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| >> It's seven o'clock. Good morning, everyone. >> Morning. >> Nice to see so many people. Heather, should I go ahead and share the screen? >> Yeah, go ahead. You should be able to do that. >> Okay. Let me just -- And then. | Two women and a man appear on a video call as they sit in an office. Then, another woman joins the call. |
| Can you see that, Helen? Great. Well, welcome everybody to an informal but hopefully useful workshop that really more of a discussion that myself and Mike Derie, who's sitting next to me, through various discussions of the couple of years that the Stop the Rot Project thought might be helpful to present and engage with members of our Stop the Rot team. So first of all, I'm Lindsey du Toit. I see some faces and names on the screen that I'm less familiar with. So I'm Lindsey du Toit. I'm a vegetable seed crop pathologist at Washington State University. I'm also the director of the Stop the Rot onion bacterial disease project. Next to me. >> Mike Derie. I'm a research technician for Lindsey. >> And if -- We call him Dr. Death because he's amazingly good at creating plant diseases so. And he's very instrumental to some of the discussion we'll have today. So I'm really pleased that Mike's willing to do this. I rubber-armed him into it. And also Heather, do you want to introduce yourself? >> I'm Heather MacKay. I'm the project manager on the Stop the Rot project. So most of you have corresponded with me by email I believe. Yeah. >> All right, so what I think we'll do is we'll start with -- I prepared a set of just a few slides to kind of guide some talking points about inoculation to create disease pressure in field trials. And, obviously, not all of you are involved in the onion Stop the Rot project. So we apologize if some of the discussion might get into terms, or trials, or specific aspects of that project that you're not familiar with. So please feel free at any point to raise your hand, ask questions. We want this discussion to be useful. The objective here is to see how those of us on the Stop the Rot team who are doing field trials can try to be more effective at creating a good level of disease pressure for differentiating treatments that we're evaluating in these trials. So it's -- We realize that everyone has different resources, equipment, they have different field circumstances but some of the principles, I think, are really common for those of us who are crop pathologists as to recognizing what it takes to try and create good disease pressure. And understanding the disease cycle. Understanding the growth stage of the crop. And the periods of susceptibility. And trying to create disease pressure that mimics as close as possible what might happen in that environment in which onions are grown. Because if you create too artificial a situation, you could skew data in some form or another. So we'll discuss this as we go through. Heather also sent a protocol that we as a team, Stop the Rot team, developed. Well, Mike Derie and I sort of put together the protocol that we had used to do inoculations of bacterial diseases in our field trials. So that Word document that Heather sent, it's a three-page document that kind of goes through what our objective was in that case to try and create good disease pressure in the field using a backpack spray inoculation method. It's not necessarily the only method out there. There's lots of various ways you can inoculate plants, but that was sort of the impetus for developing the set of slides to kind of break out some of the key principles to think about. So if you want to -- For those of you not familiar with the Stop the Rot project, on the slide that I have on the screen there's a link to alliumnet.com which is the website where our Stop the Rot project posts. Many of our reports, our resources that we're developing as part of this project. So -- And we can also post this recording of this presentation. So as you've probably noticed, the Zoom meeting is being recorded, so be careful what you say. It's going to go live. All right. So I'll move forward to the first part of this and let me see if I can get this going. | A new slide titled, stop the rot, onion bacterial inoculation workshop. Lindsey du Toit and Michael Derie, Washington State University, first June twenty twenty two. |
| So, yeah, I think almost everyone on this call, not everyone but many of the folks on this call, are plant pathologists. And not to go back over 101 plant pathology but, as you know, in order to get disease development, you have to have three essential components. You have to have the pathogen present. You have to have an environment that favors the development of that pathogen and infection. And you have to have a host that's susceptible. In the case of the Stop the Rot project, when we're planting these field trials we're clearly putting out onions. There are no completely resistant onion cultivars, but we do try to select varieties that are common for the area where the trials are taking place. And obviously, these are varieties that growers are experiencing losses to bacterial rot. And therefore we do have a susceptible host. The question is for many of us in doing these trials, is do we always have the pathogen present? Here in Washington State, most of our annual production is in the Columbia Basin, which is semi-arid. Bacteria don't like dry conditions. They favor by moisture. So you'd think, well, why do you have bacterial problems when you have a semi-arid region of production? Well, the reality is our onions have to be provided with water in order to grow. And particularly if that water is provided through overhead irrigation, we immediately are creating more favorable conditions. So one of the things you think about when you're trying to inoculate a trial is you try to think about the exact opposite of what you'd recommend to grow for management. So everything we tell everyone to do, we want to do the opposite in order to create good disease pressure, except for the treatments we're evaluating. So how do we create those favorable environments? So some of you, like Claudia, the folks at Oregon State and others, are working in areas where onion production is always drip irrigation. And it's drip irrigation, you're going to have far less favorable conditions because you don't get that moisture on the canopy. And yet that moisture is a primary driver of the disease. The other question is whether or not the pathogen is present. So that's part of what we'll talk about today is providing the pathogen there in case you don't get a good enough natural infection. And how do you provide -- When do you provide it? What do you provide? And what form do you apply it? | New slide titled, conditions needed for disease development. On the screen, a large red triangle with text in the center that reads, amount of disease. On one side of the triangle, text reads, environment. On the second side of the triangle, text reads, pathogen. On the third side, text reads, host. |
