<Emphasis Type="Italic">Spongospora subterranea</Emphasis> soil contamination and its relationship to severity of powdery scab on potatoes

We investigated soil contamination by Spongospora subterranea f. sp. subterranea (Sss) and disease severity of powdery scab in 29 potato fields in Hokkaido, Japan, using a hydroponic culture method with tomato seedlings as bait plants. The quantity of Sss infection on the roots of bait plants was evaluated using the polymerase chain reaction (PCR) and expressed in terms of the infection potential in the soil. The infection potential was positively correlated with the disease severity of harvested tubers, whereas the spore ball density determined using PCR had an indistinct relationship with disease severity. The infection potential can be useful in evaluating soil contamination and in applying countermeasures against powdery scab.     

Identification of differentially expressed genes in tolerant and susceptible potato cultivars in response to Spongospora subterranea f. sp. subterranea tuber infection

Powdery scab caused by Spongospora subterranea f. sp. subterranea (Sss) has recently become one of the most devastating potato diseases of economic importance in South Africa. The use of resistant cultivars has long been considered the most effective and sustainable strategy to manage the pathogen. However, little is known about the molecular mechanisms underlying resistance of potato tubers to Sss. Using RNA-sequencing (RNA-seq), 2058 differentially expressed genes (DEGs) were identified from two potato cultivars (tolerant and susceptible) in response to Sss infection. Analysis of the expression patterns of 10 selected defence-response genes was carried out at two different stages of tuber growth using RT-qPCR to validate the RNA-seq data. Several defence-related genes showed contrasting expression patterns between the tolerant and susceptible cultivars, including marker genes involved in the salicylic acid hormonal response pathway (StMRNA, StUDP and StWRKY6). Induction of six defence-related genes (StWRKY6, StTOSB, StSN2, StLOX, StUDP and StSN1) persisted until harvest of the tubers, while three other genes (StNBS, StMRNA and StPRF) were highly up-regulated during the initial stages of disease development. The results of this preliminary study suggest that the tolerant potato cultivar employs quantitative resistance and salicylic acid pathway hormonal responses against tuber infection by Sss. The identified genes have the potential to be used in the development of molecular markers for selection of powdery scab resistant potato lines in marker-assisted breeding programmes.     

Biocontrol of powdery scab of potato by seed tuber application of an antagonistic fungus, <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>, isolated from potato roots

An environmentally friendly measure to control potato powdery scab caused by a protozoan pathogen Spongospora subterranea f.sp. subterranea (Sss) was developed by focusing on antagonistic microorganisms that were considered compatible with potato root. Five hundred and eight soil fungi, isolated from potato root cultivated in soil suspensions from four potato fields in Hokkaido, were screened for suppressiveness of root infection by Sss in a hydroponic culture system and for powdery scab severity in greenhouse and field experiments. Antagonistic isolate Im6-50, identified as Aspergillus versicolor, was selected as a potent biological control agent. In a 3-year field test, A. versicolor Im6-50 suppressed powdery scab with a protection value of 54–70 (100?=?complete protection) when applied directly on seed tubers compared with a protection value of 77–93 by the synthetic fungicide fluazinam. A. versicolor Im6-50 was detected from the surface of daughter tubers and from the soil in which the inoculated seed tubers were cultivated by PCR using species-specific primers. The establishment of A. versicolor Im6-50 on the stolon of inoculated potato plants and in the rhizosphere is considered to contribute to the mechanism for disease suppression.     

Root infection of potato by Spongospora subterranea: knowledge review and evidence for decreased plant productivity

Information is reviewed on root infection of potato by the plasmodiophorid Spongospora subterranea f. sp. subterranea. This pathogen has long been recognized as the cause of root galls (hyperplasia) and the economically important disease powdery scab on tubers (modified stolons). The significance for plant productivity of the zoosporangium stages of the pathogen in potato roots has only recently begun to be documented. Two experiments are described that assessed effects of S. subterranea root infection on potato plant root function and productivity. A greenhouse experiment measured root function and plant parameters for eight potato cultivars with markedly different susceptibilities to tuber powdery scab. Water uptake and plant growth were reduced by S. subterranea inoculation in all eight cultivars. The magnitudes of these negative effects, and intensities of root hyperplasia, differed among the cultivars, but were not related to respective susceptibilities to tuber powdery scab. A field trial assessed root function and plant productivity for a cultivar (Iwa) that is very susceptible to Spongospora tuber and root diseases. Soil water content beneath uninoculated plants was consistently less than for inoculated plants, indicating that inoculation reduced water uptake (root function). Inoculation reduced shoot and root dry weights, and reduced weight of tubers per plant by 42%. Spongospora subterranea causes three diseases of potato: root membrane dysfunction, root hyperplasia and tuber powdery scab. The root diseases caused by the pathogen are likely to be important both for powdery scab management and for deleterious effects on potato crop yields.     

The potential for the Rapid Screening of Potato Cultivars (Solanum tuberosum) for Resistance to Powdery Scab (Spongospora subterranea) using a Laboratory Bioassay

Powdery scab of potato, once established in a field, is difficult to control because of the longevity of the resting spores (cystosori) of the causal organism, Spongospora subterranea f.sp. subterranea. Host resistance is likely to be the most efficient in a long-term control strategy for preventing build-up of field inoculum and spread of the disease. Resistance screening of potato cultivars is mostly done in laborious field trials where disease development is likely to be unpredictable. A bioassay with potato tissue cultured plantlets and cystosori as inoculum is described and was tested for its potential to screen potato cultivars at an early stage for their relative susceptibility to powdery scab by comparing the lab results with field data. With cystosori inoculum of Swiss origin, the laboratory test showed clear differences between the potato cultivars in the severity of zoosporangial root infection which correlated better with ranked tuber infection data, compared to root galling. There are apparent differences in the relative trends in susceptibility between roots and tubers of five selected cultivars when using naturally infested soil instead of prepared cystosori as inoculum in the lab bioassay. Furthermore, differences in the severity of zoosporangial root infection of two selected cultivars were found when cystosori from different countries where used as inoculum. A possible host genotype × pathogen interaction is discussed. The bioassay has the potential to screen and select for resistant material at an early breeding stage thus making field trials not unnecessary but more economical. It will allow the use of a standard set of pathogen collections and facilitate testing for inoculum virulence in infested soils.     

The importance of the infected seed tuber and soil inoculum in transmitting Potato mop‐top virus to potato plants

Potato mop‐top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV‐infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real‐time RT‐PCR that planting PMTV‐infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.     

Effect of soil inoculum level and environmental factors on potato powdery scab caused by Spongospora subterranea

The effects of soil inoculum level and three environmental factors (soil type, soil moisture regime and temperature) on the incidence and severity of powdery scab caused by Spongospora subterranea were investigated in potato plants grown under controlled environmental conditions. Symptoms of powdery scab on tubers were assessed visually, after which DNA was extracted from tuber peelings and quantified in a real-time polymerase chain reaction assay using primers and a TaqMan® probe specific to S. subterranea to establish tuber infection levels. Soil inoculum concentration of S. subterranea did not significantly affect the incidence and severity of either tuber infection or powdery scab symptoms at maturity. No significant differences in disease incidence and severity were found between sandy, loamy and clay soils, although the two lighter soils yielded more powdery scab than clay soil. Constant dampness of the soil resulted in significantly more disease than a fluctuating moisture regime. Infection and disease levels were high at all three temperatures tested (9, 12 and 17°C), but symptoms were most severe at 12°C. The percentage of plants with infected tubers did not increase after tuber initiation, although the amount of S. subterranea DNA detected in tubers and the severity of powdery scab symptoms increased in mature plants. Latent tuber infections were found to be common, especially under conditions suboptimal for disease development. This new information may be important for the prevention of powdery scab in potato-growing areas around the world.     

Somaclonal selection in potato for resistance to common scab provides concurrent resistance to powdery scab

Variant somaclones of potato cultivar Russet Burbank, selected for resistance to common scab using in vitro cell selection techniques, were tested for resistance to powdery scab, another important disease of potato caused by Spongospora subterranea. This pathogen also invades roots, producing root galls. Most variants consistently showed increased resistance to powdery scab, both in field and glasshouse challenge, when compared to the parental cultivar, several significantly so. On average, the best variant reduced powdery scab incidence by 51% and severity (tuber surface coverage) by 64%. In contrast, no improvement in the extent of root infection and root galling was seen. These results suggest host interactions during root and tuber infection are distinct. Correlation analyses of disease indices amongst the selected variants showed no association between Sp. subterranea root infection and gall scores, nor between root infection and tuber disease severity. However, a weak positive association was found between root gall score and tuber disease, and a strong correlation between tuber disease incidence and severity scores. Interestingly, positive correlations were also found between the extent of powdery and common scab resistance expressed and both incidence and severity of these diseases within the variants, suggesting a common defence mechanism. The role of thaxtomin A in selecting for concurrent resistance to both diseases is discussed.     

Detection and Quantification of Spongospora subterranea f. sp. subterranea in Soils and on Tubers Using Specific PCR Primers

PCR-based methods were developed for the detection and quantification of the potato pathogen Spongospora subterranea f. sp. subterranea (S. subterranea) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were chosen from the internal transcribed spacer regions. These primers amplified a 391bp product from S. subterranea DNA but did not amplify DNA from potato or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25×10^–5 cystosori or 1 zoospore per PCR. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenically micropropagated potatoes. A rapid method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3h and facilitated the detection of 1–5 cystosori per gram of soil. A PCR quantification technique was developed involving comparison of product ratios obtained after co-amplification of S. subterranea DNA along with an internal standard (competitor DNA fragment). This quantitative technique was also adapted for use in soil. PCR detection of S. subterranea in soil was considerably more sensitive than previously reported immunoassays and was quicker and easier than conventional bait plant bioassays. Such an assay could be useful for developing disease risk assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.     

Powdery scab resistance in Solanum tuberosum: an assessment of cultivar × environment effect

Powdery scab of potato caused by Spongospora subterranea is one of the main disease problems in many potato production regions of the world. However, no efficient and economically sound control method is currently available. Host resistance will be a key component of the integrated management of powdery scab, but there are discrepancies in published powdery scab resistance ratings of cultivars between countries. In order to identify the main factors causing such discrepancies, 10 reference cultivars thought to have a range of susceptibility to powdery scab and potato mop‐top virus were cropped over 4 years in four to six locations across Europe and disease levels on roots and tubers were assessed using standardized scoring scales. Soil contamination was tested using real‐time PCR and ELISA. The cultivars performed as expected according to previous characterization, with one exception. No relationship was found between tuber and root susceptibility. Assessment of powdery scab symptoms 1 month before harvest gave results comparable to those assessed 2 months after harvest. Neither real‐time PCR nor ELISA soil test results were closely related to disease index data. The field trial results indicate that different scoring methods are the main factor for the discrepancy in resistance ratings, and that environmental conditions and/or soil inoculum level play a minor role. Furthermore, there was either no difference between the pathogen populations in each location or the resistance of most of the cultivars is polygenic.     

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.     

The effect of post‐harvest storage conditions on the development of black dot (Colletotrichum coccodes) on potato in crops grown for different durations

The effects of post‐harvest curing and storage temperature on severity of black dot, caused by Colletotrichum coccodes, were investigated for potato crops grown for different crop durations (days from 50% emergence to harvest) in soils that posed a low, medium and high risk of disease. In field trials over four growing seasons (2005–8), black dot severity at harvest increased with increasing crop duration, within the range 103–146 days from 50% emergence to harvest (P < 0.05). In field trials over three growing seasons (2006–8), black dot severity on tubers at harvest increased significantly with increasing soil inoculum in each year, within the range 43–4787 pg C. coccodes DNA/g soil (P < 0.05). Storage trials were conducted to measure the influence of accumulated post‐harvest temperature on black dot. In 2005, no difference in black dot severity was observed on tubers stored for 20 weeks at 2.5 and 3.5 °C. In 2006 (but not 2007), increasing the duration of curing after harvest from 4 to 14 days increased black dot severity on tubers from 8.9 to 11.2% (P < 0.01) in long duration crops (>131 days after 50% emergence) grown under high (>1000 pg C. coccodes DNA/g soil) soil inoculum. The number of days of curing did not affect disease severity for shorter duration crops grown at high soil inoculum, or on crops grown at medium or low (100–1000 and <100 pg C. coccodes DNA/g soil, respectively) soil inoculum concentrations. Soil inoculum and crop duration together provided a reasonable prediction of black dot severity at harvest and after a 20‐week storage period.     

云南省马铃薯粉痂病的发生与危害调查分析

粉痂病严重影响马铃薯的产量和品质,是我国马铃薯产业健康发展的重要制约因素之一.为进一步探明马铃薯粉痂病在云南省的发生、分布及危害情况,本研究于2018年-2019年,在云南省内马铃薯不同生态种植区,开展了马铃薯粉痂病的发生与危害情况调查.调查范围涉及19个县(区)、涵盖了19个当地马铃薯主栽品种(系).调查结果显示:粉...     

Improved immunological detection of S<Emphasis Type="Italic">pongospora subterranea</Emphasis>

The genus Spongospora has two members which are important pathogens of vegetables, S. subterranea f.sp. subterranea (Sss) and S. subterranea f.sp. nasturtii (Ssn). The close taxonomic relationship of these formae speciales is based on similar cystosori morphology. The potato disease powdery scab, caused by Sss, is difficult to control. The key control measure is avoidance, aimed at planting clean seed in clean soil. For the development of routine tests for the presence of the pathogen on tubers and in soil, a monoclonal antibody (MAb) was developed using Sss cystosori as immunogen. It detected less than one Sss cystosorus and recognised Sss material from many parts of the world. No cross-reactions with other Plasmodiophoromycetes including Plasmodiophora brassicae, Polymyxa betae, Polymyxa graminis and different Streptomyces species causing common and netted scab of potatoes were observed. A novel tuber sample test method was developed using a kitchen peeling machine. This detected two tubers with one powdery scab lesion each in a sample including eighteen uninfected tubers. When soil samples spiked with cystosori were tested with the MAb, different Sss infestation levels could be discriminated. Ssn cystosori gave absorbance values in ELISA as high as Sss cystosori, whereas fresh crook roots of watercress containing Ssnzoosporangia and plasmodia or mud from an Ssninfected watercress bed gave low absorbance values or no reaction. The potential of these findings for the development of a disease control management are discussed.     

Factors affecting the incidence and severity of Spongospora subterranea infection and galling in potato roots

The incidence and severity of root infection and root galling caused by Spongospora subterranea were assessed in potato plants (cv. Estima) grown under controlled environmental conditions. The effects of temperature, soil type, soil moisture regime and soil inoculum level on infection and root gall development were determined by molecular and visual methods at two plant growth stages. Root gall severity was scored at harvest, after which DNA was extracted from the roots and quantified in a real-time polymerase chain reaction (PCR) assay specific for S. subterranea . Root galling was severe at 17°C, with a disease score of 3·1 on a 0–4 scale, low (0·6) at 12°C, and did not occur at 9°C. The level of inoculum in soil, in the form of artificially added sporosori, had no effect on the incidence and severity of visual symptoms, with 21%, 41% and 33% incidence observed at 5, 15 and 50 sporosori g⁻¹ soil, respectively. Incidence of infection, as detected by the real-time PCR assay, was greater with increasing soil inoculum concentrations, ranging from 48% at 5 sporosori g⁻¹ to 59% (15 sporosori g⁻¹) and 73% (50 sporosori g⁻¹) of plants infected at maturity, but this effect was not statistically significant. No correlation was found between the occurrence of galls on roots and powdery scab on tubers of the same plants.     

Infectivity,inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system1

Spongospora subterranea, causal agent of powdery scab of potatoes and vector of potato mop-top furovirus, survives in the soil as balls of resting spores (cystosori). So far, the factors affecting longevity, germination and infectivity of cystosori have not been investigated. A rapid and versatile bioassay with tomatoes as bait plants has been developed to quantify the infectivity of cystosorus inoculum or infested soil. The intensity of root infection, as a measure of infectivity, was determined by evaluating the quantity of zoosporangia present in epidermal cells and root hairs of the whole, stained root system. A correlation was obtained between the intensity of root infection and the cystosorus inoculum density in nutrient solution. Sterile soil suppressed the inoculum potential of pure cystosori. Infectivity of untreated soil decreased with increasing time of storage. Root infection was not influenced by the pH level of the nutrient solution.     

Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab

Infection by Spongospora subterranea of roots of two potato (Solanum tuberosum) cultivars, either very resistant or very susceptible to powdery scab on their tubers, was studied in a glasshouse experiment. Plants grown in sand/nutrient solution culture were inoculated with S. subterranea sporosori 2 weeks after planting. Plant parameters, the intensity of zoosporangium infection in roots, numbers of Spongospora root galls and amounts of Spongospora DNA in roots, measured using quantitative PCR (qPCR), were assessed at sequential harvests. Inoculation with S. subterranea reduced water use (56 days after planting) by 26% in the tuber resistant cultivar compared with uninoculated plants, and by 60% in the susceptible cultivar. Inoculation did not affect growth of the resistant cultivar, nor shoot mass of the susceptible cultivar, but caused a 38% reduction in root mass of the susceptible cultivar. The intensities of zoosporangium development in both cultivars were similar. The susceptible cultivar had approximately four times more Spongospora root galls g^?1 root mass than the resistant cultivar. Quantitative PCR detected S. subterranea DNA in roots 1 week after inoculation, and indicated a twofold greater amount of pathogen DNA in roots of the susceptible than the resistant cultivar. This study suggests that the S. subterranea zoosporangium stage in host roots is affected differently by host resistance factors than the sporosorus (root gall and tuber scab) stages. The study has also demonstrated the usefulness of qPCR for sensitive and consistent detection of S. subterranea across the duration of potato root infection.     

Effect of temperature on the transmission of Potato mop-top virus from seed tuber and by its vector, Spongospora subterranea

Potato mop-top virus (PMTV) causes disease in both the growing plant and tubers (spraing) of potato and is transmitted by the plasmodiophorid Spongospora subterranea , the cause of powdery scab. The effect of temperature during plant growth on the transmission of PMTV from infected seed tubers and from infested growing media was investigated in a series of glasshouse experiments. Symptoms developed on foliage of plants derived from infected seed tubers but none developed when PMTV was transmitted by S. subterranea in soil. The incidence of foliar symptoms was greatest on plants grown at 12°C, less at 16°C, few at 20°C and absent at 24°C. The transmission of PMTV from infected seed tubers was not significantly affected by temperatures between 12 and 24°C, but when the virus was transmitted by S. subterranea , minimal tuber infection occurred at 24°C and no differences were recorded at temperatures between 12 and 20°C. The incidence of powdery scab on tubers was greatest at 12 and 16°C and very low at 20 and 24°C. However, the incidence and severity of root galling caused by S. subterranea , was greatest at 20 and very low at 24°C. The incidence of powdery scab was greater on tubers of plants derived from infected seed tubers grown in a fluctuating temperature regime of 12 h at 20°C followed by 24 h at 12°C than on those grown at a constant 20°C, whereas the incidence of tuber infection by PMTV and spraing was similar for both regimes. This demonstrates that infection of roots can occur at a higher temperature than that for powdery scab on tubers and that this root infection can enable the transmission of PMTV into the potato plant.     

Evaluation of the <Emphasis Type="BoldItalic">Sss</Emphasis> AgriStrip rapid diagnostic test for the detection of <Emphasis Type="BoldItalic">Spongospora subterranea</Emphasis> on potato tubers

Spongospora subterranea, f.sp. subterranea (Sss), which causes powdery scab, is mainly spread through infected seed tubers and survives in contaminated soil for many years. The visual assessment of tuber lots by inspectors carries the risk of misidentification due to the difficulty of distinguishing lesions caused by either Sss or Streptomyces spp.. To avoid this, the “Sss AgriStrip”, a rapid and lab-independent test tool based on a lateral flow immunoassay has been developed, and we assessed its accuracy and sensitivity for detecting Sss. The Sss AgriStrip performed as well as other lab-based identification methods. The Sss AgriStrip, microscopy, ELISA, PCR, and real-time PCR techniques identified infection with S. subterranea in all tubers with typical powdery scab lesions. When lots with tubers showing a mixture of typical and atypical (suspicious) symptoms were tested, the presence of S. subterranea was confirmed in all lesions by all methods. The DNA content was generally lower in atypical than in typical lesions. Diverse and suspicious symptoms, which were difficult to assign to either powdery or common scab, tested negative with Sss AgriStrip and the other methods. This was despite microscopic observation of sporosori-like structures in some samples. Isolation and molecular identification confirmed that these lesions were mostly caused by Streptomyces spp. The Sss AgriStrip is as sensitive as DAS-ELISA with a detection limit between 1 and 10 sporosori per ml buffer. It is ideal for rapid and selective detection of Sss on farms and border inspection points to prevent spread of the pathogen.     

Relative importance of seed‐tuber and soilborne inoculum in causing black dot disease of potato

Controlled‐environment and field experiments were done to quantify the individual contribution of seed‐tuber and soilborne inoculum of Colletotrichum coccodes in causing black dot disease of potato tubers. Seed‐tuber and soilborne inocula of C. coccodes were quantified using an existing real‐time PCR assay and related to subsequent incidence and severity of disease. In four field trials, a controlled‐environment experiment and through the monitoring of 122 commercial crops, seed‐tuber inoculum was found to be relatively less important than soilborne inoculum in causing black dot, and the level of seed‐tuber inoculum did not significantly affect either the incidence or severity of disease or the percentage of progeny tubers deemed unmarketable. By contrast, soilborne inoculum had the potential to result in high levels of disease and the level of C. coccodes soil infestation (pg DNA g^?1 soil) was found to have a significant effect. At soil infestation levels below 100 pg DNA C. coccodes g^?1 soil, 7% of commercial crops had an incidence of black dot greater than 20%, increasing to 40% and 57% of crops at levels of 100–1000 pg g^?1 and >1000 pg g^?1 soil, respectively. These arbitrary threshold levels for soilborne inoculum related to disease risk are discussed. Interpretation of disease risk based on inoculum levels must, in the future, be informed by agronomic variables and potential control strategies.