Rpp-Gene pyramiding confers higher resistance level to Asian soybean rust

Asian soybean rust (ASR) causes large reductions in soybean yield, affecting the entire grain market. With low fungicide efficiency, the use of resistant cultivars can be an economical, safe, efficient, and sustainable control alternative. However, the great variability and aggressiveness of ASR and the use of Rpp genes are limited. Thus, gene pyramiding is a promising strategy for the development of cultivars with high resistance to a greater number of isolates. Thus, the objective of this study was to evaluate sister lines, previously evaluated by Meira et al. (2022). https://doi.org/10.1007/s10681-020-02667-x), presenting different Rpp-pyramided genes for resistance to Phakopsora pachyrhizi to clarify the pyramiding effect of two originally developed Rpp-pyramided lines compared to two existing lines or lines possessing only a single Rpp of resistance under field conditions. Rpp-pyramided lines 52117-1 (Rpp2 + Rpp1-b), 52117-57 (Rpp2 + Rpp1-b), 52117-59 (Rpp2 + Rpp1-b) + 52117-60 (Rpp2 + Rpp4) showed high resistance levels compared to resistant sources and resistance control, carrying a single Rpp gene PI 200487 (Rpp5), PI 200492 (Rpp1), PI 230970 (Rpp2), PI 459025A (R pp4), and PI 506764 (Rpp3 + Rpp5) with significant reductions in sporulation levels (SL), number of uredinia per lesion (NoU), and frequency of lesions with uredinia (%LU). Only, the line 52117-54 (Rpp2 + Rpp1-b), and 52117-63 (Rpp2 + Rpp4) showed resistance level smaller than PI 594723 (Rpp1-b) and similar resistance levels than PI 230970 (Rpp2), respectively. Rpp-pyramided lines carrying Rpp2 + Rpp1-b (52116-54, 52116-74, 52117-21, 52117-59 and 52117-60), and Rpp2 + Rpp4 (52117-60), and single gene Rpp1-b were classified as “highly resistant” and “resistant”. Furthermore, one sister line, 52117-57 (Rpp2 + Rpp1-b), showed immunity under field conditions. The Rpp-pyramided genes are an alternative for achieving high resistance levels against ASR.


