Introduction

Rice blast caused by filamentous ascomycete Magnaporthe oryzae [1], is a devastating disease affecting rice production every year worldwide [2]. The blast fungus infects all aerial parts of the rice plant at every stage of its growth starting from seedling to grain filling stage, often resulting in total loss of rice grain [3]. Many rice varieties with resistance to blast have been developed but their resistance is rapidly reduced by the emergence of strong virulent races which are ascribed to the high genetic instability in the avirulence/virulence genes of the pathogen [3, 4].

Magnaporthe oryzae, a filamentous ascomycete, is a model organism for the study of host pathogen interactions as it has a small genome size (~40 Mb). It can be cultured in defined media, facilitating biochemical and molecular analyses [5]. Resistance to M. oryzae follow the classical gene-for-gene relationship in which a major resistance gene prevents infection by races of pathogen containing the corresponding avirulence (Avr) gene [6]. Differential races of Magnaporthe oryzae were used earlier to identify resistance gene(s) in landraces, cultivars and wild relatives [7]. Detection of resistance genes in rice germplasm by traditional pathogenicity test using differential races is time consuming, less efficient and requires strict environmental control. Availability of gene specific or tightly linked DNA markers has helped overcome these constraints and represent a significant advantage for identification and incorporation of resistance gene in a breeding program [8–10]. During the past decade, genetics of blast resistance has been extensively studied. So far, 100 race specific resistance genes and about 350 QTLs have been identified [11, 12]. Most of the blast resistance genes and their analogues are located on chromosomes 4, 6, 11 and 12. Out of 100, 22 resistance genes have been cloned, among which 20 are major (Pib, Pita, Pi9, Pi2, Piz-t, Pid2, Pi36, Pi37, Pik-m, Pit, Pi5, Pid3, pi21, Pb1, Pish, Pik, Pik-p, Pi54, Pia, NLS1) and 2 minor (Pi25 and Pi54rh) [13]. Most of the blast resistance genes encode NBS-LRR type of resistance protein except Pid2, which encodes a receptor-like kinase protein with a predicted extracellular domain of a bulb-type mannose specific binding lectin (B-lectin), and pi21, which encodes a proline-rich protein that includes a putative heavy metal-binding domain and protein–protein interaction motifs. Though these genes are of the same gene families, their efficiencies against different pathotypes are different, some exhibiting more broad spectrum resistance than others.

Pi-2 gene is a member of a multigene family, located on the short arm and near the centromere of chromosome six. At least eight blast resistance genes were identified from the Pi2/9 locus [Pi9, Pi2, Piz-t, Pi26(t), Pigm(t), Piz(t), Pi40(t) and Pi50(t)] [14–20]. Among them Pi9, Pi2, Piz-t were successfully cloned. The Pi-2 gene was introgressed from the highly resistant O. sativa spp. indica cultivar 5173 into the susceptible cultivar C039 and the derived isogenic line was named C101 A51 [21]. Greenhouse inoculation revealed that C101 A51 was resistant to 455 isolates collected from Philippines and most of the 792 isolates from China [22]. Extensive inoculation tests in several other countries also indicated that Pi-2 is one of the broad-spectrum blast resistance genes [23]. Pi2 gene was also found resistant to 66 out of 72 isolates from different regions of eastern and north eastern India, by virulence analysis [24], indicating that Pi2 gene is resistant to most of the pathotypes prevalent in the region. India is having rich biodiversity in rice, but no serious effort has been made to exploit this extensive bioresource. Local landraces and wild germplasm of rice play an important role in maintaining this diversity as they are thought to be at an intermediate stage in the domestication process from wild ancestors to cultivated rice [25]. They represent a unique and critical source of genetically valuable traits for tolerance to biotic and abiotic stresses, particularly disease resistance. Molecular screening of Pi2 gene among local landraces might provide rare/superior alleles of Pi2 with broad resistance spectra. The objectives of this study were to identify the existence of Pi2 gene in landraces using a gene based marker and its validation by virulence analysis.

