 Can you hear me? Yes, somebody had muted me. Is the screen there? You're able to see the shared screen. Great. Okay, so thank you very much for asking me along. I have to all the people organizing this. Nelofar, Beck, Liz, Rebecca, and Miss Louisa. And I'm going to talk about my favorite topic. Now that I prepared this thinking, people have heard this before, but I realized not everyone's heard this before, so apologies to those who have heard bits of it, or perhaps all of it, and hopefully those who have never heard it before will enjoy the world of Pusarium, wilt of banana. Now, just in case I forget to mention the main people at the end, I'm going to put their names up front, and so much of the work I'm going to talk about today is that which has been conducted by Andy Chen, Liz Joslowski, Jay Anderson, Rachel Mildrum, Nolene Warman, Sam Fraser Smith, and also Andrea and Matthews and name Chen. So I'm just going to split the talk up into sort of four sections, so I'm going to apologize now. UQ is just one big building site, and there's a cement truck that keeps going back and forward outside, so I hope it's not going to disturb us too much. So I'm going to split it up into looking at initially banana cultivation, just for those to enlighten those that aren't so familiar with it. Then we're going to talk about the pathogen of interest, a little bit about the interaction on the host, and then we're going to talk about the host a little bit more. And banana has a convenient, you know, everyone's familiar with the shape of banana fruit, but the pathogen that we're looking at has the spores the same shape, which are very banana shaped. So, okay, now in the last four or five years, I think it is, we've had funding from a Gates program, which goes through the International Institute of Tropical Agriculture based in the East African nodes of that institute. And because banana is an incredibly important crop as a subsistence crop in East Africa. So when we think of banana, we think of banana growing in North Queensland and how important it is to the Queensland economy or certainly culturally to us as well. But if we look at the statistics here, Australia really just ranks 48th in the world as when it comes to banana production. Uganda's 10th, but when we compare that with consumption per capita, the countries of East Africa, Tanzania, Uganda, Kenya, they rate very high in consumption because to them, banana is a particularly in Uganda is a, you know, their main carbohydrate source. So we can look, for instance, at the sort of diet that they might have in the Kassava as well and other things. But banana is a very important part of their main diet, it's a more of a cooking banana. And you know, it's a bit of a problem getting statistics sometimes on banana because for certain reasons, they split banana and plantain and cooking banana into different groups, you know, when they're looking at agriculture commodities, but it's all the one. So for that purpose, you know, when I talk about banana, I might be talking about the cooking, starchy banana, not necessarily cavendation stuff, but they're all considered banana. I mean, gosh, it's got as low diversity as possible as it is, so why you would split it up any further. So in East Africa, they call the cooking bananas Motoki and Meshari. Being Scottish took me a while to work out that Meshari, I used to call it mick hair, but it's actually called Meshari. And they do lots of things with banana, they make juice with it, they make banana beer, they make, which is a little bit, well, not very pleasant in my taste buds, but they also make banana gin, which was quite good. But we look at banana, we see it's seedless, we see it's partner carpet, and we think, how heck do you breed it? But people are breeding banana. And this is an example of production from the IITA program in East Africa. And we see on the left, the two parentals. And here on the right, we have a much higher yield. So banana breeding is possible. And I'm going to talk about that as we progress. Okay, so basically, that's in Africa, they've probably been eating banana for quite, you know, several thousand years. It was probably brought in by early traders from Southeast Asia. And, you know, there's lots of books and theories written on that. But banana, as we know it in Australia, and certainly in Europe and North America, wasn't really consumed much until the end of the 19th century, 20th century. In Australia, slightly different, the bananas were probably brought by the Chinese into Australia in the 19th century. But certainly in Europe and North America, they didn't actually start consuming bananas until the advent of the steamship when trading vessels were able to get to these markets fast enough before the bananas had ripened on board. So it was only then that they started growing. And at that point, most of the banana production for those markets was in Central, Southern, South and Central America. And the cultivar at that time was called Big Gross Michel, or otherwise known as Big Mike. And that was a principal cultivar of trade. And there's a very good book by, oh gosh, Dan Kopel, it's called The Fate of the Fruit that changed the world. And banana production in the Central American Republics was heavily politicized basically because of fusarium wealth. Because