 Well, good morning, good evening, good afternoon to everybody, here it's about a quarter past midnight in Australia. And so I'm sure people around the world are at different time zones. So what I'm going to talk about today is a case study that we've been doing in Australia on tear fortyte diagnostics. And this is part of a much bigger project that we're doing on the development of resistant cul-de-vas. Next slide, please. OK, so we have a field trial site up in in near Darwin in the Northern Territory in Australia. It's a large commercial plantation area. TR4 has been in the area probably for around about 20 years. This particular plantation was established over 10 years ago and very quickly TR4 became established there. So it has a very high level of endemic TR4 in the soil. So where our field trial site is previously was a commercial planting of Cavendish and is now adjacent to those commercial plantings. The important thing is that we have a complete history of every plant in that trial. And all of the infections that we record from a visual inspection are confirmed by PCR and in most instances by sequencing. And I'll go into that at a later time. Invariably, we do sequencing, as I say. This trial is providing a huge amount of very, very important data on the rate of infection, the rate of movement around the field, the spread, also the recovery phenotype that we see. And we've got a large collection of samples from that site already. Next slide, please. So this is where the site is at the place called Lambels Lagoon. So I'm at Queensland University of Technology, which is in Brisbane and Australia. That's a subtropical environment where the field trial is at Lambels Lagoon is a tropical environment right up in the tip of Australia. That's about 2,500 kilometers between QUT and our field trial. So we regularly visit there. We go there probably around about once a month. Next slide, please. There we are. Oops, we're missing. Can we go back? Go back. Back. Oops, we're going back. There we are. Thank you. Go back one more. OK, now we should go forward from there. OK, so as you'll see from this slide, it's a very large field trial. It was established in March 2018 and it continues today. The results we've taken so far are up to or presented today up to the end of March 2021. So that's three years after the initial planting. Next slide, please. OK, this is an aerial view of the field trial site. It's a four-hectare 10-acre site, as you'll see there, with a commercial banana planting right next door. This is just before we planted that site. We have large screenhouses there where we can do two things. One is that we can acclimatise plants we take up, but also we can do small plant studies in those screenhouses again. We have a small office area and we've got a lab onsite as well. This is quite a remote site, so we've got a small lab there that we can conduct relatively simple experimentation, particularly around DNA diagnostics, but we can also isolate TR4 from soil or plant samples there as well. Next slide, please. OK, so this is a trial which involves our genetically modified Cavendish lines. As I say, the trial will run over a period of five years. It's a randomised block design planted in March 2018. So we've got two controls, both Cavendish, Grand Nane and Williams. And then we've got four genetically modified lines. We had a previous field trial with these same four lines, and these are in Grand Nane. And one of those lines in that first trial appeared to be immune to TR4 again over a three-year period, and three other lines with very, very high levels of resistance. So this trial has those same four lines. The results we got are essentially identical to the phase one trial, but on a much, much larger scale. And for this trial, we're collecting most of the agronomic data as well as the disease resistance data, including yield cycle time. And as I said, we have this complete history of every plant in the trial over a three-year period. And at the moment, we're just preparing a publication over those first three years of that trial. Next slide, please. Just to give you an idea, these are some of the non-GMs. So these are some of the wild-type plants in that field which are very, very typical symptoms, yellowing of leaves. And when you cut open the shooter stem, areas of significant vascular discoloration, visually high levels of vascular necrosis. Next slide, please. OK, our diagnostic approach. So our diagnostic approach is all based on the six genes, the secreted enzyme genes, which encode small proteins. And all of these genes are based, are located on the FAC pathogenistic chromosome. So when you go in sequence, and it doesn't matter whether it's tropical race 4, subtropical race 4, or race 1, they all have these six genes, but they have different six genes. And that's very important. For instance, in TR4, there are three sequences of 6.1, one sequence of 6.13, et cetera. And you can differentiate TR4, STR4, and race 1, depending on the six genes and the sequences of those six genes that they have on their chromosome number 13, the pathogenistic chromosome. So what we've done is we've completely sequenced our TR4 isolate from the Darwin field, as well as local isolates, Australian isolates of subtropical race 4 and a number of race 1 isolates as well. So we've got a very good sequence database. And we've also got GenBand sequences. And from these sequences, we've designed our diagnostic primer sets. Next slide, please. OK. So all of the plants that have symptoms are screened by PCR. So this is quite important. So what we do is we've got a set of six primer sequences. And they're all 6.1 gene sequences. And they're all unique. So we've got three for tropical race 4, TR4. We've got one for subtropical race 4 and one for race 1, as I said, all from the 6.1 gene. And what we do is, and I'll go into a little bit more detail in a moment, we extract DNA on site. And then we transport that DNA to our labs in Brisbane. We do PCR on those, not multiplex PCR. We do each of those PCRs in a separate tube. And then we normally sequence by Sanger sequencing either the PCR product directly or we clone and then sequence that PCR product. Next slide, please. OK. So this is actually how we do it on site and back at the lab. So wherever we have an infected or a suspected infected plant, and we also from time to time take healthy banana plants at our Darwin fruit farm field trial site. As I said, we have a small lab there where we isolate genomic DNA. So what we do is we always collect tissue from the vascular necrosis areas within the shear to stem, and I'll certainly talk about that in a moment. So that's the tissue that we collect. We use DNA's OLS and liquid nitrogen. It's one step method. And we only need ethanol and no fume hood. And that's quite important because we don't have a fume hood up in our small in our small Darwin lab. So this methodology is particularly designed so we can take samples at remote locations and where we don't need very sophisticated facilities to be able to do that. So we dry those DNA pellets on site and then fly them back to Brisbane. We do PCR there with a music housekeeping gene. We always use the 3TR4 6-1 gene sequences, the PCR for those, and randomly do STR4 and race 1 amplifications as well, just to make sure that we're not picking up anything else. These are then electrophrys through agarose. We cut out and purify those bands and send those off for sanga sequencing. We can either do that within our own facilities at QUT or we can also do it with external labs. We use Genius as our gene comparison software. So we import those sequences into Genius and we can do analysis basically using our long read assembly of TR4. We can also use next-gen sequencing and comparisons with non-TR4 sequence assemblies of those published. So what I thought would be useful is to go through some of the results that we're getting and you'll see over the next group of slides the sorts of results we're getting. And I think this is quite important. We've seen lots of examples in the literature about the sorts of results people are getting. These are the results that we're getting and this is in a field trial where we know the history of every plant. Okay, so this is a very early stage infection. This is where you see just one leaf showing some symptoms. And this is very, very common in our trial. Next slide, please. So what happens when you cut that plant down the pseudostem and you'll see at the base of the pseudostem, so we'll go all the way up the pseudostem taking samples at the base of the pseudostem, even at that very, very early stage you see very significant vascular discoloration. And as you go up mid-stem and you'll see right there in the center of that you'll still get some flecks of vascular discoloration and even right up towards the top of that pseudostem. So this is even in very, very early infections you get that vascular necrosis right up through the pseudostem. But you do not see or we don't see any necrosis in the peduncle and we don't see any necrosis in the fruit. So next slide, please. So the results from that we get our PCR positive in the base, PCR positive mid-seudostem and PCR positive at the top of the pseudostem, but