Okay, good afternoon everyone. I think we'll get started. Our first talk of the afternoon session is managing Sir Cosper Leaf Spot by missing cultivars. Authors are Joachim and Dr. Mohamed Khan. Well good afternoon everyone. I hope you had a good lunch and thank you very much for being here today and listening to the results of all the research that's being conducted despite everything that's going on in the world. Hopefully this year things will get much better and we can meet again in person and at meetings and in person plot tours and other events. So here's hoping that 2021 is a much better year for all of us. Anyways, my name is Peter Huck. I'm Dr. Mohamed Khan's technician here at North Dakota State University in the Plant Pathology Department. And I want to talk about a trial that we've done for a few years now looking at controlling Sir Cosper Leaf Spot in sugar beets. So we've obviously gone over this before but just a quick overview but Sir Cosper Leaf Spot in sugar beet is an extremely damaging disease and is the most destructive foliar disease on sugar beets in both North Dakota and Minnesota and it's a major problem in all growing districts but especially the further south that you go. It's a fungal disease and infects the leaves of sugar beets typically starting when the rows close around the first of July give or take a week or two. It thrives on days and nights with higher humidity and rainfall. It kills the leaves and can possibly cause the roots to have more impurities when the time comes for harvest and processing in addition to causing a lower sucrose concentration. We know there are several ways to control this disease including fungicide applications, cultural methods such as longer crop rotation and trying to keep beets away from a field planted with beets the previous year and using tolerant varieties. And so thinking about tolerant varieties was the basis for this trial. A few years ago we started a trial looking at the possibilities of mixing seed varieties to help control diseases in this particular sense Sercospora leaves spot in combination with fungicide use and to see if there's a certain percentage of mixtures that leads to better disease control. So variety selection is a method that's been used to control diseases in the past over many different crops maybe even all the crops. However solely relying on one genetic variation can be a risk. In the 1960s a single genetic strain of corn became more and more widely adopted and by 1970 85 percent of the corn planted in the United States and Canada had the same genetic background. Southern corn leaf blight was not a new disease at that time it had been observed since the 1920s but there had been increasing reports and scattered areas around the corn belts in the late 1960s and especially starting in 1969 of larger outbreaks of the disease due to a mutation. Starting in early spring of 1970 reports came early and came often from southern states of serious leaf damage to corn helped along because of an unusually wet spring. Well by mid-July the disease had stretched up the Mississippi Valley all the way to far southern Minnesota and into Wisconsin and when the year was done an estimated 15 percent of the entire corn crop in the United States and Canada were destroyed and some individual growers especially in the south lost 100 percent of their corn on their farms and in the aftermath of this outbreak geneticists decided that the industry couldn't solely rely on one single genetic line as the only means to control a disease. So in the years and the decades since then a more balanced approach has occurred in crop production that looks at multiple ways of controlling disease and raising a good crop and varieties and genetic variation are definitely part of that. Researchers at the Agriculture and Agri-Food Canada Research Centre Ontario Canada a few years ago said that they were looking at possible ways to mix corn hybrids in a hopper and not just planting different strips of varieties in a field. They said there's a potential in mixing varieties to allow for a certain variety to avoid insect damage, disease development, lack of moisture or excess moisture and allow a field to succeed if one variety is susceptible to those outside influences. However they also noted that this is not about possible drawbacks. Different varieties would have different moisture contents that would complicate harvest and pollination could take longer if varieties are mixed together also delaying harvest among other issues that they brought up. So it's something that is still being looked at. However mixing varieties is something that is sometimes used by wheat growers. A 2001 survey in Kansas found that winter wheat blends were the fourth most popular variety planted by growers there. However later studied by the University of Minnesota tested whether several blends had a yield advantage and they found no increase in yield in protein but it is something that's done by some growers mixing different varieties that is. So we looked at mixing different varieties of sugar beets with varying levels of cls tolerance at different percentages. However like what I mentioned earlier there are drawbacks to using two varieties at one time like what we mentioned with corn. There are different times to maturity so the canopy will develop at different times with different varieties and also different varieties have different amounts of the beet crown exposed above the soil line. So at harvest time defoliation will be difficult because the topper might be set perfectly for one variety but you might be severely damaging the other variety or vice versa you might not be taking enough of the green material off. However we wanted to give it a try so we picked four different varieties from two companies and mixed the ones from similar companies at rates of 25 75 50 50 75 25 and then each variety separately and then applied standard fungicides to one set and then left the other set untreated. We started this trial back in 2017 but the first two years it didn't really turn out for us. In 2017 we planted it our Hickson research site and despite inoculating it the same way that we inoculate a regular boxhomes or prosper leaf spot site we just didn't get good disease development that year. CLS didn't really set in until the very end and so we ended up seeing more of a variety effect than testing for CERCOSPR leaf spot effects on those varieties. So the next year we also tried to plant it at the Hickson site and we were starting to get some good disease growth but then in mid-August this happened.  It honestly looked kind of like a bad defoliation job and there was so much hail that there are actually still some hailstones left there the next morning so that year was obviously lost again. You have to do a study looking at leaf disease when you don't have much for leaves left late in the year like that. So again like in 2017 we just couldn't use this data because we ended up testing more of the variety than the effective CERCOSPR leaf spot on those varieties. So starting in 2019 we planted it at our Foxhill Minnesota CLS site and here is some information from that year. So last or I guess now two summers ago we planted it on May 17th. We harvested the plots on September 24th and we inoculated them on July 12th. We applied four rounds of fungicide sprays on July 22nd on August 1st, August 15th and then again on August 29th. For the first set of varieties the CLS ratings were 5.2 and 4.2 and the second set of CLS ratings were 5.6 and 4.4. So in 2019 we had good but not overwhelming CLS pressure and here are the results for the plots that did not have fungicides applied and you can see that the 100% resistant variety really stood out. In the top set you can see that the 100% resistant variety was better than all other treatments in the CLS ratings and statistically better than all of the treatments in the sugar percentage and sucrose per ton columns and it was the same story on the bottom half of the chart in variety set number two except that the CLS ratings were more even but by the time you move over to the sugar percentage column and the sucrose per ton column the 100% resistant variety really stood out above all the other treatments. So when you move over to the fungicide treatments you can see that there was only one treatment that was significantly different from the 100% susceptible variety in the top set when it comes to CLS ratings and that was the 100% resistant variety. When you look at the yield column there were significant differences but they were with the susceptible varieties being better and that's because since we control the disease so well with fungicides and essentially we essentially took the disease out of the equation and the susceptible variety was quite a bit better absent outside influences. When it comes to sugar percentage the resistant variety and blends at at least 50% were the best and then you can carry that over the sucrose per ton column where the two worst performing treatments were the susceptible variety and the 75% susceptible variety. In the second set there were less significant differences overall but it was the same pattern in the sugar percentage in the sucrose per ton columns with the susceptible variety and the 75% blend being significantly less than resistant variety and the 75% resistant variety. So in 2020 we did the trial again at the Fox home sites we planted the plots on May 4th we harvested them on September 28th and this year we inoculated the plots on July 6th. The plots were fungicides were applied to the plots on July 22nd, August 3rd, August 13th and August 26th and the first set of CLS ratings were 5.2 and 4.1 and the second set of CLS ratings were 5.0 and 4.7 and in 2020 we had overwhelming CLS pressure. In late June, July and early August it seemed like it was just always raining at our Fox home Minnesota plots and we had standing water and at least parts of our plot area for the entire month of July so controlling CLS was a challenge this past summer. In the non fungicide treatments as you can see here you can really see the effects of the high CLS pressure year that we dealt with where all the ratings in both sets of varieties were at or above 8.5 and then when you get to the sucrose percentage and sucrose per acre and sucrose per ton columns the treatments under 50% resistance were the lowest and were significantly different from the treatments with more tolerant varieties in both sets. So looking at the data for the treatments that received fungicides there were significant differences in the top set of data between almost all of the treatments and there was a gradually descending trend as you go from the 100% susceptible to the 100% tolerant variety. Following the trend in 2019 the 100% susceptible and 75% susceptible were significantly different from the resistant variety in the sugar percentage column but this year the 50-50 blend was also different and also in the sucrose per ton column if you look at the sucrose per acre column there's a pretty sharp cutoff between the 75% susceptible variety and the 50-50 treatment. In the second set of treatments we see the same trend in the CLS ratings where they gradually decrease from the susceptible to the resistant variety and we also see the same trend in the sucrose columns and the sucrose per acre columns and the sucrose per ton column where there's a noticeable drop off when you get under the 50-50 blend. And so here's some pictures that we took of the plots in mid-August. First of the plots without fungicides you can see the progression from susceptible to tolerant varieties especially in that top set. Circosper just really did a number on really both of the varieties and the mixtures in between. The bottom set did a little bit better but it was as we mentioned earlier very heavy year for circospera. And now here's the plots with fungicides and you can see the same thing just obviously better control since we were actually using the fungicides. So what does all this mean? So we've looked at a lot of the numbers and pictures and the takeaways that as you increase the percentage of seed that is tolerant of circospera leaf spot the yield statistics do go up if there's disease presence. It shows that fungicides are very helpful and that tolerance to CLS is also needed under severe conditions and that's somewhere around the 50% mixture is where you can start to see an increase in yield over the 100% susceptible variety. The varieties that were the most resistant perform the best especially in 2020 because of the rain that made fungicide applications difficult. So we need fungicides to control this disease but we also need genetics as a backstop when rainfall is high like in 2020. So we'll look at this for another year to see if what happened in 2019 and 2020 is confirmed. And this trial is just being performed for academic reasons and not to make a recommendation for growers to mix seed. It's simply to answer a question that we had. And so with that I want to say thank you to all the sugar beet seed companies that have helped us over the years giving a seed not just for these trials but for all the trials that we've done. Same thing for the chemical companies that have given us products to use. We're really really thankful for both of those. And also to Kevin Etzler our Fox home cooperator, Vince Alston, Terry Compton up at Hickson and then the whole NDSU and U of M Sugar B team for helping with everything from planting to spraying to harvest. So thank you very much and thank you for listening. Hope you have a good rest of the day. Hello Peter. This is Kimberly Webb. The question I have is one of the benefits of using mixtures is not just for protecting against the fungicide resistance of Sercospora but also maintaining the resistance that you're using to protect as well within the sugar beet plant. Were you using only a single source of resistant line or did you have