| So the disease triangle, you know, as you all know, the size of the triangle, the area inside that triangle, represents the amount of disease. So if you have a favorable environment and a susceptible host but the pathogens not present, you have a straight line. There's no area. That means no disease. If you have the pathogen present and a susceptible host but the conditions are highly unfavorable, and you use drip irrigation, it's a super hot, dry season, you're going to have one side of the triangle is going to be really short. Now you have a smaller area. Or if you have very little disease present. So we have a favorable environment. We have the host. But we don't provide the [inaudible], again one side is small so now you have a smaller triangle. Well, if all three are less favorable, you're going to have less disease. So as we think about inoculating, we try to think about maximizing all three sides of the triangle as best we can, or at least the ones we can control. And that's really important. In field conditions you sometimes have less control of the environment. Last year we had our hottest summer on record. We got up to 117 Fahrenheit, which is over 40 degrees Celsius for those of you who think metric. And that means really, really dry. So 10% humidity, 15% humidity, which is not good for bacteria. But if we can provide overhead irrigation, we can counter that. The heat is actually good for many of the bacteria that we struggle with in central Washington, because most of our bacterial pathogens are thermophilic. So the heat was good but the dryness was a problem we had to counter. And that's why we use overhead irrigation in our field trials. We do have the ability to set up overhead irrigation. | Text appears that reads, environment. Under it is a red line, and below the red line, text reads, host. Another triangle appears on screen, this time with two longer sides and one shorter side. Text on the longer sides reads, pathogen and host. Text on the shorter side reads, environment. Text in the center of the triangle reads, disease. Then, two more of the same triangles appear on screen. For the second triangle, the two longer sides read, environment and host, and the shorter side reads, pathogen. For the third triangle, the two longer sides read, pathogen and host, and the longer side reads, environment. |
| So how do you create favorable conditions? So I deliberately put these two photos on the slide. The bottom left one -- Both of these are -- Mike Derie features remarkably in my slides because I'm the one taking photos. So this is Dr. Death at his finest. So on the bottom left is Mike inoculating our field trials, this was a couple of years ago, for the Stop the Rot project. This is a cultivar trial. You can see different heights of growth of the plots representing the different varieties. But I strategically put this picture in here because, for one thing, you can notice I'm standing right behind a center pivot irrigation. So you can see the sprinkler. You can see water still coming out of that nozzle. We -- One of the things we do when we're trying to get disease established in our plots is we run the sprinkler for about 15, 20 minutes that the pivot -- over the plots before we inoculate to get good moisture in those necks. Okay? So we had run the pivot through the fields to get good moisture in those necks, get that canopy nice and hydrated. And then we came through and inoculated. The other thing in that picture, I'm not sure if you noticed, but there's a long shadow to the right of Mike. So we're inoculating at about eight o'clock in the evening. Because this is late July. And it's very, very hot in central Washington in late July, as I said. And there were cases we were inoculating when it was 40 degrees Celsius, or 110. Not very pleasant with the backpack sprayer on and a bunch of liquid on your back. But we do that because if we can inoculate in the evening when it's starting to cool off, that moisture that we've applied through that irrigation prior to inoculating and then the inoculum that we are spraying on will not dry out quite as quickly as if we inoculate in the morning or in the middle of the day. So Mike and I -- My lab is about a five hour drive from where our field trials are located. So Mike grows inoculum on the day prior, the morning of -- He prepares the inoculum with a spectrophotometer. He quantifies it. We get it aliquoted out. We put it in a cooler with ice. And we drive five hours over to the basin. And then we inoculate that evening. And the picture on the right is one of those days where we inoculated. It was a bit later in the season. It gets darker a little earlier. And we were inoculating by 11. But that was much better than inoculating in the middle of the day when that inoculum would desiccate and dry out much too quickly. | New slide with text that reads, create favorable conditions for onion bacterial growth. On the left side of the screen, an image of a machine working to inoculate a field of crops during the day time. On the right of the screen, an image of a machine working to inoculate crops at night. |
| So moisture is a huge driver for bacterial diseases. So we're specifically talking bacterial pathogens here. Some of this is relevant to fungal pathogens, but since our project is focused on bacteria these are the key things. All of these bacterial pathogens that impact onions are favored by moisture. And that's something we really have to consider in how we create good disease pressure. And one of the things we ran into when we were discussing some of these field trials with various members of the project is when do you provide that moisture? So you want the inoculum to land on a nicely hydrated canopy. But you don't want to provide moisture right off your inoculation in terms of overhead irrigation because you can wash that inoculum off. So it's really important to think about the timing. You want to get those -- the canopy nice and moist and humid, [inaudible] or open. In our case, where most of the bacterial infection happens is when you have maximum neck size, upright leaves, full size neck, the water collects in that neck. We want to provide at that time we can get moisture into that neck. So the inoculum, when it's applied, will land on that nice dehydrated canopy. There's still a film of moisture on the leaves that it's less likely to dry out as rapidly when it's 35 degrees Celsius. So we don't want to inoculate immediately after -- I mean irrigate immediately after inoculation. But one of the things we do in our trials is the day after inoculation, so the next afternoon, we will turn the sprinklers on again every other evening for about 15 to 20 minutes. So that you continue to provide a period of hydration of those necks, moisture in those necks, so that the inoculum that you did apply can continue to multiply and cause infection. And, you know, what source of overhead irrigation you use, it could be rain if you're in a rain fed area. It could be a center pivot or sprinklers. Claudia at Utah State University actually uses a watering can because her trials are under drip irrigation. They -- She doesn't have the means to set up overhead sprinklers. So she will actually fill a watering can and go over the plots before inoculation with a watering can. And then come back days after that and continue to put some moisture over the top of the canopy with a watering can. That