at that point, it got into there in the late 19th century and started causing problems because it's a soul born pathogen. The banana companies needed new land. And so they moved to new land and they sort of took it over and caused lots of issues. And I'm not going to go into politics today, but really good read, I would certainly look into it. But anyway, the steamships allowed banana to be transported to North America and Europe from Central and South America. And there was a real boom in banana production, banana growth by these companies. And often the companies that owned the land owned the steamships as well. So it was a real export market. And so through that cultivation, unfortunately, fusarium wealth got in. Now, fusarium wealth in banana was first described actually in Brisbane. But as not to say it came from Brisbane, it was just that somebody, I guess, with a Western education was able to sort of identify and stuff like that. But it was in the 1890s, it became really, really serious in tropical America and Central America. And it was so named Panama disease. So the reference to Panama disease, because obviously it was recorded in Panama, it was recorded in other parts of Central America as well, of course, where it was affecting gross Michelle, I particularly don't like calling Panama, Panama disease, but that's my own preference. And certainly with the new strains, I wouldn't really refer to them. But it seems to have taken off that way. It's caused by the fungus fusarium oxysporum. You probably all heard of fusarium oxysporum. It's a root infecting fungus enters via the roots, gets into the xylem, travels up through the xylem, causes a wilt and eventually leads to death. And so this image here on the right is one poor little plant that's affected by fusarium wilt. You can see the collapsing of the leaves. The leaves have gone all pleurotic. And if we will look in a minute at some more symptoms associated with it. So the first wave of fusarium wilt was in the mid 20th century, starting off from those infections that occurred in late 19th century. And when gross Michelle was then trade cultivar trade, so it was completely susceptible. And it became, it was succumbed by other cultivars that were resistant. So yeah, we have the thing, yes, we have no bananas. I'm not 100% sure if that relates to the fusarium wilt or not, but certainly those little songs around the time with them, the, the non availability of bananas. So then rose Cavendish. Now Cavendish became the cultivar trade. Now it's important to mention at this point that Cavendish is a dessert banana. It is becoming increasingly cultivated even in subsistence agriculture, but it's 85% of banana production worldwide is not for trade. It's for subsistence agriculture. So 15% is certainly mostly based on that Cavendish, but even within subsistence or domestic productions, Cavendish is increasing in production, goodness knows why, because it's replacing a lot of interesting cultivars that would be particularly in Southeast Asia and places like that. So Cavendish became the cultivar trade. It was resistant to fusarium wilt at that time. So let's look a little bit of fusarium moxiborm has many forms. And, you know, I, you know, when I'm talking about plump in lectures, plump, I often say plant pathogens of poor sapropites. And so they are outcompeted by sapropites once the pathogen invades the tissue. That doesn't work for fusarium moxiborm. It's a good sapropite. It's everything. I don't know why every were not overtaken by it. It's an asexual member of the Ascomi quota. It has a wide host range, but as host specific strains. It's also an endophyte. You can find endophytic strains within banana. You can find endophytic strains within many crop plants and non-crop plants. It's a sapropite. So once the tissue dies, it can colonize it. It can colonize the tissue of other plants too. So we see fusarium moxiborm as a lot of studies being done on tomato. The fusarium moxiborm species is like a persimmon because tomato is quite easy plant to work at, particularly on the genetics. But we also see it in strawberries. We see it in ginger and cotton. And it's caused huge issues in cotton in the 90s and cultivars. And we still don't have a truly resistant cultivar. We have tolerant cultivars. And of course it's an issue on banana. These are some ladyfinger plants that have fusarium wilt here. When we look at the symptoms, particularly on banana, you see necrosis at the leaf margins. You can see leaf discoloration, petiole collapse, sort of forming a sort of skirt. And stem splitting is pretty strong characteristic. You can see down at the base, but it's only when you really cut into the plant, you see the very reliable vascular discoloration where you've got phenolics, etc, being produced within the vascular tissue in response to the pathogen. So that's very characteristic. And eventually the plant will die. Sometimes the suckers that come from it will survive. Sometimes not. And generally, you're not going to get much healed. So just looking at banana cultivation, keeping in mind the time, bananas are traditionally cultivated by suckers and vets. So you take the suckers here. Hopefully you can see my mouse here, looking at the sucker as it grows. Remember, the bananas are just basically a big herb. So we talk about