Biotechnology

Year: 2010 | Volume: 9 | Issue: 2 | Page No.: 89-105
DOI: 10.3923/biotech.2010.89.105

WITHDRAWN: Development of a Combined Molecular Diagnostic and DNA Fingerprinting Technique for Rice Bacteria Pathogens in Africa

A. Onasanya, A. Basso, E. Somado, E.R. Gasore, F.E. Nwilene, I. Ingelbrecht, J. Lamo, K. Wydra, M.M. Ekperigin, M. Langa, O. Oyelakin, Y. Sere, S. Winter and R.O. Onasanya

Abstract:

Case No: 26082013

This article has been withdrawn due to technical issue.

Keywords: Pseudomonas fuscovaginae, Pseudomonas syringae pv. syringae, Xanthomonas oryzae pv. oryzicola, Xanthomonas oryzae pv. oryzae, DNA fingerprinting, PCR technique, molecular diagnostic, Africa and genotype

INTRODUCTION

Rice (Oryza sativa L.) is the primary food grain consumed by almost half of the world’s population, making it the most important food crop currently produced (Cottyn et al., 2001). It provides 27% of energy and 20% of proteins in developing countries (Kennedy et al., 2002). More than 70 diseases caused by fungi, bacteria, viruses and nematodes have been recorded on rice. Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas fuscovaginae (Pf) and Pseudomonas syringae pv. syringae (Pss) are bacterial pathogens capable of causing disease on different rice cultivars. Bacterial Leaf Blight (BLB) caused by Xoo is a very destructive disease in Asia and Africa (Adhikari et al., 1995; Sere et al., 2005). In a more recent survey in West Africa, bacterial blight incidence ranged from 70-85%, indicating a wide spread of the disease in 14 farmers’ fields (Sere et al., 2005). Research studies have also revealed that BLB is an important rice disease in irrigated rice ecosystems in West Africa, mainly in Sahelian and soudano-sahelian countries. Bacterial Leaf Streak (BLS) caused by Xoc is restricted largely to tropical and subtropical Asia, including southern China, Thailand, Malaysia, India, Viet Nam, the Philippines and Indonesia, but it also affects rice-growing regions of northern Australia and Africa (Ou, 1985; Awoderu et al., 1991; Sigee, 1993). Although, documentation does not exist for many areas in which BLS is present, available reports suggested that yield losses due to this disease typically up to 20% depending on the rice variety and climatic conditions (Ou, 1985). Pf is a fluorescent pseudomonad that causes sheath brown rot of rice in temperate regions (Miyajima et al., 1983; Tanii et al., 1976). This pathogen has been described in countries (Japan, Nepal, Madacascar, Burundi and Colombia) where cold temperatures represent a limiting factor for rice cultivation (Duveiller et al., 1988; Rott et al., 1989; Zeigler and Alvarez, 1990). The most important damage associated with the bacterium is grain sterility and yield loss up to 58% has been reported by Jaunet et al. (1996). In Asia, America, Europe and Africa (mainly Burundi, Zaire, Rwanda and Madagascar), Pss has been reported as the cause of rice bacterial sheath brown rot and 75% of the panicle can be affected (Zeigler and Alvarez, 1990).

Early field detection and identification of Xoo, Xoc, Pf and Pss pathogens will prevent further spread of the pathogens and allow early disease control. Several indirect and direct methods aid in identification of the pathogen. The phage technique involves the incubation of seed samples with a species phage. The increase in phage number by plaque count would detect the presence of bacterium. Serological methods also serve as sensitive tools for detection of the pathogen because the outer membrane of gram negative bacteria has a variety of potentially antigenic molecules which may be detected by monoclonal antibodies. Gnanamanickam et al. (1999) demonstrated the detection of Xoo in rice seeds inoculated with the pathogen using Enzyme-Linked Immuno Sorbent Assay (ELISA), whereby the bacterial colonies that reacted positively to monoclonal antibodies specific to the bacterium were examined by direct immunofluorescence (IF). Though ELISA and IF provide conclusive evidence for the presence of the pathogen, neither technique is sensitive enough to detect low numbers of the pathogen, which necessitates enrichment (Sakthivel et al., 2001). Molecular probes, on the other hand, facilitate detection of even low numbers of the pathogen through Polymerase Chain Reaction (PCR) analyses (Sakthivel et al., 2001). Random amplified polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP) and Restriction Fragment Length Polymorphism (RFLP) markers have been used for DNA fingerprinting analyses of bacterial pathogens (Sakthivel et al., 2001; Onasanya et al., 2003).

