Introduction

Asian soybean rust (ASR) is the most devastating disease of soybean production worldwide. The disease is caused by the obligate biotrophic fungal pathogen Phakopsora pachyrhizi Sydow and Sydow1,2. Currently, the main strategy for control the disease is the use of fungicides3. Recently, however, pathogen resistance to fungicides has frequently been reported1,4. Genes conferring resistance against P. pachyrhizi (Rpps) have also been used to protect soybean from the disease, with eight Rpp loci being reported from soybean and introduced into soybean varieties to date5,6,7,8,9,10,11. For some of these Rpps, several alleles have reported11; for example, Rpp2 has been detected in PI 23097012,13 and Iyodaizu B14, and Rpp3 has been reported from FT2 (PI 628932)15, PI 46231212,16, and Hyuuga17,18. Breeding for ASR-resistant soybean by introducing Rpps is also emerging as a major strategy for disease control. Pyramiding of two or more Rpps into a single variety has produced plants showing strong resistance against the pathogen19,20, and in this regard, several soybean varieties have been used as a source of Rpps. These varieties are also used for inoculation tests to distinguish the host specificity of P. pachyrhizi isolates21,22. A set of ASR-resistant varieties are determined as “Rpp differentials” that possess a single Rpp gene. However, in some cases, differential varieties exhibit unclear susceptible/resistant reaction against the pathogens, which indicates that the genetic background of some of the differential varieties influences the susceptible/resistant phenotype19,23. Furthermore, plant characters such as growth rate and size of leaves differ among Rpp differentials, given that these varieties originate from different countries (Japan, China, India, Indonesia, and Brazil21,22).

In plant–pathogen interactions, a pathogen gene corresponds to a plant resistance gene, referred to as an “avirulence gene,” based on Flor’s gene-for-gene concept24,25. In the relationship between soybean and P. pachyrhizi, however, pathogen avirulence genes against Rpps have yet to be identified. Nevertheless, sequences of the P. pachyrhizi secretome26 and genome (https://mycocosm.jgi.doe.gov/Phapa1, accessed on January 28, 2020) have recently been released, and this will accelerate the quest to identify pathogenicity-related genes from P. pachyrhizi. With respect to gaining a more comprehensive understanding of the soybean–P. pachyrhizi interaction, differences in plant characters and genetic background among the differential varieties appear to be bottlenecks currently hampering research efforts. In this regard, it is desirable to generate genetically uniform materials to compare results of inoculation tests.

In this study, we sought to prepare genetically homogeneous plant materials harboring Rpps, for which we generated near-isogenic lines (NILs) of Rpps bred from a single genetic background. Given that these NILs retain an almost identical genome, the phenotypes of the different NILs only reflects the effect of the introduced Rpp gene. In the present study, we bred NILs for Rpp1, Rpp2, Rpp3, Rpp4, Rpp6, and Rpp1-b from the ASR-susceptible soybean variety BRS 184, which shows low valiant inoculation results compared with those from other Rpp donor varieties. These clear reactions are useful for assessing the diversity and host-specificity of pathogens based on inoculation tests. By using our leaf-culture inoculation method22 and these Rpp-NILs, we are able to ascertain a pathogen’s compatibility/incompatibility against each Rpp in only 2 weeks. Moreover, these NILs are potentially applicable for the study of gene-for-gene relationship to identify avirulence genes from P. pachyrhizi that correspond to Rpps.

Results and discussion

Development of Rpp-NILs

Nine Rpp-NILs (BC5F2 lines) carrying single Rpp genes of donor ASR-resistant varieties were successfully developed by backcrossing and SSR marker screening. The results of marker-screening from generation F1 to BC5F2 are shown in Table S1. In the BCnF1 from the backcross of BCn-1F1, 50% of the screened plants should carry the target Rpp in the heterozygous state, whereas in the BC5F2 generation, 25% of individuals should carry a homozygous target Rpp. In practice, we obtained frequencies of positive plants that were lower than these theoretical frequencies, owing crossing failure and recombination between markers of the target Rpp. In the most cases, however, an ideal number of positive plants were obtained (Table S1).

