Fingerprinting of Ustilago Scitaminea (Sydow) in Egypt Using Differential Display Technique: Chitinase Gene the Main Marker

13Abd El Fattah, A.I., Alamri, S, 2R.A.I. Abou-Shanab and 2E.E. Hafez

1Department of Sugar Crops Pests and Diseases, Sugar Crops Research Institute, (SCRI), Agricultural Research Center (ARC), 9 Gamaa street, Giza, 12619, Egypt. 2Mubarak City for Scientific Research and Technology Applications, P.O. 21934 New Borg El Arab City, Alexandria, Egypt 3Dept. of Biology, College of Science, King Khalid Univ., Abha P.O.Box 9004, Saudi Arabia.

Abstract: The sugarcane smut disease caused by Ustillago scitaminae causes the greatest yield losses and is the most difficult to control through producing host genetic resistance, in part because the pathogen occasionally produces a new race that overcomes prior host resistance. In the smut fungi, few features are available for use as taxonomic criteria (spore size, shape, morphology, germination type, and host range). DNA-based molecular techniques are useful in expanding the traits considered in determining relationships among these fungi. Approximately 17 smut infected samples from five different sugarcane cultivars were collected from different governorates in Upper Egypt. Only 12 fungal isolates were succeeded to grow on Potato Dextrose Agar (PDA), the mycelium of each isolate was subjected to RNA extraction, followed by differential display technique to classify these isolates. Determination of the identity of sugarcane smut races and maintenance of resistant commercial varieties has proven to be difficult. So, it is essential to know the fungus race to find the suitable tools for controlling. For that reasons this study focusing on the scanning of the fungal mRNA, especially the one of the functional genes like chitinase gene. This study concluded that chitinase genes are the most suitable for genotyping study between fungal isolates. Differential display had the capability to demonstrate the changes carried out in fungal genome and could be a good substitute to the DNA sequence for fingerprinting of Ustillago sp., because of it is so easy, achieved in no time, cheep and impressive.

Key words: Smut disease, Sugarcane, chitinase gene, fungi, differential display

INTRODUCTION between species[5]. Determination of the identity of sugarcane smut races and maintenance of resistant Smut disease of sugarcane, caused by the fungus commercial varieties has proven to be difficult. Ustilago scitaminea (H and P Sydow), can cause Sugarcane resistance to U. scitaminea appears to be the considerable yield losses and reduction in sugarcane result of several characteristics and is probably quality[1]. Sugarcane smut was first reported in South determined by a number of genes[6, 7]. In addition, Africa in 1877, and many observations were made in commercial sugarcane varieties are polyploid hybrids of Africa and Asia in the following decades[2, 3]. Sugarcane several Saccharum species. Genetic resistance in this smut was observed for the first time in the Ord River case does not follow the strict gene-for gene pattern as Irrigation Area of Western Australia in July 1998. seen in some pathogen-host interactions, although Australian sugarcane cultivars are currently being differences in variety susceptibility to different smut screened in Indonesia to obtain smut resistance ratings, isolates have been reported[8, 9, 10]. and results show that 70% of Australian cultivars are Successful disease management strategies and susceptible[4]. As the use of resistant cultivars is the development of resistance requires an understanding of best option for long term control of smut a high variation in the pathogen population. While neutral priority of Australian sugarcane breeding programs is genetic markers such as AFLPs may not detect changes to increase the level of smut resistance in commercial in pathogenicity genes such markers can track changes cultivars. in pathogen populations across geographic areas and Ustilago scitaminea as well as other Ustilago over time, determine whether resistance screening trials species readily hybridize between races and even are being done in the appropriate location, and reveal

Corresponding Author: Saad A. Alamri, Department of Biology, College of Science, King Khalid University, Abha P.O. Box 9004, Saudi Arabia. E-mail: [email protected]