Introduction
Year after year, soybean farmers face numerous adversities caused by biotic and abiotic stresses, with the potential to damage the crop. Plant diseases are Abstract Asian soybean rust (ASR) causes large reductions in soybean yield, affecting the entire grain market. With low fungicide efficiency, the use of resistant cultivars can be an economical, safe, efficient, and sustainable control alternative. However, the great variability and aggressiveness of ASR and the use of Rpp genes are limited. Thus, gene pyramiding is a promising strategy for the development of cultivars with high resistance to a greater number of isolates. Thus, the objective of this study was to evaluate sister lines, previously evaluated by Meira et al. (2022). https:// doi. org/ 10. 1007/ s10681-020-02667-x), presenting different Rpp-pyramided genes for resistance to Phakopsora pachyrhizi to clarify the pyramiding effect of two originally developed Rpp-pyramided lines compared to two existing lines or lines possessing only a single Rpp of resistance under field conditions. Rpp-pyramided lines 52117-1 responsible for huge crop losses and are a threat to global food security and agricultural sustainability. Asian soybean rust (ASR) is one of the most economically important diseases of crops, especially in tropical and subtropical countries, where reductions in grain yield can reach up to 80% (Godoy et al. 2016). In addition, ASR directly impacts the soy market, as it leads to a drop in productivity (grain) and consequently of its derivatives (oil, bran, protein) (Ishiwata and Furuya 2020).
ASR is caused by the fungus Phakopsora pachyrhizi (Sydow and Sydow), an obligatory biotrophic basidiomycete, which has multiple infection cycles in the same crop, with a high capacity to produce uredospores and to easily disseminate (Chander et al. 2019). In addition, it has a high intraspecific variability of isolates and wide geographic distribution and is extremely severe and difficult to control (Darben et al. 2020). These characteristics drive the efforts of scientists, research agencies, and institutions from different countries, who work in a continuous search for management strategies to control the disease (Meira et al. 2020).
Currently, fungicide use is the most commonly used method to manage ASR (Murithi et al. 2021). However, fungicide costs are extremely high ($2.2 billion per harvest), and their ineffectiveness has increased with pathogen insensitivity every cropping season (Godoy et al. 2016). Thus, the use of resistant cultivars may be a promising strategy, being more economical, safe, efficient, and sustainable (Godoy et al. 2016;Ishiwata and Furuya 2020;Murithi et al. 2021).
Genetic resistance can be characterized as the ability of a plant to prevent and/or delay pathogen entry and development in its tissues. This mechanism can occur through resistance directly or indirectly controlled by genes, which can detect the presence of pathogens and initiate a signal cascade, resulting in resistance mechanism activation (Nelson et al. 2018;Zaidi et al. 2018). Resistance can be considered qualitative, when governed by a gene with a greater effect (major genes), or quantitative when governed by several genes with less effect (Nelson et al. 2018). Seven major ASR resistance genes have been reported in soybean, known as Rpp1 to Rpp7 (Resistance to P. pachyrhizi) (Bromfield and Melching 1982;Childs et al. 2018;Garcia et al. 2008;Hartwig 1986;Hyten et al. 2007;Li et al. 2012;Yu et al. 2015). These genes interact with pathogen avirulence genes, resulting in different resistance reactions, ranging from reddishbrown lesions (RB), with little or no sporulation, to immunity (absence of lesions) according to isolate severity (Godoy et al. 2016;Langenbach et al. 2016).
In Brazil, genetic resistance to ASR has been used in soybean breeding programs through the release of resistant cultivars. Resistant cultivars have a technology named according to the breeding company such as Inox ® of TMG (Tropical Melhoramento e Genética), Shield ® of Embrapa (Empresa Brasileira de Pesquisa Agropecuária) (Aoyagi et al. 2020), and TF of GDM (Grupo Don Mario) Genética do Brasil S.A. It is known that there is huge variability in P. pachyrhizi isolates with different degrees of severity and aggressiveness, which can increase the chances of breaking down the rust resistance gene (Darben et al. 2020;Yamanaka et al. 2015). Thus, a broad, effective, and long-lasting range of resistance can be developed through gene pyramiding (Mundt 2018).
Gene pyramiding combines multiple resistance genes in a single genotype (Chander et al. 2019;Mundt 2018). Several studies have been successfully used to improve disease resistance, mainly ASR (Lemos et al. 2011;Parhe et al. 2017;Vigano et al. 2018;Yamanaka et al. 2013;Yamanaka et al. 2015;Yamanaka and Hossain 2019). However, there is little information about the effectiveness of Rpp gene combinations and the pyramiding effect in controlling ASR (Yamanaka and Hossain 2019). Furthermore, the genetic bases are individual of each plant, and the interaction of Rpp-pyramided lines with the environment can result in different resistance phenotypes. Thus, to understand the pyramiding effect, it is necessary to develop and evaluate sister lines, mainly under field conditions (Yamanaka et al. 2015).
Thus, the objective of this study was to evaluate sister lines, previously evaluated by Meira et al. (2022), presenting different Rpp-pyramided genes for resistance to P. pachyrhizi and to clarify the pyramiding effect of two originally developed Rpp-pyramided lines compared to two existing lines or lines possessing only a single Rpp of resistance under field conditions.

Plant material
Seven sister lines from three populations, carrying two Rpp-pyramided genes, four resistance sources (PI 594723-Rpp1-b, PI 594538A-Rpp1-b, PI 230970-Rpp2, PI 459025A-Rpp4) and four resistance control (PI 200487-Rpp5, PI 200492-Rpp1, PI 506764-Rpp3 + Rpp5, and PI 587880A-Rpp1b) (Plant introduction: PI), and five resistant and six susceptible commercial cultivars were evaluated in this study (Table 1; Fig. 1). The Rpp-pyramided lines were developed from double crosses between F 1 plants, obtained from susceptible Brazilian commercial cultivars (63I64RSF IPRO and 55I57RSF IPRO) and four different ASR resistance sources (PI 594723-Rpp1-b, PI 594538A-Rpp1-b, PI 230970-Rpp2, PI 459025A-Rpp4) (Table 1) obtained from previous studies by Meira et al. (2022). These lines were selected through marker-assisted selection in the F 2 and F 3 generations to confirm the presence of two Rpp genes. Information on molecular markers of all strains used in the present study is available at Meira et al. (2022), and they can see Supplemental Table 1. The F 4 generation of the lines was evaluated under field conditions. These Rpp-pyramided lines were developed by the breeding company GDM Genética do Brasil S.A.