Therefore, the present study aimed at developing a combined molecular diagnostic and DNA fingerprinting PCR technique for rice bacteria pathogens in Africa in order to reveal the distribution, movement and epidemiology of Xoo, Xoc, Pf and Pss rice pathogens in Africa.

MATERIALS AND METHODS

Research location: Bacterial isolate propagation and primer design were carried out at Plant Pathology Unit, Africa Rice Center (AfricaRice), Cotonou, Benin Republic, while the molecular PCR analysis was carried out at Central Biotechnology Laboratory, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. This study was conducted between February and May 2009.

Bacterial isolates and propagation: Ninety five bacterial isolates (Table 1, 2) used in this study were obtained from Plant Pathology Unit, Africa Rice Center (AfricaRice), Cotonou, Benin Republic, where they were isolated from different diseased plants in West and East Africa. Two bacterial isolates from Asia were obtained from International Rice Research Institute (IRRI) in the Philippines and used as control isolates during the study. The bacterial isolates were first propagated using a modified procedure developed by Kado and Keskett (1970). Nutrient broth (75 mL; pH 7.5) was prepared inside a 250 mL conical flask. Each Xoo isolate (200 μL) from storage was transferred into 75 mL of nutrient broth and kept under constant shaking at 30°C for 24 h for bacterial growth. The bacterial cells were removed by centrifugation, washed with 0.1 mM Tris-EDTA (pH 8.0) and kept at -20°C for DNA extraction.

Genomic DNA extraction: DNA extraction was according to Roeder and Broda (1987) and Thottappilly et al. (1999) with some modification: 0.3 g of washed bacterial cell was suspended in 200 μL of Cetyl trimethylammonium bromide (CTAB) buffer (50 mM Tris, pH 8.0; 0.7 mM NaCl; 10 mM EDTA; 2% hexadecyltrimethylammonium bromide; 0.1% 2-mercaptoethanol), followed by 100 μL of 20% sodium dodecyl sulfate and incubated at 65°C for 20 min.

The DNA was purified by two extractions with phenol: chloroform: isoamyl alcohol (24:25:1) and precipitated with -20°C absolute ethanol. After washing with 70% ethanol, the DNA was dried and resuspended in 200 μL of sterile distilled water; its concentration was measured using DU-65UV spectrophotometer (Beckman Instruments Inc., Fullerto CA, USA) at 260 nm. DNA quality was also checked by electrophoresis on a 1% agarose gel in Tris-Acetate-EDTA (TAE) buffer (45 mM Tris-acetate, 1 mM EDTA, pH 8.0). Finally it was kept at -20°C freezer before Polymerase Chain Reaction (PCR) analysis was carried out.

PCR primers design: Using Xanthomonas oryzae pv. oryzae complete genome sequence (NCBI reference sequence: NC_007705.1), Xanthomonas oryzae pv. oryzicola whole genome shotgun sequence (NCBI reference sequence: NZ_AAQN01000001.1), Pseudomonas fuscovaginae genome sequence (NCBI reference sequence: AB021381.1) and Pseudomonas syringae pv. syringae complete genome sequence (NCBI reference sequence: NC_007005.1) obtained in previous studies (Anzai et al., 2000; Feil et al., 2005; Ochiai et al., 2005; Salzberg et al., 2008), four diagnostic and fingerprinting primers (Table 3) were designed using Primer Express^TM software version 2·0 (Applied Biosystems).

The specificities of the primers were computer-tested, as was the theoretical PCR products. For direct use in PCR analysis, the designed primers sequences (Table 3) were synthesized at Eurogentec, B-4102 Seraing, Belgium.

Molecular diagnostic and DNA fingerprinting PCR analysis: The PCR analysis was according to Onasanya et al. (2003) with some modifications. Amplifications were performed in 25 μL reaction mixture consisting of bacterial genomic DNA, reaction buffer (Promega), 100 mM each of dATP, dCTP, dGTP and dTTP, 0.2 mM each for forward and reverse primer, 2.5 mM MgCl₂ and 1U of Taq polymerase (Boehringer, Germany). The reaction mixture was overlaid with 50 μL of mineral oil to prevent evaporation. All the DNA of the 95 bacterial isolates (Table 1, 2) were analyzed. Amplification was performed in a thermowell microtitre plate (Costa Corporation) using Perkin Elmer programmable Thermal Controller model 9600. The cycling program was (1) 1 cycle of 94°C for 3 min; (2) 35 cycles of 94°C for 1 min for denaturation, 55°C for 1 min for annealing of primer and 72°C for 2 min for extension and (3) a final extension at 72°C for 7 min. Amplification products were maintained at 4 ^oC until electrophoresis. The amplification products were resolved by electrophoresis in a 1.4% agarose gel using Tris-Acetate-EDTA (TAE) buffer (45 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 100 V for 2 h. A 1 kb ladder (Life Technologies, Gaithersburg, MD, USA) was included as molecular size marker. Gels were visualized by staining with ethidium bromide solution (0.5 μg mL^-1) and banding patterns were photographed over UV light using UVP-computerized gel photo documentation system.