being the pseudo stem as opposed to stem because pretending to be a stem. And you break off the suckers, or you can actually take that rhizome and chop it up. And there's lots of little buds there that you can break up and take and transplant, which is fine. You're dealing with a clonal crop, and you can cultivate it that way. And if you see that traditionally, from one plant, sorry, I got fed up doing my copy and cut and pastes and reductions. But basically, I just wanted to emphasize, from one plant, you can get this whole field eventually cultivated. Now, obviously, if you're dealing with a soil-borne pathogen, that's a real big no-no. And that was a problem that happened in Central America in the mid-20th century is that they'd say, oh, let's go to some new land that's fusarium-free. And they'd take their disease plant tissue with them and replant from there. And we think we've learned lessons. But when Tropical Race 4, which I'm going to talk about later, came in in the late 20th century, people did the same thing again. And they just spread it around. So if you start with your mother plant that's infected, everything else is going to get infected. And it's going to go into new land, new ground, and it's going to contaminate that. So the answer would be to cultivate from tissue culture plants, which is strongly advised, particularly here in Australia, where you get tissue culture plants. At least you're starting from clean material in the first place. You're multiplying it up through tissue culture plants. You take the plants out of tissue culture. You take them to a nursery, which should be clean. And then you grow them up and then you plant them into fields. So any new plantings, particularly in new land, should always be from tissue culture. But that still depends on the pathogen not being in that soil in the first place or not having been spread into it. So it's not only plant material can spread the fungus. It's other things like pigs can go and rummage in the soil that's full of spores or bits of mycelium or bits of debris of plant matter that's carrying the fungus and they spread it from one site to another. Beetles can do the same. Even then they can go and feed on it. Sorry, I didn't couldn't find a nice agricultural shovel. But what I'm meaning is spades and other equipment can spread it. Tractors, vehicles. And we know that when we go up north now, we see all these signs that you can't go into people's properties. You have to leave your vehicle at the gate and that's to stop the potential of soil embedding in the tracks that have been carrying fusarium wilt and being dispersed into the next farmer's field. Boots, et cetera, you know, change your boots between fields, between farms, certainly, you know, going on to properties because, you know, they're one of the biggest carriers, you know, all that dirt on the base of boots is going to be covered in spores and mycelium. Cane knives to a certain extent, because you're cutting cane knives in the upper stem and going from there to there, it's not likely to be a root of infection, but it's still going to have debris and soil on it. You're going to put your cane knife down on the ground, et cetera. And I'm not going to go into the details. Queensland Biosecurity can have wonderful programs trying to limit the spread of fusarium wilt, a tropical race for this waterfall is meant to illustrate flood waters, irrigation waters can move material, can move spores, but the biggest inoculum spreader of all is human beings. We're just so good at spreading things around and taking material from one thing, not doing the right thing. So, it's quarantine is very important for this pathogen. So, let's look at the types of variants of fusarium oxysporum. It's an asexual fungus, but gosh, it's got a lot of diversity. We talk about the first wave being caused by race one. Now, race one actually consists of several different vegetative compatibility groups, several different genetic groupings, but those are the ones that can affect lady in here in Australia, Ladyfinger and Grossmichelle, or but Cavendish's resistance. And we got on fine until the 1990s with the surviving with Cavendish being fine, a few Ladyfingers down in Northern New South Wales and Southern Queensland. And the Cavendish was growing happily, quite happily other pathogens affecting, of course, but not fusarium. And then in the 1990s, we first saw what we saw it in causing some what we call subtropical race for it was affecting Cavendish in suboptimal conditions and subtropical conditions in Southeast Queensland. Cavendish started collapse, you know, bad, but not, you know, you haven't certainly had a reduction in yield and it was it was an issue. But so far subtropical race for has been confined to this southern corner Queensland, Northern New South Wales. But it was with tropical race for that first came in first identified in Australia in the Northern Territory that caused the alarm bells because it's a much more aggressive form of pathogen affects the plants in tropical conditions. We don't know what subtropical race for would do in tropical conditions, but certainly tropical race for terminology why it's complicated race. There is a race to which have more or less does the same as race one race three doesn't even affect banana is just a