Evaluation of inoculation techniques and screening markers for smut (ustilago scitaminea syd) resistance in sugarcane

Abstract

Sugarcane smut 111 Kenya cause severe losses in susceptible varieties. Seedling smut screening may select for sugarcane genotypes with physiological resistance, and may therefore be of great use to the industry because genotypes with physiological smut resistance are sought for commercialization. This study was conducted in the green house first to assess the feasibility of inoculating sugarcane (Saccharum spp) seedlings with smut (Usti/ago scitaminea syd), to evaluate different inoculation methods and secondly to screen DNA markers for smut resistance. Crosses involving two populations emanating from resistant and susceptible varieties was undertaken at Sugarcane Breeding Center- Mtwapa(Mombasa).To investigate the reaction of seedlings to smut, three different inoculation methods were employed. The first method involved soaking seedlings in smut spore suspension at a concentration of 4 x 106 spores/ml for 30 minutes. The second method involved wounding the seedlings at the bud with a scalpel then applying a paste of smut made at a concentration of 2 grammes of spore for 2 ml of sterile water. The third method was a paste method that involved a paste of smut at the seedling buds. Each treatment had 30 entries (seedlings) planted in plastic bags in the glass house. Two controls of the un-inoculated progenies were included. The experiment was randomized complete block design replicated three times. There was no significant difference in whip production between the two populations. Population Co 331 X Co 945 had near significant results with wound paste method leading on whip production followed closely by paste method. Inoculation had significant effect on seedlings survival across four months under observation. There was significant difference in tiller production. There was significant difference in whip production between the two families at three months. The study recommends screening for smut resistance at first stage of selection to assess seedlings reaction to smut and to avoid carrying large numbers of clones that are eventually discarded at the advanced stage of selection. To screen molecular markers for smut resistance, tissues of the seedlings were harvested and DNA extracted, quantified and electrophoresis performed. DNA extraction method using lyophilized leaves was better than sap extraction method in terms of DNA quantity and quality. Sugarcane tissue gave a lot of DNA. The primers used in the experiment were obtained from SUCEST database with expected homology of protein like Kinase. Another primer XLRR was designed from conserved motifs of LRR, NBS and kinase domain. The pnrners were synthesized by the oligo sequencing unit at the International Livestock Research Institute (lLRI). The concentration of primers used in this study was between 200-300ng. DNA templates of 30ng gave fairly good PCR products with clear bands. Only one hot- start PCR temperature profile with a ramped decrease in annealing temperature was necessary to display amplicons of high quality for the primer pairs tested. The amplicons in the figures presented in this study represent the presence of segment that contains resistance gene analog. Empty wells without any band indicate there was no amplification and therefore the gene analog was absent. The bands were scored as present or absent depending on their intensity and clarity. DNA extracted from plants that exhibited smut whips phenotypically did not produce any band when PCR was done. Every primer pair produced scorable loci in the progeny analyzed. The size of the product from primer SCB 07 and SCC09 was around 800bp. However the size of amplification products from primer XLRR was near the expected size 900bp. Primer SCC09 produced very clear bands.