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Real-time PCR capacity building in Kenya
The East African rose industry is the major exporter of cut roses to the Dutch flower auctions and UK supermarkets. Kenya alone exported 45. 76 million kilos of roses in 2003 worth £82 million and accounts for some 60% of all roses sold in the EU.
The Kenyan flower industry is estimated to employ directly 25 staff per hectare (50,000). About 70% of the staff on rose farms are women. Current unemployment levels in Kenya are over 50% of the population. There is no social security system for unemployment benefits and every worker usually provides financial support to at least 6 other extended family members, extending the benefits of employment in rose farms to some 300,000 people at the subsistence level.
Worker welfare standards have risen significantly in recent years due to the need for rose growers to comply with EUREP GAP audits which dictate worker welfare and health and safety standards. This has set a bench mark for other local industries which have a bad reputation for worker welfare.
Nearly every rose nursery in East Africa is affected by crown gall, which is caused by Agrobacterium tumefaciens.
Pathogenic A. tumefaciens strains harbour a Ti-plasmid, a small circular piece of DNA. During infection part of this plasmid is transferred from the bacterium to the genome of a recipient plant cell. This genetic transformation induces the infected cell to proliferate, resulting in the formation of the gall, and also produce a compound known as an opine, a nutrient compound which only the infecting A. tumefaciens strain can metabolise. Therefore the gall is, in effect, an opine “factory” and can be a favourable environmental niche for A.
tumefaciens. The disease weakens plants and reduces yields. It has been estimated that this disease is causing losses of some £40 million per annum in the region. The disease is so severe that galls are often seen on leaves – a highly unusual observation in crown gall epidemiology.
In collaboration with a Kenyan company, Real-IPM Ltd. [www. realipm. com], CSL have developed a real-time PCR assay which detects pathogenic A. tumefaciens strains and, critically, have also developed a simple DNA extraction procedure which will enable the processing of many samples at once. As rose nurseries can comprise many hectares and the number of stems in a batch which require testing can number 20000 it is clearly important in being able to test more than one sample / stem at a time. After the end of this project, and ahead of a bid for funds to develop a diagnostic lab in Kenya, we became aware that a real-time PCR machine was already present in Nairobi.
This machine is owned by Biosciences eastern and central Africa (BecA) at the International Livestock Research Institute campus in the city. BecA, through Dr Rob Skilton, kindly provided access to use this machine and their laboratory facilities. The BSPP agreed to co-fund Simon Weller on a trip to train Real-IPM staff in the use of this machine and also the extraction technique.
Real-IPM employ locally trained microbiologists to produce some of their biological control agents so there was a level of scientific expertise present in the three trainees. However, PCR machines are relatively rare in Kenya so whilst the trainees understood the principles behind the technique none of them had practical experience.
Fortunately PCR is not particularly difficult to do and as the running of agarose gels is not required in real-time PCR then in many ways it is easier to train people to do real-time PCR than it is to do conventional PCR. CSL also has a strong QC culture so SOPs were readily available for use as training aids. In addition to these basic requirements, real-time PCR provides data throughout the reaction so it is easier to look at individual reactions and troubleshoot any problems. This can be used as a valuable training aid, in that different experiments can be devised during training, rather than just testing samples to provide a simple Yes / NO answer to the presence of a particular target organism.
In all we had time to set up three separate PCR runs over the two days of training on the machine, from a simple testing of positive and negative controls to the testing of actual samples from rose nurseries. Each trainee set up successful reactions and thus the training will hopefully provide the technical expertise to support a diagnostic service in the future.
Biocontrol agents, distributed by Real- IPM, and the proposed test will be combined into an effective integrated control strategy to comprise an Industry Best Practise Standard (IPBS) for rose propagation. From this point it is hoped the impact of crown gall will start to reduce. The authors thank BSPP for this travel award.
Simon Weller, Central Science Laboratory Louise Labuschagne, Real-IPM (Kenya) Ltd.