we do not get PCR positives from either the peduncle or from the fruit. So that's an important result and we've repeated this many, many times. Okay, so here's a plant, this is mid-stage, this has a number of leaves already showing symptoms. And so it's a mid-stage infection, it's still standing. And so when we go and take the pseudostem from that plant, next slide, please, we'll see that there's significantly more vascular discoloration. So we're getting a lot of vascular discoloration then in that base. We've seen it very, very obviously in the mid-seudostem region and also in the top-seudostem region. If you look at the peduncle, very careful, whoops, go back, yeah, jumping the gun there. If you look at the peduncle region, sometimes we see little flecks of necrosis. We always take those and very carefully see whether they have any fusarium fungus present. Next slide, please. However, in all of the mid-infections, we get obviously PCR positives at the base, the mid, and the top, but again, we don't see any fungal evidence in the peduncle and certainly not in the fruit. So next slide, please. Okay, so this is a late-stage infection. So this is very close to the final moments of that banana. And we have certainly a lot of those in our control plants. I should say our control plants now after three years, we're getting up to close to 80% infection. Next slide, please. Here we have very, very extensive pseudostem vascular necrosis at the base. It really is pretty well-going rotten. And when we look, there are other, there are other often saprophytes in there as well as fusarium. In that mid-stem region, very significant vascular necrosis even right up at the top. Again, in the peduncle, we see sometimes these little, little necrotic flecks, but they are just necrotic flecks and we don't see anything in the fruit. Okay, next slide, please. Okay, and again. So obviously positive at the base, positive in mid-seudostem and positive in the top of the plant, never do we find fusarium up in the peduncle and we never find fusarium in the fruit. Next slide, please. Okay, so some take-home messages from our experience. This field trial site has been a fabulous field trial site for us to develop and validate diagnostics and diagnostic protocols. So what we did over the, and we had a previous, I should say we had a previous field trial to this a much smaller one. And we tried a range of primary sex, some of which or many of which have been published. And it was using that first field trial that we came up with the primary sex that we've developed and that's been really a great opportunity at that field trial site to both validate those diagnostics and the diagnostic protocols. And we've been specifically doing that so that the sorts of protocols we've been developing can be used at remote sites like at this field trial site. The primary sets we use on the unique 6-1 genes and they've turned out to be very robust and highly reproducible results. We're pretty careful. So what we do is we tend to sequence the PCR products and we think this is highly desirable and essential I would say for any new incursion verification. And that can be done in our case. We have in-house capability for doing Sanger sequencing and Illumina and Pat Baer sequencing but from time to time we also send that off to other laboratories to make sure that we're getting the same sorts of results whether we do them in-house or we send them overseas. I think one of the important things that we've found is to sacrifice the plant that you're collecting tissue from because of that very, very early stage of infection, the vascular discoloration can be very unevenly distributed across the seater stem. And if we take, and I didn't show those results but I could have, if we take the non-vascular discolourated tissue, the tissue that looks quite healthy, very often we do the PCR product. Always get a PCR product from that vascular discolourated area. So we always sacrifice the plant to make sure that we collect that vascular discoloured tissue to ensure that we're collecting tissue that has been invaded. I should say that we've now done literally hundreds of PCR diagnostic tests from samples at Darlin' Fruit Farm and these are all field samples. So these are a pretty good example of how we can test for TR4 in the field. So very importantly, we have not identified TR4 in the peduncle of even very, very advanced TR4 infections. So that briefly is the diagnostic protocols and our experience from our Darlin' Fruit Farm. Next slide, please. We've got a large group of researchers involved in this. This is primarily a project to develop genetically modified and gene-edited Cavendish with resistance to TR4 but a very good side project data that has been the development of the diagnostics and we've had really very, very good support, funding support over the years. So thank you, Victor and Nelson. That's my presentation. Oh, thank you very much, Dr. Dale, for your presentation. I take a few points away from your presentation which of course we'll speak about a little later. We are already beyond in our agenda and our time. So we'd move on to the other presenter, Dr. Yolan. And I wonder before we give her the floor. Very interestingly that your research should show that the infection could exist in the stem, in the pseudo-stem and not in the peduncle and the fruits. So this is an important take home and thank you for that. I'll give the floor right now to Dr. Yolan Shilling-Charles, Sirad. As she would expand on her presentation. Thank you very much. Remember, just a reminder that the presentations are 10 minutes long and we will take a few questions later on. The floor is yours, Yolan. Okay, all right. Thank you, Nelson. Good morning, everybody. In the following presentation, I'm going to develop the methodology proposed by Sirad to diagnose a 40A04 in the particularly in the French overseas territories because we will see the official process which has been validated by the Plant Protection Services of the French Ministry of Agriculture. Next slide, please. The four steps we can see in this process are common to all diagnosis processes. And the first three steps in the field are very, I will tell that is there are very important to do a quick and a good diagnosis because we know that even if we have good molecular tools, if the samples are bad, we couldn't do a correct diagnosis. Next slide, please. In the French territory overseas territories, we have four principal actors in the monitoring. The producers and the technician, the Plant Protection Services and delegates. The first one are monitoring the presence of the disease on the field and send the four two symptoms to the second. And now at Sirad, we are actually developing an Android software and this tool will help the Plant Protection Services shorting the data sent by the producers and the technician and they can plan the sampling if it is necessary. Next slide, please. Thank you. Two steps in the identification of four TAO4 in the field. First, the producers and the technician using the flyers to do a good identification for the external symptoms only. And they can during this time, they have to secure the area and inform the Plant Protection Services as they are not allowed in France to do the windows in the pseudo-stem to observe the internal symptoms. Next slide, please. Thank you, Matt. The sampling is operate especially the slide before. Okay, right, no problem. The sampling is operate especially by the Plant Protection Services or delegates and the numbers of technician are limited of two person to avoid the risk of dispersion. The only sampling techniques we use is the one shown by Dr. Perez Vicente during the first FAO workshop in Trinidad. The pseudo-stem samples are dried before sending to the reference laboratory in France. Next slide, please. Okay, so since the identification of the symptoms by the growing technician, it is necessary to put the contingency action in place because the following steps in the laboratory will need time, enough time for the dispersion of the disease if the area is not secured. Next slide, please. For the French Odyssey territories, the diagnosis is operate by ANSES, which is the reference laboratory. The first steps for ANSES is the confirmation of the presence of folk in the samples by the isolation and the morphology identification of folk strengths. Next, please. The second step is the real-time PCA essay developed by Agaiou and I in 2016-17. Using plants samples. This test is the formal one. Next, please. But Sirad will propose soon at the beginning of the next year another molecular test based on the loop-mediated isothermal amplification. Especially to practice a first diagnostic in a short time in the field and do an early detection of the disease on plant which have few symptoms. Next, please. The pathogenicity essay are the third step of the process. They respond to coax postulate. They confirm that the strains isolate are in cause of the symptoms. For this essay, we use a solid inoculum with which is millet seeds inoculate with a Cornelia suspension. This solid inoculate inoculum mainly contains Clamidospore and the banana plantlet used are two months each old. Next, please. The ultimate step of