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An Improved Screening Method for Evaluation of Resistance to Phytophthora Blight in Sesame Cultivars and Accessions
Plant Breeding and Biotechnology 2018;6:305-308
Published online September 1, 2018
© 2018 Korean Society of Breeding Science.

Eunyoung Oh1,*, Sungup Kim1, Sovetgul Asekova1, Young Nam Yoon1, and Jeong-Dong Lee2

1Department of Southern Area Crop Science, NICS, RDA, Miryang 50424, Korea, 2School of Applied Biosciences, Kyungpook National University, Daegu 41566, Korea
Correspondence to: *Eunyoung Oh, lavondy10@korea.kr, Tel: +82-55-350-1232, Fax: +82-55-353-3050
Received August 2, 2018; Revised August 17, 2018; Accepted August 17, 2018.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Phytophthora blight causes serious yield losses and deterioration of quality in sesame. In this study, we modified the screening method for enhancing the evaluation efficiency of Phytophthora blight resistance. The inoculum density and seedling stage were adjusted for large-scale screening. The optimal stage for inoculation was the seedlings grown to emerged-first-true leaves. At the density of 1 × 104 to 1 × 105 zoospores per milliliter, susceptible cultivars showed disease response within seven days. To verify the screening method, we assessed sesame cultivars and accessions for Phytophthora blight resistance, and varied resistance levels such as resistant, moderate and susceptible reactions were observed.

Keywords : Phytophthora, Sesame, Resistant, Screening
INTRODUCTION

Phytophthora blight by Phytophthora nicotianae causes serious yield losses and deterioration of quality in sesame. Previous studies had reported several evaluation methods of Phytophthora blight resistance. Choi et al. (1987) reported the soil-drenching screening method, while Yeh et al. (1991) reported the foliar spraying inoculation. Among the nursery stage, 20-day-old seedlings (Chae and Park 1992) and 30-day-old seedlings (Park et al. 2004) were used for the screening in earlier studies. Based on the findings of previous studies, there was still loopholes and need to be modified the screening methods along with seedling stage and inoculation conditions to screen a large number of plants and to produce a reliable and reproducible resistant data. Herein this study, we developed a screening method by modifying the inoculum density and seedling stage.

MATERIALS AND METHODS

Plant and pathogen materials

For the development of screening method, we used four sesame cultivars, ‘Goenbaek’, ‘Yangbaek’, ‘Dodam’, and ‘Milsung (Shim et al. 2013)’. We treated four different inoculum densities from 1 × 103 to 1 × 106 zoospore/mL by ten-fold increase. We also tested four different seedling growth stages, cotyledons (VC), first true leaves emerged (V1), second true leaves emerged (V2), and third true leaves expanded (V3). Each treatment consisted of 20 plants and inoculated by isolate KACC.48120. For the evaluation of Phytophthora blight resistance, 68 sesame cultivars (Supplementary Table S1), and 73 germplasm (Supplementary Table S2) were screened by four isolates, which were collected from infected sesame plants in Gyeongju, Naju, Gunwi, and Yecheon (KACC.48120, 48121, No.2526 and 2040, respectively).

Inoculum preparation and screening

The preparation of inoculum followed the protocol of Ahonsi et al. (2007). We inoculated two pieces agar plugs to V8 broth and kept at 28°C for five days in the dark (Supplementary Fig. S1). Then, the medium was drained and mycelia were rinsed with distilled water. The unsealed dishes were incubated at 23°C under fluorescent for seven days. After adding distilled water, the dishes were placed in 4°C and in room temperature for 30 minutes, then the suspension was filtered by cheesecloth. Five mL of inoculum was soil-drenched on each cell using tube-connected dispenser. After inoculation, seedlings were irrigated to make the saturated soil moisture using the reservoir. Disease severity (DS) was estimated by index 0 to 9 (0: no symptom, 1: slight wilting of leaves, 3: wilt and minor blight on leaves, 5: minor discoloration in stem base, 7: clear discoloration of stem and wilting, 9: damping off and withering). The average DS of 0 – 3, 3.1 – 5, 5.1 – 7, and 7.1 – 9 was regarded as resistant (R), moderate resistant (MR), moderate susceptible (MS), and susceptible (S), respectively.

Modified screening method from this study

This screening method for Phytophthora blight resistance is following. (1) Seedling was grown about V1 stage; (2) Inoculum density was adjusted to 1 × 104 – 1 × 105 zoospore per mL and 5mL of zoospore suspension was soil-drenched; (3) After the inoculation, soil water should be maintained to be saturated; and (4) Disease severity is rated seven days after inoculation.