misnomer, unfortunately. And the other ones we can call rich subtropical and tropical race for because they affect Cavendish particularly in particular. Okay, so we look at the distribution of these different races within Australia. I don't dwell too much on this, but race one was present for a while. We saw it in we see it in North Queensland on Dukas, we see it here in Southeast Queensland, Northern New South Wales, and it's in Western Australia. And then came some tropical race for became an issue and then tropical race for an issue in the Northern Territory. And then more recently, unfortunately, tropical race for has gotten to the principal growing area of banana in Australia, which is in the top and North Queensland and around the Tully Valley areas and just more of that. And it's an issue. It's now on five properties, but they're doing a fantastic job in trying to contain it. And it hasn't spread yet is being spread, I should say, whether it's by pinks or beetles or whatever, but relatively slowly. So hopefully slow enough in that we can develop resistance by whichever means and have some alternative to Cavendish. I'm going to move a little bit quicker now here. So tropical race for it's now an issue. You know, we think we've got problem here. But if we look at some of the countries of Central and South America where banana commercial banana exports are such an important part of their economy, they are actually quite terrified of tropical race for their commercial export markets are based on Cavendish Cavendish is highly susceptible. So it's in Columbia. It's now in Peru. And we saw it spread in Southeast Asia in the 1990s and early 2000s. It's caused havoc in China. It's now in India. India is the biggest banana producer in the world, mostly from domestic market. They do have have Cavendish types there. And it's not just Cavendish. There are other cultivars that susceptible to it. In Africa, it's in Mozambique. It's not in East Africa yet, Uganda and Kenya. We don't know for certain if we know that the Meshari cooking banana could potentially be susceptible, whether the Motoki ones are, we're not quite sure. And it's not nor is it in West Africa and amongst the plantains. And again, we need to see whether they're susceptible or not. But it's caused havoc in the Philippines and Indonesia, particularly with the export markets. Okay. So how do you control this? It's a plant pathologist's dream. Well, not dream nightmare. I should say nightmare. How to control this persistent soil-borne fungus with saprophytic capabilities and a monoculture of single genotype, clonally propagated perennial crop, mostly grown in areas of high rainfall. You couldn't actually devise something that's more of a problem plant pathogen-wise. So how do we control it? We can control it by quarantine, by biosecurity, by implementing good containment protocols, whether at a local farm level, regional level or a national level. In order to administer good biosecurity, we need good diagnostics. For good diagnostics, you need to study the pathogens diversity. So I'm going to talk a little bit about that. Cultural control. We've known this pathogen for 100 years, over 100 years. There's still so many gaps that things that we need to find out. And the more we find out about the spread of this fungus, about its spread within the plant, between plants, its persistence, the better. And so more and more that is needed to study there and resistance. And you can breed bananas, but much of the studies in banana breeding have just gone full in, because it's such a problematic crop, breeding-wise, they've gone full in and just done the breeding without doing the background genetics. So that's one thing I like to emphasize that you need genetics and breeding in order to understand the genetics of resistance and how to introduce it into the crop. Okay. So let's move on to look a little bit at the pathogen. Now, many of you probably familiar with the term vegetative compatibility groupings and I'm not going to go into details of that. Much of the VCG analysis of fusarium oxysporum strains in Queensland are done at Boga Road and Wayne O'Neill is certainly one of the world experts, I would say, on the analysis of that in fusarium oxysporum. And basically your VCG means of identifying genetic groupings, because within a VCG, it's compatible with other isolates within that same VCG. And it's just there's a lot of trick way that you can, by bringing a couple of mutants together, we can see if there's some complementation of certain pathways. So I'm not going to go into the details of it, but it's a convenient way of identifying associations with certain races of the pathogen. So if we look at race one, we know it's got all these VCG groupings, and it's not just done for fusarium oxysporum cubensia, thanks banana, but other fusarium oxysporums and for other pathogens. They also use VCG groupings. So we can we can link the VCG groupings to certain races of the pathogen, certain races by meaning that those are the ones that can attack ladyfinger or can attack Cavendish vice versa. So we group subtropical race four is quite distinct and VCG grouping from that of tropical race four. So tropical race four generally focuses around these VCGs in particular one, two, one, three and one, two, one, six. So if you traditionally, if you