the diagnosis in the laboratory for us is the VCG test. In this test, the formation of Heterocyan obtained with net mutant of universal 40-4 strains confirm that the diagnostic of VCG 1230, 1216. Next, please. As you understood, the methodology chosen to diagnose 40-4 in the French of Heterocyan stories is a partnership process in which each partner breaks its experience and is expertise in the different step of the diagnosis. Next, please. If we have a global look on the different steps and the advantages and the challenges of those steps, we can say that the participation of the producers are very important. More eyes to survey. And the Android software, it's a good really advantage to give us a quick response and to collect the data on the symptom disease, the symptoms, but also on the geographic localization of the disease. The challenge we have now is to increase number of observation and finalize the web software. On the identification step, we have now a good sample of data base to help the plant protection services, but we have to improve the public information to reduce the risk of spreading the disease. The sampling is easy and safe because few persons are allowed to do it. But in the future, we'd like to improve the FTA codes did by Sigma because these codes can allow the safe delivery of samples between different laboratories. On the diagnosis, we can say that now is quite safe because we have both molecular tests and pathogenic tests. It can be quick, with the first diagnostic on the field with the lump assay. And for the few months in the future, we have to finalize the lump assay on soiled and water to use it for the survey. The next please. And I would like to end my presentation on this poster did by the Plant Protection Services of Guadeloupe to inform banana plantation workers on TAO4 symptoms. And I will tell thank you all for your attention. Thank you very much, Yolan, for your presentation. Of course, you said something which is important, everything was important, it stood out to me. It says more eyes for service. So diagnostics goes hand in hand with service. Thank you for that. I will just inform the group that the Q and A tab is active. You could place your questions in there and the panelists will be able to answer your questions. There are a few questions are ready for Dr. Dale and Dr. Yolan. And we will, currently, we will skip a bit the question and answer session so we could gain some time. Of course, we have four more panelists to go. We'll gain some time and then we will, if we have enough time, then we'll have this interaction, this verbal interaction between yourselves and the panelists. So right now, we'll just go on to Dr. Diane Monster from Stellenbottch University, who will be presenting to us today. Thank you very much. You have the floor, Dr. Monster. Hi, good afternoon here in South Africa. Can you guys see me and hear me fine? You are loud and clear. Great. Okay, well, thanks for the opportunity to present today. It's a great honor to me to present to this group. And I will be talking about how we characterize FOSC at Stellenbottch University. And I will refer to FOSC, of course, it's the physiarum oxysporum formispecialis and cubanes fungus that we all know and that is causing all the problems all around. So if we move to the next slide, please. So in the interest of time, I will jump right into it and explain the process we follow here to diagnose FOSC samples. And I will make the hypothesis that we are dealing with a situation where there is a possible new incursion of FOSC-TR4. So we also obviously characterize some other strains. So I'll focus this presentation mostly on FOSC-TR4. So usually when we start our process, we get a piece of infected dried plant material. And I think Yolanda has shown that as well in her presentation that we usually request from someone to send to us by courier. And depending on how urgent the answer is, and usually it is very urgent, we will do an initial extraction directly from the plant material and run a QPCR, which can give us an answer within a day of receiving a sample. And this technique obviously has the advantage that you can do it very fast and it's very sensitive and you can directly extract from the environmental material. However, it is, it can be quite expensive and you need quite specialized skill to do QPCR. And there's always a risk if you are doing your detection directly from environmental materials that you can detect false positives. So the QPCR, just like other PCRs is usually based on a single gene. So that can also be kind of a negative. And if you did not sample correctly, a negative answer might be due to your sampling bias rather than the sampling actually not harboring FOSC-TR4. So therefore we will always go ahead and isolate and purify our samples. And once we have a purified sample, then we will store it in our culture collection so that we can go back to that sample if anything goes wrong in the diagnostic process. We will extract the DNA and then we can do whatever downstream application we want to do with that. That could include obviously PCR or gene sequencing or VCG testing or pathogenously, depending on who is the person that we are doing this test for. If we are dealing with a new incursion, like I said before, we will use different types of conventional PCRs that are available so that we are sure that we are confirming this FOSC-TR4 ID correctly. So purification and subsequent PCR analysis have the advantage that it's affordable and that most labs have PCR machines. So I think Professor Dale has also touched on that. You want an assay that can be widely applied. So that is very important. And of course, if someone is skilled, they can do their first analysis, the isolation and DNA extraction themselves and then send the DNA over to our lab. And that obviously poses less of a quarantine risk so you don't have to move around this infected plant materials. Again, like I said with the QPCRs, because single-plex PCRs are based on our single gene regions, there's still possibilities that false positives can be picked up and I will elaborate on that in the next slide. We will sometimes also support our diagnosis with reference gene sequencing and we have an in-base gene database which we will then use to draw up a phylogenetic tree and the power of gene sequencing, of course, is that you can compare your sample to public databases, which is a very good pro of this. It is however expensive and you need specialized skill to interpret the data and your sequencing of your reference genes, this is an important point, can also not always distinguish between very closely related FRC-VCGs. And here I can make an example of two of the VCGs, the one that's related to tropical race four, which is the 12, 13, 16 and zero, one to one, having identical sequences for the TEF region that is used very often to identify for serum oxysporum species, but they can actually have very different virulence levels. So this is why we confirm our FOC IDs that we have with our molecular studies always with VCG testing. And although this is quite timely, we have a lot of confidence that once we have ID something with VCG testing and we combine that with molecular analysis that it's very accurate. Unfortunately, it has the added cons that it takes quite some skill to do this test and you need the standard test, which is fungal material, which might pose also a quarantine risk. And ultimately, we also would confirm a new incursion by doing pathogenicity testing on Cavendish to complete COHS postulates. And this is really the only a reliable way that you can currently confirm the virulence of a sample. However, just doing pathogenicity testing alone is not enough as you can't distinguish closely related FOC VCGs and that sometimes samples within a VCG will have different responses when you do the pathogenicity testing. Furthermore, of course, it poses a quarantine risk and you need specialized skill and access to plant material to conduct this. So we go through this whole process and we make very, very sure before we put the data out there. And once we've done all of this test, then we can confidently say, okay, cool, we do have an FOC ETR4 or we don't. So next slide, please. Okay, as I mentioned before, we always also carefully consider molecular markers that we are using to diagnose FOC. And we come from a standpoint that molecular markers are not absolutely reliable. And this was highlighted in the work by MacDalma et al, who is also in the audience today, who showed that some of the popular molecular markers were amplifying some non-pathogenic endophytes. The problem is that when you're validating a molecular marker, you cannot taste all of the species that you're going to encounter out there. So this, why this picked up on this assess is because the sequences that were used to design these markers had high homology between your true and false positive samples. And this is often the case when molecular markers are designed from genes that have housekeeping functions. From this paper, they basically concluded that diagnostic markers in the study of Lee et al, they are on your left-hand corner and colleagues were the most reliable. And this genetic locus was then used