We observed that the Rpp-NILs retained uniform plant characters such as seed color and size, whereas the Rpp donor varieties are completely different from each other (Fig. S1). The Rpp-NILs shares more than 95% of the genomic region of BRS 184 by virtue of a single cross and five backcrosses. In order to compare the severity of infection with P. pachyrhizi isolates, these NILs and the Rpp donor varieties were subjected to an inoculation test.

Disease reactions of Rpp-NILs

Before determining the results of the inoculation test, we assessed germination of the spores of each P. pachyrhizi isolate by slide culture, and accordingly obtained the following germination percentages: 75.5% (BRP-2.5, isolate from Brazil), 86.2% (BRP-2.6, Brazil), 77.0% (E1-4-12, Japan), and 85.1% (MRP-13.18, Mexico). Among all the assessed P. pachyrhizi isolates, the highest level of spores developed on BRS 184 (Figs. 1, 2).

Figure 1
figure 1

Sporulation of Phakopsora pachyrhizi on Rpp-NILs, recurrent parent, and Rpp donor varieties. Photographs of segments of the abaxial side (spore-inoculated side) of the leaflets of Rpp-NILs (NIL), recurrent parent (BRS 184) and Rpp donor varieties (Donor) inoculated with the four P. pachyrhizi isolates BRP-2.5, BRP-2.6, E1-4-12, and MRP-13.18. Bars = 1 mm. The photographs were taken before removing spores from the leaves.

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Figure 2
figure 2

Sporulation levels on Rpp-NILs, recurrent parent, and Rpp donor varieties inoculated with Phakopsora pachyrhizi. Average sporulation levels (SL) of P. pachyrhizi-inoculated leaflets. The average SL values were calculated from up to 30 lesions on leaflets. An average SL value of less than 1.5 was taken to be indicative of a resistant reaction (below the dotted line). Plots on the graph show the SL of assessed lesions. Red, BRP-2.5 (Brazil); orange, BRP-2.6 (Brazil); blue, E1-4-12 (Japan); green, MRP-13.18 (Mexico). Bars indicate the standard deviation (SD).

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Sporulation on the Rpp-NILs and Rpp donor varieties is shown in Figs. 1 and 2. In most cases, Rpp-NILs showed a lower variant level of sporulation against each of four P. pachyrhizi isolates than the respective donor varieties (Figs. 1, 2). For example, we observed differences in sporulation level on the Rpp1 donor variety PI 200492 and the NIL B1-8-3-2. Moreover, we observed that the donor variety infected with the Brazilian isolate BRP-2.5 showed differences in the levels of sporulation. However, in the case of NIL, all of evaluated lesion revealed that BRP-2.5 showed the highest level of sporulation, thereby indicating that compatible/incompatible relationships between pathogen and plant were more clearly identified using the NIL than the donor as host. In contrast, we observed that the Japanese P. pachyrhizi isolate E1-4-12 failed to form lesions on either NIL or donor (Fig. 2), which we accordingly interpret as indicating an immune response (see Table S2 for details). In the case of the Mexican isolate MRP-13.18, however, we observed lesions on the NIL, whereas no lesions could be detected on the donor, which tends to indicate the donor parental variety show a more complex resistance reaction based not only on Rpp but also other genes contributed to resistance against P. pachyrhizi.