multiple are you going to look at multiple resistances and using those as a mixture instead of a susceptible versus resistant line? For this so far we've just used yeah no we have not looked at different basis of resistance. We've just been taking market varieties and looking at those but that's definitely a good question to think about for future runs of this trial. So thank you. Okay thank you very much Peter. All right thank you. All right our next presenter is Mr. Co-author with Dr. Kahn and it is the role of adjuvants with fungicides under stimulated rainfall at controlling Sercospora particular in sugar beet. Thank you. So welcome and good afternoon everyone. My name is M.D. Z. Rahman Buya. Today I am going to talk about rule of adjuvants with fungicide under simulated rainfall for controlling Sercospora particular in sugar beet. Sugar beet is an important sugar elinthropy in United States here in North Dakota and Minnesota are two leading sugar beet producing states in United States. Here American Crystal Sugar Company mean that farmers cooperated in southern Minnesota beet sugar cooperative combinedly contributed approximately 57% of total US sugar production which is corresponding to five billion dollar economy. But sugar beet production is highly influenced my high production is highly influenced by Sercospora boricola Sercospora leaf spot disease which is a major foliar destructive disease caused by Sercospora boricola is a hemibiotropic polycyclic fungus. It has significant impact on sugar beet productions that it causes illus up to 40% or even more in severe condition reduce sugar quality and causes huge monetary loss as well as reduce its stability in the piles. But sugar beet for better management of sugar beet Sercospora leaf spot currently growers are highly recommended to use tolerant variety if available tillage operation crop rotation with non-host crop for two to three years and as well as timely spraying of fungicide for better control. But application of fungicide and efficacy of fungicide losses due to many reasons especially environmental factor like wind, heat, solar radiation, rain and irrigation. Among them rain is an important factor that immediate after spraying fungicide causes fungicide losses be due to washing off redistribution deposition and residual activity of fungicide. And sometimes after spraying of fungicide if rainfall happens that fungicide takes away to the near runoff to the near water wise that causes serious environmental pollutions too. Last year and this year the rainfall pattern in foxone that indicating that mostly in the green season there was less rain compared to this year in 2020 and at least 41% precipitation chance daily precipitation chance was in foxone in this year. So adjuvants is a product adjuvants kind of product that other than fungicide formulation fungicide that added in spraying improve the efficacy of fungicide. When the spray solution without adjuvants added because of higher surface tension it has lower wetting capacity but when added with adjuvants it improves the it improves the reduce the surface tension and improves the better coverage on the spray surface. So we set the objectives to study the effect of simulated rainfall for evaluating the effectiveness of fungicide and adjuvants for controlling sarcosperl hip spot. The research was conducted both at AES and ESC greenhouse and failed at foxone Minnesota. We use such a variety sugar beet variety which is highly susceptible to sarcospera beticola. So we use different amount of rainfall 0.1 inch quarter inch 0.5 inch and one inch rainfall and then rainfall was simulated artificially using a spray book chamber at NDSC greenhouse at one hours after fungicide spray one days three days and five days. Evaluation we evaluate the sarcospera CLS severity using one to 10 scale after 14 days post-inuculation of sarcospera beticola. We use three different type of fungicide from AESL group we use we use Inspire XT from EVDC group we use Bencogep and from copper fungicide we used HSC. We use two different kinds of adjuvants complex and transfix complex especially known for its penetrator penetrating and transmitting activity. Transfix is mostly a spreader or a sticker that has waterproofing protection from the rain. So in the greenhouse we spray fungicide or fungicide adjuvant mixture followed by rainfall simulation different amount of different time and then after that we inocular sarcospera beticola and incubated in humidity chamber for four days and then transferred the plan to the greenhouse then after 14 days post-inuculation we evaluate the disease severity. For spraying fungicide in adjuvant mixture we use a special nozzle that's star between jet's TT 660 160 100 degree that has two 110 degree flat fan is sprayed that can move 60 degree back and forth that provide wider spray depth compared to conventional flat fan nozzle that and as well as reduce the treat with desired size of droplets on the spray surface. We also use water sensitive spot card ensure how the droplets distributions indicating the best coverage and uniform distribution using carbon to inject. That with fungicide droplets seems uniform globular on the surface but when added with adjuvants indicate the spray droplets wider and become large and ensure excellent coverage to the leaf surface. When we use inspire and after the different amount of rainfall simulations one hour after fungicide spray one day 3D and 5D and then after and then when you edit complex and adjuvants with inspire we see all the treatment treatment one is treated check and treatment 18 is non-treated inoculated checks immediate after spraying fungicide that how about the amount of rainfall simulated it reduce the effectiveness of fungicide but for all up to up to one days one hour and one days and five inch one inch rainfall that reduce the effectiveness of fungicide increase high disease severity but over time when when we spread rainfall did rainfall simulation at three days and five days that reduce the disease severity that's little improved the efficacy but all the treatments they are not significantly different but they are significantly different from the treated check when added complex that improves the efficacy of fungicide activity and initially when at one hour after fungicide spray when we added the amount of rainfall at one inch quarter inch half inch and one inch that decrease the effectiveness of fungicide but over time when we spray fungicide that show more or less similar than except one inch rainfall show the differences likewise for PENCOJ brain fall and PENCOJ plus complex from that two combination PENCOJ and PENCOJ plus complex improved efficacy of fungicide and showed lower disease severity only at one inch rainfall and five inch rainfall initially at one hour showed that this higher disease severity that indicates fungicide lose its efficacy controlling sarcos proliferate over time one days three days and five days showed more or less similar turn that increasing the amount of rainfall decreasing the increasing the disease severity still at one days only at one inch rainfall to reduce the efficacy of fungicide improving using complex that improve efficacy of fungicide it indicates only one inch and one inch and five inch rainfall didn't show that much effect complex that treated checks what and five inch and one one inch rainfall show a big difference compared to and one inch and quarter inch rainfall when we added complex and rainfall uh then badge SC from copper fungicide when we added rainfall and added with complex shows about the amount of rainfall added after fun fungicide is sprayed it shows efficacy of fungicide that reduce that shows higher disease severity over time of one inch and point five two five inch rainfall show more or less similar effect for the rest of the time one day three day and five and five inch and one one inch rainfall show the similar trend of uh the severity and effectiveness of fungicide of when added complex with badge fungicide that shows uh improve efficacy of fungicide little bit but still higher amount of on this uh rainfall simulation like point five inch and one inch show a big difference at one after one from one days three days and five days only one inch rainfall show the differences other but up to point five inch the fungicide showed good efficacy with reduced disease severity we added also we also use other uh another type of adjuvants transmits with inspire and badge so it's indicating uh inspire provided better control competencies at uh for inspire and cojet at five days they showed more or less similar trend of effectiveness in both cases when rainfall simulated up to uh and one inch and quarter inch but in all other cases increasing the amount of rainfall simulation increase uh increasing the disease severity compared to panko jab and badge badge perform better compared to panko jab point use uh transmits in all cases initially up to one for badge for one uh three uh one days higher disease a higher amount of rainfall rainfall simulation increases the disease severity but later on uh at three days and five days disease severity reduced indicating the fungicide works well uh if rainfall happened after three days and five days of fungicide supreme this is badge as see uh when mixed with transmits at different time point and in this year field experiment at fox home in minnesota in 2020 uh all treatments indicating that treatment are significantly different from the inoculated text uh from all the combination that only inspire xt as complex uh this showed low lowest disease severity uh low severity compared to other adjuvant use transmits with other fungicide like panko jab and badge but rotation works uh more or less similar uh this happened but initially in this year uh the disease control was fairly good but later in this season uh june july and august rainfall was uh more and more so that gives reduce uh lower disease control this year this is a non-treated checks of disease control uh of adjuvant trial in at fox home this year edge badge plus con complex panko jab panko jab complex and inspire and inspire this looks green it's provided better control on the above discussion we can conclude rainfall reduce the effectiveness of fungicide rainfall event uh immediate after fungicide spray resulted higher cls severity compared to treated check rainfall amount or 0.1 inch at quarter inch uh cannot uh affect that much of fungicide ill efficacy higher disease severity observed if rainfall simulated 0.5 inch and one inch uh disease severity reduced uh over time especially at three days after fungicide spray in five days uh inspire x t better than common panko jab if is sprayed alone or mixed with complex transmits perform better high and added with badge than panko panko jab plus transmits mixture and field experiment inspire plus complex showed better efficacy than panko jab and badge as the fungicide so i would like to thank my research advisor professor dr. mohammed khan for his continuous support and guideline and would like to thank uh for the technical support for dr. khan slap ember dr. yangshilu and peter hag and i would like to again thanks sugar beet research education board for funding research and last uh my and uh department and dc plant pathology department thank you very much everybody for your patience theory thank you for the presentation uh do you have two questions in the q&a box uh the first question is how did you simulate rainfall uh uh thank you very much we did uh rainfall simulation uh we use a sprayer boot at n dsu greenhouse we calibrated the amount of rainfall per hour uh like one inch point two five inch uh five inch and one inch uh uh to see uh uh to see the uh effectiveness of fungicide that how much fungicide loss and how dizzy severity either fungicide effects dizzy severity or not so john will be talking about protection of sugar beet ceilings from b and y vv using double standard rna technology um so i guess starting off with you know we've heard a lot about sarcastra this morning now and if if sarcastra is the disease that needs no introduction then certainly rise mania is the one that has gotten its own youtube channel is approaching is approaching quickly from behind so but that said i think everybody knows that rise mania is a is a global disease of very high importance uh this is partially because uh the fact that you cannot uh have any fungicides or any virus sides or any bacteria sides that can work to control the disease rise mania gets its name from the symptoms that are seen in this photograph right here at the bottom from kws uh rise mania essentially translates to crazy root instead of getting a nice smooth tap root that is high in sugar high in yield you instead get this proliferation of side roots that and root hairs that essentially compromise the disease uh inside of that hairy root mass if you do a cross section of the beet root you'll see a high amount of necrosis that prevents accumulation of sugar and accumulation of yield um so this disease is quite severe quite concerning and as you'll hear throughout the talk although there is some genetic resistance that they use in the crop to control rise mania in fields that have the disease there are some concerns about uh the uh durability of some of that resistance its effectiveness um and yet at the same time I don't think there's cause for alarm at this point so just a little bit of background on the disease for those who may not have been introduced to it much in their in their past um it is caused by a virus called beat necrotic yellow vein virus so even though it's uh rise mania disease as a root disease and affects the roots um it was initially discovered and characterized based on a leaf necrosis that is caused by the disease when uh it infects up through the root and up into the veins the virus itself does not exist by itself in the soil it actually exists within these uh cystosauri