Podcasts about subtilis

Managing pests like powdery mildew, downy mildew, botrytis, and sour rot can be a complex challenge. Andy Fles, Vineyard Manager at Shady Lane Cellars in Michigan, shares insights from his USDA Sustainable Ag Research Education producer grant project. The project compares two pest management approaches: a ‘soft' pesticide program and a conventional one. Andy conducted the experiment using his on farm sprayer, providing real-world results. Despite climate variability and fluctuating pest pressures, the soft pesticide program proved effective. The project underscores the potential of using softer chemistries to manage disease while maintaining fruit quality. Resources:         REGISTER: April 25, 2025 | Fungicide Spraying: Evolving Strategies & Grower Insights 80: (Rebroadcast) The Goldilocks Principle & Powdery Mildew Management 117: Grapevine Mildew Control with UV Light 197: Managing the Sour Rot Disease Complex in Grapes 219: Intelligent Sprayers to Improve Fungicide Applications and Save Money 235: Battling Fungicide Resistance with Glove Sampling Rufus Issacson, Michigan State University Shady Lane Cellars Secures $11K National Farming Grant Timothy Miles, Michigan State University Vineyard Team Programs: Juan Nevarez Memorial Scholarship - Donate SIP Certified – Show your care for the people and planet   Sustainable Ag Expo – The premiere winegrowing event of the year Vineyard Team – Become a Member Get More Subscribe wherever you listen so you never miss an episode on the latest science and research with the Sustainable Winegrowing Podcast. Since 1994, Vineyard Team has been your resource for workshops and field demonstrations, research, and events dedicated to the stewardship of our natural resources. Learn more at www.vineyardteam.org.   Transcript [00:00:00] Beth Vukmanic: Managing pests like powdery mildew, downy mildew, botrytis and sour rot can be a complex challenge. [00:00:10] Welcome to sustainable Wine Growing with Vineyard Team, where we bring you the latest in science and research for the wine industry. I'm Beth Vukmanic executive director. [00:00:21] In today's podcast, Craig McMillan, critical resource Manager at Niner Wine Estates. With Longtime SIP Certified Vineyard and the first ever SIP certified winery speaks with Andy Fles, vineyard Manager at Shady Lane Cellars in Michigan. Andy shares insights from his USDA Sstainable Ag Research Education Producer grant project. The project compares to pest management approaches, a soft pesticide program and a conventional one. [00:00:50] Andy conducted the experiment using his on farms sprayer, providing real world results. Despite climate variability and fluctuating pest pressures, the soft pesticide program proved effective. The project underscores the potential of using softer chemistries to manage disease while maintaining fruit quality. [00:01:10] If you'd like to learn more about this topic, then we hope you can join us on April 25th, 2025 for the fungicide spraying evolving strategies in Grower Insights tailgate taking place in San Luis Obispo, California. Dr. Shunping Ding of Cal Poly will share updated results from a study on the efficacy of different fungicide programs containing bio fungicides. [00:01:34] Then we will head out into the vineyard to learn about new technologies for integrated pest management and talk with farmers from different growing regions about their program. Now let's listen in.  [00:01:49] Craig Macmillan: Our guest today is Andy Fles. He is the vineyard manager at Shady Lane Cellars in Michigan. And today we're going to talk about a pretty cool little project. He's got going looking into two different pesticide programs. Thanks for being on the podcast, Andy. [00:02:03] Andrew Fles: Yeah, my pleasure, Craig. [00:02:05] Craig Macmillan: So you have a grant from the USDA sustainable agriculture research and education program. To look at what you call a soft pesticide program for your vineyard in Michigan and comparing it to what we would call a sustainable or sustainable conventional program. What do you define as a soft pesticide program? [00:02:25] Andrew Fles: Well, that's kinda just a, a term that we applied to identify it. I didn't want to use organic because I thought that there would be a good chance we would utilize things that are considered by the industry to be very soft in terms of you know, they're not a harsh chemical or a carcinogenic, a known carcinogenic compound. [00:02:49] But something, for example, like. Like horticultural grade peroxide, which goes by several different trade names. So that's just, it's hydrogen peroxide and it is a disinfectant that turns into water and oxygen. So it's pretty Soft in terms of what it does to beneficials and, and plants and, and such. [00:03:11] We utilize some of those products already in our spray program. But combined also with, we're probably 50 percent organic in terms of what we spray out. for fungicides, pesticides, insecticides. And so we're still altering in some synthetic compounds. [00:03:28] And we wanted to compare that, what we currently do, to something that was much softer, like only soft compounds. Something that could be considered a OMRI certified organic program, or, or almost, right? Like maybe there's just one or two things that are very soft, but not technically OMRI certified. [00:03:49] Craig Macmillan: Right, and I do want to , get into the weeds on that a little bit later. Cause it's a, it's an interesting, Set a program that you've got going and I have lots of questions about them. What inspired this project? [00:04:01] Andrew Fles: I think just that continued movement towards investigating what works here in the east. You know, we, of course, get more wetting events and, and wetting periods that cause more fungal issues here compared to the west coast. And so we really, you know, we have to have an eye on sustainability. Certainly at Shady Lane, we really push for that. [00:04:25] But we also need to make sure that we have a marketable crop. We need to make sure the wine quality is, is high and acceptable for our standards. And so you know, if we're talking about, you know, every year is quite different here. We can get a, like, for example, in 2024, very wet in the first half of the year, very, very dry in the second half. [00:04:51] And, and then, which was quite different from 23 and quite different from 22 and so on and so forth. so, so some years we need to kind of step in and use a synthetic product here at this key time or, you know we need to protect our, our, our wine grape quality. [00:05:07] Craig Macmillan: What are the primary pests and diseases in your area? [00:05:11] Andrew Fles: So we have issues with the usual suspects that powdery mildew, of course. That's, that's fairly, I think if you're on top of your game, that's, it's pretty controllable. Even with soft products here it's just a spray frequency and coverage issue. [00:05:27] Downy mildew is something that can be quite challenging in certain years. [00:05:31] And there's, and there's less tools in the toolbox to use for that as well. And so you gotta, you gotta be on top of that with scouting preventative, like canopy, you know, canopy management practices that deter too dense of a canopy or, or clusters that are. hidden behind several layers of leaf. [00:05:53] Those are going to cause problems for you no matter what you're spraying, synthetic or organic, right? So, so we try and utilize all those things and and then we, we can also have issues in some years with botrytis and even sour rot and tight clustered varieties. So, so we were looking at sour rot and botrytis in the, in the cluster analysis of this portion of the , project. [00:06:18] Yeah, we have some locations can struggle with grape erinium mite. That's becoming more and more prevalent here. Wasn't an issue four years ago. Not, not really up in, up in northern Michigan anyway. So that's becoming more and more of an issue. And then we always struggle with rose chafers. It's a, it's a grub that, you know, comes out for six weeks and really terrorizes the vines. [00:06:49] And for that, for that pest, we really walk the line of the economic damage threshold, right? So, so a little, you know, we're going to see rose chafers every year. Some years are better than others. And what is our acceptable damage, you know? And so, once we see the rose chafers really getting dense in number, and also, you know, munching on a few leaves is one thing, munching on the clusters and the shoot tips is another thing. [00:07:21] Craig Macmillan: That's what I was going to ask. Yeah, I'm unfamiliar with this this pest. It, skeletonizes leaves, but it also will attack flower clusters and, and grape clusters in the early stages of development. Is that right? [00:07:34] Andrew Fles: Pretty much all green tissue. Yeah, a bunch of shoot, shoot tips leaves are probably, you know, their preferred source, I think, but anything tender. And so if, if the timing is just right where the, the inflorescences are, are you know, just coming out when the, when the beetles hatch, then they can really go for those cluster tips and, and shoot tips. [00:07:59] While we're scouting for this pest, we not only do the, you know, the density numbers and annotate that, but we look at, you know, how many are actually eating leaves versus shoot tips and clusters. [00:08:13] Craig Macmillan: Interesting, interesting. What is the design of your project and what varieties are we talking about? And what kind of variables are you measuring and how are you measuring them? [00:08:25] Andrew Fles: this is a farmer grant as opposed to a research grant. , it's tailored to folks that want to do on, on farm trials. And we want to do. Something in a significant enough volume, you know, that, that some that it would apply, it would be more applicable in the real world. [00:08:45] So for example you know, at a university they might do this randomized plots, you know, and they're using a backpack sprayer because they're, they're applying you know, three vines here, three vines there, scattered all throughout the block. And we wanted to use the sprayer that we actually use. [00:09:04] Um, and we wanted to do a bigger sections. And so what we did was we broke it up into two acre sections and we did two acres of both the traditional, the conventional program that we normally would do here and the soft treatment. So we did two acres of each in pinot noir, two acres of each in a, in a