Impact of preconditioning temperature and duration period on carpogenic germination of diverse Sclerotinia sclerotorium (Lib.) de Bary populations in south-western Australia

Plant Disease ◽  
2020 ◽  
Author(s):  
Pippa J Michael ◽  
King Yin Lui ◽  
Linda Thomson ◽  
Ashmita Lamichhane ◽  
Sarita J Bennett
Keyword(s):  
Severe Disease ◽  
Population Diversity ◽  
Sclerotinia Stem Rot ◽  
Carpogenic Germination ◽  
Time Period ◽  
Broad Leaf ◽  
Mycelial Compatibility ◽  
Conditioning Temperature ◽  
Significant Yield ◽  
C 50

The soil-borne pathogen Sclerotinia sclerotorium is the causal agent of sclerotinia stem rot, a severe disease of broad-leaf crops including canola/rapeseed Brassica napus that can result in significant yield losses. Sclerotia, the hard melanized resting structure of the pathogen, requires preconditioning before carpogenic germination can occur. We investigated the effect of pre-conditioning temperature (4°C, 20°C, 35°C, 50°C and field conditions) and duration (0, 30, 60, 120, 179, 240, 301 days) on germination of S. sclerotorium sclerotia collected from five canola fields in the south-western Australian grain-belt. The ecological diversity of each population was characterised using mycelial compatibility groups (MCGs) typing. No response was observed for isolates conditioned at 4°C at any time period indicating chilling is not a preconditioning requirement for these isolates. Sclerotia required preconditioning for a minimum of 60 days before any significant increase in germination occurred, with no further increases in germination recorded in response to longer conditioning after 60 days. The highest germination was observed in sclerotia conditioned at 50°C. The MCG results indicated significant within and between population diversity suggesting local adaptation to different environments as well as ensuring the ability to respond to seasonal variation between years.

scholarly journals Carpogenic Germinability of Diverse Sclerotinia sclerotiorum Populations Within the Southwestern Australian Grain Belt

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 2891-2897
Author(s):  
Pippa J. Michael ◽  
King Yin Lui ◽  
Linda L. Thomson ◽  
Katia Stefanova ◽  
Sarita J. Bennett
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Temperature Fluctuations ◽  
Sclerotinia Stem Rot ◽  
Dark Cycle ◽  
Diurnal Temperature ◽  
Population Responses ◽  
Carpogenic Germination ◽  
Southwestern Australia ◽  
Temperature Regimes ◽  
Mycelial Compatibility

Sclerotinia stem rot, caused by the necrotrophic plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary, is a major disease of canola and pulses in Australia. Current disease management relies greatly on cultural and chemical means of control. Timing of fungicide applications remains a challenge, because efficacy is dependent on accurate prediction of ascospore release and presence on the plant. The aims of this study were to determine the optimal temperature for carpogenic germination of S. sclerotiorum populations sampled from canola and lupin fields in southwestern Australia and characterize diversity using mycelial compatibility groupings (MCGs). Sclerotia were collected from four diseased canola and one diseased lupin field from across southwestern Australia. Forty sclerotia from each population were incubated at four alternating temperatures of 30/15, 20/15, 20/4, and 15/4°C (12-h/12-h light/dark cycle) and assessed every 2 to 3 days for a 180-day period. MCG groupings for populations were characterized using 12 reference isolates. Results indicated the time to initial carpogenic germination decreased as diurnal temperature fluctuations decreased, with a fluctuation of 5°C (20/15°C) having the most rapid initial germination followed by 11°C (15/4°C) followed by 16°C (20/4°C). Optimal germination temperature for all five populations was 20/15°C; however, population responses to other diurnal temperature regimes varied considerably. No germination was observed at 30/15°C. MCG results indicate extensive diversity within and between populations, with at least 40% of sclerotia within each population unable to be characterized. We suggest that this diversity has enabled S. sclerotiorum populations to adapt to varying environmental conditions within southwestern Australia.

scholarly journals Analytical and diagnostic performances of a high-throughput immunoassay for SARS-CoV-2 IgM and IgG

