CULTIVARS WITH NATIVE INSECT RESISTANCE – POTENTIAL, ADVANCES AND CHALLENGES

– The development of resistant cultivars is one of the strategies applied in pest control. The method has the advantages of reduced cost and the lack of unwanted effects on the environment. Over the past decades, significant effort has been made toward developing the natural maize resistance to pests by evaluating germplasm and cultivar selection. This review highlights a maize breeding program, potential, advances, and challenges in addressing these characteristics. Also, it describes the main components and procedures applied in the mass rearing of insect pests of maize, artificial diets, techniques of artificial infestation employed in genotype selection, and methods to evaluate the mechanisms and causes of resistance. Studies on the inheritance of resistance, the breeding methods, and the potential for integrating classical and transgenic resistance are also emphasized.


2
In Brazil, the technology used in maize crop production systems has experienced expressive changes in recent years. This dynamic has modified the incidence of pests in the crop by using cultivars with different resistance levels and adopting new crop practices, such as the no-tillage system in the second crop in irrigated areas, with at least two crop seasons per year. Among the insect pests of maize crops, the fall armyworm Spodoptera frugiperda is of the most significant economic importance, causing losses of up to 34% (Carvalho, 1970). According to , the armyworm can cause grain yield losses from 17.7% to 55.6%, depending on the hybrid, the developmental stage of the plant, and the growing season. The lesser cornstalk borer, Elasmopalpus lignosellus, is another relevant pest that can destroy the crop Mendes, 2020).
Furthermore, the sugarcane borer, Diatraea saccharalis, and the cotton bollworm, Helicoverpa armigera, have assumed primary pest status in some maize production regions. The damage caused by the sugarcane borer feeding on corn stalk hinders the transport of photoassimilates and predisposes the plant to stalk breakage and lodging (Cruz, 2007;Mendes et al., 2014). Finally, H. armigera is a species recently identified in Brazil that has caused considerable losses in the production system. This polyphagous species feeds on the reproductive structures of plants and has caused damage to soybean, cotton, and maize crops (Ávila et al., 2013).
In addition, many other insect pests such as the corn rootworms, Diabrotica sp., the corn earworm, Helicoverpa zea, the corn leafhopper, Dalbulus maidis, the green-belly stink bug, Dichelops melancanthus, can also cause significant losses to the maize crop, depending on the region. Furthermore, although it is considered a secondary pest, the corn aphid, Rhopalosiphum maidis can also damage the crop, and its effects depend on the population (Racliffe, 2001).
The development of resistant cultivars is a method for pest control that has the advantages of The resistance of plants to insects is defined as the relative sum of hereditary qualities of the plant that affect the resulting degree of damaging that the insect causes (Painter, 1951). A plant resistance program aims to develop cultivars resistant to insects and maintain or enhance agronomic traits. The role of resistance in plants in a breeding program varies according to the crop and the pest species (Ortman and Peters, 1980).

Main components of a maize program for insects resistance
The success of a maize program for insects resistance requires a extensive knowledge of target plants and insects. Thus, pest biology and population, infestation, rearing, pest damage evaluation methods, plant germplasm, resistance, and inheritance mechanisms must be known. This level of knowledge requires a cooperative and interactive multidisciplinary team composed of entomologists, breeders, biochemists, statisticians, and other scientists considered essential for the program's success.
Information concerning the biology, habits, distribution, and control measures for maize pests is available in various publications , Viana and Mendes, 2020. Although various insects attack the crop, only some are considered economically important or primary pests. The main species that attack the initial stage of the plant are the lesser cornstalk borer, the fall armyworm, the corn rootworm, and the green-belly stink bug. During the vegetative and reproductive phases, injuries caused by caterpillars and sucking insects attacking the leaves, stalks, and ears predominate. Depending on the region, the infestation of the sugarcane borer has increased in recent years, leading to risks of losses for maize growers (Cruz, 2007).
Advances for identifying both natural resistance and the resistance of genetically modified maize depend on the ability to distinguish the most resistant genotypes during selection. For that reason, it is necessary to have a laboratory infrastructure for rearing insects that allows them to be used in the infestation of plants and an effective method for evaluating plant resistance related to pest damage. In addition, it is necessary to establish uniform levels of infestation that must be used at the appropriate phenological stage of the plant, allowing selection of resistant genotypes, reducing or eliminating the chances of escape, and allowing the accumulation of genetic resistance (Ortega et al., 1980). In the following, we discuss the main requirements for developing a program of plant resistance to insects.