Material and Methods

Plant Material

For the identification of Pi2 gene, 61 accessions of rice originating from different geographical locations of India, mainly from eastern and north-eastern region, were selected from the germplasm collection maintained at Central Rainfed Upland Rice Research Station, Hazaribag (25°58′N, 85°26′E; 604 m above sea level) (Table 1). A monogenic line IRBLz5-CA, harbouring Pi2 gene was used as positive control as it is resistant to M. oryzae isolates containing Avr-Pi2 gene. CO39 was used as a negative control as it is susceptible to M. oryzae isolates containing Avr-Pi2 gene. Five to seven seeds of each landrace and checks were germinated on moistened filter paper for 3 days in a plant growth chamber, planted in plastic pots, transferred and maintained in a green house at 25 °C with 16 h light for 3–4 weeks until the emergence of fourth foliar leaf for pathogenicity tests and genomic DNA isolation.

Table 1 Geographical origin of landraces
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Genomic DNA Isolation and PCR Amplification

Leaf samples of all the landraces with controls were collected, frozen in liquid nitrogen and stored at −80 °C for DNA extraction. Genomic DNA was isolated using Plant Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instruction. The extracted genomic DNA was estimated on 0.8 % agarose gel in 1× TBE buffer, stained with ethidium bromide and visualized under Gel Documentation System (Alpha Innotec). The quantity of extracted DNA was estimated by NANO Drop spectrophotometer (Thermo, USA) and diluted to 100 ng/µl with 1× TE buffer and stored at −20 °C. A pair of primers NBS2P3 (5′-GATTTAGTTCAGGAAAACACTC-3′) and NBS2R (5′-TGGAAGCCTCATTGATCATC-3′) specific for Pi2 was used to amplify the gene [15]. PCR reaction mixture contained: 100 ng of genomic DNA, 1× Taq buffer, 0.2 mM of dNTPs mix, 1.5 mM of MgCl2, 0.5 µM of each primer, 1 unit of Taq DNA polymerase (Fermentas Life Sciences, Burlington, Canada) and MilliQ water to make final volume of 20 µl. The PCR amplification program for this marker consisted of (i) 5 min. initial denaturation at 94 °C (ii) 35 cycles of 45 s denaturation at 94 °C, 45 s annealing at 55 °C, 2 min. extension at 72 °C, and a final extension for 5 min at 72 °C. After amplification, the PCR products were resolved in 1.5 % (W/V) agarose gel in 1× TBE buffer, stained with ethidium bromide and visualized under gel documentation system. All PCR reactions for each sample were repeated at least twice to confirm the results.

Restriction Digestion of PCR Products

PCR amplification of susceptible Co39 and monogenic resistant line IRBLz5-CA and 61 land races with NBS2P3 and NBS2R primers [15] generated monomorphic bands. However, restriction digestion of the PCR product with fast-digest EcoR1 enzyme (Fermentas Life Sciences, Burlington, Canada) generated polymorphism between susceptible Co39 and monogenic resistant line IRBLz5-CA (Pi2). Restriction enzyme digestion was carried out directly in the PCR tube. The enzyme mix contained 2 µl 10X FD buffer, 2 units of Fast digest EcoR1 enzyme, 10 µl of PCR product and 17 µl MilliQ water to make final volume 30 µl. After a brief spin (microfuge) the mixture was incubated at 37 °C at 20 min to digest the PCR product. The digested PCR product was resolved on 1.5 % agarose gel. Restriction digestion of PCR product produced a polymorphic band of 850 bp corresponding to the resistant allele of Pi2 gene but not in susceptible Co39. PCR amplification and restriction digestion were repeated twice to confirm the results.

