First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Wisconsin

Brown root rot (BRR) has been associated with winterkill of alfalfa (Medicago sativa L.) in the temperate regions of North America where winters are severe (1). Although suspected, BRR has not been associated with winterkill of alfalfa in the upper Midwestern United States. Alfalfa plants exhibiting symptoms resembling those induced by the causal agent Phoma sclerotioides G. Preuss ex Sacc. were collected from fields in Marinette, Pierce, and Marathon counties in Wisconsin during the spring and early summer of 2003. Symptoms included stunting and decline in 1- to 3-year-old plants that were slow to break dormancy in the early spring. Roots frequently exhibited dark brown lesions or were entirely decayed. Advanced lesions often formed dark bands around the circumference of tap and secondary roots. Beaked pycnidial structures typical of P. sclerotioides were also observed on many samples with advanced lesions. Plants with symptoms of BRR were also observed in Clark, Langlade, Lincoln, Oconto, Shawno, Taylor, and Wood counties. Several lesion areas of tissue on the tap and lateral roots of each sample were excised with a sterile scalpel. Total DNA was extracted using the Fast DNA kit (Bio 101, Carlsbad, CA). In addition, soil samples were collected in the root rhizosphere of symptomatic plants from four fields in two counties. Soil DNA was extracted with the Ultra-Clean DNA soil extraction kit (Mo Bio, Solana Beach, CA). DNA extractions were diluted 1:10 or 1:50, and samples were evaluated for the presence of P. sclerotioides using polymerase chain reaction (PCR)-based sequence-characterized amplified region (SCAR) markers according to the method described previously (4). Amplicons of the expected size (499 bp) were detected from alfalfa roots sampled from Marathon (4 of 4), Marinette (4 of 5), and Pierce (4 of 4) counties but not in roots from healthy controls produced in the greenhouse at Prosser, WA. PCR amplicons were also produced from all field soil samples in Marathon and Marinette counties. Proof of pathogenicity via Koch's postulates for this host-pathogen system was not attempted because of the extensive time period required (1). However, characteristic beaked pycnidia were present, and the pathogen was identified using PCR on DNA from roots symptomatic of BRR. Detection of BRR has been limited in the United States to Wyoming (2), but has been thought to occur in other states with severe winters (3). To our knowledge, this is the first report of P. sclerotioides in Wisconsin. References: (1) J. G. N. Davidson. Brown root rot. Pages 29–31 in: Compendium of Alfalfa Diseases. 2nd ed. D. L. Stuteville and D. C. Erwin, eds. The American Phytopathological Society, St. Paul, MN, 1990. (2) F. A. Gray et al. Pages 27–28 in: Proc. 10th Western Alfalfa Improv. Conf., 1997. (3) C. R. Hollingsworth et al. Can. J Plant Pathol. 25:215, 2003. (4) R. C. Larsen et al. Plant Dis. 86:928, 2002.

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First Report of Brown Root Rot on Alfalfa Caused by Phoma sclerotioides in the Continental United States

Plant Disease ◽

10.1094/pdis.1999.83.11.1071a ◽

1999 ◽

Vol 83 (11) ◽

pp. 1071-1071 ◽

Cited By ~ 7

Author(s):

C. R. Hollingsworth ◽

F. A. Gray

Keyword(s):