also to, and they think that this genetic locus is linked to virulence. And since other approaches have also considered the six genes, and I think Professor Dale has elaborated on that quite a bit. Important, like I think also Yolanda mentioned that we work together with Sirat, or ANSI is actually to develop a QPCR assay that was based actually on the genetic locus in that paper by Lee and colleagues. And we know this is very accurate and it's been very well validated, but it does pick up two of the closely related VCG. So this prompted us to design our own QPCR assay that only pick up the VCG 1316. And we validated this on different environmental samples. So we looked at can we detect it from water, soil and plant material. And this is the QPCR assay that we are using currently that's listed there in that paper. So, but when we are dealing with a new incursion, we would often use two or three different molecular markers designed from different genetic loci so that we have more confidence that our answer is correct. Okay, so next slide, please. I will go quickly through the next session, but it's just that I want to kind of cement in that there's important considerations when you're using particular diagnostic tools. So I will quickly discuss the following questions that you see on your screen now. And this is the things that we consider when we are conducting diagnosis. And this will be relevant to anyone out there in the audience today, when you are considering which tool to use and how to go about it. Next slide, please. Okay, so to answer the first question, what capacity and budget do we have available? So at Stellenbosch, obviously we can do almost everything, but you have to consider your situational with what you have available. So for molecular diagnostics, we can do PCR, QPCR, we have a sequencing unit that where we can do reference gene sequencing and whole genome sequencing. For phenotyping, we can do morphological identifications. We also do obviously VCG testing and one of the advantages that we have is that we have a characterized population that is accurately characterized from all over the world, which we can do to compare and do comparative population studies. We also have a tissue-culturing facility and we have optimized techniques to screen different varieties for pathogenicity or resistance against different FOC VCGs. And we have highly skilled, dedicated personnel that can conduct each of these analysis. You can go to the next slide, please. Okay. And now two very important things that we consider is firstly, when we get a sample, where was it isolated from and what is the history of the disease in that region? So we know, for example, if we look at that map of Africa, that's where a lot of our activities are, we know that there's a group of closely related VCGs in a single phylogenetic lineage that we call lineage six that dominate whenever we're doing surveillance out in the central and East African regions, we always get this group of VCGs that cluster in that lineage and this has prompted us to kind of optimize a multiplex PCR2 to rapidly identify this lineage. We also consider the variety that we isolate from. So of course, when we see disease on Cavendish and the tropics, we are immediately considering, okay, it could be FOC-TR4. And we will start our diagnostic process there. However, if we get a sample from South Africa, that's a Cavendish that's infected from South Africa, it would more likely actually be STR4. Or if you look at that picture on the left, that's a blecoreo type of banana. We took that in northern Mozambique, so close to where the FOC-TR4 incursion was, there it could actually be any of the races. So this kind of guides the process on how we go about our diagnostics. Okay, next slide, please. The last point that I want to make is that you should consider your situation very carefully before you spend a lot of resources on expensive tools, diagnostic tools. And I'm saying this because the first step really is, the most important is that role players are able to recognize the disease accurately in the field. And that's your first line of defense. And we know when a Cavendish shows typical FOC symptoms under tropical climatic conditions, we might have a problem. And from that point, you can already start your containment protocols. So when the symptoms show on a plant, it also means that the disease might have been there quite a while already. So now do we need to confirm our suspect plant within 30 minutes? Well, that would be very convenient, of course, but the most important is that we accurately identify the sample because rather than causing panic, if we haven't confirmed it very accurately. And I will make an example of this diagnostic that was recently marketed to the community. We don't know how accurate this is as it wasn't published in a peer review article. With this, I'm not saying that it's not accurate and obviously it can be used, but just that we don't know. And even though they are claiming that they can very rapidly IDFO CTR for, it's worth to spend our resources. Is it worth that we spend all our researchers on buying these expensive machines that we need to do this assay? Or can we just rather use our PCR machines at our research institutions? So what I'm saying with this is, look at your situation, what is your budget, what type of skills do you have available and then decide which tool is the most appropriate to your situation. And with that, you can go on to the next slide. I would just want to say thank you for some of the funding bodies and collaborators that we've been working on in recent projects on diagnostics and studying the populations of FOC worldwide. Thank you very much for this opportunity again. Very good. Thank you very much from South Africa, Diane Moncet. Thank you for your presentation and sharing with us the experience in South Africa. It's Dellenbosch University. We will go directly now to Dr. Gerd Kammer from Morgan England University and research and he will present to us their methodologies and he will do his presentation now. Thank you very much. The floor is yours, Dr. Kammer. Thank you. Can you hear me, Nelson? I can hear you loud and clear. Okay, thank you very much. Well, first of all, welcome everybody and around the world, so to say. So here it's now five o'clock in the afternoon. And I also would like to congratulate the World Banana Forum for your plus 10 years anniversary. And I was one of the inaugural members and I still remember I was actually correcting during the first meeting our first paper on the molecular diagnostic for TR4 that we developed. So that was published in 2010. So maybe we can go to the next slide. Well, here you see the internal symptoms that have also been shown by the previous speakers and I of course like to thank them for introducing various aspects of TR4 diagnostics. But what you can see in the left panel is those vestular strands and also in the right panel, those are the ones that are really dark but also the faint and yellow ones, those are the vestular strands that we usually receive. As also mentioned by one of the previous speakers which had been dried at room temperature and then these are processed in our lab. Very similar to what has been discussed already by all the previous speakers. So I'm not going to repeat that. Of course, what is very important is number one, speed. And that has to do with new incursions and it's always being validated by inoculations of plans in our greenhouse facilities at Wageningen University to indeed complete postulates. And I have to say we actually have abandoned VCG testing because we sequence all the strains that we receive. So from our perspective, sequencing has really replaced VCG testing. So we can go to the next slide. And this is a kind of the distribution over the years of TR4 and the dissemination of TR4 around the globe. So to make a long story short, in most of the green those that you see the diagnostic that we developed and published in 2010 and that was a collaborative effort with Dr. Miguel Dita who will speak later who was a visiting scientist in my lab in those days, in those years. And that's when we started to work on Fusarium and our first target was to develop a molecular diagnostic simply to have speed because until then TR4 diagnostics was entirely based on VCG testing. And it's also been mentioned by Dr. Mostet and others that really take several weeks and sometimes even month. And for the management of a disease you need accurate diagnostics to act quickly. And we see that now happening around the world. Of course also with the COVID-19 pandemic. And there you also see that the various tools that we have developed and that have been discussed also during this webinar are very important to diagnose and to manage. And that's exactly the same for TR4 and banana. So you see one red year, 2014 more or less in the middle of the screen. And that was when we used our diagnostic for the first time or when we actually diagnosed TR4 for the first time outside Southeast Asia. And that was in Jordan. And since then it has shown up in various other regions of the world as you can see. And as also be mentioned by the other speakers. And in most of those incursions after 