these polymixa uh betae uh organism resting spores and that that was this would be like a root hair of the sugar beet that has those spores inside of it so when the end of the season comes around and the beets are harvested and these are left in the soil this is where your inoculum source is coming in from the for the previous year as we mentioned polymixa nor bnyvd are controlled by any of the treatments that you normally put on the seed like apron thiram or even the tachygera and that you put on the seed for the control of um afanomyces for example um and so really genetic resistance is the the form of resistance in the form of control that we have for this disease in sugar beet shortly after rhizomania was discovered in minnesota in the mid to late 1990s the gene rz1 which was already known for resistance it's a single dominant gene that was deployed by the sugar beet seed companies in many of the varieties and that was given good resistance but eventually it did break down and it was good that at that time they already had another gene called rz2 that was waiting in the wings that they could also breed into the varieties that are sold to the growers partially and rz2 and rz1 now are deployed in quite a number of the varieties it's almost hard to find anything that is rz1 alone or a susceptible variety anymore because of the proactive desire to make sure that we keep rhizomania at bay so again um this picture down here just shows what you might see in a field that has rhizomania this would be a field that has what we call blinkers where some of the beets for one reason or another isolated beets are accumulating the virus and showing the yellowing in the leaves with the characteristic symptom in the field for this disease and many of these beets then if you were to pull them up would have this root symptom that you see over here on the lower right one of the things we wanted to do is keep monitoring what's happening with the bnyvv as i mentioned we did have isolates that were overcoming the resistance in rz1 and in recent years we are beginning to be concerned about the possibility of rz1 plus rz2 varieties also being overcome our concern is really no different than what you hear about for coronavirus for all of us you know you're constantly monitoring to see if you have new strains new variants that are going to compromise the crop that you're trying to grow so the way this is done is that whenever there are some problem fields crop up we request some soil to be sent to us and then on the i don't know if you can see my cursor here on this but on the left hand side we have pots that we plant seeds in those those pots contain soil that's mixed with sand so that we get good expression of the disease we typically look at rz1 rz2 and then rz1 plus rz2 varieties that we plant we pull those roots up at about five to six weeks post planting we prepare the roots for and allies a test an immunological test for the virus and then we go ahead and grind the samples up put them in these plates that will have an antibody react with them and if there's a virus in there we will get a yellow color in the plate at shown at the bottom right so that basically is the test we have a plate reader that we put that plate on and we're able to see quantitatively how much yellow is in those plates and that gives us some idea of the intensity or the level of the virus that was in there so next side okay so on the left there would that would be a typical field that would have some blinkers in it maybe even a little bit more severe than blinkers this is in the southern minnesota area but this year we did receive samples from all of the cooperatives just more from southern minnesota because of the severity that was cropping up in some of those fields down there at the right hand side you can see the table that we have that is illustrating the reactions based on having planted a susceptible and rz1 containing hybrid and rz2 containing hybrid and rz1 plus rz2 containing hybrid and as you can see the susceptibles we have several that are cropping up as being positive with rz1 almost all of those are overcoming rz1 as well and then with the rz1 plus rz2 which is the one the growers would be most concerned about because those are the varieties that they typically are planting in the fields we do have a couple that are of concern although not as many as if you're looking at just the susceptibles or rz1 alone so the good news actually is that even though we receive samples in that are suspect only a few of those for the rz1 plus rz2 are truly coming up as positive and yet we've been we've been witnessing this for several years now and we have yet to see any whole field just become swamped with this type of of rhizomania that would be taking this over so um so if we feel that that's actually a good a good point that as we monitor this we are still getting good control out of those varieties now for the first year with the rz2 we've always included an rz2 alone and in previous years we had not seen really any dnyvv overcome the rz2 alone that variety is not anything you would really concern yourself about because rz2 alone typically is just an experimental line they have not implemented rz2 alone varieties for commercial purposes and they went directly to rz1 plus rz2 for maximum control however the fact that there are a couple of spots where we found dnyvv that could overcome rz2 alone are something we want to keep an eye on in terms of our investigations to see how changes in the bnyvv population might be affecting that so in in the you know near future our cop priority remains that we want to examine virus for mutational changes that might be occurring additionally we want to use more sophisticated techniques these days to examine total virus population for new players new viruses that might be impacting the crop in a similar way so next slide until then genetic resistance to rhizomania disease remains the most cost effective and most efficacious method to control this disease as i mentioned several genes have already been deployed to combat the disease namely rz1 and rz2 there are additional genes that these seed companies are continually continually looking at and trying to isolate in addition to their efforts we have a new geneticist dr chang and chou in our group who you heard from earlier who will be looking into some of the wild germ plasm collections that we have in in with the possibility of finding new genes for rhizomania resistance but even given that there's a relatively long lag time any worse than six years to incorporate new gene by standard breeding and selection methods and putting them into elite hybrids so one of the things we've been doing is looking at novel methods novel approaches that might be used either as a seed dressing or some other method of application for combating rhizomania one of these methods would be the newly emerging ds RNA or double stranded RNA technologies and investigating how they might fit into this role interestingly some people have already may maybe have heard of some of the leading products in this area there is a product undergoing registration i believe right now for colorado potato beetle control that is essentially a double stranded RNA molecule used to downregulate a gene that that insect requires for its feeding reproduction irrespective of the many different ways that ds RNA can be used it as an excellent candidate for the technology for virus control so the next slide we'll kind of show how we we began using this we use it by starting with untreated seed and the untreated seed that we have is a susceptible variety if we germinate seeds in a petri dish so that we're just getting the radical so there's a tiny root piece to emerge from the seed and we mix that with double stranded RNA it did have another thing here that is missing a double stranded RNA as you all know about the double helix many of you have been introduced to use the tv shows or whatever of the dna double helix double stranded RNA looks almost like exactly like double stranded dna like the helix and we just take small snippets of that double stranded RNA that match the genome of the virus we're trying to control mix it in with the seeds put it in a cuvette which is shown on the left hand side there that cuvette has electrodes on the side of it so the seeds float to the bottom in a buffer solution in that chamber and when they're subjected to a quick pulse of electrical charge the double stranded dna can actually get into the tissue into the cells that are on the outside of the emerging seed radical those seedlings then are planted into pest soil that has rhizomania in there and after five to six weeks we pull the plants up and we look at the percentage of the seeds that are infected and as you can see that if we have plus the ds RNA in the table on the in the chart on the far right when you have ds RNA the number of seedlings that get infected are quite a bit reduced compared to when you have no ds RNA in there and that this particular chart is a summation of several experiments where we see this effect consistently revealed you go to the next slide now during this time we actually had had the chance to buy a new electroporator so this is the device that actually delivers the electric pulse it's a new more flexible electroporator and we purchased that on information from the literature that suggested that using a square wave instead of an exponential decay wave was better at actually getting dna and rna into intact cells and so that was one of our first parameters that we wanted to evaluate and so you can see that your pulse your electrical discharge can either be a sustained square wave or it can be a rather fast exponential decay and the exponential decay is what we're using previously the data below now are using the exponential square wave and although we are still seeing better control when we add the ds RNA and use electroporation of these seeds in this case the mortality is higher so if you look in both the plus ds RNA and the minus ds RNA columns you see the number of plants that were infected but you also see the number of survivors in parentheses and so you can see that out of 12 seeds electroporated if we're only getting six to five surviving then that means we need to tweak our parameters a little bit so we don't have such high elite validity but we're very glad to have this new piece of equipment to be able to investigate these because we get much more data on the pulse that was delivered on the conditions that the seeds undergo so we feel this is going to be a great addition to our next slide next okay so i'm just in in the summation i'm sorry you have just about a minute here to wrap up yep that's it so we're very glad about our inclusion of dr vanita rama shandran who's our new virologist to our group and we feel that her contributions are going to be really excellent her background experience going to be excellent in terms of contributing to the control of rise mating of the diseases we now have infectious clones that dr alissa floveness had made in the laboratory to begin to investigate the resistance breaking by the nyvv and we will continue to refine and scale up the ds RNA tests to allow the development of technology for ultimate implementation in the field most likely as a seed adjuvant um as a generalized approach ds RNA will continue to offer unprecedented potential both in pest and disease control um and it may in the future be used experimentally to ultimate metabolism noted to ship production to desired products or outcomes in the proposed uh and proposed for the induction of temporary male sterility even in plant breeding so i will just close off there and just go to the last slide just to thank some people dr bolton for hosting me in his laboratory sugar beet research and education board of missota of course for supporting this research and in addition the field and lead agronomists and the cooperatives who along with the seed companies and through the beat sugar development thank you thanks a lot john excellent talk in spite of the technological issues um there are a few questions on the q and a if you could just answer those offline that'd be great okay stop sharing so we can move on to the next speaker so i don't need to do any okay no okay thanks okay the next speaker is uh dr chukchanda uh with co-author jason brentner talking about management of full season rizak tonia and sugar beet thank you melvin can you see my screen in the presentation mode yeah you're you're good to go okay thank you very much uh again uh a lot of excellent presentations uh since this morning and i just want to shift some gears here because i think we heard a lot of uh chukchanda related talks uh but i think it's time to look at some of the rizak tonia control for 2021 but at the outset i would like to actually thank jason uh for all his contributions towards my programs since 2014 so he just decided to uh go to the american crystal sugar company it's a bittersweet feeling for me but i'm glad he's still with the sugar beet industry so thank you very much jason for all your contributions here uh you know one thing i always talk about you know when it comes to you know disease diagnosis especially with the root diseases i mean it could be tricky right number one these are below ground number two they are look alike you know sometimes it's very difficult even for us unless we played them in cultures and see whether it's exactly rizak tonia or phanomyces just looking at my uh samples from 2020 uh most of the samples actually were rizak tonia we did get some samples for epanomyces about 13 we got about 90 samples in total 89 and there are some fields you know it's always common to recover both rizak tonia and epanomyces sometimes you know from the same root or sometimes from the different roots from the same field enough from the same sub sample there the number of samples for fusarium went a little bit