works for a reasonably sized trial. But if you have a very large trial, that might be a little -- not too feasible just [inaudible]. Does anyone have any comments or questions, especially those of you on the project who've done inoculations regarding moisture? >> The watering can works in a dry climate actually surprisingly well. I mean, we got 30% rot with Burkholderia that we used for inoculations for Utah. That's really good. >> Yeah, exactly. Thank you, Claudia. Okay. >> Lindsey, just one question. I mean -- >> Yeah. >> -- like, how realistic. If we're going to be putting all this moisture on, you know, how valid are the results? Because a grower would never do that. That's the pushback I've had in the [inaudible] -- >> Yeah. >> -- set up overheads, right? >> Yeah. So James, I think, you know, in our trials, that's the reason we always have paired inoculated, non-inoculated. So we are looking at the -- you know. We're applying inoculum at a certain time. That always provides some kind of a bias about when you're applying it. But, again, I -- after 22 years of looking at onion diseases in the Columbia Basin and seeing when I encounter bacterial diseases, I've become pretty familiar with the kinds of production practices that are more favorable for bacterial infection. I see the biggest problems with bacterial diseases in our Central Washington crops when growers over irrigate, irrigate too late, irrigate excessively. Or if we have untimely rains. So in a research trial, you could say, well, I'm not going to over irrigate because it's biasing it. But we try to represent a circumstance where disease might -- when disease might occur. So if a grower irrigates too late or excessively. Or the grower of this crop experiences late -- a late season irrigation. Those are the circumstances we're trying to create so that we will get results. Otherwise you have a really high chance, James, of getting no data at all, no disease. If you've got no disease, your trial's not worth it. You've wasted it. >> Oh, I agree. I agree. It's just, you know, like most of our onions are under drip [phonetic] -- >> Mm-hmm. Yeah. >> -- so, you know, when they go and see the overhead spray set up it kind of puts us on the back foot straight away. >> Yeah, but I think -- >> I mean, I'd rather see disease than no disease, so I agree. >> Yeah. And I think, you know, what we're doing, James, is creating a worst case scenario. So if a grower didn't follow the recommended practices, this is what could happen. And this is where -- how -- the degree to which these treatments you're evaluating may or may not work under these worst case scenarios. So -- And that's part of the reason, you know, we've -- We don't keep -- We don't use that supplementary post inoculation irrigation to irrigate. We're using it just to create some moisture in the canopy. We irrigate based on the growers -- the recommended practices for irrigation. But we're adding this little bit of supplementary to create a case of disease prevalence. >> And even though we use drip irrigation, James, we do get thunderstorms every once in a while with heavy downpours. And usually you see bacterial diseases in Utah after that. >> Yeah. And I know that's true with Colorado, all those states that get these big storms that come through, the big fronts. Like, you get those convection storms. Colorado gets those fronts that come through, you know, talking to folks like Robert Sakata, those are the circumstances where you really worry. >> It's hail that worries me. >> Yeah. Yeah. Yeah. >> I wish we could simulate that. [ Inaudible ] >> This is a sand blaster. I've tried that. It works. >> Yeah. When we were -- When I was in Illinois, we used to mimic hail damage with a lawn mower set really high. This was sweetcorn, not onions, but. Yeah. But it's a really good question, James. So thank you. | Text on screen reads, number one, moisture. Onion foliar and bulb rot pathogens are all favored by moisture. Provide moisture preceding inoculation, leaves and necks wet for bacteria to survive and colonize. Provide moisture after inoculation, but not immediately after, wash inoculum off plants or dilute inoculum. Example, overhead irrigation for fifteen to twenty minutes every other evening. Source of overhead water, rain, center pivot, sprinklers, watering can. |
| All right. So the other -- So sorry. The other thing is -- to think about is temperature. And as I mentioned, most of the bacterial pathogens we deal with in the semi-arid Western states tend to be thermophilic. The Burkholderias, the Enterobacters, the Pantoeas. We tend to see bacterial problems starting mid to late season when we've got big neck size and the risk of moisture collecting in those necks. Eighty, 85% of our onion production is under center pivot in Central Washington, unlike where James is working and that's mostly drip, and Claudia mostly drip. And so we do tend to see, you know, this very high risk of ideal combination of hot and very wet conditions if they're concerned about the high temperatures and tend to over irrigate. If you talk to folks like the University of Georgia folks, where they do winter production in Vidalia region of Georgia, they do tend to see a lot more of these moderate temperature pathogens like the Pseudomonas and some of the Pantoeas. They can tolerate moderate temperatures. So they get a lot of foliar symptoms from Pseudomonas viridiflava, Pantoea ananatis that we very seldom see in our area unless, like James mentioned, we have a hailstorm where you get tremendous foliar damage and then -- and moistened from the rain and the overhead irrigation. But we don't deal a lot with these. So we don't focus on inoculations on those cooler, earlier times of the season. As I mentioned, occasionally a grower will tell me they had a hail storm come through but we don't get the frequency of hail storms that folks like Claudia and the Colorado people do. So I don't know. Beth, since you're on the East Coast with more humidity, do you have any comments regarding either moisture or temperature in more humid type of production area? >> Yeah. So I would say -- I mean here in Pennsylvania, we'll still use overhead misting to create those favorable conditions later in the day. And have had success with just these little whirly sprinklers that -- >> Yeah. >> -- we hook, we tie into our drop irrigation system. I think from a temperature standpoint, it's interesting because where I am in the center of Pennsylvania versus we have another research station in the South-east part of the state. Because it's significantly warmer in the South-east part of the state, we actually can have higher disease pressure down there than what we can have here in the center of the state. So, yeah, I think temperature really, you know, plays a pretty important role in that disease triangle. >> Yeah, thanks Beth. Is anyone from Georgia on the call? They tend to not inoculate their trials because they have such high disease pressure so. They're -- Unfortunate for the growers, but good for the researchers. | Text on screen continues. Number two, temperature. Thermophilic pathogens inoculate mid to late season. Moderate temperature pathogens, inoculate when cool or moderate. |