United States ◽

Root Rot ◽

Experimental Unit ◽

Diseased Plant ◽

Pathogenicity Test ◽

Forage Crops ◽

First Report ◽

Severity Rating ◽

Phoma Sclerotioides ◽

Brown Root Rot

Phoma sclerotioides G. Preuss ex Sacc. (previously named Plenodomus meliloti Dearn. & G.B. Sanford) is associated with root rot and extensive winterkill of leguminous forage crops, such as clover (Trifolium and Melilotus spp.), sainfoin (Onobrychis viciifolia), and alfalfa (Medicago sativa). Winterkill and root rot of irrigated alfalfa were observed for the first time in a field of cv. Multiplier in western Wyoming during the spring of 1996. Dark brown to black, sunken, rotting lesions were noted on upper secondary roots and taproots of dead and living diseased plants. Superficial and embedded beaked pycnidia and pycnosclerotia were observed near root lesions. A Phoma sp. isolated from a diseased plant in Farson, WY, was maintained on potato dextrose and half-strength V8-juice agars. Beaked pycnidia, typical of P. sclerotioides, were observed in culture when grown at 10°C for 2 months. A pathogenicity test was performed on cv. Multiplier. Two barley seeds colonized by a Phoma sp. derived from a Wyoming isolate were positioned on taproots of healthy, greenhouse-grown, 5-month-old plants ≈2.5 cm below the crown and were covered with a small piece of sterile cotton. Three replicate samples (24 plants inoculated and 24 plants uninoculated per replicate) were winter-hardened for 4 weeks (15.6°C/10°C, day/night, for 2 weeks, followed by 10°C/7.2°C, day/night, for 2 weeks) and placed outside during January 1998 in Laramie, WY, for a 4-month winter exposure period. Plants were rated for disease during June 1998. A disease severity rating of 1 to 5 was assigned to each experimental unit, where 1 = no disease and 5 = dead plant. The percentage of diseased plants at each severity rating for all inoculated plants was 1 = 19%, 2 = 33%, 3 = 31%, 4 = 13%, and 5 = 4%. Mycelium typical of P. sclerotioides was found on 99% of inoculated plant roots whether or not they had pycnidia. Pycnidia were found on the lower stems and petioles of some inoculated plants. Three percent of control plants also developed brown root rot (BRR) symptoms (taproot lesions or discoloration) by June 1998. The percentage of diseased plants at each severity rating for all uninoculated plants was 1 = 96%, 2 = 4%, and 3 through 5 = 0%. Aboveground propagule placement likely contributed to the spread of BRR by raindrop splash and wind-driven plant debris to adjacent alfalfa. Most inoculated plants had immature pycnidia or protopycnidia (94%), whereas 6.9% of the plants also had fully mature, beaked pycnidia. Pure fungal cultures were obtained from several diseased roots and compared with the original Wyoming Phoma sp. culture and a Canada isolate of P. sclerotioides (ATCC no. 56515) (2): colony, pycnidial, and conidial morphologies were identical, completing Koch's postulates. This is the first report of BRR on alfalfa in the continental United States. References: (1) J. G. N. Davidson. 1990. Brown root rot. Pages 29–31 in: Compendium of Alfalfa Disease. 2nd ed. The American Phytopathological Society, St. Paul, MN. (2) C. R. Hollingsworth et al. Phytopathology 88(suppl.):S39, 1999.

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First Report of Pythium aphanidermatum Crown and Root Rot of Industrial Hemp in the United States

Plant Disease ◽

10.1094/pdis-09-16-1249-pdn ◽

2017 ◽

Vol 101 (6) ◽

pp. 1038 ◽

Cited By ~ 7

Author(s):

J. Beckerman ◽

H. Nisonson ◽

N. Albright ◽

T. Creswell

Keyword(s):

United States ◽

Root Rot ◽

The United States ◽

Pythium Aphanidermatum ◽

First Report ◽

Crown And Root Rot ◽

Industrial Hemp

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First Report of Fusarium proliferatum Causing Root Rot on Soybean (Glycine max) in the United States

Plant Disease ◽

10.1094/pdis-04-11-0346 ◽

2011 ◽

Vol 95 (10) ◽

pp. 1316-1316 ◽

Cited By ~ 18

Author(s):

M. M. Díaz Arias ◽

G. P. Munkvold ◽

L. F. Leandro

Keyword(s):