Artificial diets and procedures used in mass rearing
One of the most critical components necessary to identify or develop maize germplasm with resistance to insects is efficiently rear the pest species in the laboratory, aiming at their use in artificial infestation (Davis, 1989;Mihm, 1989a). For the primary caterpillars that attack maize, artificial diets have been created consisting of various ingredients that can be prepared in the laboratory and allow the production of many insects for research studies using artificial infestations. For the fall armyworm and the corn earworm, the diet used in the laboratory of plant resistance to insects of Embrapa Milho e Sorgo was proposed by Burton (1967), as it is easily prepared and provides high viability. The diet modified by Chalfant (1975) and the rearing method adapted by Viana (1999) is used for rearing the lesser cornstalk borer. The same methodology and diet as in CIMMYT (MIHM, 1989a) can be used for the sugarcane borer.
For raising Diabrotica spp., maize seedlings feed the larvae and common bean plants are used to feed the adults (Ávila et al., 2000).
For any insect to be reared in the laboratory, it is essential to emphasize that each species has a specific requirement concerning the procedure used in raising it. For example, some species have cannibalism as larval development proceeds, and thus the feed requirements and the substrate for oviposition of the adults differ, affecting egg production. In addition, the requirements of temperature and photoperiod, sanitary control to avoid the appearance of fungi, bacteria, viruses, and other particular aspects vary for each insect. Therefore, each species' biology and needs must be well known to produce many insects by the rearing methods used. For example, protocols on the methods used for rearing the main pests of the maize crop are described by Burton and Perkins (1989) and Mihm (1989a).

Procedures used in artificial infestation
In addition to efficient mass rearing, the program for insects resistance also requires methodologies allowing infestation and rapid evaluation. Thus the plant selection is conducted in a greenhouse and under field conditions. It should be emphasized that damage larvae, on average, are used per plant (Mihm, 1989b).
However, in trials in a greenhouse, where there is nearly no effect of the biotic and abiotic factors, a smaller number of larvae should be used, around ten to perform the infestation in the field. In that case, it is recommended that the caterpillars be kept in the laboratory in a coffee cup containing the artificial diet for five days. Then, the diet and the caterpillars are poured on the soil beside the plant stalk (Viana, 1999).
Resistance trials carried out with the corn rootworm generally use many eggs in the infestation. Generally

