Background/context:

The technology development project was initiated to streamline diagnostic assay development for pathogens on the CRCTPP charter. Different pathogen assays have somewhat different needs from high levels of specificity, quantification, sensitivity and high throughput of samples. The use of common technologies and equipment has the potential to give rise to lower development costs due to increased experience, the use of common internal controls, lower equipment needs and shorter development times. This provides the opportunity for a larger overall diagnostic capability with the funds available.

 Aim:

The major aim of this project was to provide knowledge and technical support in the development and delivery of diagnostic tests for plant pathogens under the charter of the CRCTPP. To achieve this, information on the latest developments in diagnostic technologies and delivery formats in the agricultural, medical and veterinarian fields was evaluated for applicability to plant pathogen diagnostics.

Major Outcomes:

Using the Sigatoka leaf disease group as a model system, a range of real-time, fluorescent polymerase chain reaction (PCR) technologies were evaluated for their applicability to plant pathogens. TaqMan® MGB probe assays have been adopted by the CRCTPP as the real-time PCR platform of choice. At the conclusion of this project, TaqMan® MGB probe assays have been developed for three banana fungal leaf pathogens (M. musicola, M. fijiensis and M. eumusae) and two strains of Fusarium Wilt in cotton (Fusarium oxysporum f.sp. vasinfectum VCG 01111 and 01112). 

To address quality control, a banana internal positive control has been designed to the putative pectate lyase (MWPL1) gene. The assay is available in both gel-based and real-time PCR formats and has been validated against 57 cultivars representing all banana genotypes, and also against a range of tissue types (leaf, corm, root, pseudostem and vascular strands) from Cavendish banana. This is the first host-specific, multiple purpose internal positive control to be developed in plant diagnostics. The assay is available to banana researchers worldwide for applications in diagnostic and fundamental research. 

Automation is a logical step towards streamlining diagnostic assays for plant pathogens, particularly during eradication responses. Protocols for robotic DNA extractors and liquid handling machines to increase assay reliability, throughput and turnaround times, have been developed for use in banana Sigatoka disease diagnostics. The protocols are available for rapid deployment in the event of a Black Sigatoka incursion. This equipment effectively triples the sample throughput and reduces sample turnaround time by 40%. Automation of extraction and PCR set-up also reduces the likelihood of errors and improves sample traceability through accurate sample-tracking from the field through to final diagnosis. This system serves as a model to promote automation of diagnostic assays across all crops.

For more information contact:

Dr Juliane Henderson
Department of Primary Industries and Fisheries
Phone: +61 (0)7 3896 9341
Email: Juliane.Henderson@dpi.qld.gov.au