French American hybrid called ol, and then two acres of Riesling. [00:09:33] And we wanted to look at powdery, downy, botrytis, and sour rot. [00:09:38] In certain years, we can have quite a lot of botrytis and sour rot pressure in those three varieties. Because Pinot Noir of course is tight clustered. Vignole is even tighter clustered despite having that French American disease resistance package. It, it doesn't possess that for Botrytis or Sour Rot. [00:09:58] and then of course Riesling is a, is a very, it's probably the number one variety in Michigan. And as we all know, it's susceptible to Botrytis. [00:10:08] Craig Macmillan: Yeah. Big time. [00:10:10] Nice design. Great varieties to choose. I think that was really, really smart. How are you going to quantify these different variables? How are you going to measure the damage? [00:10:18] Andrew Fles: So for Powdery and Downey we just kind of did a scouting assessment. You know, how, prevalent is the infection based on how many leaves per, per per scouting event? I think off the top of my head, it was like 25 leaves. Per block that's more, I guess, anecdotal which we, and we did see that in the Pinot Noir, it was pretty clear cut that we struggled to control Downy in the soft treatment more so than in the conventional treatment. [00:10:50] It was, it was pretty clear there. And then as far as the Botrytis and Sour Rot, so that's really where the MSU team came in with the, the Rufus Isaacs lab and Dr. Rufus Isaacs and his master's PhD candidate. They did a lot of work there and, and then also the Tim Miles lab , so basically what they did is they took 25 clusters of each treatment and they did an assessment , for of course, how many berries were infected by, by botrytis and sour rot. [00:11:25] And then they also took those clusters and they hatched them out to see how many Drosophila species were there. [00:11:33] Craig Macmillan: Oh, okay. Yeah, good. That's interesting. [00:11:36] Andrew Fles: Wing drosophila here in Michigan and so really it was just the two species of traditional vinegar fly, drosophila, and then spotted wing. They did, you know, the, the statistics on that. [00:11:50] Craig Macmillan: interesting. And this is, this is a multi year project, right? [00:11:54] Andrew Fles: This was just one year. [00:11:56] Craig Macmillan: Just one year, okay. [00:11:58] And when will you have final results? [00:12:01] Andrew Fles: I have some of those already. We're going to do like a more formal presentation at a spring meeting here, a grower meeting, that's kind of co sponsored between MSU Extension and a local non profit that promotes grape and wine production in the area. So yeah, we're going to make a presentation in April on on the results and, and kind of, we're just continuing to, coalesce and, you know, tie my spray program with wedding events and then the results that they got as well. [00:12:37] Craig Macmillan: What other kinds of outreach are you doing? You're doing the meeting and you're doing other things? [00:12:41] Andrew Fles: I haven't discussed this with with Rika Bhandari as the PhD student. I suspect that she would use this in some of her publishing, you know, whether it gets published, I don't know, it's part of her Her main focus is sour rot, so this will be included in some of her presentations. [00:13:03] But I don't know that for a fact. [00:13:06] Craig Macmillan: That's exciting to get some information that's local. It's locally based and get it out to the local community as well as the broader community. I think that's really important if you don't mind I would like to get into some of the nuts and bolts of these two programs because I found that to be very interesting And then as we go talk about How that panned out for the different pests and diseases that you saw in these trials Let's talk about the soft program first You've got a dormant oil app in May and I assume you mean that there would be like JMS stylet oil or something like that [00:13:41] Andrew Fles: I think it was called bio cover. [00:13:43] Craig Macmillan: Bio cover and that's a pretty standard practice in your area I would guess [00:13:48] Andrew Fles: It is, yeah. [00:13:49] Craig Macmillan: and then the following month in June You, uh, have copper in the mix. In both the traditional and in the soft chemistry. I'm guessing that's also a common practice in your area. Probably for downy and for powdery. [00:14:06] Andrew Fles: Yeah, the copper is is something that we've been leaning towards and getting away from some of the synthetics. Which stick better to plant surfaces, we've been migrating that way anyway, these last numerous years now and so, yeah, , there are some similarities between the two programs at times it's really those key times of pre bloom and post bloom and variation that that we've traditionally. [00:14:34] Really locked in on some of the synthetic chemistries here [00:14:37] Craig Macmillan: And then also in June you have a Serenade Opti, which would be a Subtilis based material. And I believe that's also in your conventional in July. That's pretty standard practice, and that's an OMRI certified product, I believe. [00:14:52] Andrew Fles: Yes, yeah. [00:14:53] Craig Macmillan: There's some overlap there. It looks like the Rose Chaffer comes out around this time. [00:14:59] Andrew Fles: Yeah, probably it's not in front of me, but probably mid june [00:15:04] Craig Macmillan: Yeah, that's what you have here. In the traditional you've got a, a neonic, a sale. And then in the program, there's kind of a question mark here. What did you end up using in the, in the soft program for a roast chaffer? [00:15:19] Andrew Fles: Let me find it here [00:15:21] So we used neemix 4. 5 [00:15:26] Craig Macmillan: Nemix. I'm not familiar with that. Is that a Nemo based product? [00:15:28] Andrew Fles: Yeah, it's a neem oil [00:15:30] Craig Macmillan: And then in the traditional you have a neonic, a sail. Did you see a difference in Rose Chapter damage between the two? Because this is a pretty big difference here. [00:15:39] Andrew Fles: a pretty big difference in terms of [00:15:42] Craig Macmillan: Well, the modes of action obviously are very different. [00:15:45] Andrew Fles: Oh, sure, sure. Yeah, we had a little higher a little higher prevalence of rose chafers in mostly in the Pinot Noir treatments. Not so much in the Riesling, and I think that's largely because of black location. Traditionally the Pinot Noir block is our worst, one of our worst blocks in terms of rose chaffer rose chaffers are these beetles. [00:16:09] Of course, they're very similar to Japanese beetles for those listeners that, that may know that, but they really thrive in sandy soil, which is what we specialize here in northern Michigan, sandy based soil, right? [00:16:22] , and especially in un mowed fields. Right? We've really been trying to manage , our headland spaces like a prairie even more so upon joining SIP and, and learning more about making a comprehensive farm plan of, Of all of the land, right? And so we've really managed our, headlands and open fields like prairies which means minimal mowing, [00:16:47] like once a year is what we, we just mow to keep the autumn olive out. And and so we're trying to promote, you know, bird life and, and. All forms of life in these fields, which includes and sometimes an increase in rose chafers. [00:17:03] However, this 2024 was, was a. Fairly low pressure year. [00:17:09] And so I was very comfortable with, with sticking with this the soft insecticide. And we didn't feel like, you know, even though we saw this, this increase in pressure in the soft treatment, it wasn't surpassing the economic damage threshold that we are really keen. [00:17:27] And right. IPM [00:17:29] Craig Macmillan: So, true IPM. [00:17:31] Andrew Fles: IPM is very important, here, you know, where we have all these insects and it rains a lot and, you know, you got to really. Be ready to to, to scout and then react. [00:17:41] Craig Macmillan: Exactly. Yeah. And knowing what your economic injury limit is, I think it's huge. And your action threshold based on that. Tell me a little bit about the Spinosad based products. You have a couple in the soft that I assume are meant to be insecticides. [00:17:55] Andrew Fles: Yeah. The delegate. Yup. [00:17:56] Craig Macmillan: Yeah, Delegator and Trust. [00:17:59] Andrew Fles: I'll talk a little bit about intrepid as well. That's probably a foreign thing for any, any West coast listener, but that's intrepid is a it's a molting regulator and it's essentially for, in this case, for grapes, it's for grape berry moth. And this is an insect that is very difficult to do IPM on because there's a, there's kind of a morph that lives in northern Michigan that doesn't Go for the traps and so you can put traps out and it you just have no idea what's going on Because they just don't really care for the pheromones so they're really almost impossible to trap and I've talked numerous time with dr Rufus Isaacs about this and how do we you know get a handle on populations and you know They just can't get their traps to work up here. [00:18:50] We target with the intrepid, it's a, again, it's a molting regulator, so it just prevents them from developing, and it's very specific it's not a broad spectrum, so that goes on as a preventative where we have blocks near the woods, [00:19:05] because we see great berry moth coming in from wild, wild vines [00:19:10] that may or may not be in the woods, but we Where we see larva hatching is, is just kind of a perimeter. [00:19:16] So what we'll actually do is a perimeter spray. We don't even spray the whole block. We'll spray the outside row or two or three of each end. And then we just kind of blast it in. Along the, the other, you know, along the posts, the end posts. And that seems to work fairly well. [00:19:34] Craig Macmillan: Huh. [00:19:35] Andrew Fles: And then, as far as Delegate goes and Entrust those are Spinoza based products like you mentioned. [00:19:42] Those are primarily, you'll see that we put them on, well, I don't know if you can see timing, but we put them on. in September. Yeah, at the end of the season. September. [00:19:53] Yeah. Yeah. So, so those go on right around or right before even version and that is for drosophila [00:20:01] I think there's been some research recently from Cornell and then also Brock University in Canada. And I know also that Tim and Rufus have been doing trials here in Michigan as well. between