DOI: https://doi.org/10.21203/rs.3.rs-1707355/v1
Erysiphe pisi is the causal agent of powdery mildew disease in garden pea. So far, the resistance genes comprising two recessive (er1 and er2) and one dominant (Er3) have been reported to confer resistance to powdery mildew in garden pea. A set of 46 pea genotypes were screened against the E. pisi isolate, Ep01 in greenhouse conditions to identify resistant genotypes. Disease phenotyping and genotyping were carried to test for resistance/susceptibility through gene-specific sequence characterized amplified region (SCAR) markers. The presence of the resistant genes in the control pea genotypes; JI2302 for er1 gene, JI2480 for er2 gene and P660-4 for Er3 were confirmed through their respective gene-specific markers. The pea genotype, Arkel served as a negative control. Screening of the pea germplasm revealed three genotypes as highly resistant, six genotypes as resistant while ten genotypes were moderately resistant, thirteen genotypes were moderately susceptible, nine genotypes were susceptible and five were highly susceptible. The molecular markers for er1 resistance gene Sc-OPE-161600 was found to be segregating in most of the pea genotypes except in the susceptible control, Arkel. The marker ScX17_1400 for er2 gene was found to be in homozygous condition in the resistant pea genotypes while the marker associated with Er3 resistance, SCW4637 was found to be in heterozygous condition in majority of the pea lines except in the resistant control genotype, P660-4.
Garden pea (Pisum sativum) is an important legume crop cultivated worldwide. With an estimated amount of 87% of the total digestible fibre and 22% of total protein content, the pea crop is considered as a major source of protein, carbohydrates and dietary fibre. Besides, it also serves as a rich source of vitamins with high level of folate content along with the availability of the micronutrients like calcium, magnesium, iron, and potassium. Moreover, the presence of low levels of sodium and fat content in the pea seeds and pods renders the crop as a valuable source of food and fodder (Tiwari and Singh 2012). However, the crop is vulnerable to pathogens and powdery mildew infection caused by the fungal pathogen, Erysiphe pisi is a serious concern in garden pea growing areas worldwide.
Powdery mildew infection causes crop destruction resulting in loss of nutrients, thereby affecting yield and quality of the produce. A 50% reduction in yield was reported due to powdery mildew disease in pea, producing poor quality and quantity of green pods and dry seeds (Dixon 1987). Moreover, the coincidence of congenial environmental conditions at the time of infection promotes disease progression on pea plants (Banyal and Tyagi 1997). The damage caused by powdery midew infection is most devastating especially for the late-sown field pea crops and late-maturing cultivars which is accompanied by premature or early death of leaves, pods and other affected parts of the plants (Davidson et al. 2004).
The powdery mildew disease is more prominent in a climate with warm dry days and cool nights (Sillero et al. 2006).The powdery mildew causing pathogen, Erysiphe pisi is a member of Ascomycetes family and an obigate biotroph which propagates by both asexual (conidia) and sexual spores (ascospores). The conidial germination can occur even in the absence of water at an optimum temperature of 10–30°C with a maximum germination observed at 20–24°C (Banyal and Tyagi 1997). The initial symptoms of the disease appear as a white powdery outgrowth of mycelia and spore mass which is found on the aerial parts of the pea plant. In severe cases, the spreading of the disease is observed covering almost all parts of the infected plant.
Previous studies reported monogenic gene-for-gene inheritance for powdery mildew resistance in garden pea (Harland 1948; Heringa et al. 1969; Fondevilla et al. 2007, 2010). Genetic resistance to powdery mildew in pea has been reported to be controlled by a set of three genes, of which two are recessive genes and one is dominant. The genes, er1 (Harland 1948; Heringa et al. 1969) and er2 (Heringa et al. 1969) were detected from the Pisum sativum genetic background while the gene, Er3 (Fondevilla et al. 2007) has been detected from Pisum fulvum genetic background. So far, there is no known source of absolute resistance that has been reported against the powdery mildew worldwide. Most of the commercial cultivars are found to vary from moderately susceptible to highly susceptible, thereby necessitating identification of resistant donors to utiize them in crop breeding program. Identifying markers associated with disease resistance or susceptibility could enable effective and early detection of resistance/susceptible pea genotypes by marker assisted breeding in garden pea. Further, to identify new or potential sources of resistance, a set of 46 genotypes including few of the previously screened genotypes were tested in greenhouse conditions against the E. pisi isolate, Ep01 and the findings are presented.