want to identify that you've got tropical race four, particularly if you've got Cavendish that's come down, you have to make sure is it tropical race four or is it just subtropical race four? You would have done a VCG test, which can take up to six weeks. So for the purpose of diagnostics and also for the purpose of finding a little bit more about the pathogen, many groups have done phylogenetic analysis of various fusarium oxysporums, not just of cubensia, to see where they place with each other in relation to phylogenia. And we find that the race one and race two group together in one clade and tropical race four and subtropical race four group together in another clade but again quite distinct from each other and that's based on some core genes, on the sequences of some core housekeeping gene analysis. So it's quite distinct and in Australia we have all three variants, well all races now unfortunately, but in different locations. There's recent proposals that suggesting that we change the name of fusarium oxysporum because it's more than just one species, it's always a bit tricky how you define what species is of an asexual, a grouping of asexual fungi, I'm not going to go into the taxonomy of that. I think it's quite understandable that you would certainly the what we call clade A and B do look to be quite genetically distinct. On the plant they look the same, the mycelia looks the same, the spores look the same, some people say there's slight variations, a slight variation, volatiles etc but at this point in time I'm still going to call it fusarium oxysporum until we get all this sort of terminology sorted out. So fusarium moving on from core genes there was work done on as I said before tomatoes much easier system to work with and in the Netherlands in the 1990s they started looking at these little peptides that were expressed in the plant by the fungus and they called them secretive and xylem genes and they then did reverse genetics to identify what these peptides were and it turned out they were some of the classic avalanche or what we now call effector genes that were associated in gene for gene interaction in the tomato fusarium oxysporum pathosystem. So we looked at these six genes and wondered if I wonder if there's any in fusarium oxporum cubensi. So this was the work initially of Rachel Meldrum and then of Sam Fraser Smith where they looked at these corresponding six genes now sorry the terminologies so it means secreted in xylem and today there's a published at least 14 different six genes you know when I say 14 different distinct genes within those each gene within six six ones you're finding different variants different sequence and variation so it gets quite complicated but basically by looking at presence of absence initially of six genes of six one two through to eight we found that we could distinguish superficially between races one and subtropical race four and tropical race four by presence absence and this was done just by doing well initially by southern analysis which nobody does anymore but then by doing PCR and we could see a presence absence pattern so that was quite convenient for an initial diagnostic just to try and distinguish what was tropical versus subtropical race four. So let's just lastly has taken this took this further in our PhD studies and assessed a wider range of different isolates of FOC and non-FOC isolates to looking at the six gene pattern on that and published this work with a subsequent publication and she used what we call myseq analysis but many of you are probably familiar with it but just to to briefly go through it you compare amplified sequences within the genome and compare them against a reference and in the case initially the reference was against the tomato one and then we built our own references to look at a pattern of the the sequences that encode for these six genes in these different isolates and we get initially we saw really clear distinction if you look at presence absence of the six genes between the different races and you know there's a little variation on that but Liz took it further and she actually looked at the sequences within those six genes and was able to find that there were several copies in cases and in which case in tropical race four you can see quite a different pattern of sequence variation in the different six genes. Six one seems to be present in all of the fused air moxibore and cubensi but there's variations in it for tropical race four so does that make a difference we don't know we're still trying to find out but it's certainly a very convenient diagnostic and we can see this and if we look a little bit further yeah looking at those vcgs which are particularly problematic in the ones which have been identified as a tropical race four that's spread around the world we can see this very distinct pattern that this particularly with the six one eye as it's identified so these differences can be used as molecular targets so we look at this and then we Liz took a little bit further she looked at how these different six one sequences related to each other just looking at the sequences from the different isolates not just from the cubensi the banana infecting strains but from from other fused air moxibore and she found that that that six one which was only only unique to tropical race