the three of us out here in the, in the Northeast we're very focused on sour rot. [00:20:19] And so Michigan State along with these other folks have done these trials where they found that including an insecticide at veraison or, and then also at about 15 bricks significantly reduces sour rot infections. And that's because you're going after one of the vectors. [00:20:39] Craig Macmillan: Interesting. There's another material that I wasn't familiar with. I did a little bit of research on it. That's a product called Jet Ag, which is a hydrogen peroxide, a peracetic acid material. You have that in both the soft chemistry and your quote unquote conventional section. Is that a material you've used for a long time? [00:20:57] Andrew Fles: Yeah, we, I forget when exactly it started coming around I think probably 2015, 16 is when it was maybe released or made its way to northern Michigan and kind of coincided with with some sour rot. Issues that we have had off and on over the years with Pinot Noir or Vignole. And it's a, you know, it's a strong hydrogen peroxide. [00:21:23] It's a heavy oxidizer. It goes in and it, it, it cleans everything up. You know, it disinfects. And there's, there's some thinking as well that it, it'll kill the yeast. And some of those yeasts, the aroma is very attractive to spotted wing drosophila and regular drosophila. And so if you're, if you're kind of this is probably something that, that people, you know, that rely on native ferments might not want to hear, but you know, it really, it really disinfects the fruit which, which is key for You know, controlling sour rot. [00:21:59] And so we've used that over the years as both a preventative and a curative treatment. [00:22:05] Craig Macmillan: Right. [00:22:06] Andrew Fles: I didn't actually end up using it this year because It essentially stopped raining it was almost west coast ian here in the fall. It stopped raining in August and it didn't rain again. [00:22:19] You know, I mean, aside from like just a, you know, very, very light mist that wouldn't even penetrate the soil deeper than a centimeter. You know, so we didn't get any appreciable rain. From, I think it was maybe August 5 or 10, all the way till November 31st, or sorry, October 31st. [00:22:39] Craig Macmillan: Actually, that raises a good question. So, what is the summer precipitation like, quote unquote, in a normal year or an average year? [00:22:48] Andrew Fles: Yeah, we've been having, [00:22:49] Craig Macmillan: is it? [00:22:52] Andrew Fles: it's so variable is the, you know, we keep coming back to that. Every season is different here and it's so true even in Northern Michigan we have seen climate change affecting our summer rainfalls. So, whereas, you know, traditionally, and I say traditionally as maybe like the 80s and 90s maybe even early 2000s, you would expect to see, you know, a good four to eight inches a month. [00:23:20] you know, less, less so in, you know, in July and August is walking that more like four inch. Four inches of precipitation and you can get that sometimes in two different days [00:23:33] Craig Macmillan: Wow. [00:23:34] Andrew Fles: And that could be all or it could be spread out, you know over over several 4, 5, 6, 7, 8 different events. we had a dry June a couple years ago, I think we, I think it rained two days and amount to much. [00:23:50] And 2023, all of May we had, it rained one day. It was very dry. And so it's really been a roller coaster here in terms of what to expect as far as precipitation comes, I mean during the growing season anyway. [00:24:08] Craig Macmillan: Mm hmm. [00:24:09] Andrew Fles: It's been a challenge to know, you kind of have to have all these tools ready, right? [00:24:13] You have to have your jet ag ready. [00:24:15] If you get a bunch of infections going you got to have some of these other products ready and just , be ready for anything essentially. [00:24:24] Craig Macmillan: That, I'm just kind of reeling, I'm from California, and so like four to eight inches of rain during the growing season, it sounds like a fungal disease disaster to me. I'm impressed that you can get a crop, a vinifera crop to, to harvest with those kind of conditions. [00:24:39] Let's talk about the sustainable conventional program a little bit. Again copper appears early which would make sense. Then the insecticide portion would be a sale. It's a neonicotinoid, and then you've got a couple of fungicides in here. [00:24:55] You've got sulfur, and you've got a boscalid. Then in July again you've got a subtilis, that's serenadopty, and the, the intrepid, the IGR. August, you've got another neonic rally, and then you've got a product called ranman, or ranman. Which is a Sazofenamide, again, traditional fungicide. And then Inspire Supert, verasion, very common. And then you've got the the JetAg and Delegate, which is an antispinosid based product. When I look at this, I see a lot of very safe, very smart, very rotated fungicide chemistries here. Was this the kind of program you were using previously? [00:25:34] Andrew Fles: Yeah. And you know, it always can change a little bit. Sometimes you can't get a certain product or you can't get it in time. [00:25:42] Uh, whereas, you know, you, you're planning to use X product for your, for your kind of like You know, your, your pea sized berry spray, let's say but you, all of a sudden you have a bunch of rainfall, you know, and, and so if I was planning to use Quintech, which only covers powdery all of a sudden I have this big wedding event that was just perfect for growing downy mildew I I might switch from Quintech to and vice versa, you know, if we're, if we're into some weather, that's really favorable, it's time to push more of those serenades and you know, we've used some of the other biologicals over the years as well and, and just trying, trying to go that way as much as possible, but, you know, sometimes the weather forces your hand, like, like it did this year, you'll see in my, In my program we went into some Randman and some [00:26:35] Zampro, and those are those are very specific to to downy mildew. [00:26:41] You know, but we're still, with those products, you know, they're more expensive than something like Kaptan, you know. We Can't spray that with sip and we didn't spray it before because we don't want it on our fingers [00:26:56] The vineyard you and I don't want it in our lives So so we're always trying to go the ran man route, even though it's a little pricier, but it's very Target specific for Downey and so, you know with all the rains that we had in June and July and early July we felt like the smart play and we did start seeing some downy mildew cropping up much earlier than normal. [00:27:21] If, if we see it at all, that is. in that, at that point you want to make the call, you know, Hey, I want to get out in front of this thing. I don't want downy on my fruit. You know, if you start seeing it on growing tips, I think it was the 4th of July or the 2nd of July or something we were scouting and we were getting a lot of rain at that point and it was very humid and it was just like rain every other day for about a week there and it's like you gotta pivot and, and make the move to something that's really going to provide. control there. [00:27:52] For the soft program at that point, we were trying to use, I believe we use serenade, you know, which is more broad spectrum as far as biologicals go. We knew we wanted to keep it going after the, , the Downey with the soft chemistry. And that's why we got into the orange oil as well. [00:28:10] Craig Macmillan: Oh, interesting. [00:28:11] Andrew Fles: to, Yeah, that's, that wasn't in the proposal that I sent you, but we did pivot. I couldn't get. The cinerate it was, I was told it was on the West coast, growers were hoarding it and none of it, none of it made it over this way. I was really hoping to get my hands on some of it. [00:28:28] I've already pre ordered my 2025 cinerate. [00:28:32] Craig Macmillan: And Cinerate is a cinnamon oil based product, right? [00:28:36] Andrew Fles: Correct. Yeah. Cinnamon oil. oil. Yeah, it's another oil. [00:28:39] Yeah. Yeah. It's another one of those kind of antimicrobial oils, if you will. Um, So we pivoted to, to orange oil and thyme oil. TimeGuard has been, is a product that's been out for a number of years now. We've used it before, , we haven't really relied on it as much in the past. As, as we did with this soft treatment. [00:28:59] Craig Macmillan: Tell me a little bit more about what the outcomes have been at this point. We talked about the the pinot noir a little bit. We talked about the Rose Shafter showing up there a little bit more. At, at the end of the day, the end of the season. How did you feel about it? How did you feel about comparing the two [00:29:15] Andrew Fles: you know, it felt, it felt really good. It seemed like the soft program kept pace with the conventional for the most part. In the Pinot Noir, we had we had some more rose chaffer damage, of course, but without doing a, a full on research trial, it's hard to say that it was the treatment alone because of, as I mentioned, the location was a big factor. [00:29:38] With the downy mildew, it seemed to be a little more prevalent, certainly in the Pinot Noir on the, on the soft program that is but it never got to the point and I was, I was always ready to go in with whatever I needed to, because we don't want to have a defoliation and not being able to ripen fruit, you know, the fruit and, and especially in such a great growing year. [00:30:01] we never really resorted to. You know, breaking the glass and, and grabbing the ax and running out there and like, and it was emergency, you know, we never, we never had to do that. There was a moment there in July where, you know, where would the downy pressure we thought maybe. [00:30:19] Maybe we would have to abandon it, but then things dried up and we kept after things with with some of these, these things like thyme oil and orange oil. Getting good coverage with them is so important. But getting those on at the right time really seemed to provide enough control. [00:30:37] Craig Macmillan: Actually that's a, that's an excellent point. Let's talk about the phenology a little bit. How, for the varieties that you're growing, how big are these canopies getting? What's the spacing that they're planted on? How many gallons per acre are you using in your spray applications to get good coverage? [00:30:54] Andrew Fles: Yeah, so