Plant material and growth conditions
The pea material were grown in greenhouse conditions as described by Bheri et al. 2016. Seeds were sterilized with 0.1% sodium hypochlorite for 5 min followed by washing once with 70% ethanol for 30 s and 4–5 times with sterile distilled water. The sterilized pea seeds were soaked in autoclaved distilled water for 12–24 h and sown in pots filled with fertile field soil and manure in 3:1 ratio. The plants were maintained at 25 ± 3°C with natural photoperiod of a maximum of 14 h in greenhouse conditions.
Pathogen growth and testing on plants
Monoconidial isolate of E. pisi, Ep01 was maintained in isolation on leaves of Arkel plants in greenhouse conditions at 25 ± 3°C. Four-week-old pea plants were challenged by inoculating leaves by dusting of E. pisi spores and culturing the inoculated plants in isolated conditions. Fifteen pea plants per replication were monitored for 10 days post inoculation (dpi) for disease development and the mean over replications was calculated. The experiment was repeated at least three times with three replications each.
Disease phenotyping
The pea genotypes were phenotypically assessed for powdery mildew appearance on leaves, stem, pods and stipules.The phenotyping was carried by assessing whole plants as described by Fondevilla et al. (2006) and Katoch et al. (2010) with modifications. The percentage disease scoring ranging between 0–8 was grouped as highly resistant (HR), 9–15 as resistance (R), 16–35 as moderately resistant (MR), 36–48 as moderately susceptible (MS), 49–55 as susceptible (S) and 56 and above as highly susceptible (HS) as shown in Fig. S1.
Microscopic examination for pathogen spread
Microscopic examination was carried to assess mycelia spread in leaf tissues of each of the pea genotype by monitoring at least three leaves per replication in three replications per experiment. The visual scoring was carried according to 0–4 scale as used by Fondevilla et al. (2006) and Katoch et al. (2010) wherein the scores of 0, 1 and 2 represent resistant groups, and the scores of 3 and 4 represent susceptible groups (Vaid and Tyagi 1997) as shown in Table S1.
Trypan blue staining
Trypan blue stock solution was prepared as described by Bheri et al. 2016. 0.02 g of trypan blue stain was dissolved in a solution containing 10 g phenol, 10 ml glycerol, 10 ml lactic acid and 10 ml of autoclaved distilled water. The trypan blue stock solution was diluted in ethanol (96%) in 1:2 (v/v). Three leaf samples were collected at 3 dpi from each of the three replications per genotype. The leaf samples were transferred to Eppendorf tubes containing 1 ml of trypan blue solution and incubated at 65°C in a hot water bath for 1 min. After draining the solution, 1 ml of lactic acid was added to the contents of the tubes and incubated at 65°C for 10 min. The leaf tissues were transferred to fresh tubes containing 1 ml of absolute ethanol for de-staining at 4°C for 2–3 days. The stained leaves were examined for mycelia growth using a light microscope (Make: Motic).
Screening for morphological traits
Two-week-old pea genotypes were monitored for morphological traits of the aerial parts of the plant which include stem, internodal length, leaf, stipule and tendrils (Fig. S2).
Percent disease index
The disease severity on E. pisi-inoculated pea plants were assessed by monitoring the leaves of each of the pea genotypes to calculate the percent disease incidence (PDI). The mean percent of disease infection over replications was calculated from three replications and the experiments were repeated at least three times. The visual scoring of disease was based on a 0–5 scale given by Gawande and Patil (2003) with modifications and Plant disease index (PDI) was calculated with the formula proposed by Wheeler (1969).
The PDI values were tested for significance of variance by one-way analysis of variance (ANOVA). Testing for variance between the genotypes was carried using Holm-Sidak test at P ≤ 0.0001. Statistical analysis was performed using GraphPad Prism Vs. 9.
DNA isolation
DNA was isolated from pea leaves by the method given by Doyle and Doyle (1987) with modification. Three leaf samples per plant were collected before challenging with E. pisi. Young leaves were ground in liquid nitrogen in a mortar with a pestle and the resulting fine powder was homogenized in 800 µl DNA extraction buffer (100 mM Tris-HCl pH 8.0, 250 mM NaCl, 25 mM EDTA pH 8.0 and 1% SDS) containing 20 µg/ml of RNase A. The mixture was incubated at 65°C for 20 min and subsequently 500 µl volume of phenol:chloroform:isoamyl (25:24:1) alcohol was added to the contents of the Eppendorf tubes. The leaf suspension was mixed thoroughly and centrifuged at 12,500 rpm for 10 min. The supernatant was taken in a fresh tube and the above step was repeated. The supernatant was transferred to fresh tube containing 500 µl of Isopropanol. The samples were centrifuged at 12,500 for 15 min and the DNA pellet was washed with 70% ethanol by centrifuging at 5,000 rpm for 5 min. The tubes were dried at room temperature and DNA was suspended in 50 µl of 1X TE buffer. The DNA samples were quantified by a Nanodrop spectrophotometer (ThermoScientific NANODROP 2000) at 260 nm.