Methods for evaluating resistance
Like the method used in artificial infestation, evaluation of resistance must be easy to use and allow For the fall armyworm, a scale from 0 to 9 is used to evaluate the leaf injury caused by the caterpillar, where a score of 0 means no damage on the leaves and score 9, extensive lesions, consumed (dilacerated) parts on most of the leaves, and dead plants (Williams et al., 1983). Evaluation should be made around 14 days after artificial infestation. That time is sufficient for the larval phase to end, considering that temperatures are generally higher during the maize crop seasons.
For the sugarcane borer, the procedure initially used to evaluate resistance was through the opening of stalks and measuring the extension of the galleries.
However, that method was considered laborious and consumed much time, mainly when many genotypes were evaluated. After that, research results showed there to be a high and significant correlation between the extension of the gallery caused by the borer with the number of galleries, number of internodes bored into and leaf damage caused by the caterpillar before penetrating the plant stalk (Hinderliter, 1983).
Therefore, the procedure most used in selecting genotypes with resistance to the borer is a visual scale of leaf damage. One of the most used scales is proposed by Mihm (1989b), ranging from 1 to 9, with a score of 1 representing no damage or a few small perforations in the leaves and a score of 9 representing most of the leaves with elongated lesions. The evaluation should be performed around 14 days after artificial infestation.
To evaluate the damage from the corn earworm, a revised scale from Widstrom (1967), cited by Mihm (1989b), is used. The evaluation is performed three to four weeks after infestation by removing the husk from the ear and using a scale for determining the damages in both grains and stylesstigmas. The score 0 means no injury on the ear; score 1, damage only to the styles-stigmas; score 2, damage of up to 1 cm to the ear; and score three or more, increase the value by 1 for each centimeter of damage on the ear.
The initial studies for the selection of maize with resistance to the larvae of Diabrotica used different evaluation methods. These methods included root size, the ability to regenerate secondary roots after the attack, lodging, resistance to uprooting, and the ability of the plant to survive the attack of the larvae (Ortega et al., 1980;Branson and Sutter, 1989). Currently, evaluation of the damage caused by the larvae on the roots is the most used method: collecting roots at around 55 days after the maize sowing, then washing the roots, and evaluating the larvae severity-attack through a visual scale. The scale from 1 to 6 proposed by Hills and Peters (1971) and cited by Branson and Sutter (1989) is one of the most used. Score 1 represents no damage or only some signs of feeding on the roots, and score 6, three or more root nodules are destroyed. Another easy-to-use scale to measure the degree of root-damages ranges from 0 to 3 (Oleson et al., 2005) has also been used. the artificial infestation and be performed three times a week to prevent the attack from being confused with other pests (Viana, 1999;Viana and Mendes, 2020).
The resistance of maize to the corn leafhopper, D. maidis, has been indirectly evaluated through maize bushy stunt and determined based on the percentage of plants with symptoms of the disease. The severity is determined, and scores are attributed from 1 to 6, referring to the mean level of the symptoms on the plants, where 1: absence of symptoms; 2: plants with at least 25% of the leaves with symptoms, that is, reddish or yellowish leaves, or exhibiting chlorotic streaks at their base; 3: plants with 25% to 50% of the leaves with symptoms; 4: plants with 50% to 75% of the leaves with symptoms; 5: plants with more than 75% of the leaves with symptoms; and 6: plants with early death caused by maize bushy stunt (Silva et al., 2003).

Resistance mechanisms
Although knowledge of mechanisms, inheritance, and resistance causes is not limited to developing a breeding program aiming at insect resistance, when these parameters are clarified, they are handy for progress in the insects resistance program, contributing to the choice of the breeding method adoption, duration projections, determination of the resistance effectiveness, and assistance in the planning of new lines of activity to be followed in solving future problems (Smith et al., 1989).
The resistance mechanisms described are antibiosis, non-preference or antixenosis, and tolerance. The term "non-preference" expresses a behavioral reaction concerning the plant, whereas the other two mechanisms define a reaction of the plant to the insect (Lara, 1991).
Antibiosis indicates that when the insect feeds on the plant, it experiences an adverse effect on its biology. This effect can be manifested directly or indirectly, resulting in mortality in the young phases and prevent the transformation to the adult phase, reduction in size and weight, reduction in fertility, and change in the proportion of the sexes and the life cycle.
The non-preference mechanism is characterized when the insect uses the plant less than another under similar conditions, for feeding, oviposition, and shelter.
The tolerance is the resistance mechanism that Antibiosis was also found in a hybrid coming from lines with this mechanism (Guimarães et al., 2004). However, the hybrid developed had the highest larval mortality (32%) and the lowest mean values for weight of the larvae at 11 days of age, corresponding to 41% of that shown by Z. Chico and 21% of that observed for the hybrid BR 201, the susceptible check cultivar.