for the purpose of the project I stuck with 50 gallons an acre throughout the season. Which even, even for the conventional portion, traditionally I'll, I'll start with 30 gallons an acre aside from the dormant spray, of course, but like, you know, early season sprays until the canopy becomes a little denser, , I'll be at 30 gallons an acre and then probably mid July post bloom, right around bloom, perhaps , we'll ramp up the conventional to 50 gallons as well. [00:31:26] For the purposes of this, we just did 50 gallons across the board, both treatments. a lot of the canopy is well, it's really all VSP except for the vignole. Vignole is high wire cordone. And then we're talking nine by five spacing. The vinifera as well, which is pretty common around here. Double geo some spur pruning. We've really developed a kind of a hybrid system where we do a little bit of, we kind of mix cane and spur , , and alternate those in, in some of our venefera programs. [00:31:57] Craig Macmillan: And in, on the same plant? [00:31:59] Andrew Fles: Yeah. Sometimes. [00:32:01] Yeah. [00:32:01] Craig Macmillan: one side, gator the other. [00:32:03] Andrew Fles: What that does for us you know, where we get. Or we can at least, you know, and we can, sometimes we can lose a whole cane , or a lot of buds. I don't want to get too in the weeds on, on what that system is, but, but it's really developed around being able to quickly replace and adapt to cold damage. [00:32:24] And so if we need to go in and cut a trunk out, we've already got a cane growing from down low, if that makes any sense. [00:32:31] Craig Macmillan: No, that does make sense. And it's a practice that I'm familiar with from other areas in the Midwest, the North, the Northeast. Very, very smart. But that's a very different canopy architecture than you might find someplace that's all VSP. Or, you know, a double canopy situation maybe like in New York. [00:32:48] How comfortable are you now? After going through this, it sounds like you liked the softer program, you feel you got good control on most things. But if I'm understanding you correctly, you're not afraid to keep some other, other tools in the toolbox, basically. [00:33:05] Andrew Fles: Right. Yeah. And I think a big purpose of this program was to investigate some of these products. I want to highlight Problad Verde as well. [00:33:14] That's. Another one that's been out there and we've used it before as well. You know, I did a trial with Tim Miles's lab on and Rufus doing a sour rot trial in Pinot Noir in the past with pro, and it was just kind of a end of the season application of Problad with I believe we use delegate or in trust. [00:33:34] I can't remember. One of them and, this project, the SARE project was really looking at problad as being more of the backbone , of it. And, and so we ended up using that for the soft treatment pre bloom, post bloom. And then again, at version, because it has similar to jet egg, it's kind of a disinfectant, right? [00:33:57] It's this lupine seed extract that, that is a. That is a disinfectant and so it's going to go in, but because it, it's advertised anyway as having some systemic activity, [00:34:09] Craig Macmillan: Mm [00:34:10] Andrew Fles: systemic properties, that's, that's key for us in the east here. Because, hey, if we get a half inch of rain, well, it's still kind of in the leaf or it's still in some of that green flower tissue. [00:34:24] Before it opens up and blooms and so, really working problad in as instead of a kind of just end of the season toy it's really became, became the backbone of the tritus control for us in this, in this trial. And then again, looking at some of these oils, I think there's a lot of promise for. the orange oil in particular, I've, I've been seeing more and more research coming out about how you know, it does work on Downy and we did see that you know, even though we had an increase in Downy infection man, it could have been a lot worse. It was still at an acceptable level. [00:35:02] And so I think, I think I'm going to feel more and more comfortable using those products. [00:35:07] Craig Macmillan: You've demonstrated to yourself. And that's what the, that's how it works, and that's what everybody needs, to have some confidence. Which I think is really great, I was very impressed by the idea of trying things that maybe are not widely used, were not widely tested outside of maybe the West Coast, and to be able to show efficacy on your property, I think is really important. [00:35:27] I think it's one thing many of us have learned about softer materials. They may or may not work depending on what your pressure is. And that can vary region to region, but it can also vary within a region. It definitely can vary year to year, so having that flexibility that you've built into this program is very admirable. What would you say are the big picture benefits of the soft pesticide program at this point? [00:35:48] Andrew Fles: Hopefully just to increase awareness of, of how they can be effective for folks here in Michigan or, or similar climates, New York and Canada, I should say I don't think , this SARE project alone is, is going to be any sort of groundbreaking news, but I think it's just another verification and if we start to have more and more of them people will believe more and more in these products because it's just, it's at that point, it's word of mouth, right? [00:36:21] It's more and more growers are starting to back it. And, or experiment with it at least and, and see results, I think a lot of growers are very word of mouth oriented anyway. [00:36:34] So, uh, so it's very important, like, Oh, Hey, what did you try last year? And I think there's plenty of that going on in our area. [00:36:42] A bunch of us anyway, we seem to network pretty well and, and trust each other. , Oh, I use this at this key time and it really proved effective. So I think just bringing more and more awareness to these soft programs or these soft products, I should say. , and I can't really speak to the sustainability of. Farming lupin seed for for a fungicide product, you know, I can't, speak to that, but I want to believe that it's, it's a more sustainable product than, you know something that was made in a factory and, and might have petrochemicals in it. [00:37:19] Craig Macmillan: Well, it might have resistance issues as well, I think is one of the key things. And by the way, both programs I thought were very intelligent. I think like in terms of the frack rotations in the sustainable one, I thought that was really well done. Is, is there one thing that you would tell growers? [00:37:35] What's the one takeaway you would tell people from this project? You just kind of touched on one, but is there a message here for people? [00:37:43] Andrew Fles: I think the message is, you know, that we have to be really careful in crafting our. Spray program to the season that we have. If we were getting A lot more rain in September than what we ended up having I mean, we were, we were in pretty severe drought here. I think the soft program could still work. [00:38:03] But you have to choose the product and probably apply it much more frequently. You know, you have to go in and respond to those rains. , or even maybe perhaps be ready to pivot to something that is synthetic and systemic and curative. You know, maybe you have to go in with a hammer, but that doesn't mean that, you know, the majority of this growing season can't be done in a very soft way. [00:38:30] And so we're really just responding to that weather. But I think if this is our focus , to use these softer chemistries on things that we're going to drink or eat, even if it's vegetables, I think that these products are becoming better and better and there's becoming more and more of them, which is really encouraging to see you know, 10 years ago, maybe we had serenade and And you know, a couple of other products, but now, now there's, they're really becoming prevalent. [00:38:58] And so I think the take home is, is crafting that spray program with these new found tools that we have. Problads, , your crop, , your what, what should I call them? Like your aromatic oils, lack of better term, like orange oil, thyme oil, cinnamon oil. You know, I think these things do have a place. [00:39:17] Craig Macmillan: Where can people find out more about you? [00:39:19] Andrew Fles: Well, they can visit ShadyLaneCellers. com and there's stuff in there about our farm and in what we do and where we are, who we are a little bit. And then also there will be, and I could get you this information if you're interested, so this spring meeting where we're going to present the results of this believe we'll have a Zoom link option. [00:39:43] Craig Macmillan: As a reference date, this is being recorded in February of 2025. And so spring meeting will be coming up in a few months from here. I'm not sure when this will air, but even anything is fantastic. So I really want to thank you for being on the episode. Our guest today was Andy Fless, he's Vineyard Manager at Shadyland Cellars and you've been a great guest. Hey, thanks for being on the podcast. [00:40:03] Andrew Fles: My pleasure, Craig. Thanks a lot for having me. [00:40:08] Beth Vukmanic: Thank you for listening. Today's podcast was brought to you by Martinez Orchards. Martinez Orchards is one of the most trusted and respected names in the nursery business. They have earned that reputation through years of hard work, honesty, integrity, and a commitment to their customers. They provide support with their knowledgeable salespeople and highly experienced production team. They know successful plantings allow them to fulfill their promises, and they strive to build lasting relationships with their customers based on a foundation of mutual steadfast trust. [00:40:40] Make sure you check out the show notes for links to Andy at Shady Lane Plus. Sustainable wine Growing podcast episodes 117. Grapevine Mildew Control with UV Light 219 Intelligent sprayers to improve fungicide applications and save money. And 235, battling fungicide resistance with glove sampling. [00:41:03] If you liked this show, do us a big favor by sharing it with a friend, subscribing and leaving us a review. You can find all of the podcasts@vineyardteam.org slash podcast and you can reach us at podcast@vineyardteam.org. [00:41:16] Until next time, this is Sustainable Wine Growing with the Vineyard team.   Nearly perfect transcription by Descript

