Anastomosis Groups of Rhizoctonia solani and Binucleate Rhizoctonia Associated with Potatoes in Idaho.

A survey of the relative incidence of anastomosis groups (AGs) of Rhizoctonia spp. associated with potato disease was conducted in Idaho, the leading potato producing state in the U.S.A. In total, 169 isolates of Rhizoctonia solani and seven binucleate Rhizoctonia (BNR) isolates were recovered from diseased potato plants. The AG of each isolate was determined through real-time PCR assays for AG 3-PT and phylogenetic analysis of the internal transcribed spacer region of ribosomal DNA. AG 3-PT was the predominant AG, accounting for 85% of isolates recovered, followed by AG 2-1 (5.7%) and AG 4 HG-II (4.5%). Two different subsets of AG 2-1 isolates were recovered (subset 2 and 3). Three isolates each of AG A and AG K were recovered, as well as one isolate each of AG 5 and AG W. An experiment carried out under greenhouse conditions with representative isolates of the different AGs recovered from Idaho potatoes showed differences in aggressiveness between AGs to potato stems, with AG 3-PT being the most aggressive followed by an isolate of AG 2-1 (subset 3). The three BNR isolates representative of AG A, AG K, and AG W appeared to be less aggressive to potato stems than the R. solani isolates except for the AG 2-1 (subset 2) isolate. This is the first comprehensive study of the relative incidences of Rhizoctonia species associated with Idaho potatoes and the first study to report the presence of BNR AG W outside of China.

First Report of Late Blight Caused by Phytophthora infestans Clonal Lineage US-23 on Potato in Idaho.

Late blight, caused by Phytophthora infestans (Mont.) de Bary, is a destructive disease of potato (Solanum tuberosum) and tomato (S. lycopersicum) in the United States. Prior to 2007, the US-8 clonal lineage was the predominant genotype in the United States (4). Since 2007, a significant genetic change in the population of P. infestans occurred in the eastern United States with the appearance of new isolates with unique genotypes and epidemiological characteristics (3). These new genotypes US-22, US-23, and US-24 are sensitive to metalaxyl and represent mating types A2, A1, and A1, respectively (1,2). Prior to 2012, only US-8 had been documented in Idaho (5). In 2013, late blight was discovered in late August on potato crops (cv. Russet Norkotah) in Bingham and Madison counties, ID. Infected foliage (four samples from Bingham County and five from Madison) was sent to Michigan State University and the University of Wisconsin for confirmation of P. infestans and characterization of the isolates. Five sections from the leading edge of lesions were excised with a sterilized scalpel and placed on potato tuber slices ('Dark Red Norkotah'). Pathogen sporulation on the excised lesions was enhanced by incubation in plastic boxes lined with moistened paper towels for 5 days at 18°C in the dark. The sporulating lesions were transferred onto pea agar medium (160 g peas, 5 g sucrose, 15 g agar, 700 ml distilled water) amended with 50 mg/ml vancomycin. Ten pure cultures were obtained for each of 4 isolates per county by hyphal tipping. Cellulose acetate electrophoresis was conducted to determine Gpi allozyme genotype of the 4 isolates (4). The allozyme banding patterns were 100/100 at the Gpi locus, consistent with previously reported analyses of the US-23 genotype (1,2). Genomic DNA was extracted from 10 pure cultures using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD), and SSR analyses were performed. Microsatellite markers Pi02, Pi4B, Pi63, PiG11, and D13 were used in SSR analyses. Pi02, Pi4B, and Pi63 had alleles of 162/164, 213/217, and 270/279 bp in size, respectively which is consistent with the reference US-23 genotype (1). However, heterozygosity was detected at locus D13 in the Idaho genotype with allele size of 134/210 bp and an additional allele of 140/155/176 bp at locus PiG11. This is different from the standard US-23 genotype (homozygous alleles 134/134 at locus D13 and two alleles 140/155 at locus PiG11). These allele changes indicate the isolates may be variants of US-23 isolates as all other phenotypic characteristics were similar to those of reference US-23 isolates. The Idaho genotypes were sensitive to metalaxyl both in vitro on rye A agar medium amended with metalaxyl at <0.1 ppm, and in vivo on Ridomil treated foliage tests at <0.1 ppm (1,2). Mating type assays confirmed the pathogen to be the A1 mating type. In the 2009 and 2010 late blight epidemics in the eastern United States, US-23 was the predominant genotype, but to our knowledge this genotype has never been reported previously in Idaho. Thus, this is the first known report of P. infestans genotype US-23 causing late blight on potato in Idaho, indicating a change in the population of P. infestans. In Idaho, the source of this genotype remains unknown, although infected tomatoes have been implicated in the widespread dissemination of this genotype of P. infestans in the eastern United States. References: (1) G. Danies et al. Plant Dis. 97:873, 2013. (2) C. Hu et al. Plant Dis. 96:1323, 2012. (3) K. Deahl. (Abstr.) Phytopathology 100:S161, 2010. (4) S. B. Goodwin et al. Plant Dis. 79:1181, 1995. (5) USAblight. Recent US Genotypes. Online: www.usablight.org/node/52 , retrieved 3 January 2014.