Pea genotyping using molecular markers
Polymerase chain reaction (PCR) was carried using a set of 12 gene-specific markers to test for the presence of the three resistance genes; er1, er2, and Er3. The SCAR makers; Sc-OPE-161600 and Sc-OPO-181200 (Tiwari et al. 1998), ScOPX04880 (Srivastava et al. 2012), ScOPD10650 (Timmerman et al. 1994), ScAGG.CAA125, ScOPT16480, ScOPL13990, ScOPO061100, ScAH1R (Periera et al. 2010) were used to detect er1 resistance and susceptibility alleles. The ScX17_1400 marker (Katoch et al. 2010) was used to identify er2 resistance gene while SCW4637 and SCAB1874 markers (Fondevilla et al. 2008) were used to detect Er3 resistance and susceptibility alleles respectively.
Each reaction comprises of 1x Taq buffer, 1.5 mM MgCl2, 0.25 U Taq DNA polymerase, 200 µM dNTPs, 10 µmol each of forward and reverse primers and 10 ng of genomic DNA. The polymerase chain reaction (PCR) amplifications were carried out in a thermocycler (Arktik thermal cycler; Thermo Scientific). The PCR program was as follows: an initial denaturation at 94°C for 7 min followed by 35 cycles of 95°C for 1.5 min, the annealing temperature for 1 min, extension at 72°C for 1.5 min with a final extension at 72°C for 10 min followed by storage at 4°C. The thermal profile for PCR was same for all the SCAR markers except for the annealing temperatures which were as follows: 59.5°C for Sc-OPE-161600, 50°C for Sc-OPO-181200, 60.8°C for ScOPX04880 and ScOPD10650, 52.2°C for ScAGG.CAA125, 57.9°C for ScOPT16480, 57.1°C for ScOPL13990, 57.3°C for ScOPO061100, 56.8°C for ScAH1R, 58.7°C for ScX17_1400, 57.1°C for SCW4637, 59.5°C for SCAB1874. Electrohoresis of the PCR products were carried on 1.2% agarose gel with Tris-acetate EDTA buffer (40 mM Tris-acetate 1 mM EDTA) for 1 h at 75 V (constant voltage). The Ethidium bromide-stained gels were visualized and documented using the gel doc, (DNR Bio-Imaging Systems MiniLumi) .
Disease phenotyping of pea germplasm
Forty-six pea genotypes were screened for powdery mildew disease in controlled greenhouse conditions against the Erysiphe pisi isolate, Ep01 (NCBI GenBank Acc. No. KM096758). The pea genotypes included both indigenous and exotic germplasm along with a few established sources of resistance and susceptibility which served as controls for the study. Of the seven pea genotypes selected from our previous screening studies (Bheri et al. 2016) to check for consistency in the disease phenotyping, 5 of them showed consistency in their disease reaction similar to the previous findings. However, two genotypes varied in their disease reaction which might be resulting from the molecular differences in the genetic background of those genotypes.
The pea genotypes were categorized based on the disease scoring scale adopted by Gawande and Patil (2003) with modification. Based on the percent disease index (PDI), the pea genotypes were grouped into six classes: highly resistant (HR), resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). The PDI values were calculated as the sum total of the products of the number of leaves showing the disease pattern assigned to each of the groups and the corresponding disease score of the group, which is computed as a percentage to reflect the actual disease incidence observed on that pea genotype. A typical graph representing the disease infection pattern in pea genotypes along with the resistant and susceptible controls are presented (Fig. 1a). Further, a frequency histogram confirmed that the observed variability among the pea genotypes followed a normal distribution as expected for a randomized population which is shown in Fig. 1b. The pea genotypes were also plotted individually based on their PDI values which showed that majority of the genotypes were moderately resistant and moderately susceptible, while the highly resistant and highly susceptible phenotypes were comapartively lower in number as observed in the extreme ends of the graph (Fig. 1c).