Diagnostic band scoring and cluster analysis: Positions of unequivocally scorable amplified DNA bands were transformed into a binary character matrix (1 for the presence and 0 for the absence of a band at a particular position). Pairwise distance matrices were compiled by the Numerical Taxonomy System (NTSYS) 2.0 software (Rohlf, 2000) using the Jaccard coefficient of similarity (Ivchenko and Honov, 1998). Phylogenetic tree was created by Unweighted Pair-Group Method Arithmetic (UPGMA) cluster analysis (Sneath and Sokal, 1973; Jakoet al., 2009).

RESULTS

A combined molecular diagnostic and DNA fingersprinting PCR technique for Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas fuscovaginae (Pf) and Pseudomonas syringae pv. syringae (Pss) rice pathogens has been developed in Africa by this study.

Out of 95 bacterial isolates analyzed for Xoo diagnosis using the Xoo specific diagnostic primer pair (XooF1 and XooR1), 84 of the bacterial isolates contained Xoo pathogen from which 24 were confirmed as pure Xoo pathogen and no mixture with Xoc, Pf or Pss pathogens (Table 4, 5 and Fig. 1, 2). Besides, the DNA fingerprinting of the diagnosed 84 Xoo pathogens have been revealed by the same primer pair (XooF1 and XooR1) in the same PCR assay used for the diagnosis (Fig. 1, 2).

DNA fingerprinting of 84 Xoo pathogens using primer pair (XooF1 and XooR1) produced 216 fragments from which 162 (75%) were polymorphic with genotype index of 2.6 leading to the identification of seven Xoo genotypes, which were Xoo-1, Xoo-2, Xoo-3, Xoo-4, Xoo-5, Xoo-6 and Xoo-7 respectively (Table 6, Fig. 3). Xoo-1 genotype consists of 28 Xoo isolates from Niger, Rwanda and Philippines while Xoo-2 genotype consists of 25 Xoo isolates from Niger, Rwanda, Mozambique and Uganda, Xoo-3 genotype consists of 7 Xoo isolates from Niger and Rwanda while Xoo-4 genotype consists of 12 Xoo isolates from Niger, Rwanda, Mozambique and Uganda, Xoo-5 and Xoo-7 genotypes consist of 7 Xoo isolates from Niger and Xoo-6 genotype consists of 5 Xoo isolates from Rwanda (Table 7, Fig. 3). However, Xoo genotype occurrence and distribution among countries was between 2.4 to 33.3% with Xoo-1 genotype having the highest of 33.3% and Xoo-7 genotype having the lowest of 2.4% (Table 7).

However, the same 95 bacterial isolates were analyzed for Xoc diagnosis using the Xoc specific diagnostic primer pair (XocF2 and XocR2) and 50 of the bacterial isolates contained Xoc pathogen from which only one was confirmed pure as Xoc pathogen and no mixture with Xoo, Pf or Pss pathogens (Table 4, 5 and Fig. 4, 5). Besides, the DNA fingerprinting of the diagnosed 50 Xco pathogens have been revealed by the same primer pair (XocF2 and XocR2) in the same PCR assay used for the diagnosis (Fig. 4, 5). DNA fingerprinting of 50 Xoc pathogens using primer pair (XocF2 and XocR2) produced 69 fragments from which 33 (47.9%) were polymorphic with genotype index of 1.4 leading to the identification of four Xoc genotypes, which were Xoc-1, Xoc-2, Xoc-3 and Xoc-4 respectively (Table 6, Fig. 6). Xoc-1, Xoc-2 and Xoc-4 genotypes consist of 9, 4 and 6 Xoc isolates respectively from Niger and Rwanda, while Xoc-3 genotype consists of 31 Xoc isolates from Niger, Rwanda, Mozambique and Uganda (Table 7, Fig. 6). Besides, Xoc genotype occurrence and distribution among countries was between 8 to 60% with Xoc-3 genotype having the highest of 62% and Xoc-2 genotype having the lowest of 8% (Table 7).

