Anthracnose Resistance in Sorghum Breeding Lines Developed from Ethiopian Germplasm

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1 2010. Plant Management Network. This article is in the public domain. Accepted for publication 31 October Published. Anthracnose Resistance in Sorghum Breeding Lines Developed from Ethiopian Germplasm John E. Erpelding, USDA-ARS, Tropical Agriculture Research Station, Mayagüez, Puerto Rico Corresponding author: John E. Erpelding. Erpelding, J. E Anthracnose resistance in sorghum breeding lines developed from Ethiopian germplasm. Online. Plant Health Progress doi: /php rs. Abstract Ninety-nine dwarf and photoperiod-insensitive breeding lines developed from Ethiopian sorghum germplasm were inoculated with Colletotrichum sublineolum and evaluated for anthracnose resistance at the Tropical Agriculture Research Station in Isabela, Puerto Rico, during the 2008 and 2009 growing seasons. A resistant response was observed for 57 lines in 2008 and for 47 lines in 2009 with 44 lines showing a resistant response across growing seasons. These lines showed reddening of inoculated leaves and no acervuli. Variation in resistance across growing seasons and replications within a growing season was observed for 19 lines rated as susceptible and these lines also showed low disease severity. Mean disease severity for the susceptible accessions was similar across growing seasons with a mean of 26% for 42 lines in 2008 and 23% for 52 lines in The resistant lines showed phenotypic diversity in response to anthracnose, suggesting potential genetic variation for resistance. Background on Sorghum Anthracnose and Ethiopian Germplasm Sorghum anthracnose, caused by Colletotrichum sublineolum Henn., is found in most sorghum producing regions worldwide and reported grain yield losses range from 30 to 67% (1,13). symptoms are commonly observed on leaves, but the stalk, panicle, and seed can also show symptoms (7,12). Diagnostic signs of anthracnose are acervuli, asexual fruiting bodies, in the center of circular or elliptical lesions. The disease can be successfully managed using resistant varieties; however, the pathogen population is highly variable which reduces the longevity of resistant sources (8,14). New sources of resistance are needed and germplasm collections have been an important resource for resistance. However, the majority of sorghum germplasm from Africa is photoperiod sensitive and will not flower in the continental United States when planted during the summer growing season (6). This sensitivity to photoperiod limits the utilization of African germplasm for sorghum improvement in temperate regions. To overcome this limitation, the genes for photoperiod insensitivity have been backcrossed into African germplasm to develop new breeding lines (11). The development of these breeding lines was initiated in 1963 as a collaborative project between the United States Department of Agriculture, Agricultural Research Service (USDA-ARS) and the Texas Agricultural Experiment Station, Texas A&M University, Lubbock, TX. Population development was conducted in Puerto Rico during the winter growing season with selection of dwarf and photoperiod-insensitive lines conducted in Texas during the summer. The project developed 703 breeding lines from 1963 to 2004 (6). Since Ethiopia is an important center of diversity for sorghum, the photoperiod-insensitive breeding lines developed from Ethiopian germplasm were evaluated for resistance to anthracnose.

