African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12421

Full Length Research Paper

Assessment of Exserohilum turcicum using molecular markers for sustainable maize production in Tanzania

Onwunali Martin R. O.
  • Onwunali Martin R. O.
  • Department of Crop Science and Horticulture, College of Agriculture, Sokoine University of Agriculture, P. O. Box 3005, Morogoro, Tanzania.
  • Google Scholar
Mabagal Robert B.
  • Mabagal Robert B.
  • Department of Crop Science and Horticulture, College of Agriculture, Sokoine University of Agriculture, P. O. Box 3005, Morogoro, Tanzania.
  • Google Scholar
Massawe Deogracious P.
  • Massawe Deogracious P.
  • Department of Crop Science and Horticulture, College of Agriculture, Sokoine University of Agriculture, P. O. Box 3005, Morogoro, Tanzania.
  • Google Scholar


  •  Received: 09 June 2021
  •  Accepted: 01 June 2022
  •  Published: 31 August 2022

 ABSTRACT

The Exserohilum turcicum fungus causes the Northern leaf blight (NLB) of maize all over the world. In Tanzania, this disease is considered as a major foliar disease with yield losses of 23.9 to 62.8%. To understand the genetic variations of E. turcicum from maize fields in Tanzania, a survey was conducted in four regions, including Morogoro, Iringa, Njombe, and Mbeya. Leaf samples were collected from diseased plants exhibiting NLB symptoms. Using aseptic technique, pure colonies of fungal pathogens were isolated on selective media followed with molecular analysis. The internal transcribed ribosomal DNA (ITS), six microsatellite markers of simple sequence repeat (SSR) nature, and two specific mating type primers (MAT) designed to amplify MAT1 and MAT2 gene sequences were used in this study. With ITS marker, 14 isolates were amplified, Sanger sequenced, and their sequences were deposited to GenBank with accession numbers MT124699-MT124712. The SSR results were scored using base pairs in each genotype and subjected to power marker software to determine genetic variations. A total of four alleles across two loci were detected, with gene diversity (0.26 - 0.58). Polymorphic bands revealed 4 and 5 genotypes using SSR 06 and SSR 024, respectively. The polymorphic information content (PIC) of SSR 06 and SSR 024 loci were 0.25 and 0.52, respectively. The specific MAT markers demonstrated dominance of MAT 2 over MAT 1. The distribution of MAT 1 and MAT 2 in humid highlands of Mbeya, Njombe, and Iringa regions indicated possibility of sexual reproduction and good potential for sexual recombination than in the dry lowland of Morogoro region. This study is the first report on genetic diversity of E. turcicum in Tanzania, further studies using robust molecular and sequencing techniques is imperative in Tanzania.

 

Key words: Exserohilum turcicum, maize, microsatellite, mating type primers, ITS primers, Tanzania.


 INTRODUCTION

Exserohilum turcicum (Pass.) Leonard and Suggs (1974) of maize is an important foliar disease in Tanzania (Dong et al., 2008; Nwanosike, 2016). The disease is potentially distributed all over the world (Ramathani et al., 2011). The pathogen also infects sorghum and other related grasses (Harlapur et al., 2007; PANNAR Seed Company, 2009); however, epidemic may be sporadic depending on the environment and varieties to resist infection (Degefu,1990).

 

This disease is a threat to maize production globally (Muiru et al., 2010; Levic et al., 2008; Nwanosike et al., 2017). In Tanzania, yield losses ranged from 23.9% in Morogoro to as high as 62.8% in Mbeya (Nwanosike, 2016). Population studies on the pathogen become imperative to provide understanding of the genetic diversity (Nei, 1973; Ferguson and Carson, 2007; Haasbroek et al., 2014; McDonald and McDermott, 1993).

 

The studies of E. turcicum genetic diversity in various countries concentrated on use of RAPDs, AFLP, and isozymes (Dong et al., 2008; Muiru et al., 2010), but microsatellites markers considered as choice markers for population studies (Chambers and MacAvoy, 2000; Ellegren, 2004). However, SSR has been used primarily to detect polymorphism amongst the parental cultivated varieties (Klein et al., 2000). Ferguson and Carson (2004) reported that the population genetic structure of Setosphaeria turcica is subjective to the result of both sexual and asexual reproduction. Although the sexual phase of E. turcicum has not been discovered in the field, evidence of recombination exists especially in tropical populations (Borchardt et al., 1998b).