lower in 2020 and i'm glad to see that because we got about 11 samples you know that actually was a red flag in 2019 because the only thing we can do for fusarium right now is uh you know selecting a tolerant or resistant variety we did see some pydium we know it's not uncommon if you have some standing water in the fields you know you could get some pydium rutra and the chemical damage and then others you know it could include anything related to abiotic stress or you know even the wind damage you know some of those uh samples there but i think again my point here is uh when you see something wrong in your field you know please send your samples we will be doing this diagnosis again in 2021 so you know how to find us and looking at the overall rainfall pattern in 2020 uh eplen made you know very typical for our growing areas here but i see moraine in st thomas in june but as we went into julia in august i think a little bit of moraine in julia in august but you know that could actually reflect of what i have seen in fields in terms of sarcastra and then other speakers have shown some good pictures in september october thank god it was not uh 2019 you know we could get in and get out as quickly as possible to harvest these beats when it comes to rhizectonia you know it's a full season pathogen so when it's a full season the time we put the seed in the ground you know that's when rhizectonia gets into action so it causes free or post-emergence damping off so with the post-emergence damping off what we see basically is uh in a crosses just at the soil line um and then you know you're going to lose this particular seedling and you will lose stands early on but as you go later into the season uh the biggest risk i mean it could occur anytime during the growing season or to harvest you know that's a root rod like i said the challenge is it's underground so you just need to find it in a nice dry hot afternoon and you know scout your fields and if you see some wilting you know this is not like a reversible wilting you know typically it will deteriorate over time but all you need to you need to dig up those roots and look at look at them uh what you see if it's rhizectonia you see this distinct lesions you know really dark color it could be anywhere it could be next to the crown it could be very bottom or it could be just somewhere in the middle of the root which really depends on where they're not in which present in the soil sometimes you see a ladder like pattern for this um but what happens when these beets die in the field uh basically you know rhizectonia will take another form it's uh it makes these melanized ore wintering structures we call them sclerosia so typically these could survive in the soil for two to three years so you know by the time we get into the same field with beets you know it's it's live and kicking and then ready to infect the beets so that's the impact you have when it comes to managing rhizectonia there are several different things you know we always talk about you know there's no single thing that works for this we just need to have an integrated approach the crop rotation you know most of the growers are already doing a three two to three year rotation that's pretty good but having these small grains such as wheat or barley or anything in the rotation prior to sugar beets i think that's your best bet for keeping an alkaline low early planting is not in our hands you know if mother nature is favorable then you know just go it and do the early planting because when the soil is cooler rhizectonia is not really uh active and the three main things that we can do that are in our control or uh selecting a variety or doing some at planting fungicides whether it's a seed treatment or infrared fungicides or a forced emergence fungicide you know one thing that if you want to grow sugar beets you know rhizectonia is something i think that we have to live with but if you got to pay your bills and then you know make some money i think you just need to invest a little bit of money in taking care of this beets from rhizectonia here so that the trial that i'm going to talk about you know the major objective for this was to determine the best combination of a seed treatment or infrared and or post emergence fungicide using the two different varieties here so basically we looked at the stand counts early on during the season and also the full season effect especially when we go to the harvest so little bit of methods uh we did this same trial at three different locations i'm very thankful to main deck farmers co-operative and seramin is sort of beat sugar co-operative for their valuable help and cooperation in conducting this trial and we used a split split plot design with the four applications so our main plots were varieties you get a partially resistant variety with the rating of 3.7 you know that's the best thing you could get right now in the market and a susceptible variety for rhizectonia with the 4.4 rating when it comes to atmantic treatments we had nothing on the seed or in the furrow so basically that's nothing and then only sisteva on the seed at five grams straight partisan furrow at five nine and a half louis downs this is applied as a dribble in furrow on sisteva and there is another treatment where we only applied partisan furrow but no sisteva this was only done at nwr will say because you know that's the additional treatments we don't have room at other places and in terms of post emergence treatments uh had no post emergence application or a four leaf stage or eight leaf but we don't have a treatment with four and eight so it's either four or eight and again this was applied as a seven inch band at 14.3 flew down so if you're just curious about you know how it's applied it's not a broadcast and then prior to planting what we did was uh we uh multiplied rhizectonia on this whole barley grains and then we broadcast at 50 kilograms per hectare you know that's how we try to replicate uh and reproduce the disease pressure in the fields i'm only going to show data from the northwest rhizectonia always center in the southern minnesota location here nwr will see uh the planting times were uh just you know close to what the growers have done uh but the site from hindak farmers cooperative i think it had good rainfall and the same two varieties but i don't know just like dr sinker said no they're doing very good for stochastic they must be doing something else for rhizectonia i had a hard time getting any disease pressure at their site so between four and eight leaf applications you know it's always about june you know 10 or 12 and then june 25th again two different dates in the harvest is you know a little bit earlier compared to the growers you know uh to get everything out of the ground in terms of data collection we did the stand counts like i said earlier during the season and also the root crop ratings at the harvest we rate about 20 roots using a zero to 10 scale with a 10 increment all the way to zero to 100 percent and yield and quality parameters were done uh at the quality lab um in uh iskrand folks here and we looked at the main effects for the cultivars at planting treatment and the first emergence and also two and three way interactions for this so the the weather right that the weather is going to drive how the rhizectonia is going to pan out during the season so at the nwrlc location i think the striking thing was the rain in july that's seven and a half inches the 10 year average was about 3.3 inches right that's just going to tell something because we have the heat in the july all the humidity is the moisture to get the root crop in southern minnesota i think june july and august were a little bit favorable i think it's probably maybe close to normal or a little bit over than the normal in terms of the rainfall right so let's get to the data here the two varieties uh in crookston pretty much all the way from one week all the way up to eight weeks there is actually no difference whether it's 3.7 or 4.4 varieties in terms of the number of uh plants the stand count for 100 foot of row here i mean stands are good when it comes to southern minnesota i said you know we got good moisture early on it planted you know on may 9th uh we did see that it's a numerical difference between the resistant and susceptible variety you know higher stands for the resistant one but you know statistically it's not really significant but overall excellent stands even the susceptible one right i mean it's under 200 but about 170 or so when it comes to augmenting treatments i'm just going to walk you through quickly here uh the green line is our nothing on the seed or nothing in the furrow in crookston right so you see the lowest number of stands here and the purple one here is actually quadrice in furrow only and the red one on the top is a sistiva with quadrice in furrow and then the blue one is just a sistiva right so what happened just a week after planting really no difference but by two weeks after planting we could see some difference you know where we applied quadrice in furrow the stands were a little bit lower compared to sistiva or sistiva and quadrice but overall i think both sistiva and sistiva and quadrice had higher stands compared to quadrice which is a little surprising because in 2019 i had lower stands for both sistiva and quadrice and also quadrice statement applied in furrow so i wonder you know why this is happening we were just a little bit of drawing 2020 but we are not as cooler as uh not 2019 that's the major difference it comes to southern minnesota basically sistiva and then quadrice in furrow and sistiva you know they had almost very similar stands compared to the untreated again they did pretty good compared to the untreated one there again typically i don't see any stand reduction in the southern minnesota area there and when it comes to the effect of the post application on recoverable sucrose per acre i have the resistant variety and the susceptible varieties so we got about 350 to 450 pounds increased for the resistant variety compared to 950 to 1300 pounds for the susceptible variety here in crookston again this is applied as a band that it's a four eight leaf uh stage and we did see some two interaction between eggplant and also the post emergence application here when it comes to untreated we got about 1100 to 1500 pounds increase in rsa with the post applications with sistiva only about 1300 to 1400 pounds the sistiva and quadrice is about 700 to 900 pounds but again just a little bit of different results here with the quadrice in furrow only nothing on the seed there's not really any benefit just doing a post application again you know numerically it's a little bit lower compared to our best treatment right here and looking at the eggplant and post interaction on the root rot really you know the amount of root rot that we saw was very high when there was nothing on the seed or sistiva only but once we did four eight leaf application there was less root rot that we saw in this treatment here in crookston and between comparing the two varieties in southern minnesota basically there was less disease in the resistant variety there was a higher sugar and the amount of fruits the person of the roots with the root rot were lower and then rst was also higher for the resistant variety you know everything is statistically significant here but when it comes to them you know the biggest difference is the effect of the post emergence application on this in terms of the lower root rot for a four or eight leaf but actually eight looked even better than the four because you know the whole season was favorable for disease development again the same thing reflected in the person roots infected with myzotonia and also about seven ton increase in yield with the post applications there the same interaction was also observed at the southern minnesota location where as the response from the resistant variety was much higher you know almost up to 1300 pounds increased for the resistant variety about 2400 pounds for the susceptible variety with the four or eight but you could see no eight was even better because the variety was so susceptible for this so looking at summary the variety selection it makes a big difference but again if you have moderate to high disease pressure you know it's very critical most of the seed treatments are really good for early protection whether they're applied individually or in combinations when it comes to info application i would say the protection is from early to mid season but there could be some stand loss under drier cooler conditions but what we saw consistently with the info applications you do get some stand reduction but you know it tends to maintain during the rest of the season at least from up to the mid mid season when it comes to post emergence application i would say you know four h don't think about soil temperatures you know just go ahead and get your part this application or anything similar to that that i'll just show in the coming slides what's coming but when you're having a susceptible variety the most important thing you know if it's okay when it's a moderate disease pressure i think seed treatment and a post application should help but if you have a severe field with the rhizoctonia history i think you have to do every possible thing to protect the plants there okay thank you and then you know we'll actually take it down and deep dive into several different topics for the grover seminars so just please add them to your calendar several different interesting topics there with that i would like to thank the r&d board for funding this research uh the cooperatives for their support the seed and then the germans for doing the seed treatments and the quality labs and last but not the least you know for my team for going above and beyond in 2020 to get everything done on time so can be thankful enough for their help okay thank you