2 Anthracnose Field Evaluation Sorghum [Sorghum bicolor (L.) Moench] germplasm from Ethiopia is an important resource for the development of improved grain sorghum varieties and the USDA-ARS, National Plant Germplasm System maintains 108 breeding lines developed from Ethiopian germplasm (6). Seed samples for 99 lines were obtained from the USDA-ARS, Plant Genetic Resources Conservation Unit in Griffin, GA. The anthracnose evaluation was conducted at the USDA-ARS, Tropical Agriculture Research Station in Isabela, Puerto Rico, during the rainy season in 2008 and 2009 with the first experiment planted on 27 June 2008 and the second experiment planted on 6 April Eleven control genotypes representing diversity in anthracnose disease response were selected from previous evaluations. Five resistant control genotypes, NSL 4025 (Redlan), PI (SC110), PI (SC748), PI (SC30), and PI (SC1313), and six susceptible control genotypes, PI , PI , PI (SC170), PI (Sureño), PI (BTx623), and PI , were included in the evaluation. Four of the control genotypes, PI (SC110), PI (SC30), PI (SC170), and PI (SC1313), were also breeding lines developed from Ethiopian germplasm and were included as duplicate accessions in the evaluation. The germplasm lines were planted in single rows using a randomized complete block design with three replications. Each row was 1.8 m in length with 0.9-m row spacing. A border row of an anthracnose susceptible genotype (PI ) was planted around each experimental field and Lorsban 15G (chlorpyrifos, Dow AgroSciences, Indianapolis, IN, USA) was applied in the seed furrow at a rate of 1.2 kg a.i./ha during planting to prevent seed loss from fire ants. Weeds were controlled with mechanical tillage and hand hoeing. For the 2008 evaluation, no fertilizer was applied and the field was irrigated three times before inoculation using overhead sprinklers with 76 mm applied. In 2009, the field was irrigated six times before inoculation with 108 mm applied. Fertilization was required in 2009 and side-banded 39 days after planting at a rate of 560 kg/ha ( NPK). No irrigation was applied after inoculation for both experiments. Rainfall events occurred on 23 days before inoculation and on 32 days after inoculation in 2008 with a total of 88 mm of rainfall before inoculation and 441 mm after inoculation. The 2009 experiment received a total of 89 mm of rainfall before inoculation and 329 mm after inoculation with rainfall events occurring on 21 days before inoculation and on 41 days after inoculation. Ambient air temperature was similar for the experiments with a maximum temperature of 31 C and a minimum temperature of 17 C recorded in both 2008 and Mean temperature was 25 C in 2008 and 24 C in Inoculations were conducted by placing sorghum seed colonized by C. sublineolum in the leaf whorl of the plants for each line. Preparation of fungal cultures, field inoculation, and disease evaluation were as described by Erpelding and Prom (4). To represent the pathotypes in the pathogen population at the Isabela research station, leaf samples with disease symptoms were randomly collected from research plots and used to establish fungal cultures. Field inoculations were conducted 39 days after planting in 2008 and 37 days after planting in 2009 with disease evaluations conducted 30, 41, and 56 days after inoculation in 2008 and 14, 29, 40, and 62 days after inoculation in 2009, using a 1 to 5 rating scale. Resistant plants rated as 1 showed no disease symptoms and plants rated as 2 showed red spots or reddening of inoculated leaves and no acervuli. Moderately susceptible plants rated as 3 showed chlorotic lesions on inoculated leaves with acervuli in the center of the lesions. Susceptible plants rated as 4 showed necrotic lesions with abundant acervuli on inoculated leaves. Highly susceptible plants rated as 5 showed necrotic lesions with abundant acervuli and disease symptoms observed on noninoculated leaves including the flag leaf. severity was determined from a visual estimate of the percentage of leaf area with symptoms for susceptible plants in a row. All rows and all plants in a row were visually evaluated. However, the number of plants evaluated in each row varied, since seed germination was poor for some accessions. The number of plants within a row ranged from 5 to 30 with most rows having 20 inoculated plants. The disease severity from the final evaluation was used for statistical analysis. The Statistix 9 software package (Analytical Software, Tallahassee, FL, USA) was used for the

3 analysis of variance and mean comparisons were conducted using Tukey s standardized range test (P = 0.05). Response of Ethiopian Germplasm Lines Resistance to anthracnose was frequently observed for the breeding lines developed from Ethiopian sorghum germplasm (Table 1). In the 2008 experiment, 57 lines were rated as resistant and showed no acervuli on inoculated leaves. All lines that were rated as resistant showed red spots or reddening of inoculated leaves within 7 days after inoculation. The majority of the lines rated as susceptible showed acervuli on the flag leaf with 18 lines rated as highly susceptible across three replications. Four lines rated as susceptible showed a susceptible response for one replication and one line showed a susceptible response across two replications. Mean disease severity was less than 1% for the lines that showed variation in resistance across replications. For the 42 lines rated as susceptible in 2008, mean disease severity was 26%. Fifteen susceptible lines showed a mean disease severity greater than 25%. Mean disease severity was 48% for the 18 lines rated as highly susceptible with plant death observed for two lines, PI and PI , during the final evaluation. progression was rapid in 2008, and at 30 days after inoculation, 35 lines were rated as susceptible with 26 lines showing a susceptible response across three replications and four lines rated as highly susceptible (data not shown). Only one line of the 42 susceptible lines was not rated as susceptible within 41 days after inoculation, with eight lines showing a highly susceptible response across replications and 35 lines rated as susceptible across three replications. A lower frequency of resistant lines was observed in 2009 with 47 lines rated as resistant (Table 1). For the 52 lines rated as susceptible, 41 lines showed a susceptible response across replications with 10 lines showing a susceptible response for one replication and one line showed a susceptible response across two replications. Twenty-two lines showed a highly susceptible response across replications and plant death was observed for three lines, PI , PI , and PI , during the final evaluation. Mean disease severity was 46% for the 22 highly susceptible lines. For the 52 lines rated as susceptible in 2009, mean disease severity was 23% with 21 lines showing a mean disease severity greater than 25%. A susceptible response was observed within 14 days after inoculation for 38 lines, and 29 lines showed no variation in susceptibility across replications (data not shown). At 29 days after inoculation, 39 lines showed a susceptible response across replications with acervuli observed on the flag leaf for four lines. Three lines showed variation in susceptibility across replications, and 10 of the 52 susceptible lines showed no acervuli on inoculated leaves 29 days after inoculation. For the evaluation conducted 40 days after inoculations, nine lines were rated as highly susceptible across three replications, with 40 lines showing no variation in susceptibility across replications and seven lines showing variation for disease severity across replications. Five lines rated as susceptible for the final evaluation showed no acervuli on inoculated leaves within 40 days after inoculation and these lines showed a susceptible response in one replication during the final evaluation. All lines rated as susceptible in 2008 showed a susceptible response in 2009 with the exception of three lines that showed a resistant response in 2009 (Table 1). These lines showed variation across replications for susceptibility in For the 13 lines rated as susceptible in 2009 and resistant in 2008, nine showed variation in susceptibility across replications and mean disease severity was generally less than 1%. Thirty-nine lines were rated as susceptible across growing seasons and mean disease severity for the lines was 28% in 2008 and 30% in Lines that showed a low mean disease severity in 2008 generally showed a low mean disease severity in Approximately 54% of the susceptible lines showed acervuli on the flag leaf and were rated as highly susceptible. The disease rating and severity was as expected for the 11 control genotypes included in the evaluation (Table 1). The five resistant control genotypes, NSL 4025, PI , PI , PI , and PI , showed red spots or