United States ◽

Root Rot ◽

Morphological Characteristics ◽

Dry Weight ◽

The United States ◽

Growth Stages ◽

Species Identity ◽

First Report ◽

Soybean Roots ◽

Soybean Diseases

Fusarium spp. are widespread soilborne pathogens that cause important soybean diseases such as damping-off, root rot, Fusarium wilt, and sudden death syndrome. At least 12 species of Fusarium, including F. proliferatum, have been associated with soybean roots, but their relative aggressiveness as root rot pathogens is not known and pathogenicity has not been established for all reported species (2). In collaboration with 12 Iowa State University extension specialists, soybean roots were arbitrarily sampled from three fields in each of 98 Iowa counties from 2007 to 2009. Ten plants were collected from each field at V2-V3 and R3-R4 growth stages (2). Typical symptoms of Fusarium root rot (2) were observed. Symptomatic and asymptomatic root pieces were superficially sterilized in 0.5% NaOCl for 2 min, rinsed three times in sterile distilled water, and placed onto a Fusarium selective medium. Fusarium colonies were transferred to carnation leaf agar (CLA) and potato dextrose agar and later identified to species based on cultural and morphological characteristics. Of 1,230 Fusarium isolates identified, 50 were recognized as F. proliferatum based on morphological characteristics (3). F. proliferatum isolates produced abundant, aerial, white mycelium and a violet-to-dark purple pigmentation characteristic of Fusarium section Liseola. On CLA, microconidia were abundant, single celled, oval, and in chains on monophialides and polyphialides (3). Species identity was confirmed for two isolates by sequencing of the elongation factor (EF1-α) gene using the ef1 and ef2 primers (1). Identities of the resulting sequences (~680 bp) were confirmed by BLAST analysis and the FUSARIUM-ID database. Analysis resulted in a 99% match for five accessions of F. proliferatum (e.g., FD01389 and FD01858). To complete Koch's postulates, four F. proliferatum isolates were tested for pathogenicity on soybean in a greenhouse. Soybean seeds of cv. AG2306 were planted in cones (150 ml) in autoclaved soil infested with each isolate; Fusarium inoculum was applied by mixing an infested cornmeal/sand mix with soil prior to planting (4). Noninoculated control plants were grown in autoclaved soil amended with a sterile cornmeal/sand mix. Soil temperature was maintained at 18 ± 1°C by placing cones in water baths. The experiment was a completely randomized design with five replicates (single plant in a cone) per isolate and was repeated three times. Root rot severity (visually scored on a percentage scale), shoot dry weight, and root dry weight were assessed at the V3 soybean growth stage. All F. proliferatum isolates tested were pathogenic. Plants inoculated with these isolates were significantly different from the control plants in root rot severity (P = 0.001) and shoot (P = 0.023) and root (P = 0.013) dry weight. Infected plants showed dark brown lesions in the root system as well as decay of the entire taproot. F. proliferatum was reisolated from symptomatic root tissue of infected plants but not from similar tissues of control plants. To our knowledge, this is the first report of F. proliferatum causing root rot on soybean in the United States. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathologic Society, St. Paul, MN, 1999. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (4) G. P. Munkvold and J. K. O'Mara. Plant Dis. 86:143, 2002.

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First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Maine, Ontario, and Pennsylvania

Plant Disease ◽

10.1094/pdis-93-3-0317c ◽

2009 ◽

Vol 93 (3) ◽

pp. 317-317 ◽

Cited By ~ 1

Author(s):

M. J. Wunsch ◽

R. Kersbergen ◽

A. U. Tenuta ◽

M. H. Hall ◽

G. C. Bergstrom

Keyword(s):