2014 the diagnostic that we developed has been used to confirm quickly as indeed this is TR4. So the speed of testing is of course very important. And of course it needs to be accurate. That's very clear. And I come back to that in one of the next slides. So maybe we can go to the next slide. This is important I think because we decided upon publishing our diagnostic in 2010 to actually start working with a commercial company in the Netherlands to commercialize the TR4 diagnostic. Because we have experienced that once people start using the protocols that we published in scientific papers and that's what we have experienced in various labs around the world they start to modulate the programs that are actually published. So you know you can change the temperature or the concentrations. And of course that is a source of errors. So we consider that if you commercialize a diagnostic you have essentially one fixed protocol. So when you use that diagnostic in Latin America and our lab in South Africa it just doesn't make a difference. So you really have data that you can compare globally. So I'm strongly in favor of commercializing diagnostics. And of course we do that in collaboration with this company. So we don't have any shares in this company at all. I mean it is really to make sure that we have these tools available around the world and that everybody is using the same technology and the same procedures. So standardization essentially. And I believe that is very, very important for cooperative reasons. And the next slide I have kind of quickly listed. You can go to the next slide. Yes, I've kind of quickly listed the various protocols or the various let's say technology that they're being used so far. So also as mentioned by the previous speakers a great majority of the diagnostics is based on PCR. And of course you can use different targets, different genes on the genome of TR4. So ours that we proposed in 2010 was on the EF1 factor. And then you see the other papers you have used different genes like the six genes or other genes to diagnose TR4. And I think that is fantastic because we really have now a panel of targets that we can use. And the more different targets you use, of course the more secure your diagnosis will be. And what we have to keep in mind of course as well is that there is not a single diagnostic that will last forever because pathogens populations will change. So you have to update diagnostics frequently. And that's why we have developed for instance the LAMP as you can see in the middle photograph that LAMP diagnostic is based on another target in the fungal genome. So in our hands, in our lab, if we use the diagnostic the PCR diagnostic, we always run the LAMP in additions because we know it for sure that we already have two different targets on the same genome. And of course that is increasing the reliability of your diagnostic. I think eventually we also need to go to lateral flow devices. They're very fast. It's like a self-test that you do for coronavirus. And I'm sure these type of diagnosis will be available also somewhere in the near future. So in the next slide, I have compared the different technologies that we have available. So you see the methods. So let's say PCR irrespective of the target gene you're using LAMP and lateral flow devices. Then you have the substrates. So plants, water and soil. Of course, water is very important. If you, for instance, if you see in now in Peru with the new incursion of T4 and Peru, that's an area that is strongly irrigated. So once you have invested soil there, of course, dissemination by water is very, very important to check. And then the last four columns is, let's say the investment you have to do to get such a diagnostic running, training necessary, the speed of the test and also the throughput. And you really have to look at those different situations which diagnostic is most appropriate. In general, we are very reluctant to test T4 in soil. We can actually do it in plant tissue and in water, but soil has a very significant complexity factor already starting with the DNA isolation of soil. So I'm not sure whether we really can reliably test, let alone quantify a T4 in soil. We have done it with Tecmanusase, that's a different approach. So we focus mostly on plant and water and that's of course with new incursions also exactly the substrate that we see. Now, if you look in the investments, setting up a PCR lab is a simply expensive. It's an equipment and it's also in training people, as you see in the next column. A lamp, let's say, is cheaper and the training you can do essentially in one day. And so you really can train people quickly to use that type of technologies. It's fast, but as you can see, the throughput is lower. And so a lamp typically is a tool that we use as an alert, whereas once you have an incursion and you have diagnosed T4, you need throughput. You need to process many, many samples. And the best way to do that is simply by PCR. Even a lateral flow device, of course, is an alert tool, but you never will use that where you require a high throughput where you really have to process many, many samples. So, and then my final slide is, as I mentioned already earlier to you, we essentially sequence every strand. And of course that is very important for new incursions because you do infer diagnostic, you isolate, you make single-sport strains, you inoculate plants to confirm the disease and the greenhouse conditions. But in addition to that, and that's what you see here in panel B, we sequence those strains, compare to reference strains. And once you have the sequence, you are sure, yes, this is T4. So it's really confirming what you've diagnosed with diagnostics, but on top of that, it gives you the ability to do phylogyography. So you can relate your strain to other strains that have occurred in particular regions. So what you, for instance, see in panel D here is the incursions of T4 in Vietnam, Laos, and Myanmar were very closely associated with T4 in southern China province of Yunnan. So there was a very clear connection to the dissemination in the Macong region from Chinese-infested farms. And so I believe that having a closer look at the phylogyography of T4 is also very important and it brings a level of responsibility to the entire banana sector. And with that, I would like to thank in the next slide our partners and collaborators. And then in the next slide, I'd like to acknowledge the team that fight the pathology. Thank you very much for your attention. Thank you so much, Dr. Kema, for your presentation. I'd like to open the floor now for about three minutes as we are against time because we have two other presenters. So I'd like to open the floor for three minutes to take two questions at least. We already have within the Q&A tab, there are questions that were asked and they were answered by the panelists. So we could visit there and have the responses. I'll take one question from Antoinette. Antoinette, your hand is raised. You have the floor? Antoinette, no? Okay, then we will go to Rodrik. Your hand is raised. All right, it appears that the hands were raised, they're shy. So let's try another person. Hannah Rumein, your hand is raised. Your mic is muted. We will revisit this question and answer section at the end of the presentations. So without any further delay, we will go on to Dr. Dieter, who will present his work now. Well, presentation. All right, Dr. Dieter, the floor is yours. Okay, thanks, Nelson. Can you hear me? Loud and clear, we hear you loud and clear. Okay, thanks. Next, Mateos. Okay, many thanks for the invitation. I think it's a wonderful opportunity to discuss tools and also process. So my talk will be more related to how we link tools from the field to a official confirmation that we have a new break of the effort. Okay, so I saw the participants attend this. I see many people speak Spanish, at least for the name. So probably it was wondering if I can switch if the interpreter, no, I mean, if you don't mind, I don't know who is translating it, but probably it's better if I don't know. Absolutely, don Miguel. Adelante en Españolo, gracias compañero. Gracias. Next, please. Okay, ya esto fue dicho por los demás colegas. Aquí simplemente decirles un poco que una cosa son las herramientas y las otras cosas, ¿cómo integras esas herramientas dentro de un flujo de diagnóstico en un laboratorio oficial? Y luego, ¿cómo lo pones todo dentro de un marco legal que difiere de acuerdo a los países? Próximos, por favor. Okay, las tecnologías ayudan muchísimo, ¿sí? Y yo he puesto esta comparación aquí un poquito para ver. Pero cuando una organización nacional de protección fitosanitaria dice que Rasa 4 está presente, ya está presente, o sea, los métodos, los protocolos deben seguir, los procesos deben seguir un análisis integrado para que sea un equívio, para que sea claro, robusto y siempre basado en principios científicos. Tú puedes decir, es Rasa 4 tropical, ya lo voy a ver después, basado en BCG. Puedes decir, es Rasa 4 tropical, basado en BCG y en una PCR, como fue hecho hasta el 2010, después, o puedes decir, sí, es Rasa 4 tropical, basado en BCG, gracias, en dos, tres PCR y también en secuenciamiento. Pero al final de cuenta, cuando