| And so another really important thing to think about with inoculation is when do you inoculate? And it's something you automatically potentially introduce a bias by when you inoculate. And this is one of the reasons I do feel it's valuable to have inoculated and non-inoculated plots. So we've had this discussion, Tim Waters and myself, that, you know, if we just inoculated and removed the non-inoculated, we would reduce our workload by 50% because we have to rate, and harvest, and evaluate twice as many plots by having inoculated non-inoculated. But I think it's really important because it demonstrates the degree to which the timing of inoculation is influencing disease. As I mentioned before, for our neck and bulb infections that are the predominant type of infections we see in the Columbia basin, we inoculate when I typically have seen the worst case scenarios. When you have maximum neck size, upright tops that the waters can collect in those necks very, very easily. Right -- Just before about the time of the top starting to fall over is our peak period of susceptibility. In Georgia, they tend to see these pathogens like Pseudomonas viridiflava earlier in the season because it's favored by temperatures -- Excuse me. Temperatures at say around 70 degrees. So low 20s, high teens for Celsius. And we tend to inoculate a second time about a week to two weeks later. Because just in case we don't have good conducive conditions, we do a second inoculation. But I do think most of our infection that we get in our trials is from that first inoculation. The reason is we rate our plots every week, starting a week prior to inoculation, just to see what background infection may have been there. And, inevitably, we see symptoms the second week after inoculation. And that's about the time that we've just done our second inoculation. So our first inoculation, we don't see symptoms that week. We usually don't see them the next week. It's two weeks later that we see. So it's about the time it takes for that inoculum to get established and start to produce symptoms in the neck. So I -- We may be able to get away without that second inoculation, but we do it as a precaution to try and ensure adequate disease pressure for treatment differentiation. I know, Beth, you've done some toothpick inoculations in the past. Do you want to comment on timing with that kind of inoculation? >> Yeah. So with a toothpick inoculation, we literally have little tubes of inoculum and dip a sterile toothpick in it. And then we stick the toothpick in two of the interior leaves. And typically, we have four rows of plants and we'll inoculate two of them, two of the rows, which represents kind of a worst case scenario because you're putting the bacteria directly into the plant. So we would rate those plants. And then we would also rate the rows that are adjacent to the ones that are toothpick inoculated and consider those to be a better simulation of natural infection. That the bacteria would move from the toothpick inoculated plants to the ones -- to the adjacent rows. We typically time that with bulb initiation. So, for us, it's the end of -- the end of June. We're kind of trying to move away from the toothpick inoculation and do the backpack inoculation because it's easier. It's pretty difficult on your back to do the toothpick inoculation. And I know Brenna can attest to that because she helped me do that and for years ago too, so. >> Yeah. Thank you, Beth. Yeah. And that is the beauty of the backpack sprayer inoculation. It's much more efficient. | Text on screen reads, number three, timing, inoculate at susceptible stages of crop growth. Foliar disease, succulent, less waxy leaves more susceptible. Neck or bulb infections, upright, maximum neck size, start of tops down. Repeat seven to fourteen days later. |
| All right. So we'll move on to the next part, which is producing inoculum. And this is where Mike's the expert since he's the one who produces inoculum so expertly. But -- And we're going to review sort of what our protocol for producing inoculum, which doesn't mean it's necessarily the only way to do it, but it's worked really effectively in our trials over the years. So do you want -- Do you want to walk through this bit, Mike, or do you want to take over? >> Oh, no, you're doing fine. >> Okay. Mike doesn't like presenting. But he's my moral support at least. All right. So I think it's really important when you're growing inoculum for trials to use, you know, bacterial cultures that are not -- that haven't sat around for three months on Petri plates in sort of desiccated states. So using fresh cultures to start each batch of inoculum. Some of the bacteriologists that serve on -- I'm not a bacteriologist. But some of the bacteriologists that serve on committees for students in my program that are working on bacterial diseases, they have said it's important to go back to your stock. You store it at -80 and start a culture from your original stock whenever you're starting a new batch of inoculums. So that you don't have a risk of those isolates losing virulence. We tend to use shake cultures to -- for producing inoculum of most of our pathogens. Sorry, I forgot to close the parentheses on that broth share cultures. And one of the things we -- Mike has mentioned is he likes to use NBY broth versus NB nutrient broth. Because the NBY has yeast extract plus glucose, which favors much more rapid growth of most bacteria. Do you want to comment on that, Mike? >> Well, it's just providing more nutrition. You know, more nutrition for the bacteria. Sometimes, like I know with Xanthomonas, there's unknown growth factors. So something like yeast extract that has the vitamins, or vitamins as the British like to say, that just to help support the growth of the bacterium while it's in broth. Yeah. >> Yeah. And if -- It depends on the bacterial pathogens you're working with. But we tend to keep them on the shaker for 16 to 20 hours at most. So that you ensure when you're harvesting those bacteria, they're in the long phase of growth and haven't entered into that stationary phase where, you know, you've got breakdown products, you've got, you know, just not the healthiest culture. So we time it so that we're removing them from the shaker during that long phase of growth. So we usually do an overnight shake. RPM two -- >> Yeah, 150, 200 on our shaker. >> Okay. >> Yeah. >> And part of the reason we want that shake culture is these -- the bacteria we're dealing with are aerobic. And so we don't want that culture to be stationary because you get more and -- less favorable growth because it's more anaerobic. I know that you can produce inoculum very effectively by just washing colonies off plates. So -- I know Carolee Bull tends to use plate cultures to wash and prepare inoculum for various bacterial pathogens. We primarily have used shake cultures. I think the volume of inoculum you can get is -- seems to be pretty efficient. >> Mm-hmm. >> It's -- A lot of folks also just wash agar plates. And just make sure those plates are not old, as I mentioned. Because we're working mostly with thermophilic bacteria for our onion project, we tend to produce the shake cultures at about 28 degrees Celsius. If you're dealing