which is q pcr based detection and genome assembly of a phantom ic's cochleoides uh the speaker is jake of botkin okay co-authors uh kory hirsch frank martin and ashub chando okay so that's me again here kory jake could not be here uh he just wanted downtime just this week uh so i'm going to play the recording of his presentation so gca based detection and genome assembly of phantom ic's cochleoides for the 51st annual sb rb meeting this work was done by me kory hirsch and ashub chanda from the university of minnesota nwrc and department of plant pathology and our collaborator um california frank martin with the usda i'll be going over a background on trigger beats and a phantom ic's rubrat briefly and then diving into our first objective q pcr based detection of a phantom ic's and our second objective de novo genome assembly of phantom ic's cochleoides in the united states sugar beats are a dominant crop with 32 million tons harvested and 1.3 billion dollars produced annually the red river valley accounts for 51 of this production in the eastern parts of north dakota and northwestern part of minnesota american crystal sugar company is one of the companies that is involved with growing sugar beats and extracting the sugar from them and they estimate that 10 million dollars is lost annually due to a phantom ic's root rot a phantom ic's root rot is caused by a phantom ic's cochleoides which is a soil dwelling water mold it is in the umicode phylum and it used to be considered a fungus um and it causes several diseases the damping off of seedlings which is an acute disease and a phantom ic's root rot which is a chronic reoccurring disease this affects sugar beet spinach chard pigs weed lambs quarters and fireweed which are all in the amaranthaceae family in the soil this pathogen exists in an oospore state which can be dormant for years and germinate into sporangia in favorable conditions when a host is present these sporangia have zoospores on the end which can swim through soil water using chemotaxis attracted to root exudates and infect the roots of sugar beets and then produce the oospores in the root the symptoms of damping off are a necrotic hypocottal and the absence of wilting which leads the death of seedlings for a phantom ic's root rot the chronic phase that could reoccur throughout the growing seasons when there's enough soil moisture this results in a necrotic tap root that has the characteristic wine glass shape and water soaked lesions the foliage can be stunted yellowing and having a scorched appearance to manage this disease producers use methods like early planting improved drainage through tiling plant resistant varieties they line fields and then use crop rotation seeds are often treated with tachygarin which is a fungicide that prevents the damping off phase of this disease but there are no fungicides in use for the chronic phase our first objective is the qpcr-based detection for this pathogen phantom ic's coyboides the current assays for phantom ic's are a bio assay and water culture assay the bio assay on the left is for infested soil it is where you have a naturally infested soil and you plant susceptible sugar beet seeds in it and over four weeks you pull out the ones that are symptomatic and then diagnose them under the microscope to determine the root rot index of that soil this can miss inactive phantom ic's the other method is a water culture assay on the right which is for infected plant material and this is where we take the infected plant material and we place it in a agri-plate of water and over two to four days we observe structures that grow out of it looking for those zoes spores and zoes farangia this can miss phantom ic's coyboides in overgrown plates the rationale for our qpcr assay is that it's a dna-based culture-independent method qpcr is a technique quantitative polymerase chain reaction that allows us to detect the dna of a specific organism in a sample so we are going to use this technique to develop the qpcr assay because we believe growers have a need for a sensitive accurate and rapid assay to make informed management decisions the research outline for this project begins with getting infested soil and infected plant samples then we'll do the bio assay on infested soil samples in the water culture assay on infected plant samples to determine the amount of phantom ic's present and if it is present next we will do the dna extraction and qpcr assay we'll extract dna for both our naturally infested soil samples and from our naturally infected sugar beet samples and this is an image here of me setting up the qpcr assay on the right this will allow us to measure the amount of a phantom ic's dna present in our soil and plant samples when we did this for 10 soil samples collected from 2019 and 2020 in the red river valley area and southern minnesota our qpcr assay detected a phantom ic's in all of our samples with a ct value ranging from 28 to 36 a low ct value say 20 would mean a high amount of phantom ic's dna and a high ct value like 38 would mean a very little amount of phantom ic's dna we did the bio assay on these soil samples to get the root rot index which ranged from 86 to 100 for our samples and currently we are working to be able to correlate the ct value that the qpcr assay generates with the amount of a phantom ic's spores per gram of soil samples we also tested this qpcr assay on mature sugar beet tap roots we did this first by visually evaluating the sugar beets and determining if they had symptoms for phantom ic's root rot jason bretner at the university of minnesota sugar beet plant pathology lab did this and found that 78 percent of our samples appeared like they may have a phantom ic's root rot we next did a dna extraction from a piece of the tap root and we also did a waterculture assay from another piece of the tap root indicated by the blue and red square in the image the qpcr assay determined that 63 percent of our 60 sugar beets had a phantom ic's dna present and the waterculture assay determined that only 15 percent of the same sugar beets had active a phantom ic's growing in them in conclusion for this project we were able to validate the assay on both infected plant and soil samples the assay is sensitive detecting 0.1 pico grams of dna and fast can be done in just a few hours the cons of this are sampling issues and soil inhibitors for sampling issues sometimes you can sample the same soil sample and get different results and for soil inhibitors we often have problems doing dna extractions from soil and getting clean results our next objective was to do a de novo genome assembly of phantom ic's quick loities the rationale for this project was that there is no genome for the species currently available and this would allow us to do comparative genomic and phylogenetic studies to help further understand how this pathogen fits into the broader landscape it would allow us to understand virulence factors how does a phantom ic's create disease in plants and it would allow us to develop new diagnostic assays for this project first we need to do a dna extraction and sequencing then we need to do the genome assembly and finally the annotation for dna extraction and sequencing we first brew the tissue and then from that a phantom ic's tissue we did the dna extraction using kaiogen's genomic tips kit seen here on the right that allows us to collect very long strands of dna that we call hymolecular weight dna this resulted in 10 micrograms of hymolecular weight dna we took this dna and we used it for both long read sequencing using oxford nanopore which generated 24 gigabytes of data and then we use the same dna for short read sequencing using alumina for long read sequencing oxford nanopore technologies detects the base pair by its electrical current going through a pore called the nanopore and this is done on very small sequencing devices like the minion and this is relatively new technology that allows us to get dna sequences in millions of base pairs short read sequencing we did using alumina technology which is a little bit older technology but it is much more accurate and this detects the base pair by imaging fluorescent labels attached to the base pairs this results in dna sequences of 100 to 300 base pairs in order to do the genome assembly first we needed to filter our long read data to just take out all the poor quality sequences and the short reads then we assembled it and in order to do the assembly of the genome the assembler software first takes all of these dna sequence reads and then it finds the overlaps between them and then generates a consensus which is a segment of dna that we call a contig and the genome is consisted of contigs or segments when it's finalized finally we need to polish the assembly and then we generate our short read data filter it and use our short read data to error correct our long read assembly finally we need to annotate the genome and we annotate the genome in order to classify all the features such as what are the genes what proteins do those gene makes where are the repetitive regions and what do they look like the goal of this is to understand the biology of the organism and this will allow us to classify the genes and the products that they make and determine the virulence factors such as what proteins is a phantom ic secreting that allows it that allows it to be a pathogen and affect sugar beads the results of this project are that we estimated the genome size to be 82 megabases our assembly size is 75.9 megabases which is distributed in 109 contigs or segments with the n50 the average contig being around 2 million base pairs and the longest contig being around 4 million base pairs busco which is a measure of genome completeness was initially at 91 percent and once we polished our assembly it's at 93 percent when we do the error correction we hope to see this improve further finally we mapped reads from other phantom ic's isolates to our assembly and found that 99.89 percent of them mapped in conclusion our genome is 92 percent of the estimated genome size and the genome is similar in size to related species our n50 is large compared to other similar assemblies our cons were that the primolecular weight DNA extraction was challenging and that assembly software can be slow and resource intensive thank you well ashok i hope you can tell jacob that he did a very nice job in that presentation there is at least one question there that i'm hoping you can answer offline i think it's time to move on to our second phantom ic's presentation that and that is a phantom ic's root rod of sugar bead current and future perspectives uh given to us by samantha rude and ashok chanda as a co-author we see your presentation samantha great um so the title of my talk today is a phantom ic's root rod of sugar bead current and future perspectives first general introduction um a phantom ic's cochlear ides is the causal agent of a phantom ic's root rod it is an omice or water mold and unlike other um plant parasitic omicids a phantom ic's is in the sapro linealis lineage which just practically differentiates what chemical controls it's susceptible to a phantom ic's causes both the seedling damping off disease and a chronic root rod on this slide i have the life cycle which shows in any given season o spores in host tissue are the primary inoculum motiles o spores swim to root exudates may require high amounts of water in soil for swimming they um because they swim to root exudates a phantom ic's is a post-emergence damping off disease because it needs a plant to be there and then an infection will happen infection can happen at any point in the season as well in terms of symptomology the diagnostic symptom that we see in the um seedling disease is darkened hypocautals that have a thread-like appearance they become quite skinny because the stems and the hypocautals are very weakened and delicate plants become really susceptible to death or damage due to wind in older plants foliage will be chaotic and it tends to wilt throughout the day and then often will recover at night or under periods of high water scarring and sensing of roots is common especially malformation can also be seen in roots that have recovered from infection infection at any point in the season will lead to yield loss and decrease in extractable sucrose and increase in the amount of impurities terms of disease incidents a phantom ic's is common anywhere that sugar beets are grown especially in the red river valley area as well as southern minnesota as you can see from this map from 2018 only the dark green squares are free of a phantom ic's so phantom ic's is quite common part of that is because of the fact that the packaging can survive in fields for up to 10 years so once you have a phantom ic's it's difficult to get rid of and as you heard in the last talk a phantom ic's can sometimes be difficult to identify when it's present in a field if you don't have infection during seasons that have warm and wet conditions we can see major losses occurring so improving drainage using tiling can be a helpful way to mitigate disease risk but infection can occur at any time in the season that conditions are right even if they're only right for a small amount of time you can still see appreciable stand losses occurring due to infection so because it's an insidious pathogen that hangs out in soil waiting for the right condition it's important to continuously use control methods to mitigate how much disease you have there are a number of factors associated with the amount of loss that we see from a phantom ic's root rot the number one is probably wetter the second is what seed treatment is used cultural practices and resistance of the variety planted are also important tachygarin is the only available chemical control for this pathogen it's very effective in decreasing the amount of seedling damping off that we see however because it's a seed treatment and it doesn't last all season we tend to now see a more chronic root rot and less of the seedling phase as the tachygarin stops working throughout the season cultural controls are a really effective measure and those can include things such as incorporating factory waste line into soils which a lot of work has been done showing how long that can benefit and decreasing the amount of phantom ic's you've seen field crop rotation is also helpful and so is weed control there are over 30 species of plants that can be infected by phantom ic's and serve as reservoirs or hosts so that could be plants like spinach and other beach species or weeds such as lambs quarters or pigweed or carpet weed there are commercial varieties with resistance or moderate resistance but there are none that are completely immune and because of the fact that sometimes you can have a high resistance in a variety comes hand in hand with a yield penalty it's important to weigh decisions between how much risk you think you might have and how resistant a variety is there has been some work done in phantom ic's quickly grotes understanding how ice looks differ but there's been considerably more work done in a different species of phantom ic's phantom ic's eutikes and so briefly some of the interesting things that have been found in that pathogen which is a pathogen of legumes is that populations within a singular soil sample are mixture of phenotypes and genotypes so isolates differ in terms of how virulent they are and how aggressive they are what's more interesting and potentially more concerning is that the frequency of highly virulent isolates of a phantom ic's eutikes so that is race two isolates has been shown to be unrelated to cropping history so basically researchers have found a phantom ic's very virulent isolates of it in fields that have never been planted with a host plant so these very virulent isolates exist anywhere that a phantom ic's does and it doesn't seem that we have put any selection pressure on them to exist so there's a lot of genotypic and phenotypic diversity in isolates we assume that the same thing is probably true for phantom ic's corkulitis we do know that the phenotype of one isolate does not affect the phenotype of another isolate within the same field but we don't have a great scale of how diverse isolates are on a regional or global scale this is important because we want to be able to use information about how isolates differ to improve our control methods you want to understand how isolates might vary and how susceptible they are to chemical controls or how long a resistant variety will actually be resistant in the field based off of how isolates differ on a soil sample level so pivoting from the current state of a phantom ic's to future research objectives i'm going to be walking you through some of my objectives that i'm going to be starting as a first year phd student the first one is mapping of genomic regions of sugar b associated with resistance to a phantom ic's rubric for this experiment we are partnering with Dr. Kevin Doran who is a research geneticist of sugar beets with the USDA ARS and we're planning on genotyping and phenotyping USDA populations of sugar beets to and using either genotyping by sequencing or genome-wide association studies to identify what genes might be involved with resistance what we do already know is that there have been some resistance QTLs or quantitative trait loci identified for this pathogen however we don't have a full picture they either have a minor effect they haven't been incorporated in lines or in some cases we actually don't know what that gene codes for so there's a lot of avenues for us to research to understand more about how many genes are involved with resistance where they are what they code for and hopefully we can identify resistance genes create markers that can then be used as a proxy for screening to more quickly go through variety screenings to identify resistant lines my second objective to identify potential clades of phantom ic's quick leoities within the U.S. and on the global scale with respect to genetic structures in this case this might be experiment experiments that use a genotypic and morphological characteristics of isolates to look at diversity and population structure so in that case what might happen is a plot like the one on this slide that shows the amount of viable dormant and dead o-spores produced by different isolates you know that isolates differ in a variety of ways we want to look at isolate diversity off of practical traits like the number of o-spores or zoospores things that affect how disease progresses in the field and understand how that might be influenced by where isolates are as well as copying history or cultural practices to really get a better picture of what's going on in the soil and what a phantom ic's isolates are like my third objective is to characterize the virulence of phantom ic's isolates using USDA germ bosom with varying levels of response to a phantom ic's root rot this case we are planning to use differential lines of sugar beet to characterize our isolates so we would expect an outcome like the table on the slide showing different varieties and different isolates and by pairing them together you can see how isolates differ in terms of what plant or what variety they're susceptible on this is how we determine races in well plant pathology in general but especially in a phantom ic species yeah and understanding how isolates differ in terms of virulence is very helpful for understanding how different virulent isolates are distributed in fields on a regional or global scale and identifying areas that we need to be focusing on or isolates in particular that we should be worried about when it comes to breeding for disease resistance that way we can make sure to be focusing on isolates that have the potential to become major problems in the future and by identifying how isolates may differ that can help us to understand if there are multiple mode of actions for genetic of resistance on the side of sugar beets so you kind of need them to go hand in hand diversity in isolates and diversity in sugar beet moving forward we're hoping to deepen our understanding of a phantom ic's root rot of sugar beet by advancing our understanding of the genetics of disease resistance and improving our ability to screen plants for resistance in a more high throughput manner and by increasing our knowledge of the genetic and phenotypic diversity of phantom ic's isolates on a regional and global scale but that I'd like to acknowledge my funding from the Department of Plant Pathology at the University of Minnesota Dr. Kevin Doran and of course my PI Dr. Shakchanda I'd be happy to take questions or in the chat or on here thank you very much Samantha excellent talk you do have one question I think in the interest of time though if you wouldn't mind taking that offline that would be great because you've now gotten us right back to being on time okay and then if you could stop the screen share that would allow our next speaker to prepare himself um so this is a new face that I believe for our reporting session this is Carl Strasbaugh who is a research plant pathologist with the USDA in Kimberley, Idaho and he will be talking about incidence distribution and pathogenicity of fungi causing root rot on the top of sugary piles in Idaho Carl that's all yours okay Melvin thank you and thank Muhammad for including me including me in your reporting session today in Idaho in sugar beet piles we store approximately two-thirds of our sugar beet roots at harvest time about a third of our crop can go directly from the field to the factory a third of our crop goes into what we consider short-term storage or about 90 days of storage and the third of our crop goes into long-term storage which can last all the way to the end of March beginning of April and so you can see from this particular slide some of this this steaming fungal infested spot if you get a hot spot in your piles outside they can be related to fungi if your pile develops a hot spot and it falls about 10 feet from the original 20 feet um that those roots are no longer usable at that point and so these hot spots can be pretty good size to remove um an average size house will fit into the hole in the pile when some of these are removed and so the situation can be serious and so we've been investigating the fungi inside piles and also on the surface of piles okay well we'll proceed see fungi in outdoor piles is our objective previous research we've looked at investigating what occurs inside piles and it was obvious at that time we didn't have time to expand the the study to include the surface of piles because the surface looked completely different than what was inside and so that's the objective of this study the focus of it um so we were looking at the incidence distribution and pathogenicity of fungi and two different types of piles piles with ventilation and tarps which are our longest term piles and then our shorter storage term storage storage piles we do not utilize tarps or ventilation for those types of piles so with that you can see from the bottom of this slide that we had seven locations where we had both types of piles the tarp and ventilated piles are in the green bars the blue bars are where we have no tarps or ventilation and the vertical axis you can see that we have the percentage of root surface area with rot and what we did on top of the piles were break it up into a three by three grid and within each of the nine squares we took a one meter square area where we assessed all the root surface area for fungal rot and so the average of those nine locations is what you're looking at here and so if we look at location six you can see with the green bar we had up somewhere around an average of 40 percent of the root surface covered with fungal growth and I'll cover in a minute just what the different fungi are that we're finding but total fungal growth is pretty significant and you can see that by comparing that location six to location two that we do have significant differences between locations both within a pile type and between pile types location two ranked the best regardless of whether they were tarped and ventilated or not and you can and this was for the 2018-19 storage season so I'll switch the next slide and this is the 2019-2020 storage season and you can see we had considerably more rot if you look at location six the green bar we're topping out around an average of 90 percent of the root surface being covered by fungal growth so obviously these roots on the surface of the pile are impacted considerably by fungal growth however if you compare it versus location two again as in the previous year both pile types are sniffly better and ranked number one as the best pile both years and where we have no tarps or ventilation those ratings would have been taken around the end of December you can see we have almost no fungal growth even though it has been that fabulous year for fungal growth compared for some of the other locations and so if we find the right location and have good roots healthy roots coming into the pile it is possible to store roots under ambient conditions and have relatively little fungal growth this gives you an example of what a root might look like after 60 some days in storage typically at that point in time cladosporium species are the only thing and that's dominating the root surface and that's what you're looking at in this particular slide and so other fungi that we see don't appear until later in the season so by the end of December we would have put these roots in the pile beginning in early October through the end of October and there is more fungal growth on the roots that are piled first in a pile as opposed to those piled last and so by the end of December the roots will look like this however and this is another view of a cladosporium species on the root surface and if you contrast that with what the roots may look like typically after 120 some days in storage or twice as long we have a mixture of fungi we still have cladosporium dominating the system however we also have betritus penicillium and if you look at off the left side of the slide you see sort of a spider web kind of look a white fungus that's an aphelia like the city of my seat so here's an example or close-up of what betritus scenario looks like it's a brown powdery appearance on the root surface an indoor storage piles this fungus is the one that dominates both inside an indoor pile and on top of an indoor pile betritus will dominate in that kind of storage situation outdoors however it is less dominant and equal into about equal in its prevalence and coverage in comparison to penicillium species and the aphelia like species here's an example of penicillium what it'll look like in storage more of a blue-green powdery kind of fungus and the aphelia like the city of my seat it was mentioned earlier whether diseases in the field and could lead to issues in storage we do find a correlation between the presence of this fungus that you're looking at and the presence of rhizomania in the field and so prior to having rhizomania in Idaho we first had rhizomania in 1992 so prior to that we may not have seen this fungus in storage because it's pretty hard to imagine someone missing that root on the left looks like a ball of cotton candy it's hard to imagine someone not seeing that in an indoor storage pile under human conditions that cotton candy kind of look is what you see on the right you see more of a spider web appearance of it and the kind of a more a flat kind of crusty kind of growth still white in appearance but under drier conditions outdoors this is more typically what it looks like so if we inoculate isolates of representative isolates for each of the fungal types that we found from these roots inoculate them back in put them in storage for about 60 days and then measure the amount of root rot that we get this picture slide shows about 17 of the isolates in the study the study actually included 30 some isolates the ones not shown on this slide produced no rot and they were all cladosporium species that were being evaluated you can see here from the right side of the slide that some of the cladosporium species here produced relatively little rot almost just barely better than the non-inoculated check really if we go a little further across the slide you can see we do have one alternaria atrial isolate that we included next to that we have two penicillium cellarum isolates that are sniffling better or sniffling worse depending on your viewpoint at causing rot penicillium polonicum and expansum are the next isolates that are increasing in pathogenicity as we move across the slide and the most pathogenic isolates we had were botrytus scenario so that gives gives you a little bit of perspective comparing the different types of fungi and storage and amount of rot and that is an average of two years of studies and the studies were not sniffling different so the these data were analyzed together now if we look at the cladosporium species i mentioned that you know in those slides i showed earlier in total fungal rot um regardless of whether it was the tarp pile or the untarped pile cladosporium species dominated and you can see here by the list of species that i found and this is just from the 2018-2019 season the current year the most recent year um it's still a work in progress at this point um but 2018 and 2019 the cladosporium cladosporioides species accounted for 66 percent of our isolates and the cladosporium subtilisimum was 17 percent so those but by far the cladospora spore cladosporioides um species was the dominant species um you can see i highlighted some of them in red um these particular species are also known to be associated associated with human clinical samples and having sequenced several genes from the these particular um species um they were very close in other words the sequence identity was between 99 and 100 percent so essentially the same thing um so with that in mind um if you do have individuals that are working for you and they're immunocompromised um you would it probably be a good idea not to have them on top of some of these storage piles um particularly later in the season when there's a lot of um fungal growth present um some of these these isolates of fungi for an immunocompromised individual um might be of concern for a healthy individual these fungi are not that pathogenic and should be of little concern um penicillium species if we look at um the three that what we find typically in sugarbeet piles um we 84 percent where penicillium expands them ponicum was 10 percent um inside a sugarbeet this is on the surface of the sugarbeet piles if previous work when we looked at inside the piles cellarum was about a 30 percent of the isolates and expansum was about 60 percent in those studies so in conclusion cladosporium species um particularly the cladosporium cladosporioides was 66 percent in the primary fungus found on top of the sugarbeet piles um regardless of whether they were early season or late season location two in our study proves that roots can be stored long term under ambient conditions if you can find the right spot and put healthy roots into the pile so with that um thank you to amalgamated and bstf for assisting in in making this possible thank you thank you carl uh we have time for i say one question hi carl it's an excellent presentation uh the one that you just showed with the cotton ball the ethylia is it close to sclerogen rod ci or something else ethylia bombacina is the fungus that it's closest to um and there it likely represents um of new fungal species um it may actually from what the mycologist tell me even represent a new fungal genus so it does that's why we call it ethylia like because the closest thing to it is ethylia bombacina okay our next speaker is dr kimmerley web she is with the usda in fort collins colorado and she will be breaking down the interactions of sugarbeet with rhizoctonia selana right kim you guys able to hear me and see my slides correctly you're good to go okay perfect so it's been a couple of years since i have talked with you and so what i'm really hoping to do is just update the group on some previous projects that you had provided funding for and what we've done on these projects in the past couple of years so i'm actually combining these two projects into a single talk i apologize um because of that we're not going to be able to get into a whole lot of detail on them but if there's any questions after i get done please email me after the sessions and we can definitely talk in more details the first project was trying to detect and quantify the amount of arsenic that is necessary in the soil that is needed to cause disease and sugar beet and then the second project is characterizing the interactions of rhizoctonia selana with sugar beet and the different biological mechanisms that might be going on that could increase disease severity or also induce resistance um i'll get started with the first project which is detecting and quantifying the amount of selana and i that's necessary to cause disease i wanted to um shout out two of my collaborators on this project uh dr kirk broders who was formerly with carter state university but is now with us da in peoria and dr james woodhall who is at the university of idaho as well as some combined funding that he brought into the project from the snake river seed alliance out of the idaho group um we all know rhizoctonia diseases and sugar beets um so i'm not going to go over the symptoms as much here but i do want to highlight that what we consider as common rhizoctonia diseases are caused by different kinds of rhizoctonia selani and when you're trying to detect these pathogens it's really important to understand these differences because your detection methods may not pick everything up so in our case um we definitely see rhizoctonia selani most of our diseases are caused by ag 2-2 3b but there's another ag 2-2 romanuminal 4 that can also cause disease um and sugar beet and they can cause both the seedling damping off but they're more known for causing the rhizoctonia root and crown rot um some work out of linda hansen's group is starting to show that there really isn't too many genetic differences between these two subgroups of 2-2 so we're really not sure what the differences of are in those two different um subgroups at this point um i can talk about a little bit more when we start talking about the detection the other one that tends to be a common um concern in sugar beets is r selani ag4 and that usually is associated with seedling damping off but i'm going to reiterate that any of these species can cause either of those two disease symptoms in sugar beet as we know um seedling damping off can primarily be managed by um seed treatments when you're planting in in your crops um and you can also apply fungicides at the later stages to better manage the crowned root rot symptoms however genetic resistance is still a primary requirement for most of our production regions but one thing we have to keep in mind is that it appears that resistance for the adult stage rhizoctonia crown and root rot is different from the resistance for seedling damping off and so we need to do a lot more to better understand what that resistance is and how they are working so one of the biggest things that we have to keep in mind when you're trying to detect and quantify how much of a pathogen is soil is the disease cycle in each particular disease system for sugar beet um the main sources of inoculum typically can be from sclerosia which are these overwintering um condensed particles of mycelia or the rhizoctonia can actually colonize organic matter such as leaves leftover roots things like that in the soil and it's these particles that then overwinter and then cause disease later on in your systems each individual particle is considered an infective particle and so when you're trying to determine how many fragments of this pathogen is out there you have to be able to count the number of particles this can be complicated particularly with organic matter because it depends on the size of your piece of leaf that it's colonizing and if that leaf suddenly gets torn into two instead of one infective particle you suddenly have two infective particles we really don't know how many infective particles are needed to cause disease in our fields and this particularly comes up in our disease nurseries where we actually artificially inoculate our fields and you'll often hear a lot of our scientists talk about well we had a really hot inoculum this year well what does that mean did you just have more particles that have more colonization of the fungus or did you have a more virulent islet typically it means that you just got better colonization and so whenever you're throwing that those barley grains into your artificial inoculum into your field you're actually just getting more disease because you have more infective particles so many of us don't actually count how hot our inoculum is before we go out and do our disease studies the reason being is it's really difficult to do this you typically have to sieve the soil to get your sclerosal fragments or you actually have to do a serial dilution series of your soil plate the soil out and then count the number of fragments that actually germinate mycelium pieces and this also comes into peace when you start talking about detecting them with a PCR assay we can't really quantify how many particles there are because you're counting the amount of DNA that's there so the purpose of our study was really trying to better idea of how many particles cause disease and what is the corresponding DNA amounts that we could then detect to correlate your infection level so what we did is we originally started out using some pre-published primers that industry-wide we call the budge primers and those have been released for the Rhysoctonia cyanide complex to detect the different anastomosis groups that we're talking about the problem with the budge primers is they've been known to actually cross-react with some of the different anastomosis groups in some of their relationships and there's a very closely related subgroup to our pathogens which is ag2-1 and the budge primers are known to cross-react with these 2-1 species so we decided to go ahead and develop a new set of primers that we developed off of the genome that was genome and transcriptome that was published by Weiberg et al in 2016 where they had some putative pathogenesis genes that they found in that genome that was not in any other genome of the Rhysoctonia selenides that they looked at and so we developed a primer based off of one of those genes and screened up to 50 different Rhysoctonia selenides isolates and compared them to the budge primers and we found that we were able to detect only the 2-2 isolates and our primers did not cross-react with the 2-1s. This is just a quick subset kind of showing some of that data where with a standard PCR we were able to get a fragment amplified using only our primers and it did not pick up the 2-1 isolates. We then increased the number of isolates to 100 and we confirmed that with a broad range of species and different Rhysoctonia and osmosis groups. After developing a quantitative PCR assay that we could then use for soil we then actually wanted to try and determine can we correlate this with inoculum densities and then how much inoculum is needed to cause disease in sugar beet. So what we did is we colonized barley with Rhysoctonia selenide and ground it into small fragments. We then mix it into the soil at different rates and concentrations and then put the soil into individual flats. We then would plant them with our different sugar beets and then allowed the sugar beet seedlings to germinate and counted the ones that germinated as well as the ones that ended up surviving and not damping off. We then collected the soil from these different treatments and then used our PCR assay to determine if we could detect them. So in our original development of our PCR assays we found we could detect about 1.2 pika grams of DNA per treatment but we're not able to quantify that even though we could detect it. So the PCR reaction did amplify a fragment but we couldn't quantify the DNA react reliably and that's what you see on the left hand screen. We actually have two graphs here because the University of Idaho validated the test and that came up with basically the same curve that we did in Colorado. So we felt like the test was fairly sensitive. When they then used the assay to detect the pathogen at different concentrations in the soil we were able to reliably detect and quantify the particles at two infective particles per gram of soil which ended up being about 0.2 nanograms of DNA and that's in the top graph. In the lower infection level of only one infected particle per gram of soil we were still able to detect it but there was too much variability in the assay that we weren't able to reliably quantify the DNA that was contained within that sample. As for how much inoculum that's needed to cause disease the first study that we did we did a range of sugar beet germ plasm. Each fragment on the left hand panel on the left hand side are different germ plasm of sugar beet and we inoculated the soil at two infective particles up to 200 infective particles per gram of soil. We used this very high rate because that is what we suspect whenever we artificially inoculate our fields for our disease in our series you tend to inoculate with a much higher level than natural amounts and we wanted to determine what is the minimum level of inoculum where we could still get susceptible plants in the field and so when we did this assay and comparing it to the uninoculated treatment which is the black bar we were able to get a reduction in disease up to 10 infective particles per gram of soil on some of the varieties that we tested if we in inoculated that only two infective particles per gram of soil we still got disease so that's telling us you don't need a lot of rhizotonia in your field to actually get disease. In our second study we really found out that we had way too much inoculum going up to the 200 infective particles per gram of soil so we changed the inoculum loads to one infective particle to and 10 per gram of soil and this time we also included some resistant lines and in this study we found out that yes we could still get disease at two infective particles per gram of soil but the interesting thing was on resistant lines once you've got up to 10 infective particles per gram of soil you started to lose effectiveness of that resistance and so if you have higher concentrations of rhizotonia selenii then we could actually be breaking down our resistance. Because I'm running out of time I think I'm going to go ahead and stop here unfortunately I wasn't able to combine it all in but if you have any questions on the metabolomics please email at later date and we'll try and incorporate that at the later time. Thanks a lot Kim a very nice talk I'm happy to see there's some light chat on the molecular differences between these rhizotonia subgroups finally. Do we have time for one question I think? What type of soil did you use Kim? For these studies we did use a potting mix for this however Dr Woodhall is actually now working with field soils and trying to increase the sampling so that we can increase it to um actual being able to use this for field detection. We do lose sensitivity of the assay you can still get detection though. Thank you. Okay thanks a lot Kim. Our next speaker is Dr Linda Hansen she is with the USDA in East Lansing Michigan. So I'll be talking about something that's showing up in Michigan uh that's alternator leaf spot and comparing it to what we've been talking a lot about uh Cercospora leaf spot I do want to say that um Dr Jamie Wilbur who is the extension potato and sugar beet pathologist in Michigan and Daniel Bublitz who is the extension specialist for sugar beet also participated in some of this. Now we talked a lot about Cercospora is the most important leaf spot of sugar beet but we do have a number of other spots as well. You can see this bead here we have a lot of Cercospora but we have some others and this is the one that we're starting to see some issues with alternator leaf spot. You've seen a lot of Cercospora characteristics this is just a little reminder for comparison. A couple of things that haven't been talked as much about but when we're looking at alternator for comparison we are particularly interested in are that Cercospora tends to be more randomly distributed over the leaf surface. It's also as mentioned a warm temperature pathogen it causes disease up to about 96 we really see it slowing down anywhere above 96 Fahrenheit with most of the growth stopping around 98 to 100 degrees Fahrenheit but 65 to 96 you'll see Cercospora. In contrast, alternator leaf spot can be all over the leaf but we often see it concentrated around the leaf edges. It can cause a variety of colors for the lesions. They'll usually be a light center with a darker edge. When they coalesce you may not see those individual lesions which often are still visible for Cercospora. When they're small they can be fairly circular but they often become quite irregular. The species that can cause it include alternator bracicula which is reported in the western United States as a pathogen as well as alternator alternata and alternator tenuosoma. Now there's some question which I'll go into a little later about whether alternator alternata and tenuosoma are actually different species or not. So we're currently calling these the alternator alternata species complex. We have a wide temperature range we can see the alternator anywhere from 50 degrees Fahrenheit to 90 degrees Fahrenheit with an optimum of 68 a little lower than Cercospora. It's promoted by anything that causes leaf yellowing including yellows, viruses, fusarium, nutrient deficiencies or even host genetic yellowing. It's also found fairly commonly on leaves that have been in contact with soil. We've seen quite a bit of it when we've had drought with leaf collapse during the day and coming back in the evening. It produces these very dark club shaped spores in infected material. When we look at the spores you can see Cercospora with these silvery needles and alternator with these dark spores. They're usually formed in chains for either alternator and tenuosoma and you can see they're much shorter than Cercospora, darkly pigmented with a number of crossfalls. Historically, alternator has been a minor issue in the US as well as Western Europe but it has been a major issue in areas like Turkey, Pakistan and the Ukraine. In 2010 we started to get it on our radar in Michigan when a close to a quarter of the spots coming in for end of season fungicide sensitivity tests had alternator spores. A technician and student running the EC50 plates when they were finding anything with EC50s above 20 parts per million that year were finding alternator rather than Cercospora on their fungicide plates. We thought it was a curiosity but then over the next five years we started getting more samples to both the USDA lab and the MSU diagnostic clinic with alternator leaf spa. These were including one or two fields per year with as much or more alternator Cercospora. These were particularly coming in. This figure here shows historical risk management with the red area high risk for Cercospora, yellow area moderate risk and green area low risk and it's in these green areas where it was low risk for Cercospora that we started to really get the reports of alternator coming in. Then in 2015 we found widespread alternator leaf spot. Certain varieties were especially prone but this was found in all of these growing areas not just in the green zones. It was more common in the yellow and green zones for Cercospora that may be in part because if you have a lot of leaf lost to Cercospora it's harder to see any other leaf spots. But those problems have continued through 2018 where about 30 percent of our foliar leaf spot samples have been coming back as alternator with about 60 percent as Cercospora and maybe 10 percent some other leaf spots such as bacterial leaf spot and other things like that. And these are causing enough leaf loss to be impacting yield. 2019 and 2020 we've had less disease unlike Minnesota, North Dakota 2020 was a fairly low leaf spot year for us in Michigan. Most of the rain stopped more in the west of us and we didn't get nearly as much. We actually had some drought conditions for part of the year so both Cercospora and alternator were much lower in 2020. Some considerations we look at diagnosis. Cercospora generally has higher sporulation on the upper leaf surface and alternator on the lower leaf surface. So if you're looking for where the spores are produced you may want to turn the leaf over if looking for alternator. Now you'll see the change when the leaf edges start to curl up you'll still see it on what was the lower leaf surface. We also can have mixed or coalescing lesions with both Cercospora and alternatoria shown here you've got your nice silver Cercospora spores, your alternatoria spores and they're running right into each other. Alternatoria is able at least the strains we've looked at are tolerant to Cercospora and are able to grow in these Cercospora lesions. That can cause some complications where depending on your temperature you may see more Cercospora spores or alternatoria spores or both. This alternatoria is what we're calling alternatoria alternata species complex. Back when I first started all small sport alternatorian chains were generally called alternatoria alternata. Then these were separated into different species based largely on the branching of chains with alternatoria tenuissima, having unbranched chains, alternatoria alternata, having highly branched chains and alternatoria arboricens, having moderately branched chains and then some other species as well. However genetic evidence does not support separating alternatoria tenuissima, alternatoria alternata and some other species like alternatoria mali. These have not been genetically identified using multiple gene regions. Arboricens can be separated but some of our others cannot. This is particularly a consideration because when we want to look at host range for host crop rotation it's important to know where else our species can infect. Alternatoria alternata the old sense has been reported to have a very wide host range but tenuissima has been reported a more limited host range. But so the question comes are which way do these beet isolates go and what we found is isolates from beet can cause disease on dry beans, on potato, on tomato and on apple and isolates from bean and potato can cause lesions on beet, bean, potato and tomato. So this would agree with a broader host range unfortunately limiting what may be able to use for resistance management and crop rotation. We do have looked at impact of belief spots on yield. Unfortunately the place we did this test is in a red zone for sarcosper so we were unable to get any alternatoria alone. We did get sarcosper alone or sarcosper with alternatoria and with a lot of fungicide use our controls had less than two lesions per plant in this particular year for sarcosper or alternatoria. You can see sarcosper alone and the sarcosper and alternatoria both reduced the yield and purity but the alternatoria increased the loss of sucrose in our samples and that has been consistent with what was reported back in the UK with Buford's work. Our management now we're looking at a combination of cultural management host resistance and fungicides both sarcosper and alternatoria survive on crop residue with alternatoria of much stronger sacrifice. It's found often on dead tissue of non-hosts as well as host plants but for both bearing infected tissue is reported to degrease the inoculum levels into your next following seasons. Host resistance is present. We have varieties that vary in their response. Current testing is primarily observations when there's natural infection in the field but we're working on developing inoculation methods. We're getting separation between resistant and susceptible varieties in detached leaf assays and we are seeing increased infection with our inoculations in the field. We're trying to move into more less sarcosper susceptible or less sarcosper heavy regions for screening. Unfortunately this year with COVID-19 restrictions we were not able to move our disease nurseries. We're hoping to do that in the future. In Pakistan they have actually screened and reported resistant varieties. Most of those are not approved for use in the US but we're looking at some of those as well as USDA germplasm. There are very few fungicides labeled for alternatoria but several of the ones labeled for sarcosper do show efficacy. We used EC50s with spiral dilution gradient plates. Looking at tetraconazoles most of our alternatoria isolates show a fair amount of resistance and field testings with these have shown that tetraconazoles are not particularly effective for alternatoria management. For QOIs we see highly resistant and some moderately resistant but these are giving some of the best management to alternatoria in field trials. Tin we do see some isolates that have maybe a little bit of tolerance but the majority are sensitive at field rates and tins are also giving good alternatoria management in field trials. So we do see evidence for potential for resistance development. It's certainly been found in other crops so resistance management practices are strongly recommended if you're using any of these fungicides for alternatoria resistance and we try to reduce reliance on fungicides alone. QOIs tins and EBDCs all have moderate to good efficacy against alternatoria leaf spot with DMIs being less effective. Thus the current recommendation is to include some QOIs towards the end of the season when alternatoria becomes a greater issue in the cooler weather and keep the DMIs for early season and also consider whether if you're going into cool season look more at alternatoria management warmer temperatures your sarcosis will still tend to predominate. However alternatoria on highly susceptible varieties can still spread even in warm conditions. High humidity and prolonged dews or leaf wetness both favor disease. We also see higher alternatoria following drought and insect damage particularly leaf minor damage and consider alternate hosts such as tomatoes, potatoes, etc as rotation crops as well as in back gardens and several weed hosts. I'd like to thank our funding sources Beecher Good Development Foundation, Project Green, the Research and Education Research and Extension Advisory Council from Michigan and Michigan Sugar Cooperative. Thank you very much Linda very nice talk. We have time for a question or two. I did get one message to me and the question is does abiotic stress increase risks for alternatoria compared to CLS? Some abiotic stresses certainly do. Nutrient deficiencies are definitely a predisposing factor. Nitrogen or manganese deficiencies both when you get yellowing from those we also see increases in alternatoria issues and then drought when you actually have leaves lying on the ground we saw a lot of increase in our alternatoria on those and any leaves that had been in contact with ground for several hours during drought. Okay any final question? If not I'd like to thank the speakers for giving great talks today. It's clear from this last session that there are no shortage of pathogens of sugar beet and we had a lot of diversity and talks on various pathogens and it was all very interesting.