dice que es Rasa 4 tropical, y eso tiene otras implicaciones a nivel oficial. Próximo. Okay, y para llegar a ese nivel, ya Diana o también Yolanda, creo que mis colegas anteriores lo han dicho. Lo primero es conocer de una manera clara los síntomas de la enfermedad y discriminarlos con los complejos que vemos en campo. Es importante que se está haciendo un monitoramiento para fusario, pero recuerde que en campo hay otros, hay otros patos sistemas, inclusive complejos. Próximo. Por ejemplo, este. Este es un típico síntoma donde hay confusión muchas veces en campo y los que están muestriando muchas veces no saben muy bien lo que hay patógenos asociados. Entonces, simplemente para resaltar lo que habían dicho mis colegas, el reconocimiento de síntomas en campo es fundamental, porque al final de cuenta, estamos haciendo un diagnóstico para tomar decisiones en campo. Luego, nuevamente, recuerden que mi presentación está un poco más basada en la parte oficial y en las implicaciones que tiene una nueva, un nuevo brote terraso tropical. Próximo. El segundo es muestreo. Hay diferentes protocolos, el plan de contingencia de lo ir claramente, los colegas lo mencionaron, pero recuerden que ni siempre, esto está muy claro y que hay que llegar a las personas que hacen ese muestreo, que de manera preferencial deben ser las organizaciones de protección fitosanitaria, las únicas autorizadas a colectar una muestra sospechosa de raza cubata tropical. Tres, el next. Tresero, parece o puede parecer trivial, pero no. Es importante que ese proceso o procesamiento de muestras hayan buenas capacidades de microbiología. Hay lugares en algunos países que hemos visto que sí, que tienen capacidades en otros lugares, hay personas que tienen muy buenas capacidades en biología molecular, pero ni siquiera saben a veces hacer un medio de cultivo y parece, puede parecer trivial, pero no lo ves. Y esto es un proceso también importante. Próximo. Y aquí es algo que yo lo digo que el diagnóstico no es una herramienta. Diagnóstico es un proceso y tiene, dependiendo del país, diferentes etapas que son interconectadas. Hay gente que está en campo, colegas colectando muestras que es un equipo diferente. Muchas veces que están en laboratorio hasta aislar el patógeno que a veces es un equipo diferente que el que va a hacer el PCR. Y luego aquí que es importante también, hay diferencias entre diagnóstico de rutina, aquello que los laboratorios hacen día a día, muestras que le llegan, miles de muestras, y un diagnóstico que yo lo he puesto aquí como emergencia, donde tú tienes una sospecha de que tienes una plaga cuarentenaria de alto impacto socioeconómico, como es el caso rasecuato tropical. Entonces, yo creo que cuando interas estas partes, hay que entender eso muy bien, inclusive los laboratorios, cuando hay una muestra sospechosa, deben probablemente manejar y cambiar su rutina. Tenemos que entender que los laboratorios oficiales de diagnóstico reciben múltiples muestras, y la mayoría de las veces están sobrecargados. Entonces, esos procesos, en lo que hayos visto en muchos países, es importante que estén bien conectados, que estén armonizados, y que se diferencia entre diagnóstico de rutina con un diagnóstico que yo lo he llamado aquí de emergencia. Adelante. Y aquí ya el doctor Quema lo ha dicho. Desde que salió el método de diagnóstico de PCR, que es más rápido en 2010, en la mayoría de esos nuevos reportes ha sido utilizado. Pero eso no quiere decir que los métodos que se usaron anteriormente, en Australia, en Filipinas, etc., no sean válidos, son muy válidos también. A partir de ahí se usan estos métodos, y luego han encontrado algunos problemas. Bueno, que el ejerquema ya mostró el tic comercial, no hay un diagnóstico que va a durar para siempre, y eso estamos de acuerdo con el doctor Quema también, y hay nuevas herramientas que deben ser también utilizadas. Adelante, por favor. Diferentes papers también. El próximo nos voy a entrar en detalles. Aquí sí es importante, y por qué lo he puesto. Y luego lo habló también James. James, un saludo para ti. Y lo habló también Diane. Diane, también un saludo para ti. Porque estamos hablando de otras regiones que probablemente están relacionadas con patogenesidad. No ha sido demostrado, pero están relacionadas con patogenesidad. Aquí, por ejemplo, yo he puesto en ese cuadrado en rojo el gen 6.8. Pero recuerden que James nos habló del gen 6.1. Próximo, por favor. Vale, aquí. Aquí yo lo he puesto, porque esto me han hecho muchísimas preguntas en los últimos tiempos. Y aquí es donde se integra y donde podríamos tener problemas con cuestiones de conceptos. Porque este artículo trae. Vsg, por un lado, trae razas, pero también trae gen 6. Es un artículo super interesante. Es super... Pero cuando lo llevamos o lo ponemos al contexto del diagnóstico oficial, siempre sur es en dudas. Porque, nuevamente, por un lado está la cuestión de los Vcg, las razas y el diagnóstico. Y yo he puesto esas preguntas ahí, porque es el que ustedes hacen en the screen. Me pueden hacer preguntas aquí hoy o después. Pero hay que tener mucho cuidado cuando lo ponemos en el marco legal, porque hay mucha confusión. Porque los Vcg no están relacionados con razas, solo con algunos casos. Porque esa metodología no está relacionada con patas de ansiedad. Los gen 6, ni siempre, están demostrados que están relacionados con patas de ansiedad. Y entonces esto nos puede traer una confusión un poco en la cuestión de conceptos. Adelante, por favor. Bueno, más básicamente eso es lo que seguimos. Si tienes una planta de Cavendish, yo lo he puesto aquí, pero de otra, con síntomas típicos, externos e internos. Y ya se colecta de muestre, lo llevan a laboratorios. Tienes una DNA de calidad, superimportante. Y tú tienes una PCR, por ejemplo. Y he puesto aquí la derecha a sus pantallas, lo que se signa el plan de contingencia de lo ISA que fue ya escrito en 2013. Si eso te da positivo, ya tienes una alerta. Porque tienes Cavendish, tienes una epidemia en curso, y tienes una PCR que te está dando positivo. Haces un cross check, un cross check, porque tienes con diferentes locos. Y aquí me gustaría mucho resaltar el paper de Fred y Magdama, también, que Dayane también lo mencionó. Lo que Fred recuperó es esta metodología de Elie. Y lo puso también y después entró en el proceso, en la región de América Latín y Caribe y en Lacan, también. Y es otro locos que te está dando. Si tienes esto y todas estas situaciones, señores, es decisión de la ONPF, decir si tienes raza con otro picalino. ¿Por qué es decisión de ellos? Porque hay problemas sociales, políticos y económicos involucrados en cada nueva confirmación. Y las ONPF necesitan su tiempo, necesitan muy bien hacer esos análisis integrados que le dice al principio para poder definir si es o no es. Y luego está la presión de la prensa, etc. Cuando ya se sabe que hay un caso de alerta, hay una sospecha, etc. Adelante. Venos a esa pena para mostrarles un poquito de lo que se hizo en Perú, en la izquierda de sus pantallas, la detección, la segunda, el diagnóstico, el tercero, la contención. ¿Por qué estoy poniendo esto? Porque, al final de cuentas, el diagnóstico es importante para que para actuar rápido lo más rápido posible en contención y evitar que el patogino se disemine. Adelante, por favor. Y la pregunta que es, y lo he puesto aquí, inclusive, para provocar un poquito, para generar discusión, quizás, no ahora o después, la pregunta, ¿cómo las ONPF manejan otras enfermedades? Por ejemplo, HLEV, o la moniliasis del cacao. Y la otra parte, poniendo junto ya la parte de diagnóstico oficial y los postulados de cocos, que también se mencionaron varias veces, y las decisiones, digamos, fortalecidas o derivadas o condicionadas por la tecnología. Y aquí hay tres preguntas como para discutirlo. Los testes o las pruebas de BSEX no son lo más necesarias. Las pruebas de patogenesidad en el siglo XXI no son más necesarias porque tiene secuenciamiento completo y si el secuenciamiento completo ya no es necesario, o si o no, o si uno y la otra. Es algo para que lo discutamos hoy o para que lo pensemos. Adelante. Aquí, para ver, es para decirle que también hay varios avances aquí en América Latina y el Caribe, en Agro-Sabia, con el ICA, en Perú. Y estamos también buscando entender o diagnosticar o detectar en muestras ambientales. Y estos señores, suelo, agua, no es para ser diagnóstico oficial, porque recuerden que el patogeno tiene que estar asociado a los síntomas. Esto es investigación, esto ayuda a la contención, esto ayuda a la epidemiología, pero no es para diagnóstico oficial. Y aquí en la región se pueden hacer secuenciamiento también de Illumina, Nanopore, Pac-Bio, etc. Se están haciendo y se pueden hacer todas estas tecnologías en la región. Adelante. Aquí algo que es importante, inclusive, para poner en pipeline o en flujo de diagnóstico, cómo certificamos el material de siembra. Sabemos que es súper importante. Y aquí hay dos temas. Más temas de inócalo y un tema también. Demostría y lo pongo aquí, porque me parece que es importante que lo pensemos en el futuro. Inclusive, no tanto o igual, con suelo y agua, pero diagnóstico o certificación, indexación de material de siembra, es crítico. Adelante. Y esto también un poco una provocación. Rasa