George S. Mahuku

A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA

A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency ratios (A260/A280) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNAs compatibility with downstream applications. This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services and marker-assisted selection (MAS) breeding.

Virulence and molecular diversity within Colletotrichum lindemuthianum isolates from Andean and Mesoamerican bean varieties and regions

Virulence on a standard set of 12 common bean differential varieties, DNA sequence of repetitive-elements (Rep-PCR) and random amplified microsatellites (RAMS) were used to assess the genetic variability of 200 Colletotrichum lindemuthianum isolates collected from Andean and Mesoamerican bean varieties and regions. High levels of pathotypic (90 pathotypes) and genetic diversity (0.97) were identified among 200 isolates, revealing that C. lindemuthianum is a highly diverse pathogen. Although a significant number of pathotypes were common to Andean and Mesoamerican regions, many more were only found in the Mesoamerican region. Cluster analysis of virulence and molecular data did not separate isolates into groups that were structured with common bean gene pools. No genetic differentiation (GST=0.03) was apparent between Andean and Mesoamerican isolates of C. lindemuthianum. The diversity exhibited by C. lindemuthianum does not appear to cluster according to common bean gene pools, and the high diversity found in the Mesoamerican region seems to indicate that C. lindemuthianum originated and was disseminated from this region. Due to the high genetic variation exhibited by C. lindemuthianum, stacking major resistance genes appears to be the best option for developing cultivars with durable anthracnose resistance.

Sources of resistance to angular leaf spot (Phaeoisariopsis griseola) in common bean core collection, wild Phaseolus vulgaris and secondary gene pool

Angular leaf spot (ALS) is one of the most devastating diseases of common bean (Phaseolus vulgaris L.) in tropical and subtropical countries. The causal fungus, Phaeoisariopsis griseola(Sacc.) Ferr. is highly variable and a diverse source of resistance genes is required to manage this disease. We evaluated a common bean core collection,primary and secondary gene pools and lines derived from inter-specific crosses of P. vulgaris and P. coccineus or P. polyanthus (secondary gene pool) for resistance to angular leaf spot. Of the 1441 accessiones in the core collection, only 2.2% were resistant to both Andean and Mesoamerican races of P. griseola, 28% were resistant only to Andean and 9% to Mesoamerican races. Of the 32 resistant accessions, 68%originated from Bolivia, Colombia,Guatemala and Mexico. More accessions from these countries should be examined for P. griseola reaction. Very few wild P. vulgaris accessions (4%), were resistant to ALS. In contrast, high levels of resistance (62%) were found in the secondary gene pool. Among the 1010 lines from inter-specific crosses, 109 lines were highly resistant. These genotypes from the primary and secondary common bean gene pools resistant to Andean and Mesoamerican races of P. griseola offer a potential for developing broad and durable ALS resistance.

Sources of resistance to Colletotrichum lindemuthianum in the secondary gene pool of Phaseolus vulgaris and in crosses of primary and secondary gene pools

Use of genetic resistance is the most practical and economic way to manage anthracnose of common bean. Colletotrichum lindemuthianum, the causal agent of bean anthracnose, is a highly variabile pathogen, and there are no host resistance genes that are effective against all known races of the pathogen. To diversify sources of resistance, we screened the core collection of the secondary gene pool of Phaseolus spp. and interspecific lines derived from simple and complex crosses of primary and secondary genotypes for their resistance to anthracnose. High levels of resistance were observed in the secondary gene pool. None of the 162 accessions tested was susceptible to C. lindemuthianum. Of the two species composing the secondary gene pool, P. polyanthus displayed higher levels of resistance than P. coccineus, and all accessions tested were resistant. The response of P. coccineus was more variable, with six genotypes showing an intermediate reaction. Among the 75 lines from interspecific crosses, 49 were resistant to the three races (races 6, 15, and 3481) used in this study, and higher levels of resistance were found in lines that had P. polyanthus as one of the parents in the crosses than in the lines derived from P. coccineus. These lines constitute a valuable source of resistance and may aid in the development of stable resistance to anthracnose.

Inheritance of resistance to angular leaf spot in common bean and validation of the utility of resistance linked markers for marker assisted selection out side the mapping population

Inheritance of resistance to angular leaf spot (ALS) disease caused by Phaeoisariopsis griseola (Sacc.) Ferr was investigated in two common bean cultivars, Mexico 54 and BAT 332. Both Andean and Mesoamerican backgrounds were used to determine the stability of the resistance gene in each of the two cultivars. Resistance to P. griseola was phenotypically evaluated by artificial inoculation with one of the most widely distributed pathotypes, 63–39. Evaluation of the parental genotypes, F1, F2 and backcross populations revealed that the resistance to angular leaf spot in the cultivars Mexico 54 and BAT 332 to pathotype 63–39 is controlled by a single dominant gene, when both the Andean and Mesoamerican backgrounds were used. Allelism test showed that ALS resistance in Mexico 54 and BAT 332 to pathotype 63–39 was conditioned by the same resistance locus. Resistant and susceptible segregating populations generated using Mexico 54 resistant parent were selected for DNA extraction and amplification to check for the presence /absence of the SCAR OPN02 and RAPD OPE04 markers linked to the Phg-2 resistance gene. The results indicated that the SCAR OPN02 was not polymorphic in the study populations and therefore of limited application in selecting resistant genotypes in such populations. On the other hand, the RAPD OPE04 marker was observed in all resistant individuals and was absent in those scored susceptible based on virulence data. Use of the RAPD OPE04 marker in marker-assisted selection is underway.