For Rpp2, Rpp3, and Rpp6, we bred two, three and one NILs respectively, and all of the NILs exhibited lower variant sporulation levels against pathogens than the differential varieties (Fig. 2). However, in response to inoculation with MRP-13.18, Rpp3-NILs showed different levels of subsequent sporulation. The results obtained for Rpp1-NIL indicate that this NIL shows a weaker resistance reaction against the pathogens than the donor variety, which can be attributed to the fact that the genetic background of this NIL was P. pachyrhizi susceptible, and that the NIL lacks the benefits conferred by other genes contributed to resistance against ASR. Among the three Rpp3 donors, Hyuuga is relatively resistant to Brazilian isolates and has been reported to carry another resistance gene18. The stronger resistance of Hyuuga than its NIL and the other two Rpp3 donors is probably conferred by this additional resistance gene. One possibility explaining this variant reaction is the viability of the pathogen. The reactions of NILs reflect an effect of the introduced Rpp gene only, and pathogens compatible with the introduced Rpp will show clear viability. A similar response was observed for the Rpp4-NIL, for which sporulation varied by lesion. In contrast, we detected no differences between the NIL (B1b-4-2) and the differential variety (PI 594767A).

The uredinium is an essential structure required for pathogen sporulation, and the number of uredinia (NoU) and frequency of lesions with uredinia (%LU) are considered reliable indices for determining the ASR susceptibility/resistance of soybeans. As shown in Fig. S2, we observed no pronounced differences between the NoU on Rpp-NILs and the donor parents. However, the average of NoU on Rpp-NILs indicated a slightly weaker (susceptible) reaction against the pathogen compared with that of the donors (e.g., Rpp6, Fig. S2). As these observations were based on SL phenotype, a possible reason of this trend is the susceptible genetic background of the NILs. This tendency was also observed with respect to frequency of lesions with uredinia (%LU, Fig. S3).

On the basis of the presence of lesions, the presence of spores within lesions, SL, NoU, and %LU (Table S2)22, we categorized reaction type of the four P. pachyrhizi isolates on Rpp-NILs into five degrees. As shown in Table 1, Brazilian isolates (BRP-2.5 and BRP-2.6) were more aggressive than the other two isolates.

Table 1 Summary of the results of inoculation tests for Rpp-NILs and BRS 184.
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The genetic background of the NILs is that of the soybean variety BRS 184, which originates from Brazil27,28. Given that BRS 184 retains small plant size at the V2–3 growth stage, the Rpp-NILs bred in this study can be maintained in growth chambers, and 3 weeks of growth after sowing is sufficient to the inoculation test. Furthermore, the leaf culture inoculation method used in this study is rapid and can be used to reliably distinguish the efficacy of Rpp genes against pathogens, with lesions appearing on leaves in less than 2 weeks after inoculation. In combination with Rpp-NILs, this inoculation method represents a powerful tool for research on ASR epidemiology. Moreover, the recent release of genomic sequences of P. pachyrhizi will yield fundamental information relating to pathogen and homogeneous plant resources such as the Rpp-NILs, and will thus provide a strong incentive for further studies on ASR.

In most cases, the Rpp-NILs showed clear susceptible/resistant reactions against the pathogens (Figs. 1, 2), whereas in some Rpp differentials, genetic background influences the phenotype related to ASR susceptibility/resistance19,23. Given that, apart from the target Rpps, Rpp-NILs theoretically share more than 95% similarity with respect to genetic background, these homogenous genetic resources are applicable for studies on gene-for-gene relationships between soybean Rpp genes and the avirulence genes of P. pachyrhizi.