First Report of a Binucleate Rhizoctonia (AG-A) from Potato Stems Infecting Potatoes and Sugarbeet in the Pacific Northwest.

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.

Rhizoctonia solani Anastomosis Group 3 is Predominant in Potato Tubers in Poland.

Rhizoctonia solani is a species complex of 13 related but genetically distinct anastomosis groups (AGs). In potato, R. solani can infect the stems, stolons, and roots, resulting in quantitative losses. It can also cause qualitative losses through blemishes occurring on progeny tubers, such as black scurf and elephant hide (corky cracking). Knowledge of the AG in local populations is important because they differ in host range, fungicide sensitivity, and disease severity (2). To determine the AGs present in Poland, 54 tuber samples displaying typical R. solani symptoms were taken from six different fields in 2011. The fields were representative of five different administrative regions of Poland and from at least 10 different varieties. Rhizoctonia was isolated from tubers by placing symptomatic material on to tap water agar amended with streptomycin and penicillin and after 2 to 3 days Rhizoctonia colonies were identified and hyphal tips of these transferred to potato dextrose agar. Rhizoctonia was successfully isolated from 48 tubers displaying black scurf and two tubers displaying elephant hide symptoms. DNA was extracted from Rhizoctonia cultures using a Wizard Food kit (Promega) and the AG was determined using specific real-time PCR assays (1). All Rhizoctonia isolates were determined to be AG3 and this was confirmed for 10 selected isolates by observing hyphal fusion with a known AG3 tester isolate (Rs08) as described previously (3). Pairings were also conducted amongst the 10 Polish isolates, C2 reactions were typically observed indicating numerous vegetative compatible groups are present. This study shows that AG3 is likely to be the predominant AG in potato tubers in Poland. This is similar to other studies in Europe, which have all determined that AG3 accounts for at least 92% of isolates from potato (2,3). AG2-1, 4, and 5 have also been found in tubers worldwide and climate and certain crop rotations can influence the presence of these other AGs in potato tubers (2). However, climate and crop rotations in Poland are similar to other parts of Europe so the predominance of AG3 is expected. AG3 was also isolated from elephant hide symptoms; however, it was more frequently isolated from sclerotia. The ability of AG3 to prolifically produce sclerotia and thereby survive on seed tubers may explain its predominance in potato crops (4). Therefore, studies focusing on the management of Rhizoctonia potato disease in Poland should consider AG3 in the first instance. References: (1) G. E. Budge et al. Plant Pathol. 58:1071, 2009. (2) L. Tsror. J. Phytopathol. 158:649, 2010. (3) J. W. Woodhall et al. Plant Pathol. 56:286, 2007. (4) J. W. Woodhall et al. Plant Pathol. 57:5, 2008.

First Report of Boscalid and Penthiopyrad-Resistant Isolates of Alternaria solani Causing Early Blight of Potato in Michigan.