cuadrotropical, señor, es una enfermedad que tiene más de 30 años, por lo menos. Hacer un diagnóstico rápido de rasa cuadrotropical no debería ser un problema. Para mayoría de los países de rasa cuadrotropical, de América Latín y Caribe. Pero sabemos que no es así. Sabemos que todavía nos falta mucho. Y la otra que lo he dicho bastante durante mi presentación es siempre hacer discriminación en cuando estamos haciendo investigación o haciendo diagnóstico para temas epidemiológicos o cuando ese diagnóstico va a resultar en una declaratoria oficial de un país. Es muy diferente decir si hacemos sequenciamiento, hacemos esto el otro. O decir, cuando un jefe nacional se para en la prensa y divulga que el patón no está presente por primera vez en un país o un continente. Eso deriva en implicaciones sociales, políticas y económicas que es muy diferente de un nuevo método cuando se publica. Adelante. Y a mí me parece ya para concluir un poco que las plataformas regionales para el diagnóstico son muy importantes. Yo no creo, yo no voy a discutir esos cinco puntos que está a la izquierda de sus pantallas. Pero a mí me parece que cada vez tenemos que ir más a ese tipo de estrategia. Y no solo pensando en la secuela tropical, sino fortalecernos, en conectarnos, hacer network con los institutos avanzados en investigación en las universidades para tener plataformas bien fortalecidas, usando inteligencia artificial, etc. O por ejemplo, inclusive plataformas, integradas a plataformas de vigilancia, como CERCASO por ejemplo, de PES, Displace, que lo tenemos nosotros en la Alianza Biodiversity International CET. Adelante. Y así concluyendo, agradecer a los colegas de Incolombia y Inperu que están trabajando fuertemente en la contención de Rasa 4 tropical en la región. Adelante. Y también a todos los que han trabajado de una manera u otra y el colaborado. Y aquí me gustaría resaltar un poco el apoyo también de la universidad de Vareningin, porque antes los kits, etc. Nos apoyaron muchísimo en todo el proceso de fortalecimiento de capacidades en la región. Adelante. Y luego mandaron un apoyo fortísimo a los colegas que están hoy el pie de combate haciéndoles frente a Rasa 4 tropical en todas las partes del mundo junto con la epidemia de COVID. Muchos se han contaminado y no es una situación muy fácil, pero sabemos que un día van a reconocer ese trabajo que están realizando porque las situaciones de hoy enfrentando el COVID no son realmente fácil. Adelante. Y con esto ya decirle gracias por su atención, ahí tiene mi email y también me puede contar por otras ideas, otras plataformas. Gracias por su atención. Thank you very much, Dr. Dita, for your presentation and of course showing linkages between the diagnostics process and that of the response with national plant protection organizations. We will move on to Dr. Joong Peng from the Chinese Academy of Tropical Agriculture Science and he will present his, he will do his presentation right now. Go ahead, Dr. Pen, the floor is yours. Okay, it's my player here to give an presentation about the a lot of field area, mobile tropical risk for detecting and controlling strategy in China. I will, next slide. Please, next slide. Yeah. Yeah, I will report in the following order. First, briefly introduce the carries and the distribution of TR4 in China and secondly, the TR4 detection method might be used in China. Finally, is how to control the field area of Bala in China. Next slide. Thanks. First, I will introduce the carries and distribution of TR4 in China. Next, next slide. Yeah. TR4 or the first identified in China is Guangdong province at two thousand and one, which caused by TR4. At present, the TR4 is all over Bala producing plantation in China. Next, next slide. Next slide. Yeah. As we know, the field area of Bala caused by field area of the red cement, which caused typical similar times in China, such as leaf healing, should do stem splitting, vascular discoloration and the right room necrosis. Yeah. Okay. Next slide. Next slide. Thanks. Yeah. At present, no effect, no effective barriers for controlling TR4 once infected in the field and the low resistance cutting loss is low in Musa against the TR4. So any accurate detection of TR4 is essential to protect Bala industry and guide Bala planting. This photo is from the Yunnan province of China. The TR4 can cause a huge economic losses in plantation. Next slide. Thanks. In this part, I will introduce the developed lump method used for cognitive and cognitive detection of TR4 in infected plants and soil sample. Although many techniques and methods available for TR4 in China, the most used is lump method because the beast polymeric beast polymeric polymeric can comfort the higher tolerance to inhibitory substrates of soil sample. Okay. Next. Next slide. Thanks. This is a progress for lump detection. First is DNA extracted and then is cycling amplification and the fellow is the result judgment. Next slide. Thanks. Next slide. Yeah. The lump employed a beast DNA polymeric and a set of force partially designed primers that recognize a total of six distinct sequence on a target DNA. The out primer F3 and B3 each re-regalized one of six sites and the prime amplification of the entire region in a long cycling manner. This slide shows you the primer design and the next slide will show you how to work use this primer. Next slide. Thanks. Next slide. Yeah. The beast DNA polymeric with the strength displacement activity, the in the primer each recognize two of the six sites within the amplified sequence and form a dumpling like DNA structure used for subsequently cycling amplification. Within one hour the cycling amplification can get a accumulation at the life the life power of 10 copies of the target DNA. Next slide. Thanks. How to how to judge a judge a detection results first with the lump reaction the by product with the white precipitate will be viewed and viewed. Next. Okay. Next slide. Next. The lump products can also be viewed with axos, axos geoelectrophoresis. The cybergreen sting is a minor approach can viewed by leaky eyes. The positive samples will tend green after staining, but the negative samples remain orange. Next slide. Thanks. Cybergreen sting. Apart from the quality qualitative assay, the real-time fluorescence loop mediated as a thermal amplification can repeat and the current tattoo detects the samples. This is a real-time tebidimeter and the amplitude according to the torpedo here. So the real uncle can use the full chrome tattoo detection from the infected blunter and the soil and the water samples. Next slide. Thanks. Next slide. Next slide. Yeah. This slideshow we use the lump method to detection risk for R4 and the tropical risk for in Thailand, respectively. This is two papers we have published. Okay. Next slide. Next slide. Next slide. Thanks. Yeah. First is the primary design for the TR4 lump. Private design the back to back to one more. Yeah. For TR4 lump, private design, primary design, the IGS region is available for TR4 lump, primary design, but for other for primary a specific rapid mark sequence used for the primary design and the next next slide. Next lessons. Yeah. Next slide. This slide show the primary sequence and the relative position in the Jilu are indicated by the arrows. The left is TR4 primary and the right is R4 primary. Okay. Next slide. Thanks. For the specificity test, the lump products or implicates only from TR4 DNA and next is TR4 specific PCR and then is Cypher Green-Stainey fellow is an application care. This result can prove that the designed primary is TR4 specific. The R4 specific test is as same as TR4 specific test. So the result can or indicate or indicated that the primary is specific to each one. Thanks. Next slide. The detection limit of real ample is about four times or for about four times of 10 to the power or for minus four nanogram micro-neeter which is lower than real-time PCR. Okay. Next slide. For the feasibility test, the TR4 pure spores and pure spores mixed with soil, the detection with real lump and real-time PCR respectively, we can show from the result shown the sensitivity or real ample is not changed after the spore mixed with soil but the sensitivity of the real-time PCR is reduced after the spore mixed with soil. This, okay, stop. This result indicated that the BST in polymerase comfort this reaction highly tolerant to inhibitory substrate from the soil samples. Yes. And this slide show we successfully using lump and method detection TR4 in the soil samples. Okay. Next slide. This slide show the comparison between lump and PCR from the result which we can figure out. Lump is a simple rapid and effective method compared with the PCR method. Okay. Next slide. In this part we will introduce how to control the TR4 in China. The main measure is using the resistant cultivars and supplementary measure is biological control in the field. Very important supporting measure is strength and field management especially precise water and fertilizer management. Okay. Next slide. Thanks. Now, there are many resistant cultivars used in Thailand but the most widely planted is Bao Dao Balala cultivar which characterized with red shielded stems and red bars. Another resistance cultivars is Nan Tian Huang Balala cultivar which