8 Res. J. Agric. & Biol. Sci., 6(1): 8-13, 2010 evidence of pathogen genotype by host genotype using arbitrary primers, chi 25 (5- GAY TTR GAT interactions[11]. TGG GAA TAY CC -3), chi 15 (5- GGY GGY TGG Mycoparasitic Trichoderma species secrete AAT GAR GG-3), A2 (5- GAA ACG GGT GGT GAT chitinases and glucanases enzymes that attack cell wall CGC-3) and A4 (5-GGA CTG GAG TGT GAT CGC- polymers in other fungi and have been exploited in the 3). The PCR reaction was performed as following; a 25 development of bio-control strategies[12]. In other µl reaction mixture containing; 2.5µl 10x Taq DNA studies, differential display (RNA fingerprinting) has polymerase buffer {10 mM Tris HCl (pH 8.3), 25 mM been used to identify transcripts that are aberrantly KCI}, 2.5 µl 50mM MgCl2, 2µl primer (40 Pmol/µl), regulated in human tumors[13, 14], differentially expressed and 0.25µl of Taq polymerase (AmpliTaq, Perkin- during mouse brain development[15], or differentially Elmer, 5u/µl), 2.5 µl from the cDNA, 2.5 µl dNTPase [16] expressed during peroxide stress in Salmonella . 4mM and 12.75 µl of dH2O. The PCR reaction was The aim of this study is genotyping screening of performed in 9700 thermal cycler (Perkin-Elmer) and the races of Ustilago species in Egypt using the the PCR conditions was performed as following: initial chitinase gene primers. denaturation 95°C for 5 min, followed by 40-cycles (94°C for 1 min.; 53°C for 1 min. and 72°C for 2 min; MATERIALS AND METHODS final extension, 72 oC for 10 min). Percentage of similarity between the 12 smut fungal isolates based on Samples Collection: A total of 17 smut infected the differential display band pattern was carried out samples from five different sugarcane cultivars were using 3 random primers according to[20]. The results collected from Giza, El-Mania, Souhag, Qena and were analyzed according to[21, 22]. Aswan Governorates in Upper Egypt (Table1). RESULTS AND DISCUSSIONS Fungal Isolation and Identification: Teliospores were obtained by shaking the whip on a sterilized plastic Results: A total of 17 smut infected samples from five sheet. Fungal spores were suspended for 24 hours in different sugarcane cultivars were collected from Giza, 1.5% copper sulphate solution for sterilization[17]. A El-Mania, Souhag, Qena and Aswan Governorates in loop of fungal suspension was streaked on Petri plates Upper Egypt. A total of 12 Ustillago scitaminae fungal containing (PDA) and then incubated at 30NC for 10 cultures were isolated from these different smut days. Tips of fungal mycelium were taken and infected sugarcane cultivars. Three, one, two, five and transferred to tubes containing PDA medium, incubated one isolates were collected from Giza, El-Menia, at 30oC for 10 days after that kept in a refrigerator at Souhag, Qena and Aswan Governorates, respectively as 4oC as stock cultures for further studies. To identify the shown in Table 1. fungal isolates, teliospores were microscopically DNA-based molecular techniques are useful in examined and measured according to their expanding the traits considered in determining morphological characteristics using Carl zeies eyepiece relationships among these isolates. This work was micrometer as described by[18](1952). conducted to determine the variability and distribution of sugarcane smut races in Egypt by differential display Molecular Characterization of Ustillago Scitaminae: technique using chitinase gene as specific primers and RNA Extraction and cDNA Synthesis: Total RNAs another 20 mers arbitrary primer as were used in this of the fungal spores were prepared using RNA-easy kit study for more confirmation. according to manufacturer's instructions (QIAGEN). The results presented in figure 1 showed The RNA was dissolved in DEPC-treated water, and differential display for 12 fungal isolates using total RNA analyzed on 1.2% agarose gel. For cDNA chitinase 25 and 15 as arbitrary primers. Primer chi 25 synthesis 2.5µl from RNA was combined with 5µl of grouped the isolates into 9 groups, group one contains a 2x reverse transcription mixture containing (50 mM G1and C1 whenever group two contains B1 & B2 and

Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, 20 mM group 3 contains B8 and B9 (Fig.1). In addition, the dithiothreitol), 2.5µl dNTPs (4 mM), 1µl oligd T rest of the isolate each one formed a separate group. primer (Promega), 13µl of RNAs free water and 1µl Whenever, the primer chi 15 was able to differentiate (50unit/µl) of Murine Leukemia Virus (MLV) reverse between the all examined isolates except the isolates transcriptase and incubated at 37°C for 1 hr, followed B4 and B8 which they were much closed to each other by one cycle for killing the enzyme at 75 oC for 10 (Fig.2). Whenever, the results presented in figure (3) minutes[19]. revealed that primer A2 succeeded to distinguish between the 12 isolates and there was no similarities Differential Display for the Collected Fungal observed among them. In case of the primer A4 as Isolates: The cDNA was subjected to second PCR shown in figure (4) the same result was obtained in compared with the primer A2.