52117-21
Rpp2  (Alvares et al. 2013). Four weeks before sowing, the border area was sowed to increase pathogen occurrence. Sowing was realized on a non-preferential date (December 1st 2020) to enable the natural occurrence and development of ASR, and no fungicide was used to control the disease. The field experiments were performed using a randomized block design with three replications. Each plot was composed of two 3-m rows spaced 0.5 m apart, totaling 3 m 2 , with a seed density of 14 seeds m −1 . Fertilizer management and pest control were performed in accordance with the technical recommendations for soybean crops, and weed control was performed manually.

Resistance evaluation
Ten leaflets from the middle third of the soybean plants in each plot were collected at the R5 growth stage (Ferh and Caviness 1977). Leaflets were analyzed in the laboratory to determine the number of lesions (NL), sporulation level (SL), frequency of lesions with uredinia (%LU), and number of uredinia per lesion (NoU) in 1 cm 2 of leaf tissue. These evaluations were performed using a binocular stereo microscope with a magnification of 4 × and 10 × objective lens, resulting in a magnification used of 14 ×. According to the data obtained from each plot, the classification criteria to determine the resistance of ASR were by Yamanaka et al. 2020 (Tables 2 and 3).
Collected data of NL, %LU, NoU, and SL were submitted to analysis of variance, and when a significant effect to genotype factor was detected using test F (p < 0.01), the mean was grouped using Skott Knott test (p < 0.05). Data analysis was performed using ExpDes.pt package (Ferreira et al. 2014

Results
The variance analysis showed significant effects on all evaluated resistance characteristics: number of lesions (NL), frequency of lesions per uredinia (%LU), number of uredinia per lesion (NoU), and sporulation level (SL) ( Table 4). The heritability ranged from 0.91 to 0.97, showing the highest genetic effects.
The border sowed a few times before the Rpppyramided lines, resistance sources, and commercial cultivars contributed to the presence of pathogen inoculum in the area. The presence of ASR in the experimental area was confirmed by susceptible lesions with abundant sporulation (TAN) in the susceptible commercial cultivars (NK6201, NS6700, 95R51, 95Y72, BMX Zeus IPRO, and BMX Raio IPRO) (Fig. 1, Table 5). The resistance characteristics to ASR on Rpp-pyramided lines, resistance sources, resistance control, and resistant and susceptible commercial cultivars are presented in Fig. 1      showed higher values of SL = 0.63 compared to its sisters.
The resistant source carrying single gene Rpp2 and resistance control carrying single gene Rpp5, and the Rpp-pyramided population carrying Rpp2 + Rpp4 were classified as slightly resistant (SR) to ASR. Furthermore, the susceptible commercial cultivars (carrying no Rpp genes) and resistance control carrying Rpp4, Rpp3 + Rpp5, and Rpp1 showed susceptible (S) phenotypic classification under field conditions.