characterized with cane stems and cane bars. The two cultivars is planted in Thailand is the most popular. Okay. Next slide. Okay. Next. This picture show the field more performance or for resistant cultivar Nan Tian Huang. From the picture we can figure out these cultivars come for resistant or tolerant to TR4 in the field. Okay. Next slide. Thanks. Dr. Pen. Excuse me. Dr. Pen. Yes. We are we are going beyond the assigned time for the webinar. Would you be able to end your presentation within the next couple moments please? One minute more is okay. Okay. Thank you. Okay. Another method is biological control. The usual biocontrol agents with anti-fungic activity promote bar growth or enhance resistance to TR4. Multi multiple agents can co-firmation and applied in field which will affect you, affect you. Next slide. Next slide. Thanks. We are effective reduce the disease in cities. Okay. Next slide. Thanks. Yes. The final slide showing the precise precise water and fertilizer measurements in Greek integration of water and fertilizer in one water piping. You can see the picture. One spring belt and two drip tubes in each plain lines. The two picture showing the maglite, the balala plantation construction, and the third picture shows a field secondary co-firmation the fermentation technology of liquid biogonic bacterial manure. Okay. Sorry for the time limits. Many works have not shared to everyone. Maybe next time we have the opportunity. Thanks for your attention. This is external view of our building of environment and the pronged protection institute. Welcome to Thailand. Thank you. Thank you. Thank you very much Dr. Penn for your presentation. As I mentioned we we are sort of we have gone beyond the allotted time for webinar but thank you very much for your presentation and sharing the information on Kata's and its and its work. Thank you very much. We we have a slight change to our agenda. We will invite for some brief comments. Dr. or Mr. Mauricio Guzman who will share a few words with us and then we'll go on to Mr. Prather the secretary of the world panel forum and he will give close and remarks. Mr. Guzman, the floor is yours. Buenos dias, buenas tardes, buenas noches. Me escucha bien Nelson? Si, te escucho muy bien. Gracias por la oportunidad voy a voy a hablar en español porque hay traducción y tenemos también mucha gente que habla en español y es nada más lo que hago mejor. Primero a felicitar a FAO y también al World Barana Forum por esta actividad que ha sido sumamente buena y por supuesto a todos los conferencistas por compartir sus experiencias y todos sus resultados que son sumamente valiosos. Yo quería comentar algo que quizás es más un comentario que una pregunta pero podría algunos de los participantes de los conferencistas referirse a ello y es sobre el diagnóstico de campo porque quedó muy claro todos los procesos para el diagnóstico y del laboratorio, la análisis molecular, la colecta de muestras, sin embargo si nos vamos a los casos de Colombia y de Perú, las más recientes apariciones de ER4D y analizamos el caso en mayor profundidad nos damos cuenta que ambas tienen algo en común y es que fueron realizados los diagnósticos de campo fueron mal realizados y fueron realizados tardíamente. En el caso de Colombia se confundió con Moco, le hecho le llamaban Moco Negro a esa patología que observaban en el campo al final este se terminó que era ER4D y en el caso de Perú si revisan también un poco la historia van a encontrar que del año 2016-2017 se venían presentando casos de falso mal de Panamá y cuando realmente ahora se revise el caso es la enfermedad está muy distribuida es decir están ahí en ambos casos desde hace mucho tiempo o sea que estamos fallando un aspecto clave que es el diagnóstico de campo y ahí ocupamos por supuesto mayor fortalezas aumentar las capacidades de los países y de los grupos de trabajo en este aspecto porque de lo contrario aunque el diagnóstico laboratorio sea tan bueno como lo tenemos ahora porque realmente si me voy a las preguntas que hizo el doctor de tal final comparto muchas de las respuestas ahí en el sentido de que ya casi con diagnóstico directos o leculares tenemos una gran herramienta para poder decir que sí pero si eso está aunado a un diagnóstico de campo bien hecho por personal capacitado yo estaría muy seguro de dar un veredicto casi que esto respondiendo un poco Miguel a tu pregunta o o al comentario que lanzaste en el sentido de que de qué manera podríamos o si podríamos usar el diagnóstico de laboratorio directo de nuestro molecular para decir si hay o no el recuatro de eso yo estaría de acuerdo solamente si el diagnóstico de campo fue realizado adecuadamente porque eso es clave y es donde creo que estamos este tenemos una debilidad importante y quizás Fajo puede retomar ese tema para un futuro webinar muchas gracias por la por la oportunidad Nelson y también a victor gracias por sus palabras gracias ahorita vamos con el señor victor brother victor you have the floor muy bien muchas gracias voy a hablar en castellano porque también es es mi lengua primero dar las gracias nuestros destacados panelistas por estas intervenciones que nos han dado tanta claridad y tanta información necesaria hace muchos meses que mientras preparamos la la red global en r4t que escuchamos que el tema del diagnóstico es un tema importante en ocasiones controversial y que requería su tiempo entonces esto es el motivo para hacer este primer webinario continuaremos con estos webinarios para dar más información y sobre todo en diagnóstico porque tantas cosas que debemos tratar para con respecto al fursarium que tenemos que separarlo en varias secciones pero me doy cuenta de que tenemos que volver a este tema seguramente será para septiembre o principios de octubre y bueno para seguir alimentando esta red global de la fao en r4t que pretende ser ese punto neurálgico neutral para dar esa información y que podamos compartir cuáles son las mejores prácticas entonces esperamos también que toda esta información haya sido muy útil para todos los participantes de la reunión de hoy y como saben el nuestro el mandato de la fao es que establezcamos estas conexiones entre distintos actores que trabajan quizás con distintas metodologías o distintas iniciativas para que podamos evaluar cómo podemos colaborar así que creo que esto ha sido un buen webinario la información ha sido muy útil pero no quita para que tengamos otros webinarios y como saben todos ustedes probablemente la semana que viene tenemos tres días de webinarios más largos que el de hoy el día ha sido de dos horas y soy consciente de que tenemos además interpretación que está contratada hasta hace unos minutos así que muchas gracias a la intérprete por la ayuda la excelente interpretación y por el tiempo extra que está dedicando y únicamente quiero unos minutos para recordarles que la semana que viene días 27 28 29 seguimos con las jornadas de la fao con respecto al fortalecimiento de capacidades y sensibilización en respuesta a la amenaza de la marcha y tez por usuario entre banano el r4t así que este proyecto como saben es parte de las actividades del proyecto de emergencia de la fao en américa latina y el caribe capitaneado por por raíz ayaogre y esterper alta y en colaboración con el ibtc el la la plataforma internacional para la protección vegetal y el foro mundial banalero pues estas sesiones que tendremos la semana que viene días repito 27 28 29 de cuatro de la tarde a 7 y media hora italia 8 de la mañana 11 y media hora costa rica 9 de la mañana 12 y media hora ecuador se basaran principalmente el martes 27 en el contexto actual donde estamos como están el tema del riesgo y el impacto y la comunicación al respecto porque también es de cesarios que todos podamos colaborar para que no suceda lo que comentaba don mauricio comentaban anteriores panelistas no está falta de desinformación o de activa de acción rápida entonces eso se va a tratar el día 27 les invito a todos a participar el 28 tardaremos temas de bioseguridad y aplicaciones móviles que apoyan a la vigilancia imágenes atiritares drones etcétera como podemos facilitar esas actividades y el jueves 29 de julio alternativas de manejo que incluye prácticas agrícolas clones promisorios y procedimientos para su introducción básicamente hay hay una agenda completa que ya está disponible en nuestra página web les acaba de compartir la página web en el en la poste aquí también mi compañero mateus todas las informaciones está disponible tanto en la página web del foro mundial manero como de la red global de r4t y actualizaremos todos panelistas en el día de hoy y el de mañana porque faltaba algún panelista por confirmar pero por lo menos todas las áreas temáticas han sido cerradas y la agenda está disponible para todos ustedes así que sin más dilación muchas gracias por su tiempo y espero hayan encontrado la información compartida hoy de utilidad para sus actividades y de nuevo muchísimas gracias tanto la intérprete como al chair al señor la bile amar a mateus por la colaboración para temas logísticos y con la plataforma y sobre todo a nuestros panelistas que han dedicado el tiempo a para compartir todo ese conocimiento con nosotros así que muchas gracias por la información y que pasen ustedes un buen día