RESULTS AND DISCUSSION

Modification of screening method for the Phytophthora blight resistance

Based on DS of cultivars, four cultivars were divided into resistant and susceptible groups in both experiments (Table 1). Goenbaek and Dodam did not show any disease reaction throughout all treatment, whereas Yangbaek and Milsung resulted in the high DS throughout all treatments (Table 1). Therefore, we decided the adequate screening conditions by the results of two susceptible cultivars (Table 2). On day three, DS went higher on the seedlings inoculated with high-density of inoculum. Likewise, on day seven, 1 × 106 zoospore/mL (T4) showed the highest DS followed by 1 × 104/mL (T2) and 1 × 105/mL (T3), while T1 showed the lowest DS. All inoculated densities except T1 rated over eight, which were regarded as susceptible, therefore, the suitable inoculum densities were remained for three densities except for T1. We decided to inoculate zoospore suspension adjusted between T2 to T3 to evaluate the resistance within seven days after inoculation. Even though the highest inoculum density of T4 showed significance for screening (Table 2), we considered T4 as not suitable for large-scale screening for the reason of inoculum preparation.

Similarly, two susceptible cultivars showed significantly low DS when seedling was older at the seedling-growth-stage experiment (Table 2). On day seven, almost seedlings of both cultivars showed the DS close to nine, regardless of the seedling stage. Therefore, we selected V1 stage, which was curtailed from 30-day-old seedlings from the previous report of Park et al. (2004). While VC was inadequate for screening because of feebleness and comparably short hypocotyls to observe the severity.

Evaluation of sesame cultivars and accessions for Phytophthora blight resistance

Among 68 cultivars, nine cultivars were resistant to four tested pathogen isolates, while 45 cultivars were found to be susceptible. The remaining 14 cultivars showed variable responses against the isolates (Supplementary Table S1). In the case of 73 sesame accessions, nine accessions showed resistance to four isolates, 35 accessions showed susceptibility, while the rest of 29 sesame accessions showed a variable response by different isolates (Supplementary Table S2). Some of sesame cultivars and accessions demonstrated the diversity of disease response by different isolates. These discrepancies in resistance might suggest the possibility of diverse interaction between pathogen and host as it had been reported in other Phytophthora species (Vleeshouwers et al. 2000; Mo et al. 2014), or the diversity of the resistant gene from different genetic sources. This modified method showed some of high screening efficiency of the Phytophthora blight resistance that was required about 20 days from sowing to evaluation. It also will provide the reliable data, which could be used for the selection, bioassay, and the further genetic studies.

ACKNOWLEDGEMENTS

This study was conducted with the grant of the Agenda Project (Project No. PJ01253601) of Rural Development Administration, Republic of Korea.

Supplementary Information
Tables

Effect of inoculum density and seedling stage on the reaction to Phytophthora blight in four sesame cultivars.

CultivarsDAIz)Inoculum DensitySeedling Stage


T1y)T2T3T4LSD0.05Vcx)V1V2V3LSD0.05
Goenbaek30.0w)0.00.00.0ns0.10.100.2ns
70.00.00.00.0ns0.7 a0.0 b0.3 b0.0 b0.33
Yangbaek32.2 b2.7 b3.4 b6.4 a1.339.0 a9.0 a8.5 b6.2 c0.47
77.8 b7.8 b8.2 ab8.9 a0.759.09.09.08.8ns
Dodam30.00.00.00.1ns0.10.00.00.0ns
70.00.00.00.1ns0.8 a0.3 b0.1 b0.3 b0.45
Milsung35.1 b6.1 b7.9 a8.2 a1.359.0 a9.0 a9.0 a7.0 b0.25
77.4 b8.5 a8.4 a8.7 a0.549.09.09.09.0ns

Rows followed by the same letter were not significantly different (ANOVA, LSD P ≤ 0.05).

z)Days after inoculation.

y)T1 to T4 corresponds to 1 × 103, 1 × 104, 1 × 105, and 1 × 106 zoospore/mL, respectively.

x)VC: cotyledons, V1:first true leaves emerged, V2: second true leaves emerged, V3: third true leaves expanded.

w)Average disease severity: 0 = no symptom, 1 = slight wilt of leaves, 3 = wilt and minor blight on leaves, 5 = minor discoloration in stem base, 7 = clear discoloration of stem and wilting, 9 = damping off and withering.


Average severity for Phytophthora blight to different inoculum density and seedling stage in cv. Yangbaek and cv. Milsung.

FactorsTreatment3 DAIz)7 DAI
Inoculum densityT1y)3.3 cx)7.7 c
T24.1 c8.2 b
T35.6 b8.3 b
T47.4 a8.8 a
LSD0.050.940.46
Seedling stageVCw)9.0 a9.0
V19.0 a9.0
V28.8 a9.0
V36.5 b8.9
LSD0.050.27ns

Column followed by the same letter were not significantly different (ANOVA, LSD P ≤ 0.05).

z)Days after inoculation.

y)T1 to T4 corresponds to 1 × 103, 1 × 104, 1 × 105, and 1 × 106 zoospore/mL, respectively.

x)Average disease severity: 0 = no symptom, 1 = slight wilt of leaves, 3 = wilt and minor blight on leaves, 5 = minor discoloration in stem base, 7 = clear discoloration of stem and wilting, 9 = damping off and withering.

w)VC: cotyledons, V1: first true leaves emerged, V2: second true leaves emerged, V3: third true leaves expanded.


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