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Table 1: Source of Ustilago scitaminea Syd. isolates used in this study Isolate No. Sugar cane variety Locality Governorate 1 (G1) Gt54-9 *SCRI Giza 2 (C1) NCO310 SCRI Giza 3 (C3) Gt54-9 Hawamdieh Giza 4 (C6) Gt54-9 Abo-Korkas El-Menia 5 (B1) Gt54-9 Gerga Souhag 6 (B2) Gt54-9 Dar-Elsalam Souhag 7 (B3) Gt54-9 Nag-Hammadi Qena 8 (B4) G85-37 Nag-Hammadi Qena 9 (B6) G99-165 El-Mattana Qena 10 (8) G54-9 El-Mattana Qena 11 (9) G99-103 El-Mattana Qena 12 (11) Gt54-9 Kom-Ombo Aswan *Sugar Crops Research Institute

Fig. 1: Differential display for the smut fungal isolates using chitinase 25 primer.

Fig. 2: Differential display for the smut fungal isolates using chitinase 15 primer.

Fig. 3: Differential display for the smut fungal isolates using chitinase A2 primer.

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Fig. 4: Percentage of similarity between the 12 smut fungal isolates based on the Differential display band pattern.

Data presented in figure 4 showed that the economy in Egypt from such disease. We tried in this differential display technique classified the twelve study to evaluate most of sugarcane cultivars cultivated isolates into 10 different groups. Group1 contains G1 in five governorates and to what extent these cultivars and C3, group 2 include B9 and B11, whenever, the are resistance and or susceptible to smut disease. other isolates each one made one group. In addition, Whenever, selecting the most resistance cultivars for all the similarity between the isolates was demonstrated Ustillago genotypes will solve the problem. based on the band pattern obtained by the AP-PCR. The similarity percentage between G1 and C3 was Due to the increasing areas infected with smut in 100% and the same percent was obtained in case of B9 the sugarcane fields in Egypt, we decided to fingerprint and B11. The lowest similarity 30% was observed the races presented in Egyptian fields in this study. The between B8, B9 and B11. spores were collected from infected sugarcane plants from different five different governorates in Upper Discussion: RNA fingerprinting using arbitrarily primer Egypt. The results obtained by differential display PCR (RAP) allows the semi-quantitative simultaneous techniques showed that there are at least 10 different comparison of the abundances of several hundred races from the Ustillago sp. in Egyptian field, these randomly sampled RNAs. In RAP first-strand cDNA results come in agree with[25] who used specific PCR synthesis by reverse transcriptase is initiated from an primers for amplification of 459 bp fragment, followed arbitrarily chosen primer at sites in the RNA that best by band elution, RFLP and finally sequencing to match the primer. Second-strand synthesis is initiated distinguish among microbial isolates.[26] make a by extension of the same arbitrary primer at sites of fingerprinting for the sugarcane cultivars resistance and adequate match on the first-strand cDNA product by susceptible for the smut disease. The primers chitinase using Taq polymerase. The products of cDNA synthesis 25 gave 20-24 different band patterns with different are then amplified by PCR and displayed on a gel as molecular weights (50bp-3000kbp), whenever, 10-15 a fingerprint representing between 10 and 50 RNAs, band patterns were obtained with primer chitinase 15 depending on the choice of arbitrary primer. Any and the molecular weights of the obtained bands was differences in the pattern produced by a primer in ranged from 100bp to 10kbp. The differences in the different RNA populations reflect abundance differences band patterns revealed how many RNAs had been in individual RNAs or between the individuals[13, 23]. scanned when we used these primers to differentiate Sugarcane smut disease caused by Ustillago between the fungal isolates. Chitinase gene was scitaminae (Sydow) causes the greatest yield losses and considered as a good guide to tell us how the fungal is the most difficult to control through producing host react with the environment, i.e the fungal able to genetic resistance, in part because the pathogen generate a new mutation for the new environment. occasionally produces a new race that overcomes prior Through the reaction between the chitinase gene and host resistance[24]. However, it is become necessary to the environmental conditions we can obviously identify how many fungal isolates distributed in Egypt. demonstrated the differences between the obtained As well as, studying which cultivars are susceptible for isolates. In conclusion the Chitinase gene is a infections with such pathogen and which genotype. functional gene but at the same time it is considered as Many question needs to answerers to protect our a one of the conditional genes.

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