 

Simple sequence repeats are sections of DNA repetitive units (2-6 bp in length) inside all eukaryotic organisms (Quellar et al., 1993). Identification of nucleotide sequences in the adjoining regions of the microsatellite facilitates development of specific primers (generally 20-25 bp) to amplify the SSR by polymerase chain reaction. Ramathani et al. (2011) reported the type of mating primers that amplified the MAT1 and MAT2 idiomorphs of E. turcicum with fragment size of 154 and 197 bp, respectively. The efficacy of such primers was used to test maize and sorghum derived E. turcicum in Uganda (Ramathani, 2010). However, in South Africa, Haasbroek et al. (2014) designed and developed mating types idiomorphs of the pathogen with higher fragment size for mating type 1 and mating type 2, which was used to screen fungal isolates of maize and sorghum. It has a single locus and two-allele mating system and as such exhibits two forms of mating type (Borchardt et al., 1998a; Klix et al., 2010).

 

NLB is a destructive foliar disease in maize growing areas of Tanzania and is reflected as a limiting biotic factor hampering successful cultivation of maize. Despite efforts of the poor resource for farmers to improve yield, it remains low 1.3 to 1.5 tons/ha (Nkonya et al., 1998). Therefore, this study will apply SSR loci and species-specific primers designed based on internal transcribed ribosomal DNA (ITS) and 5.8s rDNA sequences to determine genetic diversity and mating types of Tanzania isolates, respectively.


 MATERIALS AND METHODS

Leaf samples showing lesions were collected in 38 fields during a survey in 2012 to 2013. Twenty-eight (28) samples were collected from Southern highland regions including Iringa (10), Njombe (8), and Mbeya (10), and ten samples from Eastern region (Morogoro), using the Global Positioning System. Samples were surface sterilised and incubated for 2 days at 25±3°C, in laboratory. The fungus were isolated on V8 agar medium (Harlapur, 2005). E. turcicum cultured in a liquid media, the mycelia were collected and preserved at 4°C (Muiru et al., 2010).

 

DNA was extracted using Cetyltrimethylammonium bromide (CTAB) protocols by Moller et al. (1993) and adapted by Muiru et al. (2010). The quality and quantity of extracted DNA was measured on NanoDrop Spectrophotometer (Thermofisher, Waltham, MA) at ratio of 260/280 wavelength. The extracted DNA was stored at -20°C for use in PCR experiments.

 

PCR reactions and cycles

 

The identity of the isolates was confirmed through PCR sequence information from product targeting of the internal transcribed spacer 1 and 2 (ITS) of the 5.8S ribosomal RNA region using specific primers that were previously used by Ramathani (2010) (Table 1). The PCR amplicons were Sanger sequenced and blasted to the NCBI GenBank to identify isolate with more hits to our sequences.

 

Six microsatellite primers (Table 2) obtained from several related loci of ascomycetes and two specific MAT markers; MAT1 and MAT2 (Table 1) were used to test for polymorphism of 20 E. turcicum maize isolates using the protocol described by Ramathani (2010) and Ramathani et al. (2011), with amendments.

 

 

Amplifications were performed in a thermo cycler (Gene Amp PCR system 9700 Applied Bio Systems, 850 Lincoln Centre Drive Foster City, CA 94404 USA) using the following program: 1 cycle of 1 min at 95°C followed by 30 cycles of 30 s at 95°C, 30 s at 45 to 50°C and 45 s at 72°C, with a final extension of 7 min at 72°C, for SSR while the cycling condition for MAT primers was 1 cycle of 4 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C, with a final extension of 9 min at 72°C.

 

Amplified products were separated by electrophoresis (Bio-Rad, model 96, Bio-Rad Laboratories, Inc. Life Science Research Group 2000 Alfred Nobel Drive Hercules, CA 94547 USA) at 110 V for 2.0 h on 1.5% agarose gels and stained with intercalating ethidium bromide (0.5 μg ml-1) using 1.0 × TBE buffer (89 mMTris, 89 mM borate, 2 mM EDTA pH 8.3). Gels were documented under UV-Trans-illuminator 2000 (Sagrate, Milan Italy). Isolates that were amplified with ITS primers, were submitted to Molecular and Cellular Imaging Center (MCIC), Wooster Ohio for Sanger sequencing.