QuantifyingVerticillium dahliae in soils collected from potato fields using a competitive PCR assay

A quantitative PCR assay based on the competitive PCR technique was compared to the classical soil dilution (SD) method for its ability to estimateV. dahliae propagules directly in soils collected from fields under potato production. A strong correlation (r = 0.97) was observed betweenV. dahliae propagules estimated using the quantitative PCR assay and those using the SD method. Coamplification ofV. dahliae DNA with competitor DNA provided accurate quantification in the range of 102 to 107 spores and 1 to 100 microsclerotia/g of soil. The number ofV. dahliae propagules detected in PEI soils ranged from 4.9 to 15.6 and 0.06 to 0.5 microsclerotia/g of soil for PCR assay and SD method, respectively. The strong correlation between PCR assay and SD method and the non significant differences between replications of PCR estimates ofV. dahliae propagules in soils (P< 0.05) show that the PCR assay is reliable and reproducible, and comparable to the SD method. This method is fast, does not depend on the subjectiveness of the traditional plating method, and offers an improvement in speed and precision over currently used methods. In addition, it can be extended to estimateV. dahliae propagules in other pathosystems and finds immediate and practical use in epidemiological studies to determine the effects of various crop management strategies on the dynamics and level of fungal propagules in the soil in order to establish threshold levels for assessing disease risks and develop disease prediction systems.ResumenSe comparó un ensayo cuantitativo de PCR basado en la técnica competitiva de PCR con el método clásico de dilucón del suelo (SD), por su capacidad de estimar propágulos de V. dahliae directamente en suelos colectados de otros campos de producción de papa. Se observó una fuerte correlación (r = 0.97) entre los propágulos de V. dahlia estimados usando el análisis cuantitativo de PCR con los usados en el método de dilución (DS). La co-amplificación del ADN de V. dahliae con el ADN competidor proporcionó cuantificación exacta en el rango de 102 a 107 esporas y de 1 a 100 microesclerotias por gramo de suelo. El número de propágulos de V. dahlie dectados en suelos PEI fluctuaron entre 4.9 a 15.6 y entre 0.06 a 0.5 microesclerotia/g de suelo por análisis de PCR y método de DS, respectivamente. La sólida correlación entre el análisis de PCR estimadas de los propágulos de V. dahlie en suelos (P<0.05), muestra que el análisis de PCR es confiable y reproducible y comparable con el método DS. Este método es rápido, no depende de las subjectividades de los métodos tradicionales de siembra y ofrece una mejoría en cuanto a velocidad y precisión respecto de otros métodos actualmente en uso. Además, puede expandirse par estimar los propágulos de V. dahlie en otros patosistemas y encontrar un uso inmediato y práctico en estudios epidemioloógicos par determinar los efectos de diversas estrategias de manejo de cultivos en la dinámica y nivel de propágulos de hongos en el suelo, con el fin de establecer niveles de inicio para evaluar los riesgos de la enfermedad y desarrollar sistemas de predicción de la enfermedad.

Harmonizing the agricultural biotechnology debate for the benefit of African farmers

The intense debate over agricultural biotechnology is at once fascinating, confusing and disappointing. It is complicated by issues of ethical, moral, socio-economic, political, philosophical and scientific import. Its vocal champions exaggerate their claims of biotechnology as saviour of the poor and hungry, while, equally loudly, its opponents declare it as the doomsday devil of agriculture. Sandwiched between these two camps is the rest of the public, either absorbed or indifferent. Biotechnology issues specific to the African public must include crop and animal productivity, food security, alleviation of poverty and gender equity, and must exclude political considerations. Food and its availability are basic human rights issues—for people without food, everything else is insignificant. Although we should discuss and challenge new technologies and their products, bringing the agricultural biotechnology debate into food aid for Africa where millions are faced with life-or-death situations is irresponsible. Agricultural biotechnology promises the impoverished African a means to improve food security and reduce pressures on the environment, provided the perceived risks associated with the technology are addressed. This paper attempts to harmonize the debate, and to examine the potential benefits and risks that agricultural biotechnology brings to African farmers. Key words : Agriculture, biotechnology, biotechnology debate, biotechnology and Africa, biotechnology issues, food security, poverty alleviation. African Journal of Biotechnology Vol.2(11) 2003: 394-416