4 reddening of inoculated leaves within 7 days after inoculation and no acervuli were observed on inoculated or non-inoculated leaves during the final evaluation. For the six susceptible control genotypes, PI , PI , PI , PI , PI , and PI , reddening of inoculated leaves was observed within 7 days after inoculation with acervuli observed on inoculated leaves within 14 days after inoculation. At 30 days after inoculation, acervuli were present on most leaves including the flag leaf, and by 40 days after inoculation, most of the susceptible control genotypes showed acervuli on the flag leaf across three replications with senescence of lower inoculated and noninoculated leaves. Mean disease severity was similar for the susceptible control genotypes across growing seasons with a mean of 53% in 2008 and 51% in Two genotypes, PI and PI , showed plant death during the final evaluation with extensive stalk breakage and lodging. Table 1. rating and mean disease severity (%) for the 99 breeding lines developed from Ethiopian germplasm and evaluated for anthracnose resistance during the 2008 and 2009 growing seasons in Isabela, Puerto Rico (continued). SC number x rating y severity z rating severity Accession w NSL 4025 RC PI SC h r PI SC h qr PI SC b-d o-q PI SC a-c j-o PI SC108 4\5\ a-c 4\5\5 8.3 a-d PI SC c-e e-l PI SC110 PI RC PI SC126 PI SC a ab PI SC171 PI SC173 PI SC e-g l-o PI SC e-g d-k PI SC226 PI SC227 2\2\4 0.3 a a PI SC6 PI SC a 2\2\4 0.3 a PI SC19 PI SC29 PI SC a-c 4\4\ a-f PI SC ab a-f PI SC44 PI SC112 PI SC ab a-c PI SC a-d 4\5\ b-i PI SC a 2\2\4 0.2 a

5 Table 1. (continued). Accession w (continued). SC number x rating y severity z rating severity PI SC165 PI SC166 PI SC167 PI SC224 PI SC557 PI RC PI SC508 PI SC gh r PI SC a ab PI SC a-d g-m PI SC a 4\5\5 3.7 ab PI SC a a PI SC e-g g-m PI SC7 PI SC a-c a-e PI SC a 2\4\4 0.1 a PI SC13 PI SC15 PI SC20 PI SC fg i-n PI SC23 4\4\ a-c a-f PI SC24 4\4\ b-d f-m PI SC28 4\5\ a-d 4\5\ a-g PI SC30 PI RC PI SC b-d c-j PI SC35 PI SC37 PI SC a-d 4\4\ a-h PI SC a 2\2\4 0.2 a PI SC e-g m-o PI SC ab a-d PI SC135 PI SC a 2\2\4 0.3 a PI SC137 PI SC139 PI SC141 4\5\5 7.0 ab c-j PI SC142 4\5\ a-c j-o PI SC146 PI SC155 PI SC a ab