Root Rot ◽

Control Treatment ◽

Cortical Lesions ◽

Eastern Canada ◽

Diagnostic Pcr ◽

First Report ◽

South Central ◽

Phoma Sclerotioides ◽

Single Amplicon ◽

Brown Root Rot

Brown root rot (BRR), caused by the fungal pathogen Phoma sclerotioides G. Preuss ex Sacc. (synonym Plenodomus meliloti Dearn. & G.B. Sanford), is associated with yield loss of alfalfa (Medicago sativa L.) in regions with severe winters (1). In the spring of 2007, 9 to 69 alfalfa plants were collected from each of five production fields in Maine, 10 fields in Ontario, and nine fields in Pennsylvania. All alfalfa stands existed at least two winters. P. sclerotioides was isolated from alfalfa root and crown lesions from five fields in Maine (Penobscot, Somerset and Waldo counties), seven fields from southwestern (Woodstock and Niagara), south-central (Lindsay and Belleville), and southeastern Ontario (near Ottawa), and four fields in Pennsylvania (Columbia, Crawford, and Jefferson counties; 41.1 to 41.6°N). BRR incidence was 9 to 29% in Maine, 5 to 29% in Ontario, and 8 to 22% in Pennsylvania. In Ontario, some lesions girdled the crown; in three fields in Maine, large pycnidia characteristic of P. sclerotioides were present on alfalfa crowns and overwintered stems. On potato dextrose agar, conidia (5 to 8 × 2 to 3 μm, unicellular, hyaline, and ovoid) and pycnidia (0.33 to 1.15 mm in diameter with multiple beaks) of single-conidium isolates were characteristic of P. sclerotioides (2). Diagnostic PCR (3) of isolates resulted in a single amplicon of expected size (500 bp). The internal transcribed spacer (ITS) 1, 5.8S, and ITS2 of the rDNA were sequenced for 12 representative isolates, and sequences (GenBank Accession Nos. FJ179151 to FJ179162) were 95.5 to 100% identical to P. sclerotioides ATCC isolate 56515 over a 488-bp alignment. Eight months after seeding, potted ‘Vernal’ alfalfa was inoculated (4), kept at 4°C for 8 weeks, 0 to –2°C for 12 weeks, 4°C for 8 weeks, and 10 to 15°C for 7 weeks. Of 108 plants inoculated with the Maine isolates, 35 developed severe cortical lesions and 16 died. Of 18 plants inoculated with the Ontario isolates, 16 developed severe cortical lesions and eight died. Of 18 plants inoculated with a Pennsylvania isolate, 11 developed severe cortical lesions and five died. Lesions were typical of BRR: light to very dark brown, sometimes with a darker border, and often containing abundant pycnidia. Plant mortality was associated with lesions that girdled the root and crown. Of 18 plants in the control treatment, three developed severe cortical lesions and none died. BRR is common in Alberta, Saskatchewan, and Manitoba, but in eastern Canada it has been reported only in Nova Scotia. To our knowledge, this is the first report of BRR in Maine, Ontario, and Pennsylvania and the southernmost report of BRR in eastern North America. References: (1) B. Berkenkamp et al. Can. J. Plant Sci. 71:211, 1991. (2) G. H. Boerema et al. Persoonia 15:431, 1994. (3) R. C. Larsen et al. Plant Dis. 86:928, 2002. (4) M. J. Wunsch et al. Plant Dis. 91:1293, 2007.

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A Rapid Method Using PCR-Based SCAR Markers for the Detection and Identification of Phoma sclerotioides: The Cause of Brown Root Rot Disease of Alfalfa

Plant Disease ◽

10.1094/pdis.2002.86.9.928 ◽

2002 ◽

Vol 86 (9) ◽

pp. 928-932 ◽

Cited By ~ 21

Author(s):

R. C. Larsen ◽

C. R. Hollingsworth ◽

G. J. Vandemark ◽

M. A. Gritsenko ◽

F. A. Gray

Keyword(s):

Root Rot ◽

Random Amplified Polymorphic Dna ◽

Dna Amplification ◽

Pythium Ultimum ◽

Soil Samples ◽

Root Tissue ◽

Aphanomyces Euteiches ◽

Amplification Product ◽

Phoma Sclerotioides ◽

Brown Root Rot

A rapid technique for identification and detection of Phoma sclerotioides, the causal agent of brown root rot of alfalfa, has been developed using polymerase chain reaction (PCR). Amplification products obtained from random amplified polymorphic DNA (RAPD) reactions were cloned and sequenced, and two extended primer sets were designed from the resulting data that were used to detect sequence-characterized DNA markers. A single 499-bp DNA amplification product was consistently obtained from primers PSB12499 that was specific for 19 isolates of P.sclerotioides but was not produced from Phoma medicaginis or Phoma betae, or from other soilborne pathogens including Aphanomyces euteiches, Rhizoctonia solani, Fusarium oxysporum, Pythium ultimum, or Phytophthora infestans. A 499-bp amplification product was also produced from root tissue known to be infected with the fungus as verified by microscopic examination. A similar PCR product was obtained from soil samples collected from fields with an established infection of P. sclerotioides on alfalfa. This PCR-based assay enables detection of P. sclerotioides from alfalfa root tissue and in soil samples in a single day, including extraction of DNA, compared with standard methods that require up to 100 days for identification using agar media.