2020 ◽  
Author(s):  
Andrea Padoan ◽  
Chiara Cosma ◽  
Paolo Zaupa ◽  
Mario Plebani
Keyword(s):  
Sensitivity And Specificity ◽  
Symptom Onset ◽  
Severe Disease ◽  
Onset Time ◽  
Time Frame ◽  
Serum Samples ◽  
Time Period ◽  
Analytical Imprecision ◽  
Range Of Values

BackgroundAbstractReliable SARS-CoV-2 serological assays are required for diagnosing infections, for the serosurveillance of past exposures and for assessing the response to future vaccines. In this study, the analytical and clinical performances of a chemiluminescent immunoassays for SARS-CoV-2 IgM and IgG detection (Mindray CL-1200i), targeting Nucleocapsid (N) and receptor binding domain (RBD) portion of the Spike protein, were evaluated.MethodsPrecision and linearity were evaluated using standardized procedures. A total of 157 leftover serum samples from 81 hospitalized confirmed COVID-19 patients (38 with moderate and 43 with severe disease) and 76 SARS-CoV-2 negative subjects (44 healthcare workers, 20 individuals with rheumatic disorders, 12 pregnant women) were included in the study. In an additional series of 44 SARS-CoV-2 positive, IgM and IgG time kinetics were also evaluated in a time-period of 38 days.ResultsPrecision was below or equal to 4% for both IgM and IgG, in all the studied levels, whilst a slightly significant deviation from linearity was observed for both assays in the range of values covering the manufacturer’s cut-off. Considering a time frame ≥ 12 days post symptom onset, sensitivity and specificity for IgM were 92.3% (95%CI:79.1%-98.4%) and 92.1% (95%CI:83.6%-97.0%). In the same time frame, sensitivity and specificity for IgG were 100% (95%CI:91.0%-100%) and 93.4% (95%CI:85.3%-97.8%). The assays agreement was 73.9% (Cohen’s kappa of 0.373). Time kinetics showed a substantial overlapping of IgM and IgG response, the latter values being elevated up to 38 days from symptoms onset.ConclusionsAnalytical imprecision is satisfactory as well as the linearity, particularly when taking into account the fact that both assays are claimed to be qualitative. Diagnostic sensitivity of IgG was excellent, especially considering specimens collected ≥12 days post symptom onset. Time kinetics suggest that IgM and IgG are detectable early in the course of infection, but the role of SARS-CoV-2 antibodies in clinical practice still requires further evaluations.

scholarly journals First Report of Sclerotinia Stem Rot and Watery Soft Rot Caused by Sclerotinia sclerotiorum on Sand Rocket (Diplotaxis tenuifolia) in Italy

Plant Disease ◽  
2005 ◽  
Vol 89 (11) ◽  
pp. 1241-1241 ◽  
Author(s):  
A. Garibaldi ◽  
A. Minuto ◽  
M. L. Gullino
Keyword(s):  
Relative Humidity ◽  
Sclerotinia Sclerotiorum ◽  
Severe Disease ◽  
Soil Surface ◽  
Soft Rot ◽  
Sclerotinia Stem Rot ◽  
First Report ◽  
Initial Symptoms ◽  
Stem Necrosis ◽  
White Mycelium