Causes of resistance
Even when resistance may be present in a genotype, its causes are not always known and are There are still many aspects to be investigated related to the causes of resistance in maize, especially in the level of identification of substances involved in the phytochemical mechanisms of resistance as an instrument for entomologists, breeders, and biotechnologists in the search for new cultivars with resistance to pests (Reesse, 1989;Bergvinson et al., 1997;Arnason et al., 1997;Snook et al., 1997;Warnock et al., 2001;Prates, 2002). Studies performed by Niemeyer (1988) showed that maize lines and varieties had exhibited phytochemical properties that limit the damage brought about by insects. The hydroxamic and phenolic acids of natural origin have proven to reduce reproductive potential and, consequently, in the damage brought about by phytophagous insects (Philogène and Arnason, 1995). Hydroxamic acids are present in maize roots (Xie et al., 1991) and leaves, constituting up to 10% of the total dry weight of the plant. The concentration varies according to the line, varieties, altitude, and longitude (Philogène and Arnason, 1995). Two compounds, DIMBOA ((2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one) and MBOA (6-methoxy-2-benzoxazolinone) are active against other relevant maize pests in other countries, such as Ostrinia nubilalis (Guthrie et al., 1986;Barry et al., 1994), Diatraea grandiosella (Hedin et al., 1984), and Diabrotica virgifera (Niemayer, 1988;Bjostad and Hibbard, 1992). The maysin flavonoid (luteolin 6-rhamnosyl-4-ketofucoside), isolated from the styles-stigmas, is reported as having activity against Helicoverpa zea (Snook et al., 1989). Lopez et al. (2007) reported that the herbivory of Lepidoptera in the resistant Mp708 maize genotype resulted in a rapid accumulation of the defense cysteine protease enzyme (Mir1-CP) in the vascular tissues.
The chlorogenic acid described in the literature as a natural metabolite with feeding deterrence activity was identified in some leaf extracts of maize with resistance to S. frugiperda (Machado et al., 2014).

Inheritance of resistance and breeding methods
The gene action conditioning resistance for most of the insect pests of maize appears to be additive, indicating that procedures such as mass selection and various recurrent selections effectively accumulate the genes desirable for this trait (Ortega et al., 1980). According to Santiago et al. (2008), recurrent selection can be determinant in changing

Integration of classic and transgenic resistance
Up to the advent of genetic engineering, prospecting sources of resistance to insect pests was carried out using only the plant species diversity. With transgenics, it can be affirmed that all the ecosystem's biodiversity is available for prospecting (Waquil et al., 2019).
The bacterium Bacillus thuringiensis (Berliner) (Bt) has been used as a bioinsecticide for decades (Feitelson et al., 1992) and is registered, without limitation for use, for control of various pest species of Lepidoptera. Various Bacillus species were found, and within these species, many populations and hundreds of isolates from the most diverse regions are now registered in the literature. The active fractions produced by Bt, which are the accumulated proteins in crystal form within the cells, can constitute more than 30% of the total proteins of the cell (Hermstadt et al., 1986).  Villela et al., 2002). Incorporating Bt genes in elite public lines are considered strategic for developing resistant cultivars, also bringing the possibility of developing more resistant hybrids by combining parental lines with classic and transgenic resistance (Willians and ). The strategy adopted by CIMMYT, through the IRMA project, is transfering the resistance of genetically modified maize, based on Bt, to existing populations with multigene resistance to pests, aiming at increasing the level of the resistanace durability. (Mugo et al., 2001).