Likewise, significant level of correlation was observed between the disease phenotypes of the pea germplasm and the amount of mycelial growth in their leaf tissues as shown in Fig. 2. The microscopic examination of trypan blue-stained leaves, sampled randomly from the pea germplasm which were challenged with E. pisi at 3 dpi in the greenhouse conditions revealed variation among the pea genotypes. A rapid spread of mycelium with effusive sporulation was observed in highly susceptible (HS) pea genotypes while the infection was relatively lower in susceptible (S) and moderately susceptible (MS) genotypes respectively. Conversely, the highly resistant and resistant genotypes showed no infection while moderately resistant revealed limited mycelial spread in E. pisi-infected leaf tissues.
The pea genotypes were grouped into the different disease categories based on their PDI values (Table. 1). The PDI value was found in the range of 5–66% in the pea genotypes. The pea genotypes; JI2480, JI2302 and P660-4 showed lower level of powdery infection with 5, 7 and 8% disease incidence respectively and were used as resistance controls. The highest mean PDI value was recorded in the susceptible pea genotype, Arkel with 66% disease incidence. In addition to Arkel, four pea genotypes; IC424887, IC424892, IC424591 and Azad were also found to be highly susceptible while nine pea genotypes (IC424588, VKG30162, IC208368, IC424888, IP-3, IC417837, VLSM-11, IC417586 and EC387113) were susceptible and thirteen genotypes (EC398589, VLSM-14, EC507771, VLSM-15, EC499976, EC414485, IC356304, IC356391, IC417880, EC398602, IC267169, EC398611 and IC424592) were moderately susceptible. The moderately resistant group comprised of 10 genotypes (IC208364, IC356203, DMR-11, EC398612, VLSM-13, VLSM-12, EC507770, IC356318, KPMR-526 and IC398604) and the resistant group included 6 pea genotypes; EC6620, IC267181, PSM-4, DDR-61, IC267152 and EC499762 suggesting them as potential sources of resistance to powdery mildew disease. The research findings were observed in agreement with the visual scoring carried according to 0–4 scale as used by Fondevilla et al. (2006) and Katoch et al. (2010) wherein 0, 1 and 2 represent resistant groups while 3 and 4 represent susceptible groups (Vaid and Tyagi 1997) as shown in Table S1.
Screening with gene-specific markers for er1, er2, and Er3 resistance
The pea genotypes; JI2302, JI2480 and P660-4 which are reported to carry er1, er2 and Er3 genes respectively were used as the resistant controls for molecular screening while Arkel served as susceptible control. The representative gel image of the SCAR markers linked to all three resistance genes along with their expected band size is presented in Fig. 3a. The SCAR marker, Sc-OPE-161600 has been reported as a resistance marker and found to be in cis-phase with er1 gene, thereby amplifying a band of size 1600 bp in the resistance control, JI2302 and absent in the susceptible control, Arkel. However, the susceptibility marker, Sc-OPO-181200 amplied the expected band of 1200 bp in Arkel along with a 1400 bp band which has been reported to be associated with susceptibility to powdery mildew disease as shown in Fig. 3b. Similarly another susceptible genotype, IP-3 amplified both the bands, 1200 bp and 1400 bp as reported for susceptible phenotype. On the contrary a 1200 bp band along with 1400 bp was amplified by the marker, Sc-OPO-181200 in the resistant control, JI2480 while 1400 bp band alone was observed in JI2302 and P660-4 suggesting that these genotypes might be exhibiting allelic variation at the er1 locus.
Eight Er1 allele-specific SCAR markers were used for screening the pea genotypes of which ScOPT16480 amplified a band size of 480 bp in Arkel, while it was not found in the resistant controls, JI2302, JI2480 and P660-4 suggesting its association with susceptibility. Similarly this marker did not amplify in the other three resistant genotypes (EC499762, EC6620 and IC208364) indicating the marker to be tightly linked with susceptibility. Of the remaining susceptible markers, ScOPL13990 marker amplified a band size of 990 bp not only in the susceptible genotypes, IP-3 and Arkel but were found in all the three resistant control genotypes as well as other disease phenotypes indicating the lack of specific polymorphism for this marker. The susceptible marker, ScOP0061100 which amplified a band size of 1100 bp was found in the resistant control, JI2480 along with the susceptible control, Arkel. Likewise, SCAR markers ScAH1R and ScAGG.CAA125 amplified bands ranging from 250–300 bp and 125–250 bp respectively which were found in all three resistant controls along with the susceptible control indicating them as unlinked to the er1 locus. Likewise, the susceptible markers, ScOPD10650 and ScOPX04880 amplified the bands of sizes 650 bp and 880 bp in all three resistant controls along with Arkel suggesting the lack of specific polymorphism for the marker.
