5 Development and Implementation of Rapid and Specific Detection Techniques

Transcript

Hello!! welcome to the last talk of the week that is, Detection and Implementation of Rapid diagnostic techniques.

We know that lot of plant pathogens that they come to a new area through transmission via seeds, and leafy vegetables are one of the major source of such transmission and several pathogens has been reported to occur in new areas and new countries through this seed export and import. So, commercial significance of seed industry also heavily dependent on this seed lot whether, it is contaminated with a pathogen or not and that is why rapid detection of such seed lots has become very very important.

Commercial significance of seeds and seed borne pathogens

Fast and sensitive diagnostic tools are necessary to screen seeds and transplants used for commercial purpose. Due to the role of seed transmission, detection methods are also important for the production of pathogen-free seeds and for their certification. It was during the past one and half decade it was observed that several new diseases were introduced in European countries in the production sector through infected seeds. Molecular methods proved helpful in the detection of several formae speciales of Fusarium oxysporum, Verticillium dahliae and several other causal agents of foliar diseases of leafy vegetables. So, already there is a base that is why it needs to be investigated and this investigation needs to be done very quickly so that the commercial value of the seed lot is retain and the industries are not suffered and at the same time a new pathogen is not introduced to a new land. Fast and sensitive diagnostic tools are necessary so that primary inoculum vectors, such as seeds and transplants, can be screened promptly. Pathogen’s detection on seeds can be a difficult task because in most cases infected seeds can be asymptomatic, making visual detection difficult or impossible. Moreover, infected seeds may be present in a limited percent, and nonuniformly distributed within a lot. Different detection assays exist for different seed-borne pathogens but only a few respect the minimum requirements for adequate seed tests. Traditionally, seed assays have been developed based on – visual examination, selective media, serological techniques, Polymerase Chain Reaction. Several new diseases of leafy vegetables for example, in case of (lettuce, wild and cultivated rocket, lamb’s lettuce, cichory, endive, basil, spinach) were introduced in Europe through infected seeds. Some of them were reported for the first time in Europe or worldwide. Identifying the source of inoculum is of critical importance for effective disease management. Due to the role of seed transmission, detection methods are also important for the production of pathogen-free seeds and their certification. So, it is highly essential that such seed lots should be screened very quickly and promptly to establish whether it is carrying any pathogen or it is pathogen free. Among diseases, Fusarium wilts on lettuce, then wild and cultivated rocket , lamb’s lettuce, cichory and endive (Cichorium endivia) were newly observed. This was not early observed in certain European countries prior to this. Also several species of Alternaria, of which there is evidence of being seed transmitted, are reported on leafy vegetables. For example, Alternaria cichorii is reported on lettuce, endive and scarola, while Alternaria japonica has been recently detected on both wild and cultivated rocket. So, these are certain examples along with along with other pathogens such as, Verticillium dahliae, it was reported on lettuce, cichory, spinach and Plectosphaerella cucumerina on wild rocket, and so etc. So, lot of evidence have been gathered recently to establish that lot of new diseases has been introduced to certain parts of European countries and mostly they have been entered through the seed lots of leafy vegetables or through the transplant.

Fusarium oxysporum: Let us take the example of Fusarium oxysporum

The search for molecular techniques has been particularly intensive and effective in the case of several formae speciales of Fusarium oxysporum. So, Fusarium oxysporum has several formae speciales so it was very hard to identify which formae Fusarium is newly introduced. The detection threshold of F. oxysporum in seeds and propagation material could be increased by using molecular techniques, such as the PCR. So, PCR comes to play in such cases where the pathogen level is very low. So, PCR can multiply the pathogenic DNA to significant level for its detection.

In the case of Fusarium wilt of basil and lettuce a nested-PCR-based method allowed fast and unequivocal identification of F. oxysporum f. sp. basilici. The method permitted to detect 32 conidia/100 seeds and required 4 hours. DNA was extracted only from propagules present on the external surface of the seeds. So, technology variants of PCR’s like nested PCR helped in identification of the Fusarium oxysporum f. sp. Basilica and the amount of inoculums load was very low that is 32 conidia per 100 seeds but still this technology was helpful in identifying such low amount of inoculums of the formae speciales .

Verticillium dahliae

Next, example is Verticillium dahliae – A quantitative real time polymerase chain reaction (qPCR) assay was optimized and used for the detection and quantification of Verticillium dahliae in spinach seeds, resulting quite reliable and sensitive, permitting to assess values up to 1.3 % of seed infection. So, Verticillium dahlia was also able to detect through application of quantitative PCR and it was able to detect the presence of the pathogen as low as 1.3% of the total seed lot.

Similarly, V. longisporum has been described as a hybrid species that presents several genomic regions in common with V. dahliae. V. longisporum is a crucifer pathogen but never described in diseases associated with spinach, but in any case the external presence of this pathogen can result into a false positive; then it is important to exclude the presence of this pathogen from the samples.

So, certain other conditions has to be kept in mind, for example, Verticillium dahlia is a pathogen of spinach, but the spinach seeds can be contaminated with Verticillium longisporum which has certain genomic reasons common with Verticillium dahlia. So, PCR amplifications of those common reasons could be misinformation about Verticillium dahlia. So, that needs to be taken care of how to exclude those microbes which are sharing some common genomic reasons with the pathogenic ones but actually, not a pathogen of the concerned species.

Alternaria – Similarly, in case of Alternaria Distinguishing Alternaria species is always a challenge in leafy vegetables and PCR-based methods were used to detect Alternaia radicina infections on carrot. However, RAPD analysis that was helpful to distinguish Alternaria. radicina from the other Alternaria species. Additionally, PCR-RLFP was helpful to identify and distinguish three Alternaria species: Alternaria radicina, Alternaria dauci and Alternaria alternata from carrot seeds. Standard PCR and Real time PCR were useful in identification of seed contamination by Alternaria brassicae on cabbage and radish. So, different tools even the principal is same the variants of the particular tool is deployed for distinguishing between species of similar some pathogens like Alternaria to detect on the seed lot. So, we have seen that different tools and techniques has been deployed for rapid identification of pathogens for quick determination of whether their presence is there or not in the seed lot because it has the seed lots always carry a commercial value. At the same time seed lots need to be checked if t is moving to a different area or a country or to another country whether, it is carrying a pathogen that it non existing to the country concerned were it is going to be shown. So, this tool s and techniques again it is coming in a big way is helping for a rapid detection of seed lots and it is helping a way in checking some quarantine pathogens as well.

With this we have to an end of the fifth week and in the last week that is in the sixth week we will be talking about how diagnostics are helping in decision making process for plant disease management. So, with this thank you very much will see you in the next week.

Thank You.