scholarly journals Relative Contribution of Seed Tuber- and Soilborne Inoculum to Potato Disease Development and Changes in the Population Genetic Structure of Rhizoctonia solani AG 3-PT under Field Conditions in South Africa

Plant Disease ◽  
2018 ◽  
Vol 102 (1) ◽  
pp. 60-66 ◽  
Author(s):  
Norman Muzhinji ◽  
James W. Woodhall ◽  
Mariette Truter ◽  
Jacquie E. van der Waals

Understanding the contribution of seed tuber- and soilborne inocula of Rhizoctonia solani AG 3-PT in causing potato disease epidemics is an important step in implementing effective management strategies for the pathogen. A 2-year study was conducted to evaluate the contribution of each source of inoculum using an integrative experimental approach combining field trials and molecular techniques. Two distinct sets of genetically marked isolates were used as seed tuberborne and soilborne inocula in a mark-release-recapture experiment. Disease assessments were done during tuber initiation and at tuber harvest. Both inoculum sources were found to be equally important in causing black scurf disease, whereas soilborne inocula appeared to be more important for root and stolon infection, and seedborne inocula contributed more to stem canker. However, seed tuber-transmitted genotypes accounted for 60% of the total recovered isolates when genotyped using three polymerase chain reaction restriction fragment length polymorphism markers. The changes in population structure of the experimental R. solani population over the course of the growing season and across two growing seasons were investigated using eight microsatellite markers. The populations at different sampling times were somewhat genetically differentiated, as indicated by Nei’s gene diversity (0.24 to 0.27) and the fixation index (FST). The proportion of isolates with genotypes that differed from the inoculants ranged from 13 to 16% in 2013 and 2014, respectively, suggesting the possibility of emergence of new genotypes in the field. Because both soilborne and tuberborne inocula are critical, it is important to ensure the use of pathogen-free seed tubers to eliminate seed tuberborne inoculum and the introduction of new genotypes of R. solani for sustainable potato production in South Africa.

Plant Disease ◽  
2015 ◽  
Vol 99 (12) ◽  
pp. 1790-1802 ◽  
Author(s):  
N. Muzhinji ◽  
M. Truter ◽  
J. W. Woodhall ◽  
J. E. van der Waals

A survey of anastomosis groups (AG) of Rhizoctonia spp. associated with potato diseases was conducted in South Africa. In total, 112 Rhizoctonia solani and 19 binucleate Rhizoctonia (BNR) isolates were recovered from diseased potato plants, characterized for AG and pathogenicity. The AG identity of the isolates was confirmed using phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA. R. solani isolates recovered belonged to AG 3-PT, AG 2-2IIIB, AG 4HG-I, AG 4HG-III, and AG 5, while BNR isolates belonged to AG A and AG R, with frequencies of 74, 6.1, 2.3, 2.3, 0.8, 12.2, and 2.3%, respectively. R. solani AG 3-PT was the most predominant AG and occurred in all the potato-growing regions sampled, whereas the other AG occurred in distinct locations. Different AG grouped into distinct clades, with high maximum parsimony and maximum-likelihood bootstrap support for both R. solani and BNR. An experiment under greenhouse conditions with representative isolates from different AG showed differences in aggressiveness between and within AG. Isolates of AG 2-2IIIB, AG 4HG-III, and AG R were the most aggressive in causing stem canker while AG 3-PT, AG 5, and AG R caused black scurf. This is the first comprehensive survey of R. solani and BNR on potato in South Africa using a molecular-based approach. This is the first report of R. solani AG 2-2IIIB and AG 4 HG-I causing stem and stolon canker and BNR AG A and AG R causing stem canker and black scurf on potato in South Africa.