Several species of Diplotaxis (D. tenuifolia, D. erucoides, and D. muralis), known as wild or sand rocket, are widely cultivated in Italy. Rocket is used in Mediterranean cuisine as salad, a component of packaged salad products, and as a garnish for food. In winter 2003, a severe disease was observed on D. tenuifolia grown in unheated glasshouses on commercial farms near Albenga in northern Italy. Initial symptoms included stem necrosis at the soil level and darkening of leaves. As stem necrosis progressed, infected plants wilted and died. Wilt, characterized by the presence of soft and watery tissues, occurred within a few days on young plants. The disease was extremely severe in the presence of high relative humidity and mild temperature (15°C). Necrotic tissues became covered with white mycelium that produced dark sclerotia. Diseased stem tissue was disinfested for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 100 ppm streptomycin sulfate. Sclerotinia sclerotiorum (1) was consistently recovered from infected stem pieces. Sclerotia observed on infected plants measured 1.23 to 3.00 × 1.40 to 5.38 mm (average 2.10 × 2.85 mm). Sclerotia produced on PDA measured 1.00 to 4.28 × 1.00 to 6.01 mm (average 2.38 × 3.23 mm). Pathogenicity of three isolates obtained from infected plants was confirmed by inoculating 30-day-old plants of D. tenuifolia grown in 18-cm-diameter pots in a glasshouse. Inoculum, 2 g per pot of wheat kernels infested with mycelium and sclerotia of each isolate, was placed on the soil surface around the base of each plant. Three replicates of five pots each were used per isolate. Noninoculated plants served as controls. The inoculation trial was repeated once. All plants were kept at temperatures ranging between 10 and 26°C (average 15°C) with an average relative humidity of 80% and were watered as needed. Inoculated plants developed symptoms of leaf yellowing within 12 days, soon followed by the appearance of white mycelium and sclerotia, and eventually wilted. Control plants remained symptomless. S. sclerotiorum was reisolated from inoculated plants. To our knowledge, this is the first report of infection of D. tenuifolia by S. sclerotiorum in Italy as well as worldwide. The disease currently has been observed in the Liguria Region but not yet in other areas where sand rocket is cultivated. The economic importance of this disease for the crop can be considered medium at the moment, but is expected to increase in the future. Reference: (1) N. F. Buchwald. Den. Kgl. Veterin.er-og Landbohojskoles Aarsskrift, 75, 1949.

scholarly journals Favorable Bioactivity of the SDHI Fungicide Benzovindiflupyr Against Sclerotinia sclerotiorum Mycelial Growth, Sclerotial Production, and Myceliogenic and Carpogenic Germination of Sclerotia

Plant Disease ◽  
2019 ◽  
Vol 103 (7) ◽  
pp. 1613-1620 ◽  
Author(s):  
Xue-ping Huang ◽  
Jian Luo ◽  
Yu-fei Song ◽  
Bei-xing Li ◽  
Wei Mu ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Mycelial Growth ◽  
Disease Incidence ◽  
Stem Rot ◽  
Sclerotinia Stem Rot ◽  
Baseline Sensitivity ◽  
Carpogenic Germination ◽  
Alternative Product ◽  
Application Potential ◽  
New Generation

Sclerotinia sclerotiorum, which can cause Sclerotinia stem rot, is a prevalent plant pathogen. This study aims to evaluate the application potential of benzovindiflupyr, a new generation of succinate dehydrogenase inhibitor (SDHI), against S. sclerotiorum. In our study, 181 isolates collected from different crops (including eggplant [n = 34], cucumber [n = 27], tomato [n = 29], pepper [n = 35], pumpkin [n = 32], and kidney bean [n = 25]) in China were used to establish baseline sensitivity to benzovindiflupyr. The frequency distribution of the 50% effective concentration (EC50) values of benzovindiflupyr was a unimodal curve, with mean EC50 values of 0.0260 ± 0.011 μg/ml, and no significant differences in mean EC50 existed among the various crops (P > 0.99). Benzovindiflupyr can effectively inhibit mycelial growth, sclerotial production, sclerotial shape, and myceliogenic and carpogenic germination of the sclerotia of S. sclerotiorum. In addition, benzovindiflupyr showed good systemic translocation in eggplant. Using benzovindiflupyr at 100 μg/ml yielded efficacies of 71.3 and 80.5% for transverse activity and cross-layer activity, respectively, which were higher than those of acropetal and basipetal treatments (43.6 and 44.7%, respectively). Greenhouse experiments were then carried out at two experimental sites for verification. Applying benzovindiflupyr at 200 g a.i. ha−1 significantly reduced the disease incidence and severity of Sclerotinia stem rot. Overall, the results demonstrated that benzovindiflupyr is a potential alternative product to control Sclerotinia stem rot.

scholarly journals Effect of some fungicides against the growth inhibition of Sclerotinia sclerotiorum mycelial compatibility groups

2015 ◽  
Vol 55 (4) ◽  
pp. 354-361 ◽  
Author(s):  
Alireza Dalili ◽  
Saeed Bakhtiari ◽  
Hossein Barari ◽  
Majid Aldaghi
Keyword(s):  
Growth Inhibition ◽  
Sclerotinia Sclerotiorum ◽  
Integrated Management ◽  
In Vitro Tests ◽  
Sclerotinia Stem Rot ◽  
Mazandaran Province ◽  
Brassica Napus L ◽  
Northern Iran ◽  
Mycelial Compatibility