with things like Pseudomonas viridiflava, you may want to go down to temperatures that are favorable for those pathogens. Any comments, Mike, on that? >> Not really. Just, you know, just whatever published optimum temperatures for the growth of the bacterium we're working with. >> Yeah. And for our bacterial inoculations, we produce both Burkholderia gladioli and Pantoea agglomerans. Burkholderia gladioli is definitely affected more by cooler temperature of inoculum production than Pantoea agglomerans. So we go at 28 because it makes sure that our Burkholderia is growing fast enough to get the concentration we want in that 16 to 20 hour period. So, you know, we always try to quantify inoculum to make sure we know what we're putting on, the approximate concentration we're putting on. We tend to try to aim at applying at 10 to the 8 CFUs per ml of inoculum that actually is in the backpack sprayer. We prepare our stock bacterial suspensions before we leave. So Mike calculates the exact volume we need plus the buffer. And this is going to be determined by the type of nozzle you're using. The boom you're using. How many nozzles are on that boom. The gallons per acre you're applying. The pressure you're applying it at. The plot size. The number of plots. And we prepare more than we need because if we get -- if we drive five hours and get out of the truck and start getting ready and accidentally drop a flask of inoculum, we can't drive five hours back to Washington, Mount Vernon and start again. So we usually have one and a half to two times the amount of inoculum we need in case of accidents. So Mike always has extra containers in the cooler of ice in case stuff happens while you're out there. Mike, you want to say anything further on that? >> I could go through just kind of a calculation that we did. So say, for instance, I grew Burkholderia overnight. I put it on an spectrophotometer and so the ratio I got is -- with a 1 ml cuvette in the spectrophotometer. Say it took me 130 microliters to achieve the 0.3 OD. So that's 13%. Or 150 micros [phonetic] would be 13% of the volume. So when you're preparing your -- preparing your volume for inoculation. So 13% of that will be your -- the broth culture that you generated. >> And the -- >> -- on the -- >> And the -- >> -- on the shaker. >> And the rest -- >> And the rest will be -- >> -- will be the buffer. >> -- the buffer. >> Yeah. >> Yeah. And then, like Lindsey said, a lot depends on your nozzles and your booms. For instance ours, I have an 18 inch nozzle spacing using 8003 tips. It amounts to about 30 -- In our situation, about 34 and a half gallons per acre that we're applying. In one case it did 125 -- we did 125 plots, it amounted to about 0.13 acres total of plot area that we're applying. So I needed about four and a half gallons or 17 liters of inoculum to be able to apply that. >> Jennie, you have your hand raised. Would you like to ask a question or make a comment? >> I would. Before we move on from this section, this might be horribly specific but, Mike, are you growing your broth cultures in glass tubes or are you using something else that you then spin down before you -- and decant before you take your spec measurements? Or are you just taking your spec measurements from your turbid broth culture and just adding all that broth culture to your inoculum? >> That's what I do. I don't -- I don't do a spin down or clean the broth out of the -- out of it. So I don't -- I don't resuspend the bacteria, spin it down, or anything like that. One of the things is -- >> So -- Yeah -- >> -- there's too much volume there and too much time. >> Yeah. >> We've got to get across the state and -- >> Yeah. And that's -- >> -- get this done. Yeah. >> But it's a good point, Jennie. Because I know talking to Carolee Bull, they tend to spin down their inoculum and rinse off the buffer, and then prepare their suspension at the concentration they want. We've never done that and we've had very good success with inoculation. So not to say you shouldn't do it, but we haven't seen a limitation by not spinning down and rinsing off our inoculum prior to inoculation. Brenna, you had your hand raised. >> So when you transport it, it's -- >> Concentrated. >> -- already combined with a buffer? Or you do that combination with the buffer on the field? >> So what I usually do is I make those calculations -- >> Yeah. >> -- before I go. And I determine, say I need 330 mls per two and a half liters of buffer, say. So I'll aliquot out into a flask about 330 and put that in the cooler. >> Yeah. >> Then make the buffer separately and then we'll mix on site. >> Okay. >> We mix on site. Yeah. That's a really good question, Brenna. We never mix prior to starting the trip to the -- Central Washington. We put the buffer in a large carboy, because of the volume. You know, we're talking about gallons of buffer. >> Yeah. >> And we put the bacteria in on ice in the cooler. >> Okay. >> Yeah, really good point. It's easier to transport too. We don't have coolers big enough to carry gallons and gallons of. So we keep the inoculum cold to prevent it from going past the log phase and changing inoculum concentration too much. Obviously, you get -- you're going to get a little bit, but on ice there's not much growth happening with Burkholderia and Pantoea. If we're -- Because we inoculate with more than one pathogen, we're dealing with two isolates. We mix them in the spray tank in the field. We don't mix them prior to, you know, transporting the inoculum. So we mix -- We just pour them into the tank and add the buffer. | New slide titled, onion bacterial inoculum production. Text reads, number one, bacterial cultures. Use fresh cultures to start each batch of inoculum. Broth shake cultures. N B Y versus N B, yeast extract and glucose in N B Y favors faster growth. Harvest at log phase of growth, sixteen to twenty hours on shaker for most onion pathogens. Colonies washed off agar plates, no more than three to five day old cultures. Temperature of incubation, twenty eight degrees Celsius for thermophilic bacteria, twenty four to twenty five degrees Celsius for others. Number two, inoculum concentration and preparation. Quantify with spectrophotometer, zero point three O D at six hundred N M, equals, 10 C F U per milliliter. Prepare stock bacterial suspensions before you leave the lab based on area to inoculate. Calculate volume of inoculum plus buffer needed, nozzle, boom, G P A, P S I, plot size, number of plots. Prepare two times the aliquots what you need in case of accidents. Keep inoculum cold in transit, ice in a cooler. |