Le subterfuge mis en place par Rébecca a fonctionné : Jacob a obtenu la bénédiction de son père, en lieu et place d'Ésaü. Le retour de celui-ci va dévoiler la supercherie aux yeux aveugles d'Isaac, avec ses conséquences dramatiques.   NOTES ·        BIBLIOGRAPHIE | CARTES & ILLUSTRATIONS ·        Plateformes d'écoute | Réseaux Sociaux | @Contact | Infolettre | RSS  ·        Épisode enregistré en Vendée (85, France), 03/2025. Image de couverture : Isaac bénit Jacob, Gioacchino ASSERETO, v. 1640 (L'Ermitage) - source : Wikimédia Commons. Génériques : Erwan Marchand (D.R.). «Au Large Biblique» : podcast conçu et animé par François Bessonnet, bibliste. Sous Licence Creative Commons (cc BY-NC-ND 4.0 FR)Soutenez le podcast avec Tipeee  ou Ko-fi  CHAPITRES 00:00 Générique et présentation 01:20 (1) Le retour d'Ésaü 27,30-33 06:20 (2) L'affaire dévoilée 27,34-38 11:00 (3) L'oracle d'Isaac 27,39-41 15:00 (4) La colère d'Ésaü 27,42-45 21:20 (5) Conclusion 24:08 Générique de fin