The SCAR marker, ScX17_1400 which is reported as er2 gene-specific marker amplified a band size of 1400 bp in the resistant control JI2480 alone indicating that it is specific to the genotype, JI2480. The SCAR marker, SCW4637 reported as specific to Er3 resistance, amplified a single band of size 637 bp in P660-4 alone indicating the presence of homozygous alleles at the Er3 locus. However, the resistant marker along with the susceptible marker, SCAB1874 appeared in heterozygous condition in majority of the pea genotypes including the susceptible control, Arkel.
Genotying pea germplasm for powdery mildew resistance
Comparing the screening of the pea genotypes for powdery mildew resistance using gene-specific markers for er1, er2 and Er3 genes and their PDI values led to the identification of the few of the pea genotypes as resistant donors which are listed in Table 2. The expected band size of 1600 bp amplified with the marker Sc-OPE-161600 was observed in 26 pea genotypes including the resistant control, JI2302. However, the susceptible marker Sc-OPO-181200, amplified a band of 1200 bp alone in two pea genotypes (EC49976 and VLSM-11), while majority of the pea genotypes showed either 1400 bp band alone or in combination with 1200 bp band. The SCAR marker, ScOPD10650, amplified a band size 650 bp in most of the pea genotypes baring two genotypes; IC417837 (S) and VLSM-11(S). Though this marker was reported as a resistant marker previously to screen pea resistant and susceptible lines (Timmerman et al. 1994), the marker was found to amplify in all the genotypes irrespective of the disease phenotype (Tiwari et al. 1998). The marker, ScOPX04880, amplified a band size 880 bp in all the pea genotypes except IC208364 (MR) and IC417837 (S) pea genotypes. The marker ScOPO061100, amplified a band size of 1100 bp in eighteen pea genotypes. The marker ScOPL13990, amplified a band size of 990 bp in 44 pea genotypes including resistant and suscetible genotypes except two of the pea genotypes, IC208364 (MR) and IC417837(S). The marker ScOPT16480, amplified a band size 480 bp in 29 pea genotypes while it was absent in all three resistant controls. The SCAR markers, ScAH1R and ScAGG.CAA125, amplified a band size of 250–350 bp and 150–250 bp in all 46 pea genotypes suggesting that it as monomorphic in all the pea genotypes.
| S. No. Disease Score (%) | Disease Reaction | Pea genotypes | |
|---|---|---|---|
| 1 0–8 | Highly Resistance | JI2302, JI2480, P660-4 | |
| 2 9–15 | Resistance | EC6620, IC267181, PSM-4, DDR-61 IC267152, EC499762 | |
| 3 16–35 | Moderately Resistance | IC208364, IC356203, DMR-11, EC398612, VLSM-13,VLSM-12, EC507770, IC356318, KPMR526, IC398604 | |
| 4 36–48 | Moderately Susceptible | EC398589, VLSM-14, EC507771,VLSM-15, EC499976, EC414485, IC356304, IC356391, IC417880, EC398602, IC267169, EC398611, IC424592 | |
| 5 49–55 | Susceptible | IC424588, VKG30162, IC208368, IC424888, IP-3, IC417837, VLSM-11, IC417586, EC387113 | |
| 6 >56 | Highly Susceptible | IC424887, IC424892, IC424591, Azad, Arkel |
| S. No. | Germplasm/cultivar | er1 Gene Markers | er2 Gene Marker | Er3 gene Markers | Percent Disease Index | Disease reaction | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1. | IC208364 | Sc-OPO-181400 ScOPD10650 ScAH1R ScAGG.CAA125 | A | A | 16 ± 3.6 | Moderate Resistant | ||||||
| 2. | EC398611 | Sc-OPE-161600 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R Sc-OPO-181200 Sc-OPO-181400 ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 48 ± 0.5 | Moderate Susceptible | ||||||
| 3. | IC424592 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 48 ± 2.9 | Moderate Susceptible | ||||||
| 4. | EC6620 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 9 ± 1.5 | Resistant | ||||||
| 5. | IC424888 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 53 ± 6.7 | Susceptible | ||||||
| 6. | DDR-61 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 10 ± 2.0 | Resistant | ||||||
| 7. | IC417837 | ScAH1R ScAGG.CAA125 | A | A | 54 ± 3.1 | Susceptible | ||||||
| 8. | EC398602 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 46 ± 1.9 | Moderate Susceptible | ||||||
| 9. | IC267181 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 9 ± 2.2 | Resistant | ||||||
| 10. | EC414485 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 41 ± 3.3 | Moderate Susceptible | ||||||
| 11. | IC208368 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 52 ± 4.4 | Susceptible | ||||||
| 12. | EC507771 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 38 ± 5.0 | Moderate Susceptible | ||||||
| 13. | IC267169 | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 47 ± 2.1 | Moderate Susceptible | ||||||
| 14. | EC499762 | Sc-OPE-161600 Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 11 ± 2.2 | Resistant | ||||||