Advances, potentialities, and challenges in genetic resistance of maize to insect pests
Improving maize cultivars with durable multiple resistance to insects and diseases is considered of prime importance (Miedaner and Juroszek, 2021) and constitutes a significant challenge (Kim et al., 2021).
Broadly, breeding for resistance has been conducted to develop hundreds of resistant cultivars, increasing the yield and stability in production, associated with economic savings and good production standards, minimizing the damage caused by pests. In order to improve plant resistance to insects, it is essential to identify sources of genes conferring resistance. As sources of variability, the primary gene pool is the first choice of the breeder, as that may not only improve the crop agronomically but also confer resistance to insects. Transferring resistance from a secondary gene pool to the desired genotype is frequently timeconsuming and laborious (Sandhu and Kang, 2017).
Various sources of resistance to the attack of pests have been identified for the maize crop. Genotypes with the trait called "bitter" have been reported as the most promising for resistance to S. frugiperda (Bertels, 1956). Materials of the "Antigua" group are also reported as sources of resistance to this pest (Wiseman, 1985;Wiseman and Davis, 1990 (Wiseman, 1985). Boiça Jr. et al. Viana and Gama (1988), Viana and Potenza (1992), Viana and Guimarães (1997), and Costa et al. (2007) found resistance to S. frugiperda in tropical maize. In the United States, various maize cultivars have been registered and released for public use that has resistance to H. zea, S. frugiperda, and D. grandiosella (Wiseman and Davis, 1990). For E. lignosellus, few studies have been performed. Viana and Gama (1991) showed that this pest least attacked the variety Zapalote Chico and the CMS 15 population. New populations were selected as sources of resistance to the lesser cornstalk borer (Viana and Guimarães, 1997). Studies performed with 15 lines derived from backcrossing were evaluated for resistance to the main maize caterpillar pests (Abel et al., 2000).
The lines selected with S. frugiperda resistance The leaf damage caused by S. frugiperda in these lines and the susceptible check Ab24E were 4.6, 5.5, and 7.9, respectively (Willians and . The USDA-ARS program, Georgia, USA, uses a recurrent selection of S1 progenies in two populations, and in one of them, the mass selection was also applied, which proved to be ineffective. CIMMYT, Mexico, in a multiple resistance program, improved the MBR (multiple borer resistance) composites through two primary lines: recurrent selection of full-sib progenies evaluated in international trials; and obtaining and evaluating "per se" lines for the formation of synthetics as new sources of lines, and in crossestest for determination of heterotic groups and hybrid formation (Smith et al., 1989). Dekalb-Pfizer selected lines from elite germplasm introgressed with the resistant lines were synthesized by the USDA-ARS, Mississippi (Overman, 1989). Kumar and Kumar (2002)  and MIRT populations (Guimarães and Viana, 1994;Viana and Guimarães, 1994 (Widstrom et al., 2003). For E. lignosellus, it was shown that the variety Zapalote Chico and the CMS 15 population were less attacked by this pest (Viana and Gama, 1991). After that, new populations were selected as sources of resistance to the lesser cornstalk borer (Viana and Guimarães, 1994). Currently, there are options for developing maize cultivars resistant to S. frugiperda through Bacillus thuringiensis (Bt) genes codifying insecticide proteins Vilella et al., 2002). In addition, there is the possibility of developing more resistant hybrids by combining parental lines with classic and transgenic resistance (Willians and Davis, 1999). This procedure may retard the breakdown of resistance of transgenic Bt maize to the pests, as later reported by Tabashnik et al. (2009) andStorer et al. (2009) . For H. armigera, which has a history of a rapid selection of resistance to chemical insecticides and Bt proteins in GM plants (Alvi et al., 2012;Kriticos et al., 2015), natural resistance would be a sustainable strategy for its management in the context of IPM (Integrated

Pest Management).
According to Stout et al. (2009) Tollefson (2007). According to the author, this source may be an alternative for regions with low or moderate populations of the pest and in the cases that genetically modified maize is not permitted or not preferred by the growers.
There is little information in the literature regarding resistance to the corn leafhopper and methods for evaluations of this resistance. Maize seedlings have been used to evaluate resistance to D. maidis (Silva et al., 2003). A collection of maize hybrids evaluated for resistance to the corn leafhopper showed a significant difference among the genotypes evaluated. A more extended period of development was found for the nymphs developed in the hybrid Pioneer 3027 (27.15 days) in contrast with those developed in the other five hybrids (mean of 24.82 days) (Zurita et al., 2000).
In recent years, outbreaks of infestation of maize by the aphid R. maidis have been recorded in Brazil (Pereira et al., 2006). Studies have shown that maize hybrids show differences in the degree of constitutive resistance to R. maidis. Field evaluations have shown that the hybrids P30F53H, STATUS VIP, BM9288, DAS2B587HX, DKB175PRO, AS1633PRO, and DKB390PRO2 had the lowest percentages of plants with aphids, indicating that these hybrids were resistant to R. maidis (Bôer, 2017).
According to Carena and Glogoza (2004), plant breeding aiming at the resistance of maize to the aphid R. maidis continues to be a challenge due to dependence on the natural infestation. Nevertheless, new methods of analysis and maintaining colonies and artificial infestation that are being developed may change this scenery, making both selection of resistant types and support for breeding programs viable to avoid commercialization of more susceptible cultivars.
In addition to these advances in research, the genetic resistance of maize to insect pests has enormous potential to improve resistant lines or varieties with desirable agronomic traits and high yield. The big challenge is to make these products effective and available for additional studies and evaluations, developing and recommending these materials for research for insects resistance and breeding programs of the local, national, and multinational seed companies, intending to provide the farmer with high-yielding cultivars with genetic resistance to the main pests without causing impacts on the environment.  CIMMYT, 1989. p. 37-45.