Abstract Sclerotinia sclerotiorum (Lib.) de Bary, the causal agent of Sclerotinia stem rot, is one of the most important pathogens of Brassica napus L. in northern Iran. In this study, 13 mycelial compatibility groups (MCGs) of the fungus were identified among 31 isolates sampled from four regions of Mazandaran province, Iran. Effective fungicides are useful in the integrated management of the disease. So, the effect of tebuconazole, propiconazole, cyproconazole, and Rovral-TS at five doses (0.0001, 0.001, 0.01, 0.1, and 1 ppm) was studied on the growth inhibition of S. sclerotiorum as in vitro tests. Maximum inhibition (100%) of S. sclerotiorum mycelial growth was obtained by the highest dose (1 ppm) of all tested fungicides, as well as by the doses of 0.1 and 0.01 ppm of propiconazole, cyproconazole, and tebuconazole. In this investigation, the reaction of S. sclerotiorum isolates belonging to different MCGs was evaluated against tebuconazole, propiconazole, cyproconazole, and Rovral-TS at their EC50 ranges. The results revealed that there was high variation of S. sclerotiorum MCGs against different fungicides. The inhibition percentage varied between 4.29% and 71.72%.

scholarly journals Mycelial Compatibility Grouping and Aggressiveness of Sclerotinia sclerotiorum

Plant Disease ◽  
2004 ◽  
Vol 88 (4) ◽  
pp. 325-332 ◽  
Author(s):  
L. S. Kull ◽  
W. L. Pedersen ◽  
D. Palmquist ◽  
G. L. Hartman
Keyword(s):  
Population Structure ◽  
Disease Management ◽  
Sclerotinia Sclerotiorum ◽  
Stem Rot ◽  
Management Systems ◽  
Sclerotinia Stem Rot ◽  
Pathogen Population ◽  
Causal Organism ◽  
Soybean Field ◽  
Mycelial Compatibility

Population variability of Sclerotinia sclerotiorum, the causal organism of Sclerotinia stem rot of soybean, was determined by mycelial compatibility grouping (MCG) and isolate aggressiveness comparisons. MCG and aggressiveness of S. sclerotiorum isolates from diverse hosts and geographic locations (Diverse Set, 24 isolates), from a soybean field in Argentina (Argentine Set, 21 isolates), and from soybean fields in DeKalb and Watseka, Illinois (DeKalb Set, 124 isolates, and Watseka Set, 130 isolates) were assessed. Among 299 isolates tested, 42 MCGs were identified, and 61% were represented by single isolates observed at single locations. Within the Diverse Set, 17 MCGs were identified; 1 MCG consisted of six isolates, and 16 MCGs consisted of one isolate each. Nine MCGs were identified within the Argentine field with two MCGs composed of either five or six isolates, two MCGs composed of two isolates, and the remaining composed of one isolate each. Each Illinois field was a mosaic of MCGs, but MCG frequencies differed between the two fields. Common MCGs were identified among the Diverse, DeKalb, and Watseka Sets, but no MCGs within the Argentine Set were observed with other sets. MCG 8 was the most frequently sampled and widely dispersed MCG and occurred at a frequency of 29, 36, and 62% in the Diverse, DeKalb, and Watseka Sets, respectively. Variation in isolate aggressiveness was assessed using a limited-term, plug inoculation technique. Isolate aggressiveness varied (P = 0.001) within the Diverse, Argentine, DeKalb, and Watseka Sets. Within widely dispersed MCGs, isolate aggressiveness varied (P ≤ 0.10); however, within locally observed MCGs detected only in single fields, isolate aggressiveness did not vary. Additionally, individual MCGs within the DeKalb and Watseka Sets differed in isolate aggressiveness. Using six soybean cultivars and six S. sclerotiorum isolates, no cultivar-isolate interaction was detected, but resistant and susceptible cultivars performed similarly when inoculated with either less or highly aggressive isolates. Pathogen population structure and variability in isolate aggressiveness may be important considerations in disease management systems.

scholarly journals Infection Courts in Watermelon Plants Leading to Seed Infestation by Fusarium oxysporum f. sp. niveum