four wasn't found and it was quite distinct from all the other six gene sequences and wasn't found in any other of the banana infecting strains but was found in other fused air moxibores for instance ones from strawberry or the writing's too small for me to read but so it's talking about and I mean rep has published and others have published that there it does seem to be strong evidence of horizontal gene transfer going on between fused air moxibores how frequent it is it doesn't seem to be that frequent but it's certainly an interesting issue to look at in regards to what makes something pathogenic or not pathogenic whether six one is absolutely essential there have been some knock knockout studies doing it and seen it does seem to some of the six one genes when they've been knocked out it does seem to affect pathogenicity so that there's there's a lot of interest in this area not just in banana interaction but in other crop plants but in the meanwhile it's very useful diagnostic and lilia covelas has looked at this in more detail and developed our diagnostic for distinguishing between different races based on the six genes okay so in summary of this work on the six genes it's six one eye is not found in any other banana infecting strains but similar sequences are found in other strains although we are strawberry pea and watermelon and the interesting thing is that initially we didn't identify any 16 in the limited number of endophytes we looked at but uh liz then did a subsequent study and found quite a lot of the endophytes do actually possess six genes so it asks the question are these six genes although I should say they're the protein the peptides that they're producing are they essential for pathogenicity or are they just essential for colonizing banana tissue so there's a lot of work that can be done on that and to basically to try and determine what six genes are involved in pathogenicity and what are involved in and in just making it uh suitable to live in a banana environment so I think it's it's quite an interesting topic to look at so I'm going to leave the the six genes there and I'm going to leave the diversity and then I'm going to move on to another main target within our lab is looking at genetic resistance to fusarium wilt. To control fusarium we need to know where it is uh oh no sorry Mr we'll have to skip through the epidemiology quite quickly sorry get back to resistance to what extent does the fungus colonize the plants and where are the spores produced okay so the long held theory is that chlamydus spores of fusarium oxysporum last in the soil for decades and that's what allows it to be persistent in the soil but um there's more and more evidence that they're persisting as endophytes and as saprophytes so they do they have chlamydus they do produce chlamydus spores these long lasting spores they produce macro and microkinidia so we used a gfp strain of fusarium oxysporum this uh construct and had been transformed by lian forcibe in our lab many years ago and we got it out the freezer and it's been very useful of late so with confocal technology having improved over the decade uh no lean was able to use it to to look at the infection in banana now lots of people looked at the initial infection as a fusarium gets into the plant but what we were interested to see was what was its persistence how was it traveling up through the plant and this was in in concert with the you know with the tropical race 4 having arrived in the tally region we wanted to look at ways that we were going to be able to control but where was the fungus how we were best to control it and these fantastic images I think just show initially here we got on the left it's on the roots here on the the right 70 days after inoculation we're seeing the mycelia traveling up through the xylem and producing spores but it's absolutely I used to think that the response of the vascular welp was the plant's response with closing down the vessel but you can see here it's absolutely clogged with mycelia growing up and at that point the leaves are still green I mean that's the intriguing thing the fungus is it traveling up through the xylem vessel a lot quicker than you're seeing the symptoms develop so um and here's another example that the spores in the cavendish plant and you can see if you take a cut through the quorum you can see how the the vasculator in these cases absolutely clogged with these fluorescent fungi and again now the interesting thing that we saw from the study that no lean was doing we could see spores on the leaf surface we could see sporillation happening there now it's it's pretty well established that has to get in via it doesn't seem to be get in by wounds in the pseudostem so it has to get so producing spores there maybe is less of a worry in that in the infection within the crop it's still has to fall down to the ground and I don't quite know what happens then but the fact that it is sporillating on leaf material is interesting we can see little sporidopia here on the leaf surface and and in some on decaying leaves as well so when you you've got the trash you or you you de-leaf you're getting it developing on that and you can see in chlamydus spores developing in the air chambers and outside the leaves so these are the resting spores so the conclusion from the this