Project : USDA ARS

ARS Home » Southeast Area » Canal Point, Florida » Sugarcane Field Station » Research » Research Project #432517

Research Project: Identification of Resistant Germplasm and Markers Associated with Resistance to Major Diseases of Sugarcane

Location: Sugarcane Field Station

2021 Annual Report

Objectives
1. Identify disease resistant sugarcane and energy cane clones for high yielding commercial production. 2. Develop methodologies to efficiently screen germplasm for resistance and to identify new molecular markers associated with resistance. 3. Identify pathogenic variation in sugarcane pathogens that are endemic and emerging within the United States.

Approach
Objective 1: Sugarcane clones in the cultivar development program for both sucrose and bio-energy will be screened for their disease reaction in artificial inoculation tests separate from the cultivar development plots with the major pathogens and ratings will be determined based on incidence and severity of disease. Objective 2: For Sugarcane Yellowleaf Virus (SCYLV) existing markers will be tested against our known historical clones having both resistance and susceptible reaction. Two new populations will be screened for SCYLV resistance and genotyping by sequencing (GBS) to identify new markers. For ratoon stunt disease and smut two populations will be screened for disease reaction and GBS to identify markers. For orange rust we will test published markers and perform fine mapping using a larger population and whole genome sequencing of the parents and bulked progeny to identify markers for screening the whole population. Objective 3: Orange rust spores have been collected over the last two years from different cultivars and locations in Florida and stored for determining possible pathogenic variation. Specifically, cultivars CP 80-1743 (the susceptible cultivar first observed with orange rust in Florida), CL 85-1040 (susceptible) and two cultivars CP 88-1762 and CP 89-2143 that were originally resistant but became susceptible will be used to evaluate pathogenic variation. The whorl inoculation technique (Sood et al. 2009) that is routinely used to evaluate brown and orange rust reactions will be used to evaluate variation between the isolates. The whorl technique has been shown to give consistent results, as seen by the sample data in Table 2 of the Appendix. These will be used along with the new collections described. Sentinel plots of orange rust susceptible and resistant sugarcane cultivars will be planted at 5 different locations and from grower’s fields where the “breaking down” of resistance is reported. Fields will be observed monthly from March to July when orange rust is most prevalent. Orange rust pustules that develop on previously resistant cultivars will be collected and compared using the whorl inoculation techniques to isolate the collected spores from different cultivars differing in susceptibility over several years. Mass rust spore collections from the field will be inoculated on plants of the same cultivar twice as a means to help purify the isolate. Variation in symptom development of isolates on specific cultivars will be used to identify pathogenic differences. Unfortunately, pathogen variation will be limited to isolates from Florida because isolates from outside the state are prohibited from introduction.

Progress Report
Brown and orange rusts, mosaic, ratoon stunting disease (RSD), smut, and leaf scald cause significant losses to total yield in susceptible varieties during favorable environmental conditions. Thus, the Canal Point (CP) cultivar development programs (CP programs) screen its germplasm for resistance to these diseases. Data are obtained in natural infection and inoculated trials ensuring that disease resistant or tolerant clones are advanced and released from the program. Marker assisted selection along with phenotypic evaluation of disease resistance will benefit the CP program therefore quantitative trait loci associated with orange rust resistance as well as with brown rust resistance, RSD and yellow leaf virus (SCYLV) have been identified. Diagnostic markers for orange rust resistance have been developed. To find QTLs for other diseases, ARS scientists in Canal Point, Florida, have inoculated leaf scald, RSD and smut pathogen in a population from a cross of CP 72-2086 and CP 01-2390 and collected the phenotype data. Marker-assisted selection along with the phenotypic evaluation of disease resistance will be beneficial for the CP program therefore QTL associated with orange rust as well as with brown rust and SCYLV resistance have been identified by ARS scientists in Canal Point, Florida. The developed diagnostic markers for orange rust resistance are being used in the CP program. To detect QTL for other diseases, ARS scientists have collected leaf scald (two years), and smut (one year) disease infestation data from the field and pot trials of a population originated from a cross of CP 72-2086 and CP 01-2390. ARS scientists couldn’t collect all the anticipated phenotype data due to pandemic restriction. ARS scientists have analyzed the genotype data including marker filtering for mapping and QTL analysis. Seedlings of the population derived from a reciprocal cross between CP 01-2390 and CP 72-2086 were planted for seed increase. However, ARS scientists were not able to collect leaf tissue, extract DNA, fingerprint for population segregation analysis, and complete planting for new field trial for validation. ARS scientists in Canal Point, Florida, have received RNA-sequencing data and mapped in Sorghum genome for identifying the candidate genes associated with orange rust. ARS scientists are trying to align and map sequencing data to new sugarcane reference genome LA-purple for further investigation. In conjunction with the RNA-sequencing experiments in order to make new fine mapping population, ARS scientists have planted the two lines (one susceptible another resistant to orange rust) in the crossing parental lines for making cross this year. ARS scientists were unable to test the effectiveness of the SCYLV reported markers by Debabakas et al. (2014) using our historical population as the corresponding author did not respond to our request to provide the marker sequence. However, ARS scientists were able to analyze QTL associated with SCYLV resistance using another two populations derived from crosses CP 95-1039 X CP 88-1762 and Green German X IND81-146. SCYLV phenotype data will be collected from the population of CP 72-2086 X CP 01-2390 after an exposure to natural infection of the SCYLV for an extend period of time (at least six years). Pathogenic and genetic variations occur over time in the pathogen populations. Emerging and re-emerging diseases pose additional threat to CP program. For example, sugarcane orange rust appeared in Western hemisphere in 2007 and affected the Florida sugarcane industry as well as CP cultivar development program. Therefore, developing resistance is a continuous effort in the CP programs by identifying the strains and races of pathogens with genetic and pathogenic variations and including them in the artificial inoculations. Genomic DNA and RNA were extracted from spores of Puccinia kuehnii collected from sugarcane cultivars CL 85-1040 and CP 89-2143. A draft genome has been created. The two rust isolates showed pathogenic variation. Pathogenic variation was also observed in the sugarcane mosaic virus and it was identified in the eight species of Poaceae family.