Plant Disease ◽  
2004 ◽  
Vol 88 (1) ◽  
pp. 83-83 ◽  
Author(s):  
M. Truter ◽  
F. C. Wehner

Rhizoctonia disease (black scurf of tubers and stem canker) of potato (Solanum tuberosum L.) caused by Rhizoctonia solani Kühn was first recorded in South Africa in 1918 (3). Although the sclerotial form on tubers is one of the most common potato diseases in the country, it is not known which anastomosis groups (AGs) of R. solani are involved. Between 1999 and 2001, R. solani was isolated from 28 plant and 56 soil samples collected in 7 (Eastern Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern Cape, and Sandveld) of the 14 potato-production regions of South Africa and screened for hyphal anastomosis with tester strains of R. solani AG-1 to AG-10 according to Carling et al. (1). Of the 411 isolates from tubers with black scurf symptoms, 408 were AG-3 and three were AG-5. Symptomless tubers yielded two AG-3 isolates and three AG-5 isolates. Of 39 isolates from symptomatic stems and roots, 32 were AG-3, five were AG-4, and two were AG-5. Of the 127 isolates obtained from soil, 86, 28, 7, 3, and 3 were AG-3, AG-4, AG-5, AG-7, and AG-8, respectively. More than one AG was isolated from five of the seven regions. Virulence of 40 isolates representative of the above AGs was determined in triplicate on sprouts growing from seed tubers of potato cultivar Up-to-Date in a sand/soil mixture as described by Carling and Leiner (2) but using cultures grown in cornmeal/sand instead of colonized agar disks as inoculum. Damage to sprouts (lesions, girdling, and death) was assessed after 28 days at 16 to 28°C according to the 0 to 4 rating scale (2). Chi-square analysis of the data indicated that AG-3 was the most virulent, with isolates from sclerotia on tubers and lesions on stems more aggressive than those from symptomless tubers or soil. AG-4 and AG-5 caused significantly less disease than AG-3, but none of the AG-7 and AG-8 isolates showed any virulence to potato sprouts. References: (1) D. E. Carling et al. Phytopathology 77:1609, 1987. (2) D. E. Carling and R. H. Leiner. Phytopathology 80:930, 1990. (3) E. M. Doidge. S. Afr. Fruit Growers 5:6, 1918.


Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1701-1701 ◽  
Author(s):  
J. W. Woodhall ◽  
P. S. Wharton ◽  
J. C. Peters

The fungus Rhizoctonia solani is the causal agent of stem canker and black scurf of potato (Solanum tuberosum). R. solani is a species complex consisting of 13 anastomosis groups (AGs) designated AG1 to 13 (2, 3). Stems of potato (cv. Russet Norkotah) with brown lesions were recovered from one field in Kimberley, Idaho, in August 2011. Using previously described methods (3), R. solani was recovered from the symptomatic stems and one representative isolate (J15) was selected for further characterization. Sequencing of the rDNA ITS region of isolate J15 was undertaken as previously described (3) and the resulting rDNA ITS sequence (HE667745) was 99% identical to sequences of other AG4 HG-II isolates in GenBank (AF354072 and AF354074). Pathogenicity of the isolate was determined by conducting the following experiment. Mini-tubers of cv. Santé were planted individually in 1-liter pots containing John Innes Number 3 compost (John Innes Manufacturers Association, Reading, UK). Pots were either inoculated with J15, an isolate of AG3-PT (Rs08), or were not inoculated. Each treatment was replicated four times. Inoculum consisted of five 10-mm-diameter potato dextrose agar plugs, fully colonized by the appropriate isolate, placed in the compost approximately 40 mm above each seed tuber. Pots were held in a controlled environment room at 21°C with 50% relative humidity and watered as required. After 21 days, plants were assessed for disease. No symptoms of the disease were present in non-inoculated plants. In the Rs08 (AG3-PT) inoculated plants, all stems displayed large brown lesions and 20% of the stems had been killed. No stem death was observed in J15 (AG4 HG-II) inoculated plants. However, brown lesions were observed in three of the four J15 (AG4 HG-II) inoculated plants. These lesions were less severe than in plants inoculated with the Rs08(AG3-PT) inoculated plants and were present in 40% of the main stems. In the J15 (AG4 HG-II) inoculated pots, R. solani AG4 HG-II was reisolated from the five symptomatic stems, thereby satisfying Koch's postulates. To our knowledge, this is the first report of AG4 HG-II causing disease on potatoes in Idaho. AG4 has been isolated from potato previously from North Dakota, although the subgroup was not identified (1). The only previous report where AG4 HG-II was specifically determined to cause disease on potato was in Finland, but the isolate could not be maintained and Koch's postulates were not completed (3). The present study shows that AG4 HG-II can cause stem disease in potatoes, although disease does not develop as severely or as consistently as for AG3-PT. However, as demonstrated with isolates of AG2-1 and AG5, even mild stem infection can reduce tuber yield by as much as 12% (4). AG4 HG-II is a pathogen of sugar beet in Idaho, which was grown previously in this field. This history may have contributed to high levels of soilborne inoculum required to produce disease on potato. References: (1) N. C. Gudmestad et al. Page 247 in: J. Vos et al. eds. Effects of Crop Rotation on Potato Production in the Temperate Zones. Kluwer, Dordrecht, Netherlands, 1989. (2) M. J. Lehtonen et al. Agric. Food Sci. 18:223, 2009. (3) J. W. Woodhall et al. Plant Pathol. 56:286, 2007. (4) J. W. Woodhall et al. Plant Pathol. 57:897, 2008.


Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1657-1657 ◽  
Author(s):  
T. D. Miles ◽  
J. W. Woodhall ◽  
L. A. Miles ◽  
P. S. Wharton

Rhizoctonia solani causes economically important diseases on potatoes and sugarbeet throughout the world (2). R. solani is a species complex of 13 anastomosis groups (AGs) of which R. solani AG3-PT is most commonly associated with potato and AG2-2 and AG4 with sugarbeet. However, several AGs, including AG2-2 and AG4, have been recorded causing potato diseases (2,3). In summer 2012, plants in potato fields in Idaho were sampled for R. solani. Isolations were attempted from symptomatic plants. DNA extracted from the resulting pure Rhizoctonia cultures was screened using a real-time PCR assay for AG3-PT (3). For the isolates that tested negative for AG3-PT, AG was determined by amplifying and sequencing the rDNA internal transcribed spacer (ITS) region using the primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The resulting sequences of two isolates (isolates 204 and 206, GenBank Accession No. KC782951) shared 99% identity with other AG-A isolates (AY927358 and AY927356). Koch's postulates were confirmed for isolate 206 by placing five 10-mm plugs, from 10-day-old potato dextrose agar (PDA) cultures, onto the surface of a soil-less potting mix (composed of peat moss, perlite, and sand) of 1-liter pots, where non-inoculated PDA plugs served as a control. Each pot contained a ‘Rosara’ seed tuber or three ungerminated (BETASEED – BTS 27RR10) sugarbeet seeds (n = 5). Pots were incubated in a glasshouse between 18 and 22°C for 1 month and then assessed for disease. For potatoes, a pigmented necrosis was observed at the soil interface in 88% of the stems and plants were stunted relative to the non-inoculated controls. A significant reduction in root growth was observed in 60% of the germinated sugarbeet plants. Control plants of both potatoes and beets were asymptomatic. For reisolation, 1-cm sections were taken from each potato stem and germinated beet plant, surface sterilized, and placed on alkaline water agar. The reisolated fungi were identified using morphology and a subset was confirmed by sequencing. Isolate 206 was successfully recovered from 84% of the potato stems and from 20% of the sugarbeet seedlings. In a similar experiment, 2-month-old potato and sugarbeet plants were inoculated using 50 g of autoclaved barley grains (inoculated with isolate 206) per 1-liter pot. Between 40 and 60% of inoculated plants appeared stunted in both cases. Pigmented necrosis was observed at the soil interface on 45% of the potato stems and reduced root growth was observed in the 50% of the sugarbeet plants. Control plants were asymptomatic. To our knowledge, this is the first report of the binucleate AG-A causing disease in Idaho on potato stems. BNR species have previously been isolated from potato (4) and sugarbeet plants (1). The binucleate Rhizoctonia AG-A caused disease on potato stems and sugarbeet roots and was readily reisolated. Since sugarbeet is commonly grown in rotation with potato in Idaho, such a rotation could increase the risk of soilborne infection to either crop by AG-A. It is known that AGs can differ in fungicide sensitivity (2), and thus a knowledge of which AGs may be present is important when considering disease management strategies. References: (1) C. A. Strausbaugh et al. Can. J. Plant Pathol. 33:210, 2011. (2) L. Tsror. Biology, Epidemiology and Management of Rhizoctonia solani on Potato 158:649, 2010. (3) J. Woodhall et al. Eur. J. Plant Pathol. 136:273, 2013. (4) Y. G. Yang and X. H. Wu. Plant Dis. 97:1246, 2013.