Au cœur de la nuit, les auditeurs se livrent en toute liberté aux oreilles attentives et bienveillantes d'Olivier Delacroix. Pas de jugements ni de tabous, une conversation franche, mais aussi des réponses aux questions que les auditeurs se posent. Un moment d'échange et de partage propice à la confidence pour repartir le cœur plus léger.

Skurrile und witzige Meldungen aus der Wissenschaft. Mehr davon auch in unserem neuen Podcast: Fakt ab! Eine Woche Wissenschaft. Jetzt reinhören: http://swr.li/faktab

Was habe ich gespielt?FenixRes ArcanaRondominoPagan – Fate of RoanokeAndroid: Hero RealmsBoardgamearena.com: Innovation mit Cooper77 (Einzelspiel Podcast) Mit den KindernSchachFenixMonopoly JuniorZombie Teens Evolution Print and Play BasteleckeNISEI – System Gateway Starter Set (https://nisei.net/de/products/system-gateway/)ROVE – Expansion Collection / Reconfigured (https://www.pnparcade.com/search?q=rove)Skulls of Seclec – Zealots, Champions, Merchants / Crown of Bones (https://www.pnparcade.com/search?q=sedlec) Twitter – @vintersphrostYucata.de – vintersphrostBoardgamearena.com – … Folge 168 – Bacillus subtilis weiterlesen

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.28.358507v1?rss=1 Authors: Steinke, K., Mohie, O. S., Weber, T., Kovacs, A. T. Abstract: Microbes produce a plethora of secondary metabolites that although not essential for primary metabolism benefit them to survive in the environment, communicate, and influence differentiation. Biosynthetic gene clusters (BGCs) responsible for the production of these secondary metabolites are readily identifiable on the genome sequence of bacteria. Understanding the phylogeny and distribution of BGCs helps us to predict natural product synthesis ability of new isolates. Here, we examined the inter- and intraspecies patterns of absence/presence for all BGCs identified with antiSMASH 5.0 in 310 genomes from the B. subtilis group and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in distribution for both known and unknown products. Further, we analyzed variations in the BGC structure of particular families encoding for natural products such as plipastatin, fengycin, iturin, mycosubtilin and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Uniquely, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed analysis of BGCs and the remarkable phylogenetically conserved errodation of secondary metabolite biosynthetic potential in the B. subtilis group. Copy rights belong to original authors. Visit the link for more info

On today's episode, John Ratzenberger sits down with Kemin's Technical Services Manager, Dr. Tom Marsteller. They discuss a new approach to healthier animals that is not an antibiotic but is characterized as an “active microbial”. It is fed daily but has no withdrawal. Join us for this week's episode of Nutra Blend's Animal Agriculture podcast!

Fri, 15 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19073/ https://edoc.ub.uni-muenchen.de/19073/1/Hoefler_Carolin.pdf Höfler, Carolin ddc:570, ddc:500, Fakultät für Biologie

Vincent visits the laboratories of Kit and Joseph Pogliano on the campus of the University of California, San Diego, where he learns about their work on the bacterial cytoskeleton, sporulation, and the effects of antibiotics on bacterial cells. Visit microbeworld.org/mwv for complete shownotes including links mentioned in this episode.

Vincent visits the laboratories of Kit and Joseph Pogliano on the campus of the University of California, San Diego, where he learns about their work on the bacterial cytoskeleton, sporulation, and the effects of antibiotics on bacterial cells. Visit microbeworld.org/mwv for complete shownotes including links mentioned in this episode.

Vincent visits the laboratories of Kit and Joseph Pogliano on the campus of the University of California, San Diego, where he learns about their work on the bacterial cytoskeleton, sporulation, and the effects of antibiotics on bacterial cells. Visit microbeworld.org/twim for complete shownotes including the special video version of this episode. Thanks for listening and watching!

Thu, 13 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18637/ https://edoc.ub.uni-muenchen.de/18637/1/Sohmen_Daniel.pdf Sohmen, Daniel ddc:540, ddc:500, Fakultät f

All living cells are highly organized and exhibit complex cellular machineries facilitating biochemical reactions. Compartmentalization is a prerequisite to allocate an appropriate environment for these processes. In this work, compartments that are involved in Bacillus subtilis membrane organization and cell division were studied. B. subtilis division site selection is dependent on the nucleoid occlusion and the Min system. The B. subtilis Min system consists of four components. MinC is the actual inhibitor of the tubulin homologue FtsZ that is a crucial component of the divisome, forming the so called Z-ring. MinC is bound to the ATPase MinD that is tethered via the adapter protein MinJ to DivIVA. DivIVA senses membrane curvature and was supposed to be stably tethered to the cell poles. Thereby a stable, static DivIVA / MinJDC gradient with minimum concentration at midcell is formed. Using advanced microscopy techniques like single cell time lapse microscopy, fluorescence recovery after photo bleaching and by utilization of photo-activatable / convertible fluorophores we could demonstrate that DivIVA is in vegetative cells recruited from the cell pole to mature septa. These data provide first evidence that the role of the B. subtilis Min system is not to define midcell, but prevents reinitiation of Z-ring constriction after fulfilled division. Utilizing single cell time lapse microscopy we could further demonstrate that proteins crucial to condense the chromosome are vital for correct chromosome segregation during cell division by influencing the replication fork velocity or resolution. As a second compartment B. subtilis flotillin dependent membrane microdomains were studied. These domains are likely scaffolded by the membrane protein flotillin. This protein is pinned to the membrane via a hairpin loop as shown by SNAP–tag labelling experiments. Utilizing the anisotropic dye Laurdan we could further show spectroscopically and microscopically that flotillins prevent condensation of microdomains. Flotillin deletion strains also exhibit a generally more liquid ordered membrane compared to wild type cells. Using co–immunoprecipitation experiments several proteins interacting with flotillin were identified. These interactions were confirmed with microscopical co–localization analysis. B. subtilis flotillin was additionally heterologously purified via affinity chromatography. The purified protein creates large homo–oligomers likely in mega Dalton size. Using truncation mutants it could be shown that flotillin oligomerizes via a flotillin specific domain, namely the PHB domain. Though, contrary to eukaryotic cells, B. subtilis PHB domain does not contribute to lipid binding. However, several cellular machineries that interact with flotillins, as exemplary shown for the secretion machinery, are impaired in their functionality in absence of flotillins. These data provide first evidence that prokaryotic flotillins are elements that scaffold the plasma membrane and thereby provide a lipid environment that is vital for correct functionality of diverse cellular machineries.

Many problems caused by bacterial biofilms can be traced back to their high resilience towards chemical perturbations and their extraordinary sturdiness towards mechanical forces. However, the molecular mechanisms that link the mechanical properties of a biofilm with the ability of bacteria to survive in different chemical environments remain enigmatic. Here, we study the erosion stability of Bacillus subtilis (B. subtilis) biofilms in the presence of different chemical environments. We find that these biofilms can utilize the absorption of certain metal ions such as Cu2+, Zn2+, Fe2+, Fe3+ and Al3+ into the biofilm matrix to avoid erosion by shear forces. Interestingly, many of these metal ions are toxic for planktonic B. subtilis bacteria. However, their toxic activity is suppressed when the ions are absorbed into the biofilm matrix. Our experiments clearly demonstrate that the biofilm matrix has to fulfill a dual function, i.e. regulating both the mechanical properties of the biofilm and providing a selective barrier towards toxic chemicals.

