PCR kits for the detection of Xanthomonas bacterial diseases

Development of a detection tool for Xanthomonas axonopodis pv. dieffenbachiae (Xad), agent of bacterial blight in anthurium :

Currently, no recognised curative method exists to control the disease caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). Present in anthurium crops, Xad is a pathogenic bacterium that is systematically screened for in quarantine greenhouses during plant material exchanges. It is subject to specific international regulations. CIRAD has developed a new molecular diagnostic test based on gene amplification, capable of detecting this bacterium.

• The test is easy to implement and is specific to pathogenic strains of Xanthomonas axonopodis pv. dieffenbachiae. Used directly on bacterial suspensions, it enables identification of Xad strains from any region of the world where the disease is present.
• The test is highly sensitive and can detect the presence of the bacterium in plant tissues even before symptoms appear.
• The test allows rapid processing of a large number of samples. It is particularly useful for screening large quantities of asymptomatic plant material, such as during quarantine release procedures.

Development of a detection tool for Xanthomonas axonopodis pv. allii (Xaa), agent of bacterial blight in onion :

Bacterial blight of onion is an emerging disease at the global level. In Réunion Island, it represents a major constraint on onion cultivation. The causal agent is a bacterium belonging to the genus Xanthomonas. It has been demonstrated that the bacterium can be present in onion seeds, and that as few as 4 infected seeds per 10,000 are sufficient to trigger an epidemic. It therefore became necessary to develop a detection tool capable of assessing the sanitary quality of locally produced and imported seeds.

A molecular tool based on the amplification of two markers (Multiplex Nested PCR) has now been developed, enabling the specific detection of all strains within the global collection of this bacterium. This highly sensitive tool allows detection of the bacterium in seeds after a rapid alkaline extraction, at levels as low as 5 × 10² bacteria per gram of seed.

Tests conducted on various batches of naturally infected seeds have shown that the PCR tool can reliably and consistently detect the bacterium at contamination levels as low as 1 in 27,340, which is below both the experimentally determined threshold associated with field epidemics and the standard tolerance threshold of 1 in 10,000 commonly used in certification programmes.

This makes it a high-performance detection tool that could be integrated into seed certification schemes to improve sanitary monitoring at different stages of production as well as in international trade.

Features

Diagnosis of anthurium bacterial disease in Europe and in all regions where anthurium is distributed, and implementation of certification procedures for plant material.

• Since 2010, this rapid and sensitive diagnostic method has become the reference method.
• In Réunion Island, anthurium and onion producers can have this diagnostic test performed by an accredited laboratory on imported plantlets.
• The test has been marketed internationally since 2011.

Intellectual property

CIRAD patent No. FR2848222.

The invention patent was filed in France in 2004 and extended to the Netherlands in 2006.