| We dilute the inoculum in the spray tank with buffer. We use a phosphate buffer and we add .01% Tween 20 to that buffer. So when Mike aliquots, you know, the gallons of carboys of buffer before we depart, he adds .01% Tween 20 to that buffer, so that when we add the inoculum to the spray tank and add the buffer, it prevents that osmotic shock from sudden dilution of the bacterial suspension. Brenna, you still have your hand raised. Is that just a layover or you have another question or comment? Okay, I think it's just a layover. >> Sorry. >> That's okay. I thought maybe you had another comment. Okay. So this is basically how Mike prepares our inoculum each time we are ready to go. And we apply it at 10 to the 8 CFU per ml. So that's total concentration of using a mix of isolates. That's what ends up in the spray tank. Okay. >> Mm-hmm. >> And the fact surfactant, the Tween 20 is actually really important with a crop like onions, because the leaves are so waxy. And if -- There's a really high risk it'll just roll off the foliage if you don't add some kind of a surfactant like Tween 20. You want to add any comments about that, Mike? >> You're just breaking the surface tension. >> Yeah. >> Surface tension of the water. So it doesn't beat up on the plant -- >> Mm-hmm. >> -- and it tends to -- >> Mm-hmm. >> -- adhere better. Yeah. | Text continues. If inoculating with less than one isolate, mix isolates in the spray tank in the field, not in the lab. Dilute inoculum in spray tank with buffer, example, phosphate buffer, plus surfactant, example, zero point zero one percent Tween twenty. Do not dilute with water, osmotic shock. Apply at ten C F U per milliliter, total, if using a mix of isolates. Surfactant limits amount of inoculum rolling off waxy foliage. |
| >> Okay. And so just kind of now we're in the field. We've got the inoculum in the spray tank ready to go. These, again, are photos of Mike inoculating our plots. The top picture shows Mike inoculating at -- just at, you know, maximum neck size, upright tops, a few tops starting to go down in the plots. The bottom picture shows him inoculating 14 days later when more of the tops are down. We think this probably has less impact on creating good disease pressure than the first round. But we do this as a backup. So our timing is really important to think about the disease you're working with, the types of symptoms in the area where you produce -- you know, are doing the trials. For foliar infections like the Pseudomonas viridiflava, younger leaves are more susceptible. They're more succulent growth, less waxy, less wax has formed. And so you're going to get more take on younger leaves. But for the types of infections we typically see in the Columbia Basin, we're talking about late season neck and bulb infections are the primary type of disease we see in our area. We've talked about repeating it. So one of the really important things is coverage and placement of that inoculum. In our case, or any foliar disease, you're trying to get the inoculum on the foliage and the inoculus in the necks. You're not trying to get it on the soil, you're trying to get it on the canopy. So this is really important as you think about coverage and placement. And a lot of this has to do with your backpack sprayer configuration. So to make sure you're putting on a consistent amount of inoculum over the plot, especially if you've got lots of plots. And you don't want to be biasing some plots over another by changing how much you apply. We always walk in relation to metronomes. So Mike has a metronome, hang on your neck when you're spraying. Turn it on and it's beeping constantly and -- to make sure that you walk at a consistent pace as you're inoculating. Because that backpack sprayer is heavy, and you get tired, and it's, you know, 35 degrees Celsius, and you're, you know, it's the end of the day, you're tired. This is -- It's really important to make sure you get consistent disease pressure across the trial as to have some way to check your pace. Mike always configures the boom to make sure we can get good coverage. You can see in this bottom right-hand picture, there's a three nozzle boom. You can see the spacing of the nozzles. You can see the height at which the boom is held to ensure that you get good coverage, uniform coverage across those three nozzles. The volume that you apply it, the nozzle type, all of that is going to affect the spray pattern out of those nozzles, and the type of coverage, and the height at which you need to hold them. If you use higher pressure, you get a -- finer droplets, which can be great for creating a good mist. But if you have any breeze and very fine misting droplets, you're going to have a lot of drift And that can have a big impact on -- interplot interference as well if you've got inoculated, non-inoculated plots in close proximity. >> When you have those finer droplets too, they evaporate faster so. >> Yeah. When it's 15% humidity, we tend not to want too fine a droplet because everything just evaporates so fast. You can -- In some cases, if you've got a dense canopy, you can use a thing called droplegs. And droplegs are just, you know, extra attachments to the ends of the booms that go down, and you can attach a nozzle so it faces sideways into the plot or angled. And we've done that with crops that have a really tall canopy. Some of the seed crops we work with. A very dense, tall canopy like carrot seed crops. The canopy maybe five to six feet tall. We want good coverage. We'll actually put droplegs on the boom to enable better coverage down in the canopy where you want that product applied. Onions are not quite so critical because you can get pretty good coverage without droplegs. | New slide titled, inoculum application. Text reads, number one, timing, susceptible stages of crop growth. Foliar infections, younger leaves more susceptible. Neck or bulb infections, upright, maximum neck size, start of tops down. Repeat seven to fourteen days later. Number two, coverage and placement. Foliage and necks, not soil. Sprayer configuration. Metronome or other instrument for consistent pacing. Boom configured to cover each plot, drop legs optional. High volume, thirty to forty G P A. Nozzle type, cone, more volume. Flat fan, inoculum goes further. Pressure, higher equals finer mist but more drift, we use thirty P S I. |
| So one of the things we do to try and minimize interplot interference, especially when you've got inoculated versus non-inoculated, and we learned this from some of our earlier onion trials, we used to put our inoculated side-by-side with non-inoculated. But we noticed they were so close that any form of breeze, the inoculum would blow over. So we were getting quite a bit of disease in our non-inoculated plots. We subsequently now placed our plots end to end. So we have -- You can see here, Mike is inoculating this plot. And there's a five foot alley at the end. And the next plot would be non-inoculated. So you have an end-to-end placement of inoculated versus non-inoculated instead of side-by-side. Because you can see how narrow that gap is that Mike's walking in. So we found that tends to really show a big difference in the amount of disease in inoculated and non-inoculated plots when we do that. And just thinking too, when you -- how you set up your design, the blocking aspect. And we'll cover this in another workshop a week from today on experimental design considerations and setting up field trials. But this is something to think about in terms of uniformity of disease pressure and maximizing uniformity within blocks. It's okay to have maximum heterogeneity between blocks as long as you have uniformity within blocks. And so if you've got sloping ground, you've got a low spot in the field and you've got overhead irrigation, you're going to get wetter areas in that low spot. So we try to block so