2017 ◽  
Vol 107 (7) ◽  
pp. 828-833 ◽  
Author(s):  
Aparna Petkar ◽  
Pingsheng Ji
Keyword(s):  
Fusarium Oxysporum ◽  
Real Time ◽  
Field Experiments ◽  
Severe Disease ◽  
Transmitted Disease ◽  
Field Conditions ◽  
Pcr Analysis ◽  
Immature Fruit ◽  
Significant Yield ◽  
Seed Infestation

Fusarium wilt incited by Fusarium oxysporum f. sp. niveum is a seed-transmitted disease that causes significant yield loss in watermelon production. The pathogen may infect watermelon seeds latently, which can be an important inoculum source and contribute to severe disease outbreak. However, information regarding infection courts of F. oxysporum f. sp. niveum leading to infestation of watermelon seeds is limited. To determine how seeds in watermelon fruit can be infested by F. oxysporum f. sp. niveum during the watermelon growing season, greenhouse and field experiments were conducted in 2014 and 2015 where watermelon flowers and immature fruit were inoculated with F. oxysporum f. sp. niveum. Seeds were extracted from mature watermelon fruit, and infestation of watermelon seeds was determined by isolation of F. oxysporum f. sp. niveum and further confirmed by real-time polymerase chain reaction (PCR) analysis. Inoculation of the pericarp of immature fruit resulted in 17.8 to 54.4% of infested seeds under field conditions and 0.6 to 12.8% of infested seeds under greenhouse conditions when seeds were not surface disinfested prior to isolation. Seed infestation was also detected in 0 to 4.5% of the seeds when seeds were surface disinfested prior to isolation. Inoculation of pistil resulted in 0 to 7.2% and 0 to 18.3% of infested seeds under greenhouse and field conditions when seeds were surface disinfested or not disinfested before isolation, respectively. Inoculation of peduncle resulted in 0.6 to 6.1% and 0 to 10.0% of infested seeds in the greenhouse and field experiments when seeds were surface disinfested or not disinfested before isolation, respectively. Seed infestation was also detected in all the experiments using real-time PCR assay when pericarp or pistil was inoculated, and in three of four experiments when peduncle was inoculated, regardless of whether seeds were surface disinfested or not disinfested. Pericarp and peduncle of immature watermelon fruit and pistil of watermelon flowers could be potential infection courts for F. oxysporum f. sp. niveum leading to infestation of seeds in asymptomatic watermelon fruit.

scholarly journals Chemical and biological control of Sclerotinia stem rot in the soybean crop

2015 ◽  
Vol 45 (5) ◽  
pp. 760-766 ◽  
Author(s):  
Ciro Hideki Sumida ◽  
Marcelo Giovanetti Canteri ◽  
Douglas Casaroto Peitl ◽  
Fabiana Tibolla ◽  
Idenize Pedrina Orsini ◽  
...  
Keyword(s):  
Benzalkonium Chloride ◽  
Microbial Control ◽  
Control Agent ◽  
Field Conditions ◽  
Stem Rot ◽  
Sclerotinia Stem Rot ◽  
Germination Test ◽  
Soybean Crop ◽  
Carpogenic Germination ◽  
Ascospore Germination

It was evaluated the effect of fungicides and the microbial control agent Trichoderma harzianum on the inhibition of the carpogenic and ascospore germination of Sclerotinia sclerotiorum. This study also evaluated the chemical, fungicidal and microbial control of white mold or Sclerotinia stem rot of soybean in the field. Three experiments were conducted, as follows: 1) inhibition of carpogenic germination of sclerotia, 2) inhibition of ascospore germination, and 3) control of Sclerotinia stem rot in a soybean crop under field conditions. The treatments evaluated were fluazinam, procymidone, iprodione, thiophanate-methyl, carbendazim, benzalkonium chloride + fluazinam, and T. harzianum. Procymidone resulted in an inhibition of 13.5% and benzalkonium chloride in an inhibition of 13.9% in an ascospore germination test. Fluazinam and procymidone were the most effective in reducing the production of ascospores/apothecium, representing 65.6% and 82.4% of inhibition. Procymidone and fluazinam if combined or not with benzalkonium chloride were the most effective in controlling sclerotinia stem rot under field conditions when applied at the onset of flowering and 15 days later. In the 2009-10 harvest, these two fungicides reduced the incidence of Sclerotinia stem rot by 73.1 and 71.6% and in the 2010-11 harvest by 75.7 and 77.6%, respectively.