| 15. | IC424892 | Sc-OPE-161600 Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 58 ± 4.5 | Highly Susceptible | ||||||
| 16. | IC398604 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 35 ± 1.5 | Moderate Resistant | ||||||
| 17. | IC424887 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 57 ± 5.7 | Highly Susceptible | ||||||
| 18. | IC424588 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 51 ± 3.0 | Susceptible | ||||||
| 19. | IC417586 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 55 ± 3.7 | Susceptible | ||||||
| 20. | EC499976 | Sc-OPE-161600 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R Sc-OPO-181200 ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 40 ± 1.9 | Moderate Susceptible | ||||||
| 21. | IC424591 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 60 ± 2.3 | Highly Susceptible | ||||||
| 22. | KPMR526 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 34 ± 1.1 | Moderate Resistant | ||||||
| 23. | VKG30162 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 51 ± 4.3 | Susceptible | ||||||
| 24. | AZAD | Sc-OPE-161600 Sc-OPO-181400 Sc-OPO-181200 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 65 ± 2.4 | Highly Susceptible | ||||||
| 25. | EC507770 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 33 ± 2.7 | Moderate Resistant | ||||||
| 26. | IC417880 | Sc-OPO-181400 Sc-OPO-181200 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 44 ± 6.4 | Moderate Susceptible | ||||||
| 27. | IC356318 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 33 ± 1.9 | Moderate Resistant | ||||||
| 28. | EC398589 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 36 ± 2.3 | Moderate Susceptible | ||||||
| 29. | EC387113 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 55 ± 6.8 | Susceptible | ||||||
| 30. | PSM-4 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1 ScAGG.CAA125 | A | SCW4637 SCAB1874 | 9 ± 1.7 | Resistant | ||||||
| 31. | DMR-11 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 24 ± 0.8 | Moderate Resistant | ||||||
| 32. | IC356304 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 41 ± 1.2 | Moderate Susceptible | ||||||
| 33. | IC267152 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 10 ± 0.7 | Resistant | ||||||
| 34. | EC398612 | Sc-OPE-161600 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R Sc-OPO-181400 Sc-OPO-181200 ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 27 ± 3.5 | Moderate Resistant | ||||||
| 35. | IC356203 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 23 ± 0.8 | Moderate Resistant | ||||||
| 36. | IC356391 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 42 ± 0.7 | Moderate Susceptible | ||||||
| 37. | IP-3 | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 53 ± 1.9 | Susceptible | ||||||
| 38. | VLSM-11 | Sc-OPE-161600 ScOPO061100 ScOPL13990 ScOPX04880 ScAH1R Sc-OPO-181200 ScAGG.CAA125 | A | SCW4637 SCAB1874 | 54 ± 1.6 | Susceptible | ||||||
| 39. | VLSM-12 | Sc-OPE-161600 Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCW4637 SCAB1874 | 32 ± 1.2 | Moderate Resistant | ||||||
| 40. | VLSM-13 | Sc-OPO-181400 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 30 ± 0.8 | Moderate Resistant | ||||||
| 41. | VLSM-14 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 36 ± 0.8 | Moderate Susceptible | ||||||
| 42. | VLSM-15 | Sc-OPE-161600 Sc-OPO181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 38 ± 0.8 | Moderate Susceptible | ||||||
| 43. | JI2302 | Sc-OPE-161600 Sc-OPO-181400 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | A | SCAB1874 | 7 ± 0.8 | Highly Resistant | ||||||
| 44. | JI2480 | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScAH1R ScAGG.CAA125 | ScX17_1400 | SCAB1874 | 5 ± 0.7 | Highly Resistant | ||||||
| 45. | P660-4 | Sc-OPO-181400 ScOPL13990 ScOPD10650 ScAH1R ScAGG.CAA125 | A | SCW4637 | 8 ± 0.8 | Highly Resistant | ||||||
| 46. | Arkel | Sc-OPO-181400 Sc-OPO-181200 ScOPO061100 ScOPL13990 ScOPD10650 ScOPX04880 ScOPT16480 ScAH1R ScAGG.CAA125 | A | SCW4637 SCAB1874 | 66 ± 2.3 | Highly Susceptible | ||||||
Of the 46 pea genotypes, 23 genotypes amplified a band size of 1400 bp with the SCAR marker, ScX17_1400 showing the presence of er2 gene including the resistant control genotype, JI2480. Likewise, 26 pea genotypes amplified a band size of 637 bp with the SCAR marker, SCW4637 which is a characteristic of the domiant Er3 allele including the resistant control, P660-4. The SCAB1874 marker showed a homozygous band of size 874 bp in 18 pea genotypes including the other two resistant controls, JI2302 and JI2480 suggesting the absence of Er3 resistant allele in these genotypes. On the contrary, two pea genotypes, IC208364 and IC417837 did not amplify any of the above two bands of Er3 indicating the absence of the Er3 locus in these genotypes.