HortScience ◽  
2000 ◽  
Vol 35 (4) ◽  
pp. 557B-557a
Author(s):  
Greta Schuster ◽  
Roland E. Roberts ◽  
David Bender

Rhizoctonia solani infection of potato causes seed piece decay and stem and tuber lesions, resulting in delayed emergence and uneven stands, low-vigor plants and unmarketable tubers. Rhizoctonia prevention effectiveness of three fungicide/fir bark dusts and nontreated tubers were compared on three varieties. Seedpieces of Viking, Atlantic and Norkotah 278 were treated and planted in separate commercial fields. A randomized complete-block design with four replications with four 26-foot rows per treatment plot included four treatments: 0.5 lb Maxim, 1.0 lb Tops MZ, and 1.5 lb Nubark Captan dust/100 lb seed. Plant stems, stolons and tubers were examined for Rhizoctonia infection at the tuber initiation stage and tubers were examined again at mature stage. Maxim and Tops MZ reduced stolon infection in Atlantic and Norkotah 278 and reduced stem infection in Norkotah 278. Plants from Maxim treated tubers produced more stems/plant in Norkotah 278 and produced more weight of tubers weighing less than four oz in all three varieties. Differences in seed tuber conditioning and mechanical damage in seed handling increase variability of treatment effects. Repeated testing of potato seed piece treatments in Texas High Plains conditions is the best way to confirm potential of beneficial effects of seed treatment.


2019 ◽  
Vol 109 (11) ◽  
pp. 1957-1965 ◽  
Author(s):  
Norman Muzhinji ◽  
Jacquie E. van der Waals

Spongospora subterranea f. sp. subterranea, causal agent of powdery scab and root galls of potatoes, occurs worldwide and is responsible for quality and yield losses in potato production in South Africa. Despite being one of the most important potato pathogens in South Africa, little information is available on the genetic structure and diversity of S. subterranea f. sp. subterranea, which could provide insight into the factors shaping its evolution and the role of inoculum sources in disease development. A total of 172 samples were collected from four potato growing regions in South Africa. An additional 27 samples obtained from Colombia were included for comparative purposes. The samples were screened against six informative microsatellite (simple-sequence repeat) markers. Of the 172 samples obtained from potato growing regions in South Africa, there were 75 multilocus genotypes (MLGs), only 16 of which were shared between potato growing regions, indicating substantial gene flow and countrywide dispersal of the pathogen. The presence of common MLGs among the root- and tuber-derived samples indicated a lack of specialization of S. subterranea f. sp. subterranea to either tuber or root infection. Nei’s unbiased estimates of gene diversity for the clone-corrected data were low and ranged from 0.24 to 0.38. Analysis of molecular variance and discriminant analysis of principal components showed no population differentiation between different potato growing regions in South Africa and between root- and tuber-derived genotypes. The presence of MLGs, high considerable genotypic diversity, and failure to reject the null hypothesis of random mating in most populations are indicative of some kind of recombination, either sexual or asexual, in these S. subterranea f. sp. subterranea populations. Information from this study provides new insights into the genetic structure and diversity of S. subterranea f. sp. subterranea in South Africa. Continuous monitoring of the pathogen population dynamics will be helpful in implementing effective region-specific management strategies for the pathogen, especially in the development of resistant potato cultivars.


Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 460-460 ◽  
Author(s):  
J. W. Woodhall ◽  
A. R. Belcher ◽  
J. C. Peters ◽  
W. W. Kirk ◽  
P. S. Wharton

Rhizoctonia solani is an important pathogen of potato (Solanum tuberosum) causing qualitative and quantitative losses. It has been associated with black scurf and stem canker. Isolates of the fungus are assigned to one of 13 known anastomosis groups (AGs), of which AG3 is most commonly associated with potato disease (2,4). In August 2011, diseased potato plants originating from Rupert, ID (cv. Western Russet) and Three Rivers, MI (cv. Russet Norkotah) were received for diagnosis. Both samples displayed stem and stolon lesions typically associated with Rhizoctonia stem canker. The presence of R. solani was confirmed through isolation as previously described (4) and the Idaho and Michigan isolates were designated J11 and J8, respectively. AG was determined by sequencing the rDNA internal transcribed spacer (ITS) region using primers ITS5 and ITS4 (3). The resulting sequences of the rDNA ITS region of isolates J8 and J11 (GenBank Accession Nos. HE608839 and HE608840, respectively) were between 97 and 100% identical to that of other AG2-2IIIB isolates present in sequence databases (GenBank Accession Nos. FJ492075 and FJ492170, respectively). Koch's postulates were confirmed for each isolate by carrying out the following protocol. Each isolate was cultured on potato dextrose agar for 14 days. Five 10-mm agar plugs were then placed on top of seed tubers (cv. Maris Piper) in 1-liter pots containing John Innes Number 3 compost (John Innes Manufacturers Association, Reading, UK). Pots were held in a controlled environment room at 18°C with 50% relative humidity and watered as required. After 21 days, plants were removed and assessed for disease. Typical Rhizoctonia stem lesions were observed and R. solani was successfully reisolated from symptomatic material. To our knowledge, this is the first report of AG2-2IIIB causing disease on potatoes in the United States. In the United States, AGs 2-1, 3, 4, 5, and 9 have all been previously implicated in Rhizoctonia potato disease (2). AG2-2IIIB should now also be considered a potato pathogen in the United States. Knowledge of which AG is present is invaluable when considering a disease management strategy. AG2-2IIIB is a causal agent of sugar beet (Beta vulgaris) root rot in Idaho (1). Sugar beet is commonly grown in crop rotation with potato and such a rotation could increase the risk of soilborne infection to either crop by AG2-2IIIB. References: (1) C. A. Strausbaugh et al. Can. J. Plant Pathol. 33:210, 2011. (2) L. Tsror. J. Phytopatol. 158:649, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990. (4) J. W.Woodhall et al. Plant Pathol. 56:286, 2007.


Zootaxa ◽  
2019 ◽  
Vol 4658 (2) ◽  
pp. 251-274
Author(s):  
MILAD RASHIDIFARD ◽  
HENDRIKA FOURIE ◽  
MIEKE STEFANIE DANEEL ◽  
MARIETTE MARAIS

Accurate identification of Meloidogyne spp. is crucial and the first step to apply suitable management strategies to combat these nematode pests. Perineal-pattern morphology of female specimens is one of the most common characteristics used for identification. However, for some species various morphological characteristics are similar which makes it challenging to correctly identify species. In this study different morphological and morphometrical characteristics were used to identify 37 populations of Meloidogyne obtained during 2015 and 2016 from various crop production areas situated across different geographical regions in South Africa. A comprehensive study of females, males and second-stage juveniles (J2) of the 37 Meloidogyne populations isolated was conducted, revealing the presence of Meloidogyne enterolobii, M. hapla, M. incognita and M. javanica. Although three perineal-pattern characteristics proved to be useful in discriminating particularly between M. enterolobii and M. incognita females, most of the morphometric characters used to identify female, male and J2 individuals overlapped among the different species. Substantial intraspecies variation was also evident among different populations. The use of classical identification approaches alone could therefore not clearly distinguish among the 37 Meloidogyne populations studied. Therefore, the use of molecular techniques in combination with morphological and morphometrical analyses is suggested to be more accurate and reliable in discriminating between Meloidogyne spp.


