melacanthus was observed by Cruz et al. (2016), a value higher than those obtained in this study, in which reduction in yield ranged from 1.09 to 8.77% in the tested genotypes. Additionally, Fernandes et al. (2020) reported a significant reduction in grain yield from the BRS 1055 maize cultivar when plants were infested at the V1 and V3 stages with D. melacanthus adults. However, the duration of infestation differed from that in our study, with plants being kept infested for 14 days, resulting in a damage score of approximately 3. This finding reinforces the necessity for effective monitoring and prompt management of this species, as prolonged coexistence periods can result in substantial damage and yield reduction.

Chlorophyll plays an important role in maize by capturing and transforming light energy into chemical energy for the conversion of inorganic matter to organic matter through photosynthesis (Wang et al. 2020). As for carotenoids, they act as accessory light-harvesting pigments, increasing the range of light absorption, and also play a very important role in photoprotection, preventing light from damaging crop foliage (Swapnil et al. 2021). As Zhang et al. (2022) have demonstrated, photosynthesis and plant production are significantly affected by biotic disturbances, such as insect feeding. This tendency was verified in our study, in which the synthesis of these pigments was significantly lower in infested maize plants.

POD and PPO enzymes play a pivotal role in plant defense against herbivores by catalyzing the oxidation of phenols, which results in the formation of quinones that bind to leaf proteins and inhibit protein digestion in insects. In addition to disrupting insects’ nutrition, these antioxidative enzymes can cause indirect toxicity to insects (War et al. 2012). Although the biotic effects (antibiosis) on D. melacanthus were not evaluated in the present study, the increased enzymatic defense by POD and PPO was more evident in the hybrids 2B512PW and 2B610 in comparison to the open pollination varieties, suggesting a possible decrease in insect fitness in these genotypes.

No significant variation in the reduction caused by D. melacanthus on the phenological and productive parameters evaluated was observed among genotypes. However, the results obtained regarding infestation density, infestation period, and the plant parameters, as well as biochemical responses of infested maize plants, are critically important for future studies aiming to evaluate tolerance-type resistance in maize genotypes to this insect. Therefore, for the selection of genotypes to be used in breeding and IPM programs, further studies involving a larger number of genotypes across diverse locations and growing seasons are necessary to obtain more precise information on maize resistance to this pest.

CONCLUSION

The density of two stink bugs per plant and a coexistence period of one week are appropriate conditions for the fast screen of tolerant maize genotypes. In addition, the plant height is the most affected phenological parameter by D. melacanthus attack, whereas biochemical effects encompassed chlorophyll and carotenoids reduction, and POD and PPO increase.

ACKNOWLEDGMENTS

The authors express their sincere gratitude to the field technician Luiz Dalcin and his team for their assistance in the establishment and management of corn cultivation; to laboratory technicians Carmen dos Santos, Michelle Silva, Neusa Maciel, and Zelinda Meneguzzi (all from Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina) for their support with insect rearing.

• How to cite:
  Castilhos, R. V., Bueno, N. M., Baldin, E. L. L. and Ribeiro, L. P. (2026). Tolerance in maize genotypes to the green-belly stink bug: methods of screening and biochemical responses in infested plants. Bragantia, 85, e20250138. https://doi.org/10.1590/1678-4499.20250138
• FUNDING
  Conselho Nacional de Desenvolvimento Científico e Tecnológico
  Grant No.: 310385/2022-9
• DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE TOOLS
  No artificial intelligence tools were used in the elaboration of this manuscript.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

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Edited by

• Section Editor:
  Luis Garrigós Leite https://orcid.org/0000-0001-7947-5698

Publication Dates

• Publication in this collection
  20 Mar 2026
• Date of issue
  2026

History

• Received
  24 Mar 2025
• Accepted
  05 Dec 2025