scholarly journals Baseline Sensitivity and Control Efficacy of Various Group of Fungicides against Sclerotinia sclerotiorum in Oilseed Rape Cultivation

Agronomy ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1758
Author(s):  
Nazanin Zamani-Noor
Keyword(s):  
Oilseed Rape ◽  
Sclerotinia Sclerotiorum ◽  
Fungicidal Activity ◽  
Management Strategies ◽  
Sclerotinia Stem Rot ◽  
Baseline Sensitivity ◽  
Cultural Management ◽  
Significant Yield ◽  
And Control

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a devastating disease of oilseed rape that may cause significant yield losses if not controlled by cultural management strategies and fungicide applications. Studies were conducted to evaluate the efficacy of different group of fungicides as well as a biopesticide, including azoxystrobin, boscalid, fludioxonil, prothioconazole, tebuconazole, azoxystrobin/tebuconazole, boscalid/pyraclostrobin, prothioconazole/fluopyram and Bacillus amyloliquefaciens, on baseline sensitivity of S. sclerotiorum isolates under in-vitro conditions as well as control of SSR in the field. Artificial inoculation and mist irrigation prompt to reproducible SSR infection in oilseed rape cultivation. All compounds significantly reduced 36.7% to 86.9% SSR severity and increased 55.2% to 98.7% yield, 1.5% to 7.0% thousand grain-weight, 1.5% to 5.9% oil and 0.1% to 1.3% protein content. Fludioxonil, boscalid/pyraclostrobin and fluopyram/prothioconazole achieved strongest fungicidal activity against SSR. The biopesticide provided 36% of disease control. Under in vitro conditions, B. amyloliquefaciens not only strongly inhibited mycelial growth but also the formation of sclerotia in all concentrations. Boscalid and fludioxonil exhibited the highest level of fungicidal activity against S. sclerotiorum, with mean EC50 values of 1.23 and 1.60 μg a.s. mL−1. The highest variability of EC50 values between isolates was observed towards prothioconazole and azoxystrobin.

scholarly journals Mucoadhesive Potential of Whey Protein Concentrate in Miconazole Mucoadhesive Prolong Release Tablets

2020 ◽  
Vol 11 (2) ◽  
pp. 1403-1410
Author(s):  
Khairnar Akshay ◽  
Salunkhe Kishor ◽  
Chavan M J ◽  
Chintamani Ravindra
Keyword(s):  
Drug Release ◽  
Whey Protein ◽  
Antifungal Agent ◽  
Severe Disease ◽  
Immunosuppressive Agents ◽  
Hydroxypropyl Methylcellulose ◽  
Immediate Release ◽  
Whey Protein Concentrate ◽  
Protein Concentrate ◽  
Time Period

Orophygial candidiasis (OPC) is one of the most frequent Candida infections of the oral cavity this condition is common in patients with malnutrition, having chemotherapy, administrating immunosuppressive agents, or severe disease such as HIV. Prolong local administration of Miconazole is a must to treat OPC conditions in patients, which required the prolong mucoadhesion of the dosage form to provide local delivery of drugs for an extended time period with good mucoadhesion. Miconazole is synthetic imidazole antifungal agent, various prescription and over the counter product available like Miconazole Gel, Topical cream, Immediate release Tablet available in the market but that lack in the prolonged period of action to the local mucosa. In the present study combination of Whey protein concentrate (WPC), Hydroxypropyl methylcellulose (HPMC) and Lactose were evaluated as a potential buccal adhesive tablet. The various combination of Whey protein concentrate with release controlling polymer was tested using a Miconazole as a potential antifungal agent. The mucoadhesive potential of WPC evaluated using Tensiometer by calculating the detachment force and drug release of Miconazole was performed to study the prolong release activity of Tablet. The optimized combination of WPC/HPMC/Lactose through a statistical study showed significant bioadhesion to the porcine mucosa and prolong drug release for 10hrs.

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