 

Amplicons separated on a gel were scored using base pair (bp) size, in reference to the molecular marker for each genotype. Estimates of similarity among strains were calculated from the data matrices in the form of dissimilarity units and expressed as Euclidean genetic distance (Hintze, 1998). Polymorphic bands of SSR06 and SSR024 were subjected to power marker software for genetic variation calculation amongst the isolates.

 

Sanger sequenced products (forward and reverse) per isolate were assembled using MacVector software version 17.5.2 (Apex, NC, USA) to create a consensus sequence that was later blasted to the NCBI database for identification.


 RESULTS

Selection of E. turcicum DNA by species specific primers

 

The rDNA ITS specific primers were detected in 20 out of 38 isolates from maize samples, yielding the expected 344 base pairs (Figure 1). Fourteen isolates were Sanger sequenced and had 98  to 100% nucleotide identity with MN918438.1, MN918291.1, and query coverage of 94 to 100 with reference sequences in the NCBI Genbank. The sequenced isolates were annotated and deposited in the NCBI GenBank. The isolates, locations, altitude, and NCBI accession numbers are indicated in Table 3.

 

 

 

Genetic variation of E. turcicum based on SSR analysis

 

Molecular variations were observed among isolates based on microsatellite repeat makers used in this study (Figure 2). Two microsatellite regions, SSR 06 (Figure 2a) and SSR 024 (Figure 2b) were polymorphic, while SSR01, SSR10, SSR14 and SSR36 loci were monomorphic on the 14 E. turcicum isolates (Figure 2). A total of four alleles were identified in each locus (SSRs), and the genetic diversity ranged from 0.26 (SSR06) to 0.58 (SSR24). The analysis of the polymorphic bands using power marker software detected 4 and 5 genotypes for SSR 06 and SSR 024, respectively (Table 4).

 

 

The genotypes were distinguished by the type of polymorphic bands (Figure 2). The amplicons of isolates 2, 5 and 10 (Figure 2a) and 2, 5 and 6 (Figure 2b) were distinctly different genotypes, 4 and 8 are different from that of 9 and 13 genotypes while the other isolates genotypes were of the same strain, indicating five genotypes of the pathogen. Polymorphic information content of SSR06 and SSR 024 loci were 0.25 and 0.52, respectively, with mean of 0.39 (Table 4). Although the two loci were genetically informative, SSR 024 indicated high heterozygosity (0.23) and gene diversity (0.58) compared to SSR 06.

 

Genetic variability of E. turcicum based on mating type genes

 

Gel electrophoresis identified both MAT 1 and MAT 2 (Figure 3). Amplification of isolates that were MAT 1 (α-clone locus) yielded a fragment size of 190 bp (Figure 3a) while isolates for MAT 2 (MAT 2- specific locus) produced fragment size of 195 bp (Figure 3b). Of the 14 isolates, four MAT 1 (MBY 5, IR 8, IR 19 and MBY 13) and 10 MAT 2 idiomorphs were observed. All Morogoro samples were MAT 2 gene, while MAT 1 and 2 existed in Mbeya, Njombe, and Iringa regions, however in an unequal proportion.

 


 DISCUSSION

Information from the sequenced DNA and mating types indicated evidence of gene flow between populations of E. turcicum in the different maize fields of Tanzania. The microsatellites SSR06 and SSR24 loci and MAT type markers (MAT1 and MAT2) polymorphism revealed strains of E. turcicum. The microsatellite regions of SSR06 and SSR24 loci fragment analysis of the 14 isolates revealed 4 allele. Genetic diversity of 0.42 and polymorphic information content of 0.39 (Table 4) demonstrated that sexual reproduction may have been responsible for the genotypes. Such results showed that five different strains of E. turcicum existed in the population. In South Africa, Haasbrock et al. (2014) reported 90 alleles across 13 SSR loci and gene diversity of 0.074 to 0.929 per locus, with average diversity of 0.602 in maize and sorghum E. turcicum. The study also showed minimum detection of 2 and maximum of 19 allele with amplification length of 191 to 493 bp, respectively.