material study was that fuzermak's mom's confined to the xylem and healthy looking leaves but as a leaf senesce it starts to colonize extensively as a sacrifice now the implications from that immediately would be that trash from de-leafing subsequently becomes a source of anoculum including chlamydus spores so that just lying on the ground it can then break down even when you de-leafed it the plant might not look infected that then becomes a source of anoculum that's going to get mixed up within the soil so Jay Anderson then did some work continuing on with the GFP strain looking at alternate posts because it said we know it's an endophyte what's it doing on other weeds and we could find it colonizing the roots of weeds but it wasn't really substantial it was just you know a few infections here and there and that was with deliberate inoculations that we were doing but more to the point after we treated the weeds with herbicide we're creating a nice amount of tissue that the fusarium can go into saprophytic mode and colonize it and that's what happened we saw an increase in growth and therefore potential inoculum after herbicide treatment of the weeds so then you question what happens if you put herbicides onto a disease plant because in disease plant obviously you want you identify fusariums out there you want to get rid of it quickly so we did the same thing I say we Jay did the same thing and she she inoculates some pot plants and then treated them with herbicide after the fusarium I got hold and what we then saw was an abundance of sporulation after spraying with herbicides certain she compared different herbicides and they all really ended up with the same thing so and even treating them with a fungicide then a herbicide we still we still got an abundance of sporulation so this has implications for containment controls and protocols thereof okay now I'll get on to the horse and I better do it quickly banana basically banana is a freak it's a it's a it's only the banana that we eat and consume is only there because we propagate it because it's it's sterile most of the banana commercial certainly the commercial bananas but even in some of the subsistence agriculture you sometimes get some seeded ones but not prolific seeders generally the sterile polypoids are partner carpet and freak they produce fruit without seed there's no benefit to the plant and it's relies on clonal propagation and classically we talk about a and the b genomes being done having been a hybrid between these two species but resistance is present resistance fusarium milk is present it's in wild seeded deployed bananas but I'm not quite sure of edibility it's a correct word but whatever it is that what makes it favorable to us are the bananas that are partner carpet and the ones that sterile so that even what breeding you need to do at the end of the day you want to make it partner carpet and sterile so it makes it a little bit problematic in breeding and on top of that you've got long generation time you've got large plants and the confusion that's been happened with interploidy crosses is that the genetics are really quite messy when you start looking at banana whereas if you look at wheat which is mean cultivation for 10 000 years we've been selecting and in breeding that it's it's relatively uniform if you compare it with banana which is just basically accessions have been taken from the jungle and fixed in time by clonal propagation so we're particularly interested in one line of music uh human auto subspecies malachensis because it has shown individuals that are resistant and individuals are susceptible which is convenient because if you're going to make crosses or try to look at segregation for resistance it's good to have it both ways so that you can identify the resistance the fruit is small the seeded and they're absolutely of no commercial value whatsoever but it's the resistance traits that we're interested in it's some show resistance to tropical race for a sub tropical race for and base one and so it can potentially help to understand the mechanisms of resistance by just focusing on these not necessarily breeding with them but focusing on the genetics to develop molecular markers and that's what we've been doing we take self pollinations we do embryo culture because that's although the seeds would be viable we could saw them out and look at the variations and in germinations etc but by doing embryo culture we can take them straight into propagation and clonal propagation and maintain those lines so it allows for multiplication of lines through tissue culture and so this is what banana seeds look like these are the fruit sorry i've got the scale in of the fruit but these particular fruit are about about eight centimeters long and these are the flowers that we pollinate now banana flowers the first one the first come out of a spike maybe your first varies when the cultivars maybe first six to sometimes 15 hands are going to be female only and then the male flowers start developing producing the answer so it's quite convenient in that respect for doing crosses so that you know the first few hands are going to be female only and then these are the seeds that we do the embryo rescue for so we've been doing crosses between resistance and susceptible and getting segregation and trying to