Materials and methods

Development of Rpp-NILs

The ASR-resistant soybean varieties/lines: PI 200492 (alternative name Komata; carrying resistance gene, Rpp1), PI 594767A (Zhao Ping Hei Dou; Rpp1-b), PI 230970 (No. 3; Rpp2), Iyodaizu B (Rpp2), FT2 (Rpp3), PI 462312 (Ankur; Rpp3), Hyuuga (Rpp3), PI 459025 (Bing Nan; Rpp4), and PI 567102B (MARIF 2767; Rpp6) were used as donor parents of respective resistance genes to cross with an ASR-susceptible Brazilian variety, BRS 184 (Table S3). PI 200492, PI 594767A, PI230970, FT2, PI462312, PI459025, PI 567102B, and BRS 184 were provided by the Brazilian Agricultural Research Corporation (Embrapa) in Brazil. Iyodaizu B and Hyuuga were provided by the National Institute of Crop Science (NICS) in Japan. Six PI accessions and 2 Japanese varieties: Iyodaizu B and Hyuuga can be accessed via U.S. National Plant Germplasm System (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) and NARO Genebank project (https://www.gene.affrc.go.jp/index_en.php), respectively. The F1 plants thus obtained were once again crossed with BRS 184 (backcross) to generate BC1F1 plants. In order to achieve 95% identity between NILs and BRS 184 and among NILs, we performed a total of five backcrosses. In the process of recurrent backcrossing, BRS 184 was used as an ovule parent at least once to exclude genetic influences from cytoplasmic difference between donor parents and BRS 184. BC5F1 plants were then selfed to obtain BC5F2 plants (BCF2). BC5F3 and BC5F4 plants were developed by single-seed decent (SSD) from BC5F2, Therefore, we consider that all NILs are BC5F2 lines. A total of nine NILs (Table 1) were developed and BC5F2 (for the NILs B1b-4-2 and B6-5-2) or BC5F4 plants (for the other 7 NILs) were used for inoculation with pathogens and evaluation of their reactions.

F1 plants were assessed for hybridisms using one of the simple sequence repeat (SSR) markers polymorphic between the parents. The backcrossed progenies (BC1F1–BC5F1) were also screened using SSR markers (Table S3) to determine whether they carry the target resistance genes of their donor parents in the heterozygous state. For each Rpp locus, we used at least two SSR markers polymorphic between the parents and the sandwiching target Rpp locus. BC5F2 plants were screened using the same SSR markers used in BC5F1 generation to obtain BC5F2 plants carrying resistant Rpp alleles in the homozygous state. Given that BC5F2 plants should possess Rpp genes in the homozygous state, all SSR markers used in this study were co-dominant between parents. DNA extraction, PCR amplification, and subsequent electrophoresis were performed following previously described procedures22.

Phakopsora pachyrhizi inoculation test on Rpp-NILs

To evaluate the ASR compatibility/incompatibility of Rpp-NILs, P. pachyrhizi isolates from three countries, Japan (E1-4-12), Brazil (BRP-2.5 and BRP-2.6), and Mexico (MRP-13.18) were subjected to an inoculation test. Of the aforementioned isolates, E1-4-12, BRP-2.5, and BRP-2.6 were isolated in our previous studies14,23, whereas MRP-13.18 was isolated from MRP-1329 by a single-lesion isolation method22 in the present study. The Brazilian and Mexican P. pachyrhizi isolates are Import-prohibited articles in Japan where the work was conducted (Import Permit Numbers: 20Y157 and 27Y935 from the Yokohama Plant Quarantine Office of Japan for Brazilian and Mexican P. pachyrhizi populations, respectively). Spores of P. pachyrhizi collected from soybean leaves were dried and maintained at − 80 °C for long-term storage. Frozen spores were heated to 39 °C in a water bath for 1 min immediately prior to inoculation. Inoculation of P. pachyrhizi isolates was performed following an online manual22. Briefly, leaflets were detached from the lowest trifoliate leaf of V2–3 stage soybean plants, and gently hand-rubbed with autoclaved distilled water to remove trichomes on the leaf surface. Spores of P. pachyrhizi were soaked in 0.04% Tween 20 and adjusted to a concentration of 5.0 × 104 spores/mL. For each inoculation, spore suspensions were spread onto three soybean leaves using a paintbrush. Each of three leaves were sampled from different plants. Inoculated leaves were maintained at 21 °C in Petri dishes to maintain moist conditions. Immediately after inoculation, the Petri dishes were covered in aluminum foil to maintain darkness for 24 h. Two weeks after inoculation, we evaluated the average sporulation level (SL), average number of uredinia per lesion (NoU), and frequency of lesions with uredinia (%LU) in all assessed lesions for each of up to 30 lesions per inoculation. We initially evaluated SL, after which NoU was counted following the removal of spores from the inoculated leaves using a paintbrush. Statistical analysis of each value was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).