 

The existence MAT 2 dominated MAT 1 in E. turcicum strains in an un-equal proportion, may be due to low frequency of genetic recombination, affected by location and genetic variation. The observed clones and mutants of E. turcicum isolated from Mbeya region could be due to frequent contact of sexually compatible strains, rapid and abundant inoculum and production of multiple generations and cycles of asexual reproduction. This may lead to high prevalence of disease in the Southern regions of Tanzania (Nwanosike et al., 2017). Maize is widely cultivated in Tanzania, particularly in the humid Southern Highlands, thus, the different climatic zones and cropping patterns as well as systems employed in different maize growing regions of the country may have also subjected the pathogen to different agro-ecological environments resulting in different mating types and genetic diversity.

 

The frequency and distribution of MAT 1 and MAT 2 in humid southern highlands of Mbeya, Njombe and Iringa regions indicated a possibility of sexual reproduction and a good potential for sexual recombination than in the dry lowland of Morogoro Region (Table  3). Random distribution of mating types in the field may have enhanced chances of sexual reproduction (Welz et al., 1996). Borchardt et al. (1998b) also reported that severe epidemics of NLB in tropical environment resulted in higher population densities and more sexually compatible strains of E. turcicum. Similar condition was reported in Mbeya and Iringa regions, possibly due to consistent cultivation of susceptible maize cultivars, high E. turcicum population density and favourable climatic factors (Nwanosike et al., 2015).


 CONCLUSION

The SSR and specific MAT markers used in this study explained variation in the genotypes of the 14 fungal isolates in Tanzania. The occurrence and distribution of MAT 1 and MAT2, revealed the E. turcicum collected from Southern regions of Tanzania (Mbeya, Njombe, and Iringa) to be more diverse than isolates from Eastern regions (Morogoro). Following the current findings, there is a need for further diversity study on E. turcicum isolates collected from different zones of Tanzania by using robust molecular and sequencing techniques.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.


 ACKNOWLEDGEMENTS

The authors are grateful to Tertiary Education Trust (TET) for funding and Federal College of Education, Zaria Nigeria, for facilitating the research and Sokoine University of Agriculture, Morogoro, Tanzania for permission to publish the paper.



 REFERENCES

Borchardt DS, Welz HG, Geiger HH (1998a). Genetic structure of Setosphaeria turcica populations in temperate and tropical climates. Phytopathology 88(4):322-329.
Crossref

 

Borchardt DS, Welz HG, Geiger HH (1998b). Spatial and temporal variation of genetic marker patterns in Setosphaeria turcica populations from Kenya. Journal of Phytopathology 146(8?9):451-457.
Crossref

 

Chambers GK, MacAvoy ES (2000). Microsatellites: consensus and controversy. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 126(4):455-476.
Crossref

 

Degefu Y (1990). Evaluation of Ethiopian maize germplasms for resistance against leaf blight caused by H. turcicum. Research Report, Awassa College of Agriculture, Addis Ababa University.

 

Dong J, Fan Y, Gui X, An X, Ma J, Dong Z (2008). Geographical distribution and genetic analysis of physiological races of Setasphaeria turcica in Northern China. American Journal of Agriculture and Biological Sciences 3(1):433-445.
Crossref

 

Ellegren H (2004). Microsatellites: simple sequences with complex evolution. Nature Reviews Genetics 5(6):435-445.
Crossref

 

Ferguson LM, Carson ML (2004). Spatial diversity of S. turcica sampled from Eastern United states. Phytopathology 94(8):892-900.
Crossref

 

Ferguson LM, Carson ML (2007). Temporal variation in S. turcica between 1974 and 1994 and origin of races 1, 23, and 23N in the United States. Phytopathology 97(11):1501-1511.
Crossref

 

Haasbroek MP, Craven M, Barnes I, Crampton BG (2014). Microsatellite and mating type primers for the maize and sorghum pathogen, Exserohilum turcicum. Plant Pathology 43(5):577-581.
Crossref

 

Harlapur SI (2005). Epidemiology and management of turcicum leaf blight of maize caused by E. turcicum (Pass.) Leonard and Suggs. Ph.D. Thesis University of Agricultural Sciences, Dharwad, India.