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First Report of Stem Blight Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Greenhouse-Grown Blueberries in the United States

Plant Disease ◽

10.1094/pdis-02-11-0117 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1187-1187

Author(s):

J. J. Sadowsky ◽

T. D. Miles ◽

A. M. C. Schilder

Keyword(s):

United States ◽

North Carolina ◽

Root Rot ◽

The United States ◽

Vaccinium Corymbosum ◽

Conidial Suspension ◽

Centraalbureau Voor ◽

First Report ◽

Stem Lesions ◽

Β Tubulin

Necrotic stems and leaves were observed on 2- to 4-month-old, rooted microshoot plants (Vaccinium corymbosum L. ‘Liberty’ and ‘Bluecrop’, V. angustifolium Aiton ‘Putte’, and V. corymbosum × V. angustifolium ‘Polaris’) in a Michigan greenhouse in 2008 and 2009. As the disease progressed, leaves fell off and 80 to 100% of the plants died in some cases. Root rot symptoms were also observed. A fungus was isolated from stem lesions. On potato dextrose agar (PDA), cultures first appeared light tan to orange, then rusty brown and zonate with irregular margins. Chains of orange-brown chlamydospores were abundant in the medium. Macroconidiophores were penicillately branched and had a stipe extension of 220 to 275 × 2.5 μm with a narrowly clavate vesicle, 3 to 4 μm wide at the tip. Conidia were hyaline and cylindrical with rounded ends, (1-)3-septate, 48 to 73 × 5 to 7 (average 60 × 5.5) μm and were held together in parallel clusters. Perithecia were globose to subglobose, yellow, 290 to 320 μm high, and 255 to 295 μm in diameter. Ascospores were hyaline, 2- to 3-septate, guttulate, fusoid with rounded ends, slightly curved, and 30 to 88 × 5 to 7.5 (average 57 × 5.3) μm. On the basis of morphology, the fungus was identified as Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii Peerally) (1,2). The internal transcribed spacer region (ITS1 and ITS2) of the ribosomal DNA and the β-tubulin gene were sequenced (GenBank Accession Nos. HQ909028 and JF826867, respectively) and compared with existing sequences using BLASTn. The ITS sequence shared 99% maximum identity with that of Ca. colhounii CBS 293.79 (GQ280565) from Java, Indonesia, and the β-tubulin sequence shared 97% maximum identity with that of Ca. colhounii CBS 114036 (DQ190560) isolated from leaf spots on Rhododendron sp. in North Carolina. The isolate was submitted to the Centraalbureau voor Schimmelcultures in the Netherlands (CBS 129628). To confirm pathogenicity, 5 ml of a conidial suspension (1 × 105/ml) were applied as a foliar spray or soil drench to four healthy ‘Bluecrop’ plants each in 10-cm plastic pots. Two water-sprayed and two water-drenched plants served as controls. Plants were misted intermittently for 2 days after inoculation. After 7 days at 25 ± 3°C, drench-inoculated plants developed necrotic, sporulating stem lesions at the soil line, while spray-inoculated plants showed reddish brown leaf and stem lesions. At 28 days, three drench-inoculated and one spray-inoculated plant had died, while others showed stem necrosis and wilting. No symptoms were observed on control plants. Fungal colonies reisolated from surface-disinfested symptomatic stem, leaf, and root segments appeared identical to the original isolate. Cy. colhounii was reported to cause a leaf spot on blueberry plants in nurseries in China (3), while Ca. crotalariae (Loos) D.K. Bell & Sobers (= Ca. ilicicola Boedijn & Reitsma) causes stem and root rot of blueberries in North Carolina (4). To our knowledge, this is the first report of Ca. colhounii causing a disease of blueberry in Michigan or the United States. Because of its destructive potential, this pathogen may pose a significant threat in blueberry nurseries. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) L. Lombard et al. Stud. Mycol. 66:31, 2010. (3) Y. S. Luan et al. Plant Dis. 90:1553, 2006. (4) R. D. Milholland. Phytopathology 64:831, 1974.