Early blight of potato (Solanum tuberosum) is caused by Alternaria solani and occurs annually in Michigan. If left uncontrolled, it can result in yield losses exceeding 20% and impact stored potatoes. The disease is commonly managed using succinate dehydrogenase inhibitor (SDHI) fungicides (1). Unfortunately, recent studies have shown that SDHI resistance has increased dramatically over the past 2 years in A. solani populations (1,2). To investigate the occurrence of SDHI resistance in Michigan, potato leaves with early blight symptoms were collected from fields in Montcalm and Ionia counties, MI, in 2012. To obtain A. solani isolates from leaves, small pieces of leaf tissue (5 × 5 mm) were excised from the center of lesions and transferred on to water agar. Plates were incubated at 25°C overnight to allow conidia to germinate. Single germinated A. solani conidia were transferred to potato dextrose agar (PDA) and incubated at 25°C for 7 days. The identity of cultures was confirmed by colony and conidial morphology (3). Nineteen A. solani isolates were obtained and each was screened for sensitivity to the SDHI fungicides boscalid, penthiopyrad, and fluopyram, using a 50 ppm discriminatory dose based on EC50 values previously determined (2). Mycelial plugs (~5.5 mm) were transferred to amended and non-amended PDA plates that were incubated at 25°C for 7 days. An isolate was considered highly resistant if fungal growth relative to control plates exceeded 50%, moderately resistant if it was between 35 and 50%, and sensitive if it was less than 35% (2). A sensitive A. solani isolate (AS11) from Bonners Ferry, ID, was used as a control in these experiments. Of all isolates tested, 11% were highly resistant to both boscalid and penthiopyrad and 5% were moderately resistant to both fungicides, 21% were moderately resistant to penthiopyrad alone, and the remaining isolates (84 and 68% respectively) were sensitive to the two fungicides. None of the isolates tested were resistant to fluopyram. Recently, two major mutations, H227R in SdhB and H133R in SdhD, have been identified in highly resistant A. solani isolates in Idaho (2). Because the majority of the identified mutations occur near the 3' end of each subunit, this region was amplified and sequenced using the following primer sets: SdhB (5'-TACTGGTGGAACCAGGAGGAGTA-3' and 5'-CATACCACTCTAGGTGAAG-3'), SdhC (5'-CCAAATCACCTGGTACGCCTCG-3' and 5'-TCATCCGAGGAAGGTGTAGTAAAGGCTG-3'), and SdhD (5'-CCGACTCTATTCTCTGCGCCCT-3' and 5'-CTCGAAAGAGTAGAGGGCAAGACCCA-3'). In this study, all of the isolates that were highly resistant to both boscalid and penthiopyrad were found to contain the H133R mutation in SdhD, which correlated with the strongest resistance phenotype. To our knowledge, this is the first report of resistance to SDHI fungicides in populations of A. solani on potato in Michigan. These data are concerning as they indicate that the highly resistant isolates have already developed cross-resistance between boscalid and penthiopyrad, despite penthiopyrad not yet having regular use in Michigan. Although all of the isolates tested were sensitive to fluopyram, the discovery of isolates resistant to boscalid and penthiopyrad suggests that all SDHI fungicides should be considered at high risk of resistance development in Michigan. References: (1) K. Fairchild et al. Crop Prot. 49:31, 2013. (2) T. Miles et al. Plant Pathol. doi: 10.1111/ppa.12077, 2013. (3) P. Wharton et al. Plant Dis. 96:454, 2012.

First Report of In-Vitro Boscalid-Resistant Isolates of Alternaria solani Causing Early Blight of Potato in Idaho.

Early blight of potato (Solanum tuberosum) is caused by Alternaria solani and occurs annually to some degree in Idaho. The timing of its appearance and rate of disease progress determine the impact on the potato crop. Though losses rarely exceed 20%, they can be higher and occur in stored potatoes if the disease is not controlled. Early blight is managed mainly by cultural practices such as plant nutrition, water management, and avoidance of plant stress, but also with the use of foliar fungicides. Currently, the main fungicides labeled for control of early blight are in the carboxamide and strobilurin fungicide groups. Development of resistance to some fungicide groups may contribute to the loss of control of early blight. Isolates of A. solani from Idaho potatoes were tested for resistance to boscalid in the carboxamide group. Diseased potato leaves with early blight symptoms were collected from fields near Parma, Rupert, and Aberdeen in southern Idaho in 2009 and 2010 and Bonners Ferry in northern Idaho in 2010. To obtain A. solani isolates from leaves, small pieces of leaf tissue (5 × 5 mm) were taken from the center of early blight lesions and streaked across the surface of a thin layer (3 mm) of water agar. Plates were incubated at 25°C overnight to allow spores to germinate. Single germinated A. solani spores were transferred to acidified potato dextrose agar and incubated in the dark at 25°C. Germinated spores were identified based on spore morphology. Spores of A. solani can be easily distinguished from other Alternaria spp. found on potato because they are ellipsoid to oblong and taper to a long beak that is usually as long as the spore body. The identity of cultures grown from single spores was confirmