Sugar Beet Leaf Spot Research Articles

Direct Polymerase Chain Reaction-Based Detection of Cercospora beticola in Field Soils.

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

Trichoderma as a potential biocontrol agent for Cercospora leaf spot of sugar beet

Leaf spot disease caused by Cercospora beticola Sacc. (class Ascomycota, ord. Dothideales, fam. Mycosphaerellaceae) is the most destructive foliar disease of sugar beet. Commercial varieties are partially resistant and require repeated fungicide applications to obtain adequate protection levels; this has a high environmental impact and a risk of selecting resistant pathogen strains. A way of reducing chemical inputs could be to use biocontrol agents to replace or supplement fungicide treatments. A well-known class of biological control agents is represented by the fungi belonging to the Trichoderma genus (class Ascomycota, ord. Hypocreales, fam. Hypocreaceae), but there is a lack of information about its behaviour towards C. beticola. This study reports the evaluation of several Trichoderma isolates as possible biocontrol agents of this pathogen. Preliminary in vitro and in vivo assays led to the selection of two Trichoderma isolates characterised by their ability to reduce pathogen sporulation and antagonism towards the pathogen or competence for sugar beet phyllosphere. Repeated foliar applications of the liquid culture homogenate preceded by a single treatment of difenoconazole in 2 year trials under natural inoculum in field reduced the disease incidence and pathogen sporulation from the necrotic spots. An increase in sugar yield was also obtained by means of isolate Ba12/86-based treatments, perhaps due to induced resistance effects.

Curtobacterium flaccumfaciens pv. beticola, A New Pathovar of Pathogens in Sugar Beet.

Bacterial leaf spot of sugar beet was first discovered in 1995 in Inner Mongolia of China. The pathogen was shown to be a bacterium with properties of gram-positive bacteria: small irregular rods, lateral flagella, aerobic, and catalase-positive. The colonies of sugar beet strains produced a pale-yellow pigment. The optimum temperature for the bacteria to grow was 24 to 27°C. The bacteria could utilize a wide range of organic compounds, including hydrolyzed casein, starch, esculin and Tween 80, and released H2S from cysteine, cystine, and Na2S2O3·5H2O, but could not produce urease, oxidase, or indole. The cell wall peptidoglycan was based on ornithine (type B2β). The predominant menaquinone was MK-9. Polar lipids contained several glycosyldiacyl-glycerols. The DNA G+C content of a type strain of the new pathovar, T30T, was 67.5%. DNA-DNA homology between T30T and Curtobacterium flaccumfaciens pv. flaccumfaciens (International Collection of Micro-Organisms from Plants, New Zealand [ICMP] 2584) was 70.1%. The new pathovar and C. flaccumfaciens pv. flaccumfaciens had 99.9% identity in DNA sequence of 16S rRNA. Close genetic relatedness was observed for the representatives of the species Curtobacterium flaccumfaciens, but a low level of similarity between the different pathovars was found. Based on these physiological, biochemical, chemotaxonomic, phylogenetic, and genetic characteristics, we demonstrate that the pathogen represents a new pathovar of C. flaccumfaciens, for which we propose the name Curtobacterium flaccumfaciens pv. beticola pv. nov. The type strain is T30T (=ATCC BAA-144T).

Biocontrol Elicited Systemic Resistance in Sugarbeet is Salicylic Acid Independent and NPR1 Dependent

Systemic acquired resistance is an innate defense response in plants to a broad array of pathogens. Salicylic acid (SA) and non-expressor of pathogenesis-related genes1 (NPR1) are key players in systemic acquired resistance. Previously we demonstrated the biological control agent Bacillus mycoides isolate BmJ controls Cercospora leaf spot of sugarbeet through induction of pathogenesis-related (PR) proteins and biphasic hydrogen peroxide production. Both of these factors are associated with SA- and NPR1-dependent resistance induction. Here we show BmJ treatment leads to monomerization of NPR1, a phenomenon observed with activation of defense in Arabidopsis. The identity of the NPR1 monomer was confirmed using MALDI-TOF-MS. In contrast to the Arabidopsis system, monomerization of NPR1 in sugarbeet is not preceded by SA accumulation when triggered by BmJ. The SA levels following BmJ treatment were equivalent with water and Bacillus pumulis isolate BMH5E-33 treatments. BMH5E-33 is a non-inducer of resistance. The timeline of NPR1 activation by BmJ (3 hours post application) coincides with the previously observed secondary burst of hydrogen peroxide elicited in sugarbeet by BmJ treatment. These observations suggest PR-proteins induced by BmJ are induced through a SA-independent, NPR1-dependent pathway similar to that reported for the systemic resistance inducer

Spatial pattern of Cercospora leaf spot of sugar beet in fields in long- and recently-established areas

Spatial disease pattern of Cercospora beticola was characterised during natural epidemics of Cercospora leaf spot (CLS) in sugar beet. We applied linear regression and geostatistical analyses to characterise CLS spatial patterns in three field trials, in long-established and recently-established CLS-areas, during two consecutive years. Linear regression showed a positive influence of average disease severity of within-row neighbouring plants (0.38 < \(\hat \beta_1 \) < 0.88). Semi-variograms modelled the spatial dependence of disease severity for two directions per week in both years. Disease severity displayed strong spatial dependence over time. The within-row spatial dependence was the largest, but across-row dependence was irregular and weaker. Both long- and recently established areas showed strong spatial dependence of disease severity within row, decrease in variability between years and within the second trial year and a relation between \(\hat \beta _1 \) and the relative nugget. Observed differences were more field than area specific. These spatial and temporal analyses indicated that disease severities of adjacent plants were dependent; hence, we concluded that C. beticola is dispersed mainly over short distances from plant to plant.

Comparative Efficacy, Selection of Effective Partners, and Application Time of Strobilurin Fungicides for Control of Cercospora Leaf Spot of Sugar Beet.

In this study, we attempt to optimize the use of strobilurin fungicides by testing the efficacy of azoxystrobin, kresoxim-methyl, pyraclostrobin, and trifloxystrobin under field conditions, by testing for the most efficient partners in fungicide mixtures, and by testing control efficacy of strobilurin fungicides applied at several application times to determine the better options for disease management. Results showed that trifloxystrobin was the most efficient strobilurin fungicide, followed by pyraclostrobin. Azoxystrobin provided a modest to poor control efficacy, whereas kresoxim-methyl provided only poor disease control efficacy. Mixtures of azoxystrobin and trifloxystrobin with either chlorothalonil or maneb and difenoconazole or flutriafol were tested for their efficacy in controlling the disease. The results showed that the azoxystrobin-containing mixtures provided significantly better control compared with that obtained by single applications of each mixture component. The mixtures of trifloxystrobin with maneb or with difenoconazole or flutriafol provided control efficacy similar to that obtained by single applications of trifloxystrobin, whereas the mixture of trifloxystrobin and chlorothalonil provided significantly lower control efficacy compared with the other trifloxystrobin-containing mixtures tested. For both strobilurin fungicides tested, the calculated ratio between the observed and the expected control efficacy ranged around the value of 1, suggesting additive interactions between the mixtures' components. To determine the most appropriate time for strobilurin fungicides application, trifloxystrobin was applied as the first two, the middle two, or the final two consecutive treatments of six fungicide applications. The remaining fungicide treatments in the spray schedules were carried out by applying the systemic fungicide difenoconazole. Results showed that a higher control efficacy was obtained when trifloxystrobin was applied in either of the earlier applications.

Analysis of β‐tubulin Gene Fragments from Benzimidazole‐sensitive and ‐tolerant Cercospora beticola

AbstractCercospora leaf spot of sugar beet, caused by the fungus Cercospora beticola, is a major foliar pathogen on sugar beet. Fungicide sprays have been used extensively to manage Cercospora leaf spot, including the benzimidazole fungicides. Resistance to benzimidazoles has been observed in isolates of C. beticola. The precise genetics of this resistance is not known in this fungus. We tested benzimidazole‐tolerant and ‐sensitive isolates and found a single mutation in the β‐tubulin gene of benzimidazole‐tolerant isolates that corresponds to a mutation known to confer benzimidazole tolerance in other ascomycetes. This mutation is predicted to cause a change from glutamic acid to alanine in the protein product. Isolates containing this mutation further show an increased sensitivity to an N‐phenylcarbamate, as would be predicted based on the mutant phenotype found in other filamentous fungi. Only a single mutation was found in isolates from different regions of the United States, isolated in different growing seasons.

Comparison of Visual and Multispectral Radiometric Disease Evaluations of Cercospora Leaf Spot of Sugar Beet.

Visual assessments of disease severity are currently the industry standard for quantification of the necrosis caused by Cercospora beticola on sugar beet (Beta vulgaris) leaves. We compared the precision, reproducibility, and sensitivity of a multispectral radiometer to visual disease assessments. Individual wavebands from the radiometer, as well as vegetative indices calculated from the individual wavebands, were compared with visual disease estimates from two raters at each of two research sites. Visual assessments and radiometric measurements were partially repeated immediately after the first assessment at each site. Precision, as measured by reduced coefficients of variation, was improved for all vegetative indices and individual waveband radiometric measures compared with visual assessments. Visual assessments, near-infrared singlewaveband reflectance values, and four of the six vegetative indices had high treatment F values, suggesting greater sensitivity at discriminating disease severity levels. Reproducibility, as measured by a test-retest method, was high for visual assessments, single-waveband reflectance at 810 nm, and several of the vegetative indices. The use of radiometric methods has the potential to increase the precision of assessments of Cercospora leaf spot foliar symptoms of sugar beet while eliminating potential bias. We recommend this method be used in conjunction with visual disease assessments to improve precision of assessments and guard against potential bias in evaluations.

Sugarbeet leaf spot disease (Cercospora beticola Sacc.)†

SUMMARY Leaf spot disease caused by Cercospora beticola Sacc. is the most destructive foliar pathogen of sugarbeet worldwide. In addition to reducing yield and quality of sugarbeet, the control of leaf spot disease by extensive fungicide application incurs added costs to producers and repeatedly has selected for fungicide-tolerant C. beticola strains. The genetics and biochemistry of virulence have been examined less for C. beticola as compared with the related fungi C. nicotianae, C. kikuchii and C. zeae-maydis, fungi to which the physiology of C. beticola is often compared. C. beticola populations generally are not characterized as having race structure, although a case of race-specific resistance in sugarbeet to C. beticola has been reported. Resistance currently implemented in the field is quantitatively inherited and exhibits low to medium heritability. Cercospora beticola Sacc.; Kingdom Fungi, Subdivision Deuteromycetes, Class Hyphomycetes, Order Hyphales, Genus Cercospora. Circular, brown to red delimited spots with ashen-grey centre, 0.5-6 mm diameter; dark brown to black stromata against grey background; pale brown unbranched sparingly septate conidiophores, hyaline acicular conidia, multiseptate, from 2.5 to 4 microm wide and 50-200 microm long. Propagative on Beta vulgaris and most species of Beta. Reported on members of the Chenopodiaceae and on Amaranthus. Disease symptoms: Infected leaves and petioles of B. vulgaris exhibit numerous circular leaf spots that coalesce in severe cases causing complete leaf collapse. Dark specks within a grey spot centre are characteristic for the disease. Older leaves exhibit a greater number of lesions with larger spot diameter. During the latter stage of severe epiphytotics, new leaf growth can be seen emerging from the plant surrounded by prostrate, collapsed leaves. Fungicides in the benzimidazole and triazole class as well as organotin derivatives and strobilurins have successfully been used to control Cercospora leaf spot. Elevated levels of tolerance in populations of C. beticola to some of the chemicals registered for control has been documented. Partial genetic resistance also is used to reduce leaf spot disease.

Detection of resistance to sterol demethylation-inhibiting (DMI) fungicides inCercospora beticola and efficacy of control of resistant and sensitive strains with flutriafol

Single-lesion isolates ofCercospora beticola (n=150) were collected in 1998 from sugar beet fields in the area of Serres, N. Greece. In this area, sterol demethylation-inhibiting (DMI) fungicides have been used for almost 20 years to control sugar beet leaf spot. The sensitivity of these isolates to the DMI fungicides flutriafol and difenoconazole (EC50 values) was determined on the basis of inhibition of mycelial growth at several fungicide concentrations. The relative growth (RG) of isolates was correlated at all tested concentrations with the respective EC50 values, indicating that RG provides a reliable estimate for the sensitivity of the isolates. The highest correlation coefficients were obtained for concentrations of 1 µg ml−1 flutriafol and of 0.05 µg ml−1 difenoconazole, respectively. Consequently, they are proposed for monitoring of DMI sensitivity inC. beticola populations, as single discriminatory concentrations in a simplified test method. Based on the RG values at the discriminatory concentration of 1 µg ml−1 flutriafol,C. beticola isolates were classified as either resistant or sensitive. The efficacy of flutriafol, applied at the commercially recommended dose, in controlling Cercospora leaf spot was examined in field experiments conducted during 1999 and 2000. Disease incidence in plots artificially inoculated with resistant isolates and treated with flutriafol was significantly higher than in similar plots inoculated with sensitive strains. These results suggest that poor disease control after application of flutriafol may be based on the presence of resistant strains within the pathogen population in northern Greece. This emphasizes the risk of the development of practical resistance if there is increased frequency of such strains within the population.

Spatial and temporal population dynamics of a phyllosphere colonizing Bacillus subtilis biological control agent of sugar beet cercospora leaf spot

Abstract This study examines the spatial and temporal variation of populations of the biological control agent (BCA) BacB, a Bacillus subtilis isolate, in the field and growth chamber in the presence of the fungus, Cercospora beticola , the causal agent of Cercospora leaf spot of sugarbeet. The use of the selective BCA support substrate β-glucan, applied at 0, 0.5, and 1.0% of the spray solution, did not influence differences in total population numbers (spores + vegetative cells) of a spontaneous rifampicin resistant isolate of BacB (Rif+) over a 14 day spray period. BacB Rif+, applied as a spore formulation, declined from 10,000 CFU/cm 2 on day 0.5–100 CFU/cm 2 on day 14 at the three levels of β-glucan tested. Distribution of BacB Rif+ populations was modeled on a leaf scale, with and without β-glucan. Higher populations of vegetative cells were more likely at 14 days with 1% β-glucan than with 0% β-glucan. BacB populations were more aggregated without β-glucan than with the nutrient substrate. There was no correlation between BacB density and Cercospora leaf spot disease severity, indicating that neither antibiosis nor parasitism is likely an important mechanism of disease control.

Effect of Low-Volume Application of Fungicides on Control of Cercospora Leaf Spot of Sugar Beet

Effect of Low-Volume Application of Fungicides on Control of Cercospora Leaf Spot of Sugar Beet

Cercospora beticola Tolerant to Triphenyltin Hydroxide and Resistant to Thiophanate Methyl in North Dakota and Minnesota

Triphenyltin hydroxide (TP'I H) has been used exten­ sively for control of Cercospora (Cercospora betiola) leaf spot of sugarbeet (Beta vulgaris) in Minnesota and North Dakota foll owing the development of benz­ imidazole resistant strains in t he early 19808. The discovery oftolerance to TPTH in 1994 prompted ex­ tensive sampling throughout the region in 1995 and 1996. In 1995, 60% of the leaf spots in the southern most district were tolerant to 0.2ppm TPTH and 42% tolerant to Ippm. By 1996 these frequencies had increased to 83 and 60%, respectively. More alarm­ ing than this increase in the southern district was the rapid increase in the occurrence of tolerance fu rther north where the disease is generally less severe and fungicide use is less. In four of the seven factory dis­ tricts the frequency of leaf spots tolerant to O.2ppm exceeded 35% and the frequency tolerant to 1 ppm was greater than 15%, in 1996. Resistance to thio­ phanate-methyl, a benzimidazole-type fungicide, per­ sisted in local populations even though TPTB has been the predominant fungicide for control of Cer­ cospora leaf spot for about 15 years.

Cercospora Leaf Spot of Sugar Beet: the Present State of Occurrence and Its Protection

Cercospora Leaf Spot of Sugar Beet: the Present State of Occurrence and Its Protection

CERCOPRI: a forecasting model for primary infections of cercospora leaf spot of sugarbeet1

A computerized model for forecasting the appearance of primary infections of cercospora disease on sugarbeet was worked out using meteorological and epidemiological data collected in the western Po Valley from 1980 to 1989. This program, called CERCOPRI, gives a figure for the current risk of disease appearance, a forecast of the risk on a pre‐established day or a forecast of the day on which risk will reach a pre‐established threshold.

Fungicide Efficacy and Yield Responses to Fungicide Treatments Based on Predicitons of Cercospora Leaf Spot of Sugar Beet

Fungicide Efficacy and Yield Responses to Fungicide Treatments Based on Predicitons of Cercospora Leaf Spot of Sugar Beet

Epidemiological studies on cercospora leaf spot of sugar beet

In the glasshouse, inoculation of sugar beet with Cercospora beticola followed by 16 h of high humidity produced visible disease only with at least four conidia per cm2 of leaf area. Disease became more severe after increasing periods of high humidity in the range of 0–24 h. In the field, spraying plants with water enhanced disease spread from a focus. Disease progress curves were sigmoid. Apparent infection rate declined towards the end of the season, possibly because of high temperature. In approximate agreement with prediction, epidemic development was delayed when initial inoculum was reduced. Reduced infection, resulting from either reduced initial inoculum or delayed inoculation, decreased the adverse effect of disease on sugar yield.

Field Evaluation of Various Fungicides to Control Cercospora Leaf Spot of Sugarbeet, Caused by Benomyl-Resistant Strains

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Stemphylium leaf spot of sugar beet.

A new leaf spot of sugar beet was detected in Tokoro-cho, Hokkaido in late July 1974. The disease was restricted to the sugar beet plants adjacent to garlic fields where garlic tip blight occurred severely. The causal fungus was identified as Pleospora herbarum (Fries) Rabenhorst (anamorph: Stemphylium botryosum Wallroth), and the name of “Stemphylium leaf snot” of sugar beet was proposed for this disease.

Control of Cercospora leaf spot of sugarbeet through aerial application of systemic and surface-protecting fungicides

Control of Cercospora leaf spot of sugarbeet through aerial application of systemic and surface-protecting fungicides