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MOLECULAR AND BIOLOGICAL STUDIES ON SUGARCANE SMUT

Ayman M.H. Esh (aymanesh@gmail.com)
Molecular Biology Department, Genetic Engineering and Biotechnology Research Institute, Sadat City University
June, 2013
 

Abstract
A molecular pathological study was conducted on sugarcane smut disease in Egypt. The study concerned the isolation, identification and diversity of the causal organism sugarcane smut from different sugarcane production areas in Egypt. Molecular methods, Internal transcribed region (ITS), b-East mating type gene and RAPD-PCR were used to investigate the genetic polymorphism between the twenty Ustilago scitaminea isolates. The obtained results revealed that, the combined data clustering analysis of the RAPD-PCR assigned the tested 20 isolates into two main groups, one of them included only one isolate (HAQ-1) and the other included 19 isolates. RAPD grouped most of the isolates according to its sampling places, indicating high diversity of U. scitaminea in Egypt. Alignment of the internal transcribed spacer rDNA (ITS) of 20 isolates of U. scitaminea depicting 100 % similarities between all the tested isolates except isolate (ADA-1), which has one base difference in the ITS2 region and scored 99.87% similarity with the other tested isolates. The obtained two sequences of U. scitaminea internal transcribed spacer sequence of isolates ADA-1 and HAQ-2 was submitted in NCBI Gene bank under the accession number JQ912111 and JQ912112 respectively. The Egyptian U. scitaminea isolate ADA-1 showed a close distance to Reunion isolate while the second Egyptian isolate HAQ-2 was much closer to China isolate. Blast and multiple sequence alignment of the b-East mating type gene sequences of the 20 Egyptian isolates were found identical to Sporisorium scitamineum b-East mating-type gene published in Gene-Bank from China.
Induced mutation in sugarcane resistant (GT-54-9) and susceptible (NCo310) varieties through the use of gamma radiation was used to study the mutation effects on improving sugarcane and to study molecular markers associated with smut resistance. Doses of Gamma radiation (0.5, 1, 2, 3 kr) were used to induce mutagenesis in sugarcane. The radiation treatments caused a high significant variation in all Agronomical characteristics among the GT54-9 and NCo310 mutants. A significant increase in mean values was observed for most of the quality characters as compared to the untreated GT54-9 and NCo310 control. The normal and transformed percentage of smutted stools of GT54-9 (257 mutants) and NCo310 (32 mutants) artificially inoculated (dipping and injection inoculation) with smut spores for 3 seasons were field evaluated. GT54-9 mutants were grouped into 5 groups according to the transformed percentage of infection i.e., group 1 (0 -1%, 38 mutants), group 2 (>1-5%, 29 mutants), group 3 (>5-20%, 71 mutants), group 4 (>20-40%, 65 mutants), group 5 (>40%, 55 mutants). The untreated mother cultivar was placed in group 3 (5-20% infection). The results revealed the importance of direct mutagenic treatment and selection for improvement of commercial sugarcane varieties in Egypt.
PR proteins changes were monitored in healthy and artificially infected plants. The obtained results showed the role of these enzymes in infected and non-infected clones of the susceptible and resistant mutants. A dramatic increase in the activity of the tested enzymes phenylalanine ammonia lyase, peroxidase, esterase and chitinase was noticed in the resistant infected plants compared to the susceptible infected plants and to the moderately resistant control. Generally, the levels of the tested enzymes in the healthy susceptible plants were lower than those recorded in the resistant or the moderately resistant control.
Of the 89 RAPD primers screened, only four revealed polymorphism (difference), i.e., OPA11 and OPL-14, OPN-6 and OPAM-14 between the two DNA bulks of resistant and susceptible mutants generated from sugarcane variety GT54-9. Of the four Polymorphic RAPD primers only one primers (OPL14) was able to distinguish between the resistant and susceptible sugarcane mutants. The primers OPL14 amplified a 600bp (Approx.) band in resistant mutants. Sequencing of the extracted resistance marker bands showed high homology 98.09% between the resulted sequences. Mega blast-n searches in NCBI showed 98% maximum identity with Saccharum hybrid accession number JN800059. The used SSR (27 primer) and ISSR (20 primer) couldn’t distinguish between the resistant and the susceptible clones. Of the twelve AFLP primer combination, one primer combination (ACC/CTG) showed differences between resistant and susceptible sugarcane mutants. While the other tested primer combination couldn't distinguish between them.