The same 95 bacterial isolates were also analyzed for Pf diagnosis using the Pf specific diagnostic primer pair (PfF3 and PfR3) and 19 of the bacterial isolates contained Pf pathogen (Table 4, 5 and Fig. 7, 8). However, the DNA fingerprinting of the diagnosed 19 Pf pathogens have been revealed by the same primer pair (PfF3 and PfR3) in the same PCR assay used for the diagnosis (Fig. 7, 8).

DNA fingerprinting of 19 Pf pathogens using primer pair (PfF3 and PfR3) produced 23 fragments and all were polymorphic with genotype index of 1.2 leading to the identification of three Pf genotypes, which were Pf-1, Pf-2 and Pf-3 respectively (Table 6, Fig. 9). Pf-1 genotype consists of 15 Pf isolates from Niger, Rwanda, Uganda and Philippines while Pf-2 genotype consists of 2 Pf isolates from Niger and Pf-3 genotypes consists of 2 Pf isolates from Rwanda (Table 7, Fig. 9). Besides, Pf genotype occurrence and distribution among countries was between 10.5 to 78.9% with Pf-1 genotype having the highest of 78.9% (Table 7).

Out of the same 95 bacterial isolates analyzed for Pss diagnosis using the Pss specific diagnostic primer pair (PssF4 and PssR4), 16 of the bacterial isolates contained Pss pathogen (Table 4, 5 and Fig. 10, 11). Moreover, the DNA fingerprinting of the diagnosed 16 Pss pathogens have been revealed by the same primer pair (PssF4 and PssR4) in the same PCR assay used for the diagnosis (Fig. 7, 8). DNA fingerprinting of 16 Pss pathogens using primer pair (PssF4 and PssR4) produced 27 fragments and all were polymorphic with genotype index of 1.7 leading to the identification of three Pss genotypes, which were Pss-1, Pss-2 and Pss-3 respectively (Table 6, Fig. 12). Pss-1 genotype consists of 5 Pss isolates from Niger, Rwanda and Uganda while Pss-2 genotype consists of 9 Pss isolates from Niger and Philippines (Table 7, Fig. 12).

However, Pss genotype occurrence and distribution among countries was between 12.5 to 56.3% with Pss-2 genotype having the highest of 56.3% and Pss-3 genotype having the lowest of 12.5% (Table 7).

DISCUSSION

Molecular PCR diagnostic showed that the presence of at least a band indicates positive (+) detection of a bacterial pathogen and absence of a band indicates negative (-) as no bacterial pathogen was detected, while in the same PCR assay the presence of one or more band at different position revealed the DNA fingerprint of a bacterial pathogen. Development of an effective combined molecular diagnostic and DNA fingerprinting PCR technique for Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas fuscovaginae (Pf) and Pseudomonas syringae pv. syringae (Pss) rice pathogens in Africa as revealed by this study offers unique opportunity into early field detection of these pathogens, epidemiology and control (Eisenstein, 1990; Sakthivel et al., 2001). The Xoo, Xoc, Pf and Pss specific primers developed in this study have successfully diagnosed and fingerprinted these bacterial pathogens in PCR analysis thus confirming their usefulness and potential. These Xoo, Xoc, Pf and Pss specific primers produced multiple amplified DNA fragments on respective Xoo, Xoc, Pf and Pss isolates thus combining both molecular diagnostic and DNA fingerprinting potential for effective and reliable identification and differentiation of these bacterial pathogens. This makes the present study different from previous studies where bacterial diagnostic primers do not combine both diagnostic and DNA fingerprinting potential as did in this study (Botha et al., 2001; Dreo et al., 2005; Manceau et al., 2005; Fatmi et al., 2005).

The application and use of these Xoo, Xoc, Pf and Pss specific primers developed in this study in PCR is reliable and sensitive for diagnosing and fingerprinting seedborne Xoo, Xoc, Pf and Pss pathogens, detecting Xoo, Xoc, Pf and Pss in rice diseased leaf and identification of virulence unique to Xoo, Xoc, Pf and Pss pathogens (Adhikari et al., 1999; Sakthivel et al., 2001). Besides, the application of these Xoo, Xoc, Pf and Pss specific primers in PCR, because of its speed and sensitivity, holds the promise of making a significant practical impact in rice bacterial disease seed quarantine programs and to monitor germplasm movement within and outside Africa (Adhikari et al., 1999; Sakthivel et al., 2001). Moreover, these Xoo, Xoc, Pf and Pss specific primers could be useful for identification of Xoo, Xoc, Pf and Pss isolates from culture media thus resolving the lack of consistency and precision often arise from using cultural and morphological techniques (Bonde et al., 1993; Sakthivel et al., 2001). The convenience of the PCR method makes the application of these Xoo, Xoc, Pf and Pss specific primers highly suitable for analyzing large numbers of samples, allowing for greater efficiency in the study of Xoo, Xoc, Pf and Pss diagnosis, virulence, population structure and movement (Sakthivel et al., 2001).