Accomplishments
1. Development of disease-resistant cultivars. Canal Point (CP) cultivar development programs have been releasing disease resistant high yielding cultivars for Florida sugarcane industry. Thus, all susceptible clones in the seedling, Stage I, and Stage II are eliminated if exhibiting disease symptoms based on natural infection. In two CP cultivar development programs (i.e., CP program for organic (muck) soils and CP program for mineral (sand) soils), clones in both the muck and sand programs were screened annually in inoculation tests at, Stage III increase (40 clones) and Stage IV (13 clones) for their resistance to ratoon stunt, smut, brown rust, orange rust, leaf scald and mosaic. Screened clones in Stages I and II for resistance to rusts, mosaic, leaf scald and smut by natural infection. Advanced clones with acceptable resistance levels to later stages of the programs. Released high-yielding and disease resistant or tolerant clones in the Stage 4 increases for commercial cultivation. In 2021, the Florida Sugarcane Variety Committee released five new cultivars CP 14-1377, CP14-1490, CP14-1934, CP13-4474, and CP13-4513. Of these new cultivars, the first three are for the Florida muck soils and the last two are for the Florida sand soils. Scientists at ARS Canal Point, Florida, with collaboration of University of Florida and Florida Sugar Cane League developed these cultivars. The resistant CP cultivars have contributed greatly in a sustainable production of sugarcane in Florida. Also, these disease resistant high yielding sugarcane cultivars help Florida sugarcane growers to economically grow sugarcane and produce approximately 20% of the sugar consumed in the United States.

2. Quantitative trait loci (QTL) associated with disease resistance. QTL associated with orange rust resistance, brown rust resistance and SCYLV resistance have been identified. Diagnostic markers have been developed for orange rust resistance. RNA sequencing data has been received and mapped against Sorghum reference genome. ARS scientists in Canal Point, Florida, are doing further aligned and mapped the sequence data to new sugarcane reference genome LA purple. ARS scientists in Canal Point, Florida, identified 217 nonredundant markers and 225 candidate genes to be significantly associated with the yield traits, which can serve as a comprehensive genetic resource database for future gene identification, characterization, and selection for sugarcane improvement. This study also suggests that genomic inbreeding has led to negative impacts on sugarcane yield. Identified quantitative trait loci associated with ratoon stunting disease. A total of 82 disease resistance genes were identified by searching 23 QTL on the Sorghum bicolor genome (44 genes), sugarcane R570 genome (20 genes), and S. spontaneum genome (18 genes).

3. Pathogenic variation. With the help of collaborators, ARS scientists in Canal Point, Florida, discovered genetic variation in sugarcane mosaic virus (SCMV) at Canal Point, Florida, assessed the virulent pattern of the SCMV genetic strains from Sugarcane, St. Augustine grass and Columbus grass. This information will be useful to develop mosaic resistant cultivars. The isolate from St. Augustine grass and sugarcane can cross infect each other. SCMV has been a threat to lawns in south Florida and can re-emerge as a threat to sugarcane as a different pathogenic strain. Pathogenic variation in orange rust pathogen, P. kuehnii, has been found and studied. The first genome assemblies for two isolates (1040 and 2143) of P. kuehnii from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively were generated and will be of immense value for future genomic studies.

U.S. DEPARTMENT OF AGRICULTURE

Sugarcane Field Station: Canal Point, FL