6 Table 1. (continued). Accession w (continued). SC number x rating y severity z rating severity PI SC d-f k-o PI SC b-d i-n PI SC176 PI SC a a PI SC a-c a-f PI SC d-f g-m PI SC593 PI SC1014 PI SC982 4\5\ a-c d-k PI SC1039 2\2\4 0.2 a a PI SC gh k-o PI SC1046 2\4\4 0.3 a 2\2\4 0.1 a PI SC1049 PI SC1155 PI SC1033 PI SC1160 PI SC979 4\5\ a-c d-k PI SC ab 2\2\4 0.1 a PI SC1184 PI SC1154 PI SC a 2\2\4 0.3 a PI SC a 2\2\4 0.1 a PI SC1159 PI SC1040 2\2\4 0.3 a a PI SC a 2\2\4 0.1 a PI SC e-g n-p PI SC a 2\2\4 0.1 a PI SC f-h p-r PI SC1313 PI RC PI SC gh r PI SC1316 2\2\4 0.2 a a PI SC1317 4\4\ a-c 4\4\ a-e PI SC1177 PI SC1031 PI SC1158 PI SC a 4\4\5 1.0 ab PI SC1318 PI SC1319 PI SC1320 4\5\5 8.3 ab a-e PI SC d-f h-m

7 Table 1 (footnotes). w Plant introduction numbers (6) for the breeding lines, five resistant control genotypes ( RC), NSL 4025 (Redlan), PI (SC110), PI (SC748), PI (SC30), and PI (SC1313), and six susceptible control genotypes ( SC), PI , PI , PI (SC170), PI (Sureño), PI (BTx623), and PI Accessions are arranged in ascending order based on plant introduction numbers. x Sorghum conversion number (6,11). y rating is based on a 1 to 5 scale (4): 1 = resistant with no symptoms or signs of disease, 2 = resistant with red spots or reddening of inoculated leaves and no acervuli, 3 = moderately susceptible with chlorotic lesions and acervuli on inoculated leaves, 4 = susceptible with necrotic lesions and abundant acervuli on inoculated leaves, and 5 = highly susceptible with necrotic lesions and abundant acervuli on inoculated leaves as well as non-inoculated leaves including the flag leaf. Data for the three replications are presented for accessions showing variation across replications. No plants received a rating of 1 or 3 in 2008 or z Mean disease severity was determined from a visual estimate of the percentage of leaf area with disease symptoms in a row and averaged across the three replications. Numbers followed by the same letter are not significantly different based on Tukey s standardized range test (P = 0.05). Potential of Anthracnose Resistant Ethiopian Germplasm Ethiopia is an important center of genetic diversity for sorghum and several photoperiod-insensitive lines developed from Ethiopian germplasm have been widely used in grain sorghum breeding programs in the United States. Ethiopian germplasm may also provide genetic diversity for host-plant resistance to anthracnose. In this study, 44 lines showed anthracnose resistance. These lines showed reddening of inoculated leaves with no acervuli observed on inoculated or non-inoculated leaves. This high frequency of resistance suggests that Ethiopian germplasm could be an important source of anthracnose resistant accessions. Chala et al. (2) also identified resistant germplasm from Ethiopia and indicated that Ethiopia is an important source of resistance for sorghum improvement. However, the selection of Ethiopian germplasm for the development of these breeding lines may have also favored the selection of accessions with anthracnose resistance. A high frequency of resistant germplasm was also reported for the Mali and Sudan breeders working collections with approximately 80% of the Malian accessions and 47% of the Sudanese accessions rated as resistant (3,5). These collections were established to represent approximately 10% of the accessions in the base collection with the selection of accessions based on breeding potential and phenotypic diversity. Further evaluation of Ethiopian germplasm will be necessary to determine the overall frequency of anthracnose resistance for the collection. Additionally, 22 lines rated as susceptible showed low disease severity, less than 6%, with variation in susceptibility within and between experiments generally observed. Based on the disease rating scale of Pande et al. (10), these lines may also provide a source of resistance. Furthermore, using this rating scale, 12 lines would be rated as moderately resistant, nine as susceptible, and 12 as highly susceptible. This rating system would classify 78 lines as resistant. Overall, the majority of the susceptible lines showed disease symptoms on the flag leaf. The observed variation in disease response for the breeding lines would suggest genetic diversity for resistance that would benefit sorghum improvement. Mehta et al. (9) evaluated three of the breeding lines, PI (SC166), PI (SC137), and PI (SC155), and indicated each line possessed a unique gene conferring resistance. Additionally, this diversity in resistance for the breeding lines could be used to evaluate variation in the pathogen population. Disclaimer Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the United States Department of Agriculture.

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