that within that low spot, you've got a full block of all your treatments. So everything in that block might get higher disease pressure because water accumulates in those low spots versus the high spots. So really thinking about that in terms of inoculation as well is very important. Any questions on applying inoculum? >> I had a question, Lindsey. >> Yeah. >> Do you have a whole -- Do you use a separate boom and canister for inoculum only? Or do you just clean things really well? >> We just clean things really well. >> It would be nice to have designated sprayers, Beth, but we don't. So we really -- You know, when you're done, we triple rinse everything and may rinse -- You know, you -- Not only do you triple rinse the spray tank, but then you also spray that water out under pressure to make sure the riser pipe inside the tank gets cleaned out. That the nozzles and the booms get -- >> Right. >> -- cleaned out. We're really, really diligent about triple rinsing and spraying very thoroughly. Because you're right, we will use the same backpack sprayer for pesticide applications so. >> Mm-hmm. Okay. >> Absolutely. >> Thank you. >> Yeah, really important comment. Thank you. | Text on screen continues. Number three, minimize inter plot interference. Buffers or alleys between plots, five feet minimum end to end. Inoculated versus non inoculated plots, adjacent versus end to end. Block design, maximum homogeneity within blocks, experimental design workshop on eighth June. |
| Yeah. So that was it as far as the sort of general overview of how we try to inoculate our field trials. And, again, there's many, many ways that you can approach this stuff. We just -- We've had very good success at creating pretty good disease pressure with this approach. Tim Waters, an Extension educator that we work with who maintains these trials and plants them and irrigates them, he's been really good at providing that sort of light overhead irrigation every other evening after inoculation, which has been -- Again, I still think is very important for helping maintain that good disease pressure in our situation. Are there any questions, or comments, feedback from anyone attending? I'll stop sharing and then we can see everyone. Does anyone have any feedback you'd like to provide or thoughts about your inoculations? Oh. Chakradhar, who's a plant pathologist here at our Research Center. He's a small fruit pathologist and potato pathologist. He says what's the average disease pressure you get with your protocols over the years? So Chakradhar, it's varied depending on the season and the environmental conditions of the season. But Beth and I were just going over some of our results from last year's 2021 trials. And when you look at our losses at harvest in bulbs and in storage, we're getting sometimes over 50%. It -- We -- It's varied anywhere from, say, the high teens to over 50% losses depending on the year and the conditions. But we don't have a problem creating pretty good disease pressure if we really take into consideration all these factors. And we've learned over the years how to improve our method. But we know we can get really good disease pressure. In our trials this year, when you look at our inoculated versus non-inoculated depending on the rep, if in the low spot in the field, we know those reps tended to have higher disease pressure than non-inoculated plots. But in the reps where you've got better drainage, less likelihood of water sitting, our disease pressure on the non-inoculated plots is 5% or less, versus, say, 50% on the inoculated plots. Does that help answer your question, Chakradhar? Thank you. And I think, you know, as we've learned about how to inoculate, it really drives home for me that when I talk to growers -- As we do everything we tell you as a grower not to do in order to create good disease pressure. And so moisture management is such a key factor for onion disease development and for many bacterial diseases. I know we have quite a few folks who work on other crops. Are there any comments you'd like to throw in for other bacterial diseases and other crops, like Jacquie or Khumbuzile? I see folks who work on, Teresa, other crops. >> Hi, Lindsey, Jacquie here. Thanks for a great talk. I appreciate it. Lindsey, I haven't done any inoculations in field because of the nature of the pathogens that I have been working with lately. >> Yeah. I think you just muted, Jacquie, but thank you. >> Sorry, I accidentally muted myself. Yeah, no. I've been working on Spongospora with most of my field work and one definitely doesn't want to inoculate the field with Spongospora so. >> Yeah. >> Yeah, no. And then previously before that, it was brown spot which is Alternaria alternata, and it wasn't necessary to inoculate so. >> Yeah. >> Yeah. Thanks for that. >> Teresa or Khumbuzile, you work on also some tree, and bush, and perennial type crops with bacterial inoculations. Any words of advice you want to provide? >> I've never done field inoculations. >> Okay. So this was helpful, Teresa. >> It was. >> Same here. I've only done glasshouse [inaudible]. >> Okay. Okay. Do you have any thoughts or comments with respect to your greenhouse inoculations that you want to provide for the group? Maybe not. Mike and I have done some bacterial inoculations in the greenhouse for various crops. I'm thinking one that we've done a lot on is Xanthomonas on carrots. It's a foliar disease, very different than this. But we do atomizing of our inoculum on upper and lower surfaces of the leaves. We actually enclose -- We don't have a dew chamber at our Research Center, so we enclose the bag -- the plants in plastic bags for 24 hours prior to inoculation. Then we open the bags, apply the inoculum, re-enclose them in the bags for 24 hours. And that's our sort of a poor man's, a poor women's dew chamber. As long as we can keep the heat off those plants when they're in those bags, they don't get overheated. It can work pretty successfully. Sometimes you don't need to do the 24 hour bagging prior to inoculation depending on the crop and the pathogen. But we found that that's a relatively inexpensive way to create a dew chamber-like environment, and it's worked very successfully for Xanthomonas on carrots. What else have we done this with? Fungal diseases, too. >> Fungal diseases. >> A lot of -- >> Yeah. >> -- our fungal leaf spot diseases, Stemphylium on spinach. Phoma on beets. >> Alternaria on cabbage. >> Alternaria on cabbage. We've found that that's an inexpensive and effective way to create -- Get those stomata -- those leaves hydrated, the stomata open with that incubation prior to inoculation. So when you apply the inoculum, this leaves them a really good susceptible state for inoculum to take. Any -- We're getting close to 10 minutes before the hour. I hope this has been somewhat of a useful discussion. I know it's very specific to onions and bacteria, but I think thinking about the principles of the disease triangle and creating that maximum disease triangle, what you can control, what you might not be able to control, it's just some very fundamental principles that you can use to try and improve the chance you get good disease take and good enough disease pressure to get -- be able to evaluate your treatments effectively. So we're happy to hang out and answer any questions. Folks, if you want to leave, that's fine. We'll stay on Zoom until people all disappear. But I don't want to keep people on longer than they need to be. Really appreciate seeing folks from overseas on this call and I hope it's been useful. >> I have another specific question for you. So whenever you're following this protocol, about how many tubes would you say that you're preparing of broth cultures for, like, a gallon of inoculum in the end? Like, do you have a rough figure like that based on your experiences? >> Well, like the example I gave, you know, that one time was about 13% of my final volume was broth culture. So I typically don't grow in tubes. I grow in flasks to achieve the volumes that I'm going to need in the field. Yeah, so. >> Yeah. We're usually inoculating anywhere from a hundred to 250 plots at a time. >> Yeah. >> So we need pretty high volumes of inoculum. And the plots are, you know, 10, 15 feet -- >> Yeah. >> -- long. So the flasks help. >> So what -- >> Yeah. >> So what I'll do a lot of times is we have a 4 liter -- 4 liter flask in the lab and I'll just sterilize that empty. But I'll grow inoculum in 500 ml Nalgene flasks. Add about -- I'll put about 200, 250 mls of broth in those, in those 500 ml flasks. Grow those. And then I pool them into the -- >> Sterilized. >> -- into the sterilized 4 liter flask. And then I'll measure the pooled inoculum on the spectrophotometer and go from there, yeah. >> Okay. So do you have a fitting on your shaker or something to, like, hold that flask down with such -- >> Yeah, we have clamps. We have flask clamps on our shaker. >> We're not -- So we're not incubating the 4 liter flask on the shaker, just the 500 ml flask. >> Yeah. >> Yeah. >> Yeah. Okay. >> And we -- Yeah, we have different size clamps that we use -- >> Yeah. >> -- for tubes versus flasks and, yeah. >> I have another shaker with just a deck, but it has -- >> Sliding. >> -- the sliding bars so you can clamp the flasks against each other. Yeah. >> Okay. >> Yeah. >> Thanks. I ask all these specifics because we, historically, have been doing who -- You quoted Carolee Bull. But we've been doing a method very similar to that in the past. But I will say, just to comment for anyone who might be doing a method like that, like, it just makes so much plastic waste with -- You have to -- To generate a volume like that that you need for the field, it uses so many Petri plates and, you know, I like this better because it kind of gets away from some of that plastic waste. >> Yeah. Yeah. >> Yeah. >> It's a pretty efficient way to produce large volumes of inoculum. You don't have to sit there and wash all these plates either. Yeah. Thanks, Jennie. Really good questions. Because you're at the working end of doing these applications. Beth, any other advice? You've done a lot of bacterial inoculations. >> You know, a lot of it is so much about getting the environment -- getting the environment, you know, conducive and favorable. So, yeah, you know, for us, the -- Yeah, the misters and doing it in the evening is essential. >> Yeah. >> So as dusk hits, and we have headlamps and stuff just in case we go a little long. >> Yeah, we have headlamps in all our vehicles. Janine Bisschoff, you had a question or comment? >> Hi, thank you for a wonderful talk. I just wanted to ask when you inoculate your broth, do you inoculate it with a loop by picking up a culture from a plate? Or do you first culture broth overnight and then pipette into new broths from there? >> That's what I've been doing the last couple of years is actually starting a seed broth culture the night before, and then using that seed broth to inoculate the larger flasks. Yeah. >> Okay. >> But -- >> Great. Thank you. >> But historically -- Historically, we have also picked colonies from plates and put them in -- >> Yeah. >> -- test tubes. You can do that, it's just, I think the -- >> I'm just -- I'm trying to maximize the growth as much as I can. So I feel like -- >> Yeah, because then they're already in a log phase. >> Yeah. >> Yeah, yeah. >> Okay. Thank you. >> Especially because of the volumes we need -- >> Yeah. >> -- it seems to help, yeah. Any other questions? These are -- It's really good to see questions because it means we're actually connecting on interest. >> I was going to -- Brenna, I think you used this inoculation method last year. Did you get -- You got good results? >> Yeah, we did at some locations. So we've got three locations, you know. So it worked at two out of the three. At my location, I don't have the ability to -- >> Irrigate, yeah. >> Yeah. >> Yeah. >> To turn on the irrigation when I wanted to. >> Yeah. And that's -- >> Because I'm working at a commercial field so. >> Yeah. And so that's important to think about that disease triangle. So Brenna's providing inoculum and has a susceptible host, but if you can't provide that moisture. And central California is very, very dry. You know, you're going to have a smaller triangle just because of that not having that quite conducive condition -- environment. Yeah. And that's where -- I don't know, Brenna, if you -- if a watering can, like Claudia was using a new type, that might be feasible on the scale of your trial to consider. >> Yeah, it would be -- It would be a little challenging, but it could be done, yeah. >> Yeah, hire some high schooler to come. >> Yeah. I don't want to do it but it could be done. >> Yeah. Okay. So Chakradhar said are these protocols available online, as I think this information will be beneficial for researchers working in other patho systems. Chakradhar, we -- our protocol that Heather sent the typed up one which is sort of a rambling comments back and forth. It's available on our alliumnet site for our team. Heather is recording this workshop and this discussion, and we can certainly make these slides available in a PDF format on alliumnet.com and the link to this recording can be posted on alliumnet.com if you think this would be valuable. But happy to share this. Thanks Chakradhar. All right, we've got four more minutes before the end of the hour. Again, I'm not forcing anyone to stay on, just we do want to be available to ask questions. These types of specific questions are great because we know that you're actually doing the work and having to think through the minutiae of producing and applying inoculum and creating disease pressure. All right. Well, if there's no questions. Heather, are we okay ending the Zoom? It was wonderful to see all these people on the Zoom meeting. >> Thank you. It was very helpful. >> I think -- I think so. I will email out the link to the recording to everyone who is on the session. >> Thank you, Heather. All right. Thank you everyone. >> Thank you. >> Bye bye. >> Bye. >> Thanks, Lindsey. >> Thank you. | New slide. An image on the left of the inside of an onion with yellow and grey swirls and oval shapes. On the right, an image of the painting called, the scream, by Edvard Munch, in which a figure is depicted with their hands at either side of their face, with wide eyes and their mouth open, in an intense state of anxiety. The two images look similar in shape and color. |