Plant Disease ◽  
2014 ◽  
Vol 98 (6) ◽  
pp. 853-853 ◽  
Author(s):  
N. Muzhinji ◽  
J. W. Woodhall ◽  
M. Truter ◽  
J. E. van der Waals

Black scurf and stem canker caused by Rhizoctonia solani Kühn (teleomorph: Thanathephorus cucumeris Frank Donk) are potato diseases of worldwide economic importance (4). R. solani consists of 13 anastomosis groups (AGs) of which AG 3-PT is considered the dominant causal agent of potato diseases globally (1,4). However, other AGs such as AG 2-1, 5, and 8 have been reported to cause potato diseases (1,4). In February 2013, potato stem samples (cv. Mondial) displaying dark brown lesions resembling those caused by Rhizoctonia stem canker were obtained from a commercial field in Limpopo Province, South Africa. Symptomatic tissue was disinfected with 1% NaOCl for 1 min, rinsed in sterile water, and 4-mm stem pieces excised from the margins of symptomatic tissues and plated on 2% water agar supplemented with 20 mg/l of chloramphenicol. Single hyphal tips taken from fungal isolates identified as R. solani based on morphological traits (3) were transferred to potato dextrose agar. DNA was isolated from the resulting cultures and ITS region of rDNA was sequenced as previously described (2). The resulting sequences of three of the isolates, Rh 81, Rh 82, and Rh 83 (KF712285, KF712286, and KF712287), were 99% similar to those of AG 4 HG-III found in GenBank (DQ102449 and AF354077). Therefore, based on molecular methods, these three isolates were identified as R. solani AG4 HG-III. To determine pathogenicity of the AG4 HG-III isolates, certified disease free mini-tubers (Generation 0, cv. Mondial, produced in tunnels) were used in pot trials. PDA plugs of each isolate were added to 10 g of barley grains, which had been sterilized by autoclaving for two consecutive days at 121°C for 30 min, and were incubated for 14 days until fully colonized. Ten colonized barley grains were placed 10 mm above each mini-tuber planted in 5l pots containing sterile potting mixture of sand:clay:pinebark (1:1:1). Ten tubers were inoculated with each isolate. Uninoculated, sterile barley grains were applied to the control treatment. Mini-tubers were grown in a greenhouse maintained at 22°C with light for a 12 h day. After 7 weeks, five plants for each isolate were destructively sampled and assessed for stem canker symptoms. At 120 days after sowing, the remaining five plants per treatment were assessed for blemishes on progeny tubers. The stem canker incidences of plants inoculated with Rh 81, Rh 82, and Rh 83 were 25, 25, and 50%, respectively, whereas no symptoms were observed in control plants. Sclerotia formation and blemishes were not observed on any of the progeny tubers, which might indicate that these strains are only able to infect stems, or that environmental conditions were not suitable for tuber blemish or black scurf development. R. solani AG4 HG-III was consistently re-isolated from symptomatic stems displaying brown lesions, and the identity of the re-isolates were confirmed by molecular tests as previously described, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of R. solani AG4 HG-III causing stem canker on potato in South Africa and worldwide. Knowledge of which AGs are present in crop production systems is important when considering disease management strategies such as crop rotation and fungicide treatments (3). References: (1) C. Campion et al. Eur. J. Plant. Pathol. 109:983, 2003. (2) N. Muzhinji et al. Plant Dis. 98:570, 2014. (3) L. Tsror. J. Phytopathol. 158:649, 2010. (4) J. W. Woodhall et al. Plant. Pathol. 56:286, 2007.


2021 ◽  
pp. 008124632110201
Author(s):  
Zamafiso Nombulelo Sibande ◽  
Rizwana Roomaney

Fatigue is a common, secondary symptom of endometriosis that has not been qualitatively explored. We conducted individual, face-to-face interviews with 25 women in South Africa about their experiences of endometriosis-related fatigue. Participants were recruited at a public hospital in Cape Town and through several South African endometriosis organizations. Interviews were conducted in English and Afrikaans and ranged from 30 min to 1 hr 16 min in duration. All interviews were audio recorded and transcribed. Interviews were analyzed using thematic analysis. We found that both the experience of fatigue and fatigue-management strategies were highly personalized. Participants reported using a variety of cognitive strategies, such as planning, pacing, and pushing through their fatigue to reduce the levels of fatigue. Participants also employed physical strategies such as rest, dietary changes, using supplements, and exercise. We found that while participants often tried fatigue-management strategies suggested to them by others, they struggled to maintain these strategies even when they were successful. There are currently no interventions aimed at reducing endometriosis-related fatigue. The findings of this study provide insight into the management of fatigue in women with endometriosis and may be used to develop a psychosocial intervention for fatigue among women with the disease.


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