Disease Evaluation in Uniform Blast Nursery (UBN)

An initial evaluation of all the landraces and controls (IRBLz5-CA, R Check and Co 39, S. check) were done for their reaction to blast in uniform blast nursery at Central Rainfed Upland Rice Research Station, Hazaribag, Jharkhand, during wet season of 2013 in two replications. Thirty to 35 seeds of each line were planted with a row spacing of 10 cm surrounded by two rows of highly susceptible spreaders Co39 and B40. Abundant nitrogen 100–120 kg/ha in the form of urea was applied, half at seeding and the other half 15 days after seeding. Disease reaction of each line was recorded 30 days after sowing and continued at 5 days intervals until the susceptible check had more than 80 % infection. Disease reaction was scored visually on 0–9 SES scale (Standard Evaluation System, IRRI, 1996). Scores 0–3 were considered resistant (R), 4 as moderately resistant (MR) and 5–9 as susceptible (S).

Virulence Analysis

To validate the presence of Pi2 gene in landraces virulence analysis was done with a mixture of two avirulent isolates, Mo-ei-118 and Mo-ei-241. Monogenic line IRBLz5-CA having Pi2 gene was used as positive control as it is resistant to Mo-ei-118 and Mo-ei-241. Co39 was used as negative control as it is susceptible to both the isolates. Inoculum of isolates was prepared with a concentration of 1.5 × 105 spores/ml and sprayed on to 21 days old seedlings of landraces and control. The inoculated trays were kept in darkness at ±25 °C with more than 90 % relative humidity in a specialized blast phenotyping facility. Disease reaction of all the seedlings after seven days of inoculation was measured on a 0–5 disease assessment scale [26]. Reaction types 0–3 were considered resistant, while 4–5 were considered as susceptible.

Results and Discussion

Identification of Pi2 Gene in Landraces

Molecular screening of Pi2 gene, conducted on 61 landraces of rice using gene based marker NBS2P3 and NBS2R derived from Nbs4-Pi2 candidate gene [15] generated a monomorphic band of 1.8 kb. Restriction digestion of PCR product with EcoR1 enzyme, however, revealed polymorphism between susceptible and resistant lines (Fig. 1, Fig. S1). Three different types of banding patterns of alleles of Pi2 gene were observed among the landraces by NBS2P3 and NBS2R markers as shown in Fig. 2. IRBLz5-CA and the landraces having Pi2 gene produced a unique band of 850 bp after restriction digestion but negative control of Co39 did not have this band. Out of 61 landraces, five landraces had Pi2 gene type banding pattern. The five landraces positive for Pi2 gene originated from Sikkim (four) and Jharkhand (one). Thirty four landraces had Co39 susceptible type banding pattern and the remaining 22 landraces had null allele. Failure to generate amplicons may be attributed to sequence variation, insertion, deletion or substitution in one or both the primer binding sites [27, 28]. The landraces which were not positive for Pi2 gene did not generate desired band of 850 bp (Fig. 2). Screening of Pi2 gene in 162 local landraces by Hittalmani et al. [27] using RG64 revealed 31 landraces having the null allele.

Fig. 1
figure 1

Gel analysis of PCR products after digestion with restriction enzyme EcoR1. M 1 kb ladder, Lanes 2 and 3 represent the susceptible control CO39 and resistant control IRBLz5CA, respectively. Lane 4 IRBL9-w (Pi9), 13 represent Brown gora, 18 Dahia, 48 Safed Gora, 54 Sanu Atte, 55 Atte, 56 Chirakey B, 57 Champae

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Fig. 2
figure 2

Banding pattern of alleles of Pi2 gene by NBS2P3 and NBS2R marker, lane 1 1 Kb ladder, lane 2 Co39 type susceptible allele, lane 3 Pi2 type resistant allele, lane 4 null allele

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Disease Reaction of Landraces in UBN

Disease reaction of landraces (61) and checks (2) to blast in uniform blast nursery during wet season 2013 is summarized in Table 2. Disease reaction ranged from score 0 to 7 in SES scale when susceptible spreaders (Co39 and B40) had more than 80 % infection. Out of 61 landraces, 9 landraces were resistant (Score 0–3), 25 landraces were moderately resistant (Score 4), 23 were susceptible (Score 5–6) while four landraces failed to germinate (Table 2). The resistant check IRBLz5CA having Pi2 gene had a score of ‘3’ and the susceptible check scored ‘7’. Among the five land races positive for Pi 2 gene, four recorded resistant reaction while the fifth, Brown gora (HRC No 113), was susceptible with a score of ‘5’.