Development of a reliable molecular technique for Xoo, Xoc, Pf and Pss identification and differentiation is a prerequisite into understanding the genetics of Xoo, Xoc, Pf and Pss population structure in Africa and deployment of durable resistance cultivars (Adhikari et al., 1995, 1999). In the present study the Xoo specific primer developed has detected Xoo pathogen in 84 out of 95 bacterial isolates tested as well as produced their DNA fingerprints leading to the identification of seven Xoo genotypes (Xoo-1, Xoo-2, Xoo-3, Xoo-4, Xoo-5, Xoo-6 and Xoo-7 ) among the 84 Xoo isolates thus revealed it population structure in African countries. This report supports recent isozyme fingerprints of 30 Xoo isolates from 5 countries (Mali, Burkina Faso, Niger, Benin Republic and Nigeria) in West Africa that revealed five genetic groups (Onasanya et al., 2007, 2008).

The high distinction pattern of each isolates in this study suggests possible high level of genetic variation and frequent occurrence of mutants in Xoo, Xoc, Pf and Pss isolates in different host cells (Mongkolsuk et al., 2000; Innes et al., 2001; Onasanya et al., 2003). The genetic analyses revealed that all the seven Xoo genotypes (Xoo-1, Xoo-2, Xoo-3, Xoo-4, Xoo-5, Xoo-6 and Xoo-7) might cover about 88.4% of rice bacterial pathogen population across Niger, Rwanda, Mozambique and Uganda and possibly be responsible for most sporadic cultivars infestation and epidemics in these countries. This was followed by four Xoc genotypes (Xoc-1, Xoc-2, Xoc-3 and Xoc-4) which possibly covered about 52.6% of rice bacterial pathogen population across Niger, Rwanda, Mozambique and Uganda. Pf and Pss genotypes formed the least with about 20 and 16.8%, respectively of rice bacterial pathogen population across Niger, Rwanda and Uganda. Different Xoo, Xoc, Pf and Pss genotypes were found to exist in Niger, Rwanda, Mozambique and Uganda suggesting possible pathogen migration between these countries and long-term survival (Adhikari et al., 1995).

Distinct phenotypes usually consist of isolates that are genetically less related and such identification of isolates using cultural and morphological techniques often lack consistency and precision (Bonde et al., 1993). The Xoo, Xoc, Pf and Pss specific primers developed in the study have proven particularly useful in situations where identification of Xoo, Xoc, Pf and Pss isolates using cultural and morphological techniques often lack consistency and precision. For example, in the current study using Xoo, Xoc, Pf and Pss specific primers to screen 95 bacterial isolates only 24 were confirmed pure Xoo isolates and one as pure Xoc isolates while the rest were mixture of Xoo, Xoc, Pf or Pss pathogens.

CONCLUSIONS

It is concluded that the newly developed tool which is a combined molecular diagnostic and DNA fingerprinting PCR technique for Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), Pseudomonas fuscovaginae (Pf) and Pseudomonas syringae pv. syringae (Pss) rice pathogens in Africa, is effective, highly reliable, sensitive and therefore a suitable screening assay for early field detection of these pathogens, their epidemiology, movement, control and estimation of genetic diversity. The method can also serve as a fast and specific identification test for diagnosing and fingerprinting seedborne Xoo, Xoc, Pf and Pss pathogens, virulence unique to Xoo, Xoc, Pf and Pss pathogens and resolving the lack of consistency and precision often arise from using cultural and morphological techniques. It holds the promise of making a significant practical impact in rice bacterial disease seed quarantine programs and to monitor germplasm movement within and outside Africa. Finally, this study developed a reliable molecular technique for Xoo, Xoc, Pf and Pss identification and differentiation which is a prerequisite into understanding the population structure of these pathogens and deployment of durable resistance cultivars into different rice ecologies in African countries.

ACKNOWLEDGMENTS

We are very grateful to the Germany Federal Ministry of Economic Cooperation and Development (BMZ), Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ), GmbH and Bill and Melinda GSR Project for providing funds for this research. The authors would also like to acknowledge Mr. Sotou Georges for technical support.

REFERENCES