1 DNA Barcoding of Pathogens of Quarantine

Transcript

Hello! Welcome to the last week of this course of ‘Detection Diagnosis and Detection of Plant Diseases’ and in this course will be talking about of ‘Diagnostics in Plant Disease Management’. So, the first talk of this week will be is DNA barcoding and it is very important nowadays to have a molecular signature to have proper identification of plant pathogens. Molecular signature is important because there are many closely related plant pathogens and to differentiate them from each other it is very hard to distinguish them with certain conventional technologies. So, that is why DNA barcoding has been developed to separate each other from the closely associated plant pathogens.

DNA barcoding- the necessity

So, DNA barcoding is the necessity of today and development of accurate identification tools for plant pathogens and pests is vital to support Plant Health Policies to manage plant diseases in a broader way. But there are certain Challenges: It includes increase number of plants being traded worldwide, then chance of harmful organisms spreading is also enhanced and taxonomic knowledge on harmful quarantine organisms is not adequately available. So, these are few challenges and that is why there is a necessity of DNA barcoding for proper identification of the pathogens. The economic damage is significant: Both when a harmful quarantine organism is not identified and as well as when an organism is incorrectly diagnosed as a quarantine organism. In the European Union (EU) alone there are some 275 quarantine organisms for which protective measures against introduction into and their spread within the Community needs to be taken care of.

Barcoding for diagnostics

DNA barcoding is basically a taxonomic method that uses a short genetic marker (DNA sequence signature) in an organism’s DNA to identify it as belonging to a particular species. So, this DNA sequence signature is basically the identity of that particular pathogen. Although the DNA sequences of related species are generally very similar, there are differences to be found. The part of the DNA sequence that is different is specific to that particular organism and forms a unique and specific molecular DNA barcode. One or several specific DNA barcodes are made for each quarantine organism and that is how we are now able to regulate the quarantine pathogens.

Plant Pathogen Barcode (PPB)

It is to facilitate the rapid and accurate identification of plant pathogens, a new database is developed that firmly link names and associated meta-data to a rapidly developing new standard in biology. DNA barcoding is an emerging gold-standard for species recognition. This development has already shown itself to have unprecedented power for clarifying species identities and limits, uncovering new and often cryptic species. So, it has been very useful to even identify new or even cryptic species. In response to the growing number of researchers who are using barcoding, data standards for barcode records have been developed, and an open access database has been created. The Consortium of barcode of Life, in short it is known as (CBOL) has engaged more than 125 Member Organizations from 40 countries.

QBOL (www.qbol.org)

So, QBOL is an European Union (EU) project on DNA barcoding, that started in 2009 to generate DNA barcoding data of quarantine organisms and their taxonomically relatives to support plant health diagnostics. The data are included in a database, called Q-bank (www.Q-bank.eu), which now consists of a dynamic open-access database of quarantine plant pests and look-alikes, linked to curated and publicly accessible reference collections. It contains sequence and morphological data including photographs, nomenclatural and diagnostic data of specimens available in reference collections for the benefit of the users. QBOL made DNA barcoding available to plant health diagnostics and focused on strengthening the link between traditional and molecular taxonomy as a sustainable diagnostic resource. Within QBOL, collections harboring plant pathogenic Q-organisms were made available. Informative genes from selected species on the EU Directive and European Plant Protection Organisation (EPPO) lists have been DNA barcoded from vouchered specimens and the sequences, together with taxonomic features and have been included in a new internet-based database system i.e. Q-bank: www.q[1]bank.eu.

DNA barcoding of fungi – So, DNA barcoding of fungi, incase of fungi it was mostly the ITS amplification and sequencing was done, but it was helpful to segregate some of the plant pathogenic fungi nut in certain cases ITS alone was not sufficient and for that other signature genes has been sequenced and DNA barcode has been developed accordingly. For, example incase of Didymella ligulicola it is the ACT that is required for DNA barcoding whereas in certain cases it is the TEF, TUB, or it the COI. These serve as DNA barcode for segregating these pathogens in a unique way from other related species.

DNA barcoding for bacteria

Similarly, DNA barcoding for bacteria, DNA extraction was done and it was followed by 16S rRNA sequencing and it gave us segregation of species like Clavibactor, Rolstonia, Xanthomonas, Xylella. But for other specific identification one has to go for GyrB, Muts, Gyrb and Muts and that gave us different signatures for certain other bacterial species and they were differentiated from the closely related ones.

DNA barcoding for nematode

DNA barcoding for nematodes also, it was the ribosomal DNA that was sequenced and it was able to differentiate nematodes from each other from different geographical areas.

DNA Barcode Library/Database/Informatics

So, all these informations had been deposited in the Q-bank and it has served as a library and database for other informatic applications. So, DNA barcode library, database and informatics is again a very useful source for identification of quarantine pathogens and it has been now made available to all the uses so that the global communities benefitted out of this database. The database was developed within the Dutch FES project during 2006–2010 and has been further developed during the QBOL project. Six databases were created: i.e. for fungi, arthropods, bacteria, nematodes, viruses and phytoplasmas. The total database Q-bank is freely accessible via internet (www.q-bank.eu) and a software module to export to and import from Genbank (and therefore EMBL) has also been implemented. The Internet-based software is continuously improved to comply with the needs of the end-users. So, filling of the databases has been made significantly during last stages of the QBOL project. So, it has been initiated and then it has been improved and now it is a very strong and powerful database for identification of quarantine pathogens.

So, once the database is publishes it helped internet visitors to regularly use the system. Websites are therefore not restricted to the users participating in the QBOL project anymore. Usage of the different databases are monitored by Google Analytics and that is how one can presence the importance and significance of the database by looking into the number of users. The bioinformatics and databases of Q-bank are based on the BioloMICS software (BioAware, Belgium). This tool allows specialized and scientific biological databases to be created to fit the specific needs of researchers working on any organisms for example arthropods, bacteria, fungi, nematodes, phytoplasma and plants viruses. It is used by a broad base users such as taxonomists, ecologists, human, plant pathologists, molecular biologists, pharmacists, industrial researchers, and so on. This again shows the significance and importance of database. The database is thus helping to identify quarantine pathogens and taking adequate measures to contain them.

So, in this particular talk we have seen that how DNA barcoding is helping pathogens to be detected, For that, we have to go for specific molecular signature and it has been assigned to a specific pathogenic micro organism and the information needs to be deposited in a public domain so that it can be accessed by all potential users and they can contain and again identify the quarantine pathogens and various levels and contain them and take adequate necessary measures. So, with this we come to an end of this first talk and in the next talk we will go for on-sites testing and moving decision making from lab to the field. So, till then have a good time.

Thank you very much.