Six pea genotypes; IC267152, IC208364, IC267169, IC417880, IC417837 and IC208368, did not amplify with any of the known resistant gene markers. Of these, IC267152 and IC208364 showed resitance and moderate resistance to E. pisi despite the absence of all three resistant genes, suggesting that these genotypes might be the novel sources of resistance. Whereas, the other four genotypes which also did not amplify the resitant gene markers indicated the absence of powdery mildew resistance as evident from their disease reaction as moderately susceptible and susceptible phenotypes.
Resistance to powdery mildew disease caused by E. pisi has been reported to be monogenic gene-for-gene with only a few resistant genes identified till date. However, with rapid evolution of Erysphe pathogen, there is a tendency of breaking down the existing resistance available in pea cultivars. Therefore, identifying novel resistant soures to utilize them in pea breeding programs, by screening of pea germplasm to detect potential donors is indispensable. A few of the pea germplasm with a potential for resistance against powdery mildew pathogen have been identified in our previous studies through disease phenotyping and genotyping (Bheri et al. 2016).
Earlier studies have reported DNA based molecular markers that are linked to the powdery mildew disease resistance gene loci. The SCAR marker, ScOPE-16_1600 (Tiwari et al. 1998) is mostly used to screen for resistance in pea for the presence of er1 gene while the markers; ScOPD10650 (Timmerman et al. 1994), ScOPX04880 (Srivastava et al. 2012), ScOPO181200 (Tiwari et al. 1998) were previously used to screen for assessing susceptibility in pea. Further, few more markers for er1 gene; ScOPO061100, ScOPL13990, ScOPT16480, ScAH1R, ScAGG.CAA125 (Periera et al. 2010) have been reported. The presence of er2 resistant gene was screened using the SCAR marker, ScX17_1400 (Katoch et al. 2010). The SCAR markers; SCW4_637 for resistance and SCAB1_874 for susceptibility were used for detecting the Er3 gene (Fondevilla et al. 2008).
In this study, the SCAR markers previously reported have been employed for tagging resistance or susceptibility alleles of er1, er2 and Er3 genes. Previous studies showed that er1 is the major resistant gene contributing to resistance against powdery mildew causing pathogen/s while er2 gene which confers complete resistance was observed to express under a specific set of conditions (Tiwari et al. 1997a). Studies have shown that er1 conferred resistance was overcome by E. pisi pathogen in controlled experimental conditions (Schroeder and Providenti 1965; Tiwari et al. 1997b). Likewise, the er1 resistance has been shown to be compromised against other Erysiphe members like E. baeumleri and E. trifolii on pea (Ondrej et al. 2005; Attanayake et al. 2010; Fondevilla et al. 2006, 2013). The Er3 gene identified from Pisum fulvum background is the only dominant gene identified so far. However, Er3 resitance was also demonstrated to be overcome by E. trifolii under controlled greenhouse and field conditions in the pea growing regions where temperature is prevailing above 25°C (Fondevilla et al. 2006; 2013).
The pea genotypes were grouped into various categories based on their disease reaction against E. pisi and also evaluated for the presence of the three resistant genes- er1, er2, and Er3 using the reported gene-specific SCAR markers. The markers associated with the recessive resistance genes; er1 has been reported on the linkage group VI (Tiwari et al. 1998) while er2 has been reported on the linkage group III (Katoch et al. 2010), and the linkage group for the dominant gene Er3 is yet to be mapped. A majority of pea genotypes which showed resistance to E. pisi showed the presence of the recessive gene er1 which provides long-lasting resitance in adult pea plants to powdery mildew disease caused by E. pisi (Tiwari et al. 1997; Cousin 1965). The SCAR marker, Sc-OPE-161600 has been reported as a resistance marker and found to be in cis-phase with er1 gene, thereby amplifying a band of size 1600 bp in the resistance pea line, JI2302.