 

Harlapur SI, Kulkarni MS, Wali MC, Patil BC, Kulkarni S, Hugde Y (2007). S. arundinaceaum- A new report of alternative host of turcicum leaf blight of maize. Karnataka. Journal of Agricultural Sciences 20(4):867-868.

 

Klein PE, Klein RR, Cartinhour SW, Ulanch PE, Dong J (2000). A high-throughput AFLP-based method for constructing integrated genetic and physical maps: progress toward a sorghum genome map. Genome Research 10(6):789-807.
Crossref

 

Klix V, Nowrousian M, Ringelberg C, Loros JJ, Dunlap JC, Pöggeler S (2010). Functional characterization of MAT1-1-specific mating-type genes in the homothallic ascomycete Sordaria macrospora provides new insights into essential and nonessential sexual regulators. Eukaryot Cell 9(6):894-905.
Crossref

 

Leonard KJ, Suggs EG (1974). Setosphaeria prolata, the ascigenous state of Exserohilum prolatum. Mycologia 66(2):181-297.
Crossref

 

Levic J, Stankovic S, Petrovic T (2008). The determination of E. turcicum virulence factors in Serbia. Genetika 40(3):271-281.
Crossref

 

McDonald BA, McDermott JM (1993). Population genetics of plant pathogenic fungi. Bioscience 43(5):311-319.
Crossref

 

Molina C, Kaemmer D, Aponte AS, Weising K, Kahl G (2001). Microsatellite markers for the fungal banana pathogen Mycosphaerella musicola. Molecular Ecology Notes 1(3):137-139.
Crossref

 

Moller EM, Bahnweig GG, Sandermann H, Geiger HH (1993). A simple and efficient protocol for the isolation of high molecular weight DNA from filamentous fungi, fruiting bodies and infected plant tissues. Nucleic acids research 20(22):6115-6116.
Crossref

 

Muiru WM, Koopmann B, Tiedemann AV, Mutitu EW, Kimenju A (2010). Evaluation of genetic variability of Kenyan, German and Austrian isolates of E. turcicum using Amplified length Polymorphism DNA Marker. Biotechnology 9(2):204-211.
Crossref

 

Nei M (1973). Analysis of genetic diversity in subdivided populations. Proceedings of the National Academy of Sciences 70(12):3321-3323.
Crossref

 

Nwanosike MRO (2016). Variability, epidemiology and yield loss caused by northern leaf blight of maize in Tanzania. PhD Thesis, Sokoine University of Agriculture, Morogoro, Tanzania.

 

Nwanosike MRO, Mabagala RB (2017). Influence of metrological parameters on the development of E. turcicum (Pass) Leanard and Sugg on maize in Tanzania. International Journal of Agricultural and Food Research 6(3):1-9.

 

Nwanosike MRO, Mabagala RB, Kusolwa PM (2015). Disease intensity and distribution of Exserohilum turcicum incitant of northern leaf blight of maize (Zea mays l.) in Tanzania. International Journal of Pure & Applied Bioscience 3(5):1-13.

 

Pannar Seeds (2009). Maize diseases: northern corn leaf blight. NCLB Fact Sheet, Version 1. www.pannar.co.za

 

Quellar DC, Strassmann JE, Hughes CR (1993). Microsatellites and kinship. Trends in Ecology & Evolution 8(8):285-288.
Crossref

 

Ramathani I (2010). Characterization of turcicum leaf blight epidemics and pathogen populations in the E. turcicum-sorghum pathosystem of Uganda. M.Sc. Thesis, Makarere University, Uganda P 122.

 

Ramathani I, Biruma M, Martin T, Dixelius C, Okori P (2011). Disease severity, incidence and races of S. turcica on Sorghum in Uganda. European Journal of Plant Pathology 131(3):383-392.
Crossref

 

Welz HG, Bassetti P, Geiger HH (1996). Turcicum-Blattdurre und Aleppohirse: ZweiSchaderreder auf demVormarsch. Mais 24:66-68.

 




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