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First Report of Fusarium brasiliense Causing Root Rot of Dry Bean in the United States

Plant Disease ◽

10.1094/pdis-02-18-0332-pdn ◽

2018 ◽

Vol 102 (10) ◽

pp. 2035 ◽

Cited By ~ 4

Author(s):

J. L. Jacobs ◽

K. Oudman ◽

H. Sang ◽

M. I. Chilvers

Keyword(s):

United States ◽

Root Rot ◽

The United States ◽

Dry Bean ◽

First Report

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First Report of Clubroot on Canola Caused by Plasmodiophora brassicae in North Dakota

Plant Disease ◽

10.1094/pdis-04-14-0430-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1438-1438 ◽

Cited By ~ 10

Author(s):

K. Chittem ◽

S. M. Mansouripour ◽

L. E. del Río Mendoza

Keyword(s):

United States ◽

North Dakota ◽

Plasmodiophora Brassicae ◽

The United States ◽

Soil Samples ◽

Pcr Analysis ◽

Infested Soil ◽

First Report ◽

Resting Spores ◽

Pathogenicity Tests

North Dakota leads the United States in canola (Brassica napus L.) production (4). A canola field with a distinct patch of dead plants spreading over an area of approximately 0.4 ha was detected in Cavalier County, North Dakota, in early September 2013. Numerous spots within the patch had plant mortalities >80%. Dead plants pulled from the soil had roots with severe galling and clubbing. Clubbed roots were brittle and disintegrated easily when pressed between fingers. Root and soil samples collected at several locations within and outside the affected patch were pooled in separate groups. All plants collected in the patch were symptomatic but those collected outside were not. In the lab, total genomic DNA from three symptomatic and two healthy root samples was extracted using standard procedures and freehand slices were prepared for observation with a compound microscope. Also, DNA from pooled soil samples was extracted using FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH). Round resting structures ranging from 2.2 to 4.2 μm in diameter were observed by microscopic examination of symptomatic root tissues. These structures resembled those typically produced by Plasmodiophora brassicae Woronin. This initial identification was later confirmed through PCR analysis using the species specific primers TC1F/R and TC2F/R (1). PCR products of 548 bp (TC1F/R) and 519 bp (TC2F/R) were produced in the three symptomatic and two infested soil samples, confirming the presence of P. brassicae. PCR amplicons were not detected in healthy root and soil samples. Pathogenicity tests were conducted in greenhouse to fulfill Koch's postulates. Briefly, five square plastic pots (10 × 10 × 13 cm) were filled with a 10-cm layer of Sunshine Mix #1 potting mix (Fison Horticulture, Vancouver, BC, Canada) and then 1 g of ground root galls (approximately 5 × 105 resting spores) was spread evenly on its surface and covered with 2 cm of soilless mix. A similar number of pots were filled only with soilless mix and used as controls. All pots were planted with two seeds of canola cv. Westar and incubated in greenhouse conditions at 21°C and 16 h light daily. The experiment was conducted twice. Four weeks after planting, all plants in the inoculated pots had developed galls while plants in control pots were symptomless. Presence of P. brassicae resting spores in the newly developed galls was confirmed by microscopic observations and PCR. Based on the symptoms, morphology of resting spores, PCR reactions, and pathogenicity tests, we confirm the presence of P. brassicae on canola. While P. brassicae has been reported as widespread in North America (2), to our knowledge, this is the first report of clubroot on canola in North Dakota and the United States. Clubroot became the most important disease affecting canola production in central Alberta, Canada, within 5 years of its discovery in 2003 (3); since then, the disease has been detected in Saskatchewan and Manitoba (3), Canadian provinces that share borders with North Dakota. Considering the difficulties in management of clubroot, measures should be initiated to limit the spread of the disease before it could pose a threat to United States canola production. References: (1) T. Cao et al. Plant Dis. 91:80, 2007. (2) G. Dixon J. Plant Growth Regul. 28:194, 2009. (3) S. Strelkov and S. Hwang. Can. J. Plant Pathol. 36(S1):27, 2014. (4) USDA-NASS, Ag. Statistics No. 81, 2012.

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