follow the traits so much of this work has now been done by Andy Chen he's been funded both by a combination of the the Gates project and with Hort Innovation at intervals as well and initially we looked at a smaller population we could we get a lovely clean cut difference between resistance and susceptible the susceptibles are highly susceptible in these Malachensis lines and the resistance even in when we you know you sometimes see a little bit discoloration of corn but generally that's very so then it gets into the realms of all the molecular analysis and doing sequin analysis looking at initially we did it with snips single looking for single nucleotide polymorphic calling and we were able to compare the resistance versus the susceptibles and we found a region on chromosome three that was highly associated with the resistance we've then we've since done more fine mapping and more a larger population and this Manhattan plot I think is the best one that you don't need to understand the background behind it but it just illustrates if we compare resistance susceptible we find this one region which spikes out on chromosome three which seems to associate with the the trait of resistance so this is comparing resistant versus susceptible so we then look at individuals which we know we have to go through to the next generation compare heterozygous homozygous resistance homozygous susceptibles and we're getting to a nice clean point where okay I'll go to the next slide yes where we've identified markers on the long arm of chromosome three that we believe that our resistance is there and so there are many genes there it's you know it's not not straightforward and also as I said before banana genetics is messy we compare what we see against the published pahang map even though it's another malachensis is still quite a lot variation so we're now at the point of trying to identify the genes in this region with fine mapping by looking for rare crossing over events to see if we can get the actual gene for resistance but in the meanwhile we can still use the marker associated with that so something that's it's genetically linked to the trait of resistance and that has been used in by international breeding programs by in a bad by I mean in brapa I meant sorry in brapa also the serrat breeding program and also iita so they're using our marker to select individuals carrying resistance in the absence of being able to screen with the pathogen themselves having said that we there's a little bit of caution in that because we only seem to be able to identify that marker in lines that have a background with malachensis there are other potential resistance sources and and we have you know we know that a line known as piss and jaraboya for instance have line known as calcutta four and if we look at this phylogenetic wheel of banana lines we can see that there is quite a lot of diversity from collections that this work has been done in the Czech Republic but they look at the variation of all the the banana accessions and we can see that malachensis is reasonably close to calcutta four but piss and jaraboya is quite distinct so potentially we can there may be more than one resistance source we don't know to but we need to look at that because it's important that we don't just rely on the resistance from malachensis however we're going to intergress it into breeding lines that potentially we would want to I call it pyramiding genes but certainly have a backup plan should that resistance be overcome at some point by tropical race five six or seven so so basically our marker is not detected in piss and jaraboya calcutta four so we're now working on generating new segregating populations which one thing I get to do is do the crosses unfortunately some of these plants are quite tall and it is a problem getting up to them to do the crosses but we're crossing back onto our susceptible malachensis and then following up the segregation populations okay so I'm finishing there a few years ago there was a sort of a lot of thing in the press about banana again that tropical race four was finally going to destroy banana production worldwide and these are some images taken in china that in certainly there's a heck of a lot of spread of tropical race four in some of the southern provinces of china and where they had huge banana plantations and they've also come to tropical race four and as a consequence of planting lots of dragon fruit fruits and citrus now which is I quite like dragon fruit but I don't think it is the end of banana I think we have we can find resistance there is resistance out there not all cultivars are susceptible to tropical race four but certainly most of the deserved bananas are but there is a way out and through quarantine through good diagnostics by understanding the pathogen by resistance we can get the better of it my final acknowledgement slide as there's just so many and a lots of names missed off here but this is has been a fantastic collaboration this work between labs within Australia and internationally and it's it's really great seeing all that coming together we've got through the gates project we there's lots of several international players involved and also here within Australia and we'll stop now hopefully I didn't overwhelm everybody will I stop stop sharing my screen