Discussion
Pyramidation consists of the combination of several genes in the same genotype, resulting in their simultaneous expression in the host (Chander et al. 2019). This provides broader, longer lasting, and higherlevel resistance because of the effects of multiple genes against Phakopsora pachyrhizi (Yamanaka et al. 2015;Yamanaka and Hossain 2019;Chander et al. 2019).
The lines evaluated in the present study were developed and validated by molecular markers by Meira et al. (2022). In their study, Meira et al. (2022) identified lines with different resistance reactions (IM, RB1, RB2, RB3, and TAN), and only populations of the best combinations of Rpp-pyramided genes (showing IM and RB1 resistance reactions), and with enough seed to perform field trials, with replicates, were selected to be further evaluated in this study. Sporulation levels, number of uredia per lesion, frequency of lesions with uredia, number of lesions, in addition to photographs of lesions of each line, resistance source and resistance control (PI), and susceptible and resistant cultivars were performed in the present study. Subsequently, these were classified according to Yamanaka et al. (2020). Therefore, more generous information on combinations of Rpp-pyramided genes and resistance sources and resistance control is described in the present study.
The Number of uredinia per lesion (NoU) and frequency of lesions with uredinia (%LU) are reliable parameters for determining resistance to ASR, classifying resistant and susceptible genotypes (Kashiwa et al. 2020). In our studies, Rpp-pyramided lines showed less %LU and NoU than those of their parents and resistance control. Similar results have been reported in several studies, with different gene pyramiding combinations (Lemos et al. 2011;Yamanaka et al. 2013Yamanaka et al. , 2015Yamanaka and Hossain 2019). In addition, the lines 52117-21, 52117-57, 52116-74 and 52117-60 showed no sporulation (Fig. 1, Table 5). These results corroborate those of Yamanaka et al. (2013) and Yamanaka and Hossain (2019), who observed the absence of uredinia formation and sporulation in lines with pyramided Rpp genes.
Gene pyramiding directly affects the formation of the pathogen's reproductive structures, preventing and/or reducing uredinia formation and sporulation. Uredinias are responsible for the release of spores, which spread the fungus (Kashiwa et al. 2020). With the reduction of these structures, damage to the leaf area is minimized, maintaining a larger photosynthetically active area, and improving light interception, generating a greater accumulation of photoassimilates, resulting in higher yields (Godoy et al. 2016).
Among the Rpp-pyramided combinations, lines carrying Rpp2 + Rpp1-b showed immunity and high resistance phenotypic reactions (IM, HR, and R respectively), and lines carrying Rpp2 + Rpp4 presented HR and SR levels (Fig. 2). The level of resistance of the genotype is influenced by the number of genes and the combination of genes in the plant (Nelson et al. 2018). Within the same combination, with the same genetic basis, it was possible to observe differences between the levels of resistance of the sister lines (showed in Rpp2 + Rpp1-b and Rpp2 + Rpp4, for example). Small allelic differences in resistance genes, as well as different interactions between Rpp-pyramided genes, unknown genetic factors (in addition to Rpp genes), and the interaction by Rpp-genes with plant genetic basis, besides environmental effects can influence resistance level (Yamanaka et al. 2015;Nelson et al. 2018;Kashiwa et al. 2020). All these factors may be contributing to different phenotypes in genotypes with the same genetic basis. Therefore, within the same combination of Rpp-pyramided genes, using the same parents, it is possible to observe differences in the phenotypic reactions to ASR as is between lines 52117-60 and 52117-63.
Among the resistance sources and resistance control evaluated in this study, the PI carrying Rpp1-b (PI 594538A, and PI 594723), and resistance control PI 587880A, and the resistant commercial cultivar BRS531 showed higher resistance levels (HR and R classification) (Fig. 1, Table 5). Genotypes carrying Rpp1-b have been reported to show high levels of resistance, especially against South American P. pachyrhizi isolates (Akamatsu et al. 2017;Hossain et al. 2015;Yamanaka et al. 2016) and African isolates (Murithi et al. 2021), corroborating the results obtained in this study. Thus, using the Rpp1-b gene as a resistance source can be a promising strategy for breeding programs with higher levels of resistance, particularly against highly aggressive isolates of ASR in South America (Hossain et al. 2015). However, it is worth noting that no isolated strategy can maintain the sustainability of the culture (Chander et al. 2019).
Thus, the genetic resistance promoted by simple or pyramided genes needs to be used strategically, maintaining an integrated long-term management to increase its durability and efficiency (Chander et al. 2019). The use of fungicides, for example, associated with the use of resistant cultivars, can help reduce inoculum, as well as reduce selection pressure applied by P. pachyrhizi on resistance genes (Godoy et al. 2016). Likewise, the use of resistant cultivars helps to reduce the selection pressure on fungicides by reducing the number of applications during the crop cycle. Kato et al. (2022), for example, evaluated two soybean cultivars carrying three pyramided genes for resistance to ASR under field conditions, with and without fungicide application. The authors observed higher levels of resistance of cultivars with pyramided genes compared to susceptible parents, regardless of fungicide application. In other words, the association of the two control methods further increases the field resistance against ASR. In this way, using different management methods in an integrated way, in addition to increasing resistance levels, can help prevent fungicide resistance, in addition, increase the durability of genetic resistance.
In conclusion, Rpp-pyramided lines showed higher resistance levels to ASR, with significant reductions in SL, NoU, and %LU. The line 52117-57 carrying Rpp2 + Rpp1-b showed phenotypic reaction of immunity under field conditions, and all evaluated Rpp-pyramided lines were classified as HR and R. Only the line 52117-63 showed resistance level SR, close to susceptibility. Furthermore, the different phenotypic reactions to ASR observed in sister lines highlighted the difference between genetic bases and phenotypic reactions.