Table 2 List of landraces and their reaction to blast under UBN
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Verification of Pi2 Gene in Landraces by Virulence Analysis

The five landraces identified to possess Pi2, based on amplification of the gene specific markers and the checks (IRBLz5CA and Co39) were inoculated with a mixture of M. oryzae isolates Mo-ei-118 and Mo-ei-241. The controls exhibited expected resistant and susceptible reaction for the mixture of isolates, with the Near Isogenic Line C101 A51 (Pi2) exhibiting resistant reaction and Co 39 showing susceptible reaction (Table 3). Among the five landraces harbouring Pi2 gene, four were resistant [Sanu Atte (Thima), Atte, Chirakey B, Champae] to the mixture of avirulent isolates but one landrace (Brown gora, HRC 113) exhibited susceptible reaction even though the amplicon product was positively identified indicating the presence of Pi2 gene (Table 3). Mutation in the coding region of the gene may lead to loss of resistant function [10]. Virulence analysis with avirulent isolates for Pi2 gene however, confirmed that four landraces of Sikkim [Sanu Atte (Thima), Atte, Chirakey B, Champae] have the functional Pi2 gene (Table 3).

Table 3 Summary of disease reactions of landrace, virulence analysis against mixture of avirulent isolates for Pi2 gene and PCR result of Pi2 gene
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Wild species and landraces are unique source of valuable alleles for various biotic and abiotic stresses. Local landraces can serve as donors for future breeding programme. These untapped genetic resources exist in wild species and local landraces of crop which can be utilized successfully to identify novel or superior allele(s)/gene(s). Several blast resistance genes have already been identified and cloned from wild species like Pi9 from Oryzae minuta, Pi40 from Oryza australiensis, Pi54rh from O. rhizomatis, bacterial blight resistant gene Xa21 from O. longistaminata etc. which provide resistance against a wide range of isolates. Present study confirmed the existence of Pi2 gene in four landraces of Sikkim. Most deployed R genes have been identified from indica rice, including Pi2 which was originally identified from the indica cultivar 5173 from which the isogenic line C101A51was derived by Mackill and Bonman [21]. Presence of Pi2 gene in independent indica landraces from the eastern Indian region confirms that the gene originated and evolved in indica rice existing in different allelic forms in blast endemic zones of western and eastern India. Greenhouse inoculation test in India and in several other countries also indicated that Pi2 is a broad-spectrum blast resistance gene [22–24, 29]. These findings not only reflect that Pi2 gene has been durable in India but also suggests that it has potential for broader application worldwide. Survey for identification of alleles of Pi2 gene among local landraces, therefore, might provide a valuable source for Pi2 gene with broad resistance spectrum and agronomic fitness. Landraces having Pi2 gene identified in this study can be immediately used as donor in resistance breeding program. Since the landraces possessing the gene have evolved in the eastern Indian region, it may also provide additional genes for environmental fitness. Additionally, the gene based marker used in this study can be reliably used in screening of Pi2 gene from rice landraces and subsequent selection of the Pi2 gene in a marker assisted backcross breeding program. Recent work published by RoyChowdhury et al. [10, 30] on the identification of rice blast resistance gene Pib and analysis of rice blast resistance gene Piz also suggested that DNA markers derived from the gene and pathogenicity assays can be valuable tools for detection of novel alleles and their deployment.

Conclusion

Detection of Pi2 gene in four land races of rice from blast endemic zones of eastern India using gene specific markers and evaluation of their resistance spectra with diagnostic isolates opened up possibilities of their use in varietal improvement programme for blast resistance. Pi2 is highly effective in eastern India because it confers resistance to most pathotypes prevalent in this region [24]. Usefulness of the gene specific DNA marker for germplasm characterization further demonstrated that the process of gene tracking in resistance breeding programmes can be made precise with such molecular tools. Sequence analyses of the segments identified positive for the marker would further clarify allelic variation and the evolutionary significance of this gene in different landraces.