In the present study, the presence of the markers linked to either er1 or er2 alone exhibited either moderately resistant or moderately susceptible phenotypes while the presence of both the resistant alleles demonstrated either moderately resistant or resistant phenotypes. Majority of the pea genotypes showed either 1400 bp band alone or in combination with 1200 bp band when amplified with the susceptibility marker, Sc-OPO-181200 suggesting an ambiguous primer binding of the marker to the er1 locus. Similar finding of inconsistent level of polymorphism was also reported from the previous screening studies (Janila and Sharma 2004; Bheri et al. 2016). The SCAR marker, ScOPD10650, amplified a band size 650 bp in most of the pea genotypes baring two genotypes; IC417837 (S) and VLSM-11(S). The SCAR marker, ScOPD10650 though previously reported as a resistant marker (Timmerman et al. 1994) was observed in the pea genotypes irrespective of the disease phenotype. The marker was found to be located at 3. 6 cM (Rakshit 1997) and 3.4 cM (Janila and Sharma 2004) from the er locus which co-segregated with er allele in coupling phase suggesting that it might be integrated with the dominant allele, Er locus.
In addition to the disease phenotyping and genotyping, screening for morphological traits like stem, intermodal length, leaf, stipules and tendrils also complemented the findings that pea genotypes (IC208364, DDR-61, KPMR-526 and PSM-4) with lower number of leaves and dense tendrils showed moderate resitant to resistant phenotypes. Two pea germplasm, IC267152 and IC208364 showed resitance and moderate resistance to E. pisi eventhough the markers for all three resistant genes did not produce the expected amplicons, suggesting that these genotypes might be the novel sources of resistance. Further, there is a need to assess these genotypes in field conditions to check for the consistency of the disease phenotyping and genotyping to identify highly resistant pea genotypes. Understanding the mechanism of host-pathogen interaction in these pea genotypes, may facilitate not only identifying specific resistant (R) and Avirulence (Avr) gene interaction but also novel sources of resistance. Further, developing new markers linked to er1, er2 and Er3 loci by generating mapping population/s may facilitate fine mapping of these loci for marker-assisted breeding to confer resistance against E. pisi in pea.
Acknowledgments
The authors acknowldege National Bureau of Plant Genetic Resources (NBPGR), New Delhi for pea germplasm, pea seed material of JI2302 and JI2480 (Himachal Pradesh Agriculture University, Palampur, HP, India; John Innes Centre, Norwich, UK), JI2480, JI2302 and P660-4 (Indian Institute of Pulses Research, Kanpur, UP, India and Dr. Sara Fondevilla, Institute for Sustainable Agriculture, Cordoba, Spain), pea cultivars (Indian Institute of Horticultural Research, Bengaluru, India) VLSM-11, VLSM-12, VLSM-13, VLSM-14, VLSM-15 (Dr. Nirmal Hedau, Vivekananda Parvatiya Krishi Anusandhan Sansthan, Almora, Uttarakhand, India) and for pathogen confirmation by the Department of Plant Pathology, Indian Agricultural Research Institute (IARI), New Delhi, India. Facilities at UoH which include DBT-CREBB, DST-FIST, UGC-SAP, CIL, DBT-BUILDER, UoH-IOE and Plant culture facility at School of Life Sciences, UoH. Also the authors acknowledge the BBL-fellowship provided to PS for her doctoral studies.
Funding
This work was supported by Department of Science and Technology (DST; SR/SO/BB02/2010); Department of Biotechnology (DBT; BT/PR1264/PBD/16/848/2009) Govt. of India, India; Universities with Potential for Excellence (UPE Phase II; UH/ UGC/UPE Phase-2/Interface Studies/research projects/R-29) and University of Hyderabad-Institute of Eminence (UoH-IOE-RC3-21-020) funding received by RM.
Author contribution
PS carried out the experiments as part of her doctoral program under the guidance and supervision of the corresponding author, RM at UoH. PS carried out the experiments, data analysis, molecular screening, and drafting of the manuscript. RM has contributed to planning, supervising, obtaining funding for the research studies, providing inputs, suggestions, and correcting the manuscript.
Confict of interest
The authors declare there is no confict of interest