Vincent, Elio, Michael, and Michelle discuss a symbiosis between a bacterium and fungus that increases the virulence of oral biofilms, and the assembly of amyloid fibers, which are needed for biofilm formation.  

Soil bacteria are exposed to constant changes in temperature, moisture, and oxygen content. Additionally, they have to encounter different antimicrobial substances, which are produced by competing bacteria. Those agents often target the bacterial cell envelope, which is an essential structure composed of the cell wall and cell membrane. In order to counteract such life-threatening conditions, bacteria developed signal transducing systems to monitor their environment and to respond signal-specifically to any stress conditions, mostly by differential gene expression. Different principles of signal transducing systems have been evolved: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic function (ECF) sigma factors. Bacillus subtilis is a soil bacterium, which counteracts cell envelope stress by four different 2CSs (LiaSR, BceRS, PsdRS, and YxdJK) and at least three different ECF sigma factors (σX, σM, and σW). In the course of the present thesis, the LiaSR 2CS was investigated in detail. The LiaSR 2CS of B. subtilis is a cell envelope stress-sensing system that shows a high dynamic range of induction in response to cell wall antibiotics like bacitracin. It provides no resistance against its inducer molecules. Rather, it is a damage-sensing system that maintains the cell envelope integrity under stress conditions. The membrane-anchored histidine kinase (HK) LiaS and its cognate response regulator (RR) LiaR work together with a third protein, LiaF, which was identified as the inhibitor of the 2CS. Upon induction, the target promoter PliaI is induced by phosphorylated LiaR, leading to the expression of the liaIH-liaGFSR locus, with liaIH as being the most induced genes. In the first part of this thesis, the mechanisms of stimulus perception and signal transduction of the LiaFSR system were analyzed. Therefore, the native stoichiometry of the proteins LiaF, LiaS, and LiaR were determined genetically and biochemically with a resulting ratio of 18 to 4 to 1. We found out that maintaining this specific stoichiometry is crucial for the functionality of the LiaFSR system and thus a proper response to cell envelope stress. Changing the relative protein ratios by the overproduction of either LiaS or LiaR leads to a constitutive activation of the promoter PliaI. These data suggest a non-robust behavior of the LiaFSR system regarding perturbations of its stoichiometry, which stands in contrast to quantitative analyses of other well-known 2CSs. Furthermore, a HK-independent phosphorylation of the RR LiaR was observed. This happened in each case if the amount of LiaR exceeded those of LiaS, irrespective of the presence or absence of a stimulus. By using growth media supplied with different carbon sources, acetyl phosphate was identified as being the phosphoryl group-donor for LiaR under these conditions. Moreover, by performing a mutagenesis experiment, we obtained genetic evidence that LiaS is a bifunctional HK offering both a kinase and a phosphatase activity. In the second part of this thesis, the liaI promoter was used to generate a protein expression toolbox for the use in B. subtilis, referred to as the LIKE (from the German “Lia-kontrollierte Expression”) system. PliaI is a perfect candidate for driving recombinant protein expression. It is tightly regulated under non-inducing conditions showing no significant basal expression levels. Depending on the inducer molecule concentration, it is induced up to 1000-fold reaching a maximum already 30 minutes after addition of the inducer. Two expression vectors, an integrative and a replicative one, were constructed consisting of an alternative liaI promoter, which was optimized to enhance promoter strength. Additionally, different B. subtilis expression hosts were generated that possess liaIH deletions to prevent undesired protein production. The feasibility of the LIKE system was evaluated by using gfp and ydfG as reporter genes and bacitracin as inducer molecule. As a result, both proteins were successfully overproduced.

The peptidoglycan cell wall (CW) and the actin-like MreB cytoskeleton are the majordeterminants of cell morphology in non-spherical bacteria. Bacillus subtilis is a rod-shaped Grampositive bacterium that has three MreB isoforms: MreB, Mbl (MreB–like) and MreBH (MreBHomologue). Over the last decade, all three proteins were reported to localize in dynamic filamentous helical structures running the length of the cells underneath the membrane. This helical pattern led to a model where the extended MreB structures act as scaffolds to position CW-synthesizing machineries along sidewalls. However, the dynamic relationship between the MreB cytoskeleton and CW elongation complexes remained to be elucidated. Here we describe the characterization of the dynamics of the three MreB isoforms, CW synthesis and elongation complexes in live Bacillus subtilis cells at high spatial and temporal resolution. Using total internal reflection fluorescence microscopy (TIRFM) we found that MreB, Mbl and MreBH actually do not assemble into an extended helical structure but instead into discrete patches that move processively along peripheral tracks perpendicular to the long axis of the cell. We found similar patch localization and dynamics for several morphogenetic factors and CW-synthesizing enzymes including MreD, MreC, RodA, PbpH and PBP2a. Furthermore, using fluorescent recovery after photobleaching (FRAP), we showed that treadmilling of MreB filaments does not drive patch motility, as expected from the structural homology to actin. Blocking CW synthesis with antibiotics that target different steps of the peptidoglycan biosynthetic pathway stopped MreB patches motion, suggesting that CW synthesis is the driving force of patch motility. On the basis of these findings, we proposed a new model for MreB fuction in which MreB polymers restrict and orient patch motility to ensure controlled lateral CW expansion, thereby maintaining cell shape. To further investigate the molecular mechanism underlying MreB action, we next performed a site-directed mutagenesis analysis. Alanine substitutions of three charged amino 2 acids of MreB generated a B. subtilis strain with cell shape and growth defects. TIRFM analysis revealed that the mutated MreB protein displayed wild-type localization and dynamics, suggesting that it is still associated to the CW elongation machinery but might be defective in an interaction important for MreB morphogenetic function. Thus, this mutant appears as as a good candidate to start characterizing the interactions between the three MreB isoforms and components involved in CW elongation. It might also help to understand the function of components of theCWsynthetic complexes, and how they are coordinated to achieve efficient CW synthesis. Finally, to investigate how the integrity of the CW is maintained, we studied the localization and dynamics of the LiaIH-system, which i s t he t arget o f L iaRS, a t wo-component system involved in cell envelope stress response. We found that under stress conditions, when liaI and LiaH genes are expressed, the proteins form static complexes that coat the cell membrane. LiaI is required for the even distribution of the LiaH in the membrane. Taken together, these data suggest that LiaIH complexes may protect the cell from CW damage. Taken together, the findings described in this thesis provide valuable insights into the understanding of CW synthesis in B. subtilis, which may open new perspectives for the design of novel antimicrobial agents.

Background: Standardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for Escherichia coli, such tools are still missing for the Gram-positive model organism Bacillus subtilis. The goal of this study was to develop and thoroughly evaluate such a genetic toolbox. Results: We developed five BioBrick-compatible integrative B. subtilis vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the lacZ and luxABCDE cassettes, were BioBrick-adjusted, to enable beta-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the Bacillus BioBrick Box also provides five widely used epitope tags (FLAG, His(10), cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice. Conclusion: Our genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with B. subtilis. We believe that our well-documented and carefully evaluated Bacillus BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other BioBrick-based project in B. subtilis.

Vincent, Michael, and Elio meet up with Jonathan Dworkin to discuss how bacteria form spores and how they return to vegetative growth.

Für das Überleben von Bacillus subtilis ist eine verlässliche Überwachung der Integrität der Zellhülle essentiell, um diese zu schützen und bei Schäden adäquat zu reagieren. Neben den ECF � Faktoren spielen Zwei-Komponenten-Systeme (2KS) in der Zellhüllstressantwort von B. subtilis eine zentrale Rolle. Eines dieser Systeme, das LiaRS- 2KS reagiert auf eine große Anzahl verschiedener Zellwand-Antibiotika sowie andere zellhüllstress-auslösende Substanzen. Die zelluläre Funktion und Rolle des Lia-Systems konnte bisher nicht genau definiert werden. In der hier vorliegenden Dissertation wurde das Lia-System erstmals hinsichtlich seiner funktionalen Rolle in B. subtilis untersucht. Im ersten Teil der Ergebnisse wurde eine detaillierte Analyse der LiaR-vermittelten Zellhüllstressantwort in B. subtilisvorgenommen. Transkriptom-Studien dienten zur Identifizierung des LiaR-Regulons. Hierbei wurde die Genexpression des Wildtyps mit zwei Mutanten, die den „ON“ (�liaF) und „OFF“ (�liaR) Zustand des Lia-Systems repräsentierten, verglichen. Von den dabei identifizierten drei potentiellen LiaR-Zielloci (liaIH, yhcYZ-ydhA, ydhE) konnten durch anschließende Folgeuntersuchungen nur die Gene liaI und liaH als in vivo relevante Zielgene für LiaR verifiziert werden. Umfangreiche phänotypische Analysen zeigten, dass �liaIH-Mutanten nur schwach sensitiv auf einige Antibiotika sowie oxidativen Stress reagierten. Ebenso vermittelt eine Überexpression von LiaH in einer �liaF-Mutante keine Resistenz gegenüber stressauslösenden Substanzen. LiaH gehört zur Familie der Phagenschock-Proteine. Weitere Mitglieder dieser Familie sind PspA aus Escherichia coli und Vipp1 aus Arabidopsis thaliana, die große oligomere Ringstrukturen bilden. Die strukturelle Untersuchung von LiaH ergab, dass auch dieses Protein große Ringe bildet (>1MDa). Der zweite Ergebnisteil befasst sich mit der Untersuchung der Stimuluswahrnehmung der Zellhüllstress-detektierenden Systeme in B. subtilis. Die Zellhüllstressantwort auf das Antibiotikum Bacitracin wurde hierbei mittels �-Galaktosidase-Assay sowie Western Blot- Analyse erforscht. Das Bce-System reagiert dabei am stärksten und spezifischsten auf Bacitracin-Stress. Es wurde ebenfalls festgestellt, dass der ABC-Transporter BceAB essentiell für die Stimuluswahrnehmung ist und dass das Bce-System an sich eine Resistenzdeterminante in B. subtilis darstellt. Das Lia-System hingegen wird erst bei höheren Bacitracin-Konzentrationen induziert. Zusammengefasst deuten diese Ergebnisse darauf hin, dass das Bce-System Bacitracin direkt wahrnimmt (drug sensing) und das LiaSystem in indirekter Weise auf Zellhüllstress ausgelöst durch Bacitracin reagiert (damage sensing). Im dritten Teil der Ergebnisse wurdendie zelluläre Lokalisation von LiaI, LiaH und LiaG sowie die Beziehung der Proteine untereinander mittels Fluoreszenz-Mikroskopie und biochemische Ansätze untersucht. Die Membranproteine LiaI und LiaG sind unter Stressbedingungen in der Zellmembran lokalisiert. LiaH, ein cytoplasmatisches Protein verändert unter Stressbedingungen seine Lokalisation vom Cytoplasma an die Membran. Die Funktion von LiaH scheint sich also an der Zellmembran zu vollziehen, wobei LiaI als Interaktionspartner identifiziert wurde. Da in einer �liaI-Mutante LiaH unter Stressbedingungenebenfallsnoch an die Zellmembran assoziert ist, wurde nach weiteren Interaktionspartnern von LiaH gesucht. Eine umfangreiche bacterial-two-hybrid-Analyse ergab, dass sowohl LiaH als auch LiaI und LiaG in ein Interaktionsnetzwerk eingebettet sind, in welchem das bisher uncharakterisierte Protein YvlB eine Schlüsselrolle spielt.Die ebenso in dieses Netzwerk involvierten Proteine YjoB, DnaK und HtpG üben als Proteasen/Chaperone Funktionen in der Faltung und Degradierung von Proteinen aus. Ein Zusammenspiel des Lia-Systems und des Schlüsselproteins YvlB mit den Proteasen/Chaperonen als Reaktion auf Zellhüllstress ist denkbar. Die Phagenschock-Homologe PspA in Streptomyces lividans und E. coli üben einen erheblichen Einfluss auf die Proteinsekretion sowie die elektronenmotorische Kraft der Zelle aus. Daher wurde im letzten Teil der Ergebnisse die Rolle von LiaH in der Proteinsekretion sowie im Energiestoffwechsel näher analysiert. Ein Einfluß des Lia- Systems in der Aufrechterhaltung der elektronenmotorischen Kraft der Zelle konnte nicht bestätigt werden. Durch die Analyse des Sekretoms in B. subtilis konnte gezeigt werden, dass das extrazelluläre Proteom einer �PliaI-liaIH-Mutante im Vergleich zum Wildtyp signifikante Veränderungen in der Komposition aufwies.So wurde im Sekretom der �PliaIliaIH- Mutante vor allem das Zellwand-assoziierte Protein WapAidentifiziert, welches im Wildtyp oder in einer �liaF-Mutante nicht auftrat. Das Lia-System beeinflußt somit auch die Proteinsekretion von B. subtilis, wobei die molekularen Mechanismen noch unbekannt sind.

Background: Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (P-liaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results: Based on these traits of P-liaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the "LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the P-liaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the P-liaI promoter. Conclusions: The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of P-liaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories.

Roberto Kolter is a professor of Microbiology andMolecular Genetics at Harvard’s Medical School.  Dr. Kolter’s research interests are broad, but he says his eclectic program boils down to an interest in the ecology and evolution of microbes, bacteria in particular, and on how these forces operate at the molecular level. Although he’s worked in a number of different systems, lately Dr. Kolter is spending a lot of time with Bacillus subtilis, a modest little bacterium that doesn’t get the headlines of a wicked pathogen like Salmonella or a useful industrial workhorse like yeast.  What it lacks in notoriety,  B. subtilis makes up for in usefulness.  According to Dr. Kolter, B. subtilis is an important source of industrial enzymes (as in laundry detergent) and, as a bacterial model, a prolific source of information on how some bacteria make spores and other diverse cell types.  This ability to form different kinds of cells is intriguing to Dr. Kolter: B. subtilis cells can wear any of a number of different hats, depending on what is needed at any given moment.  From spores to swimming cells to cells that wage chemical warfare on their neighbors, B. subtilis can do it all.  Dr. Kolter and his colleagues are looking at the how and the why of this multiplicity. In the interview, Dr. Merry Buckley talks with Dr. Kolter about what he finds interesting about B. subtilis, why we should start thinking about bacteria as multicellular organisms, and how he got involved in producing a book of poetry (poetry about microbes, that is). Dr. Kolter also provided the photography for the book Germ Stories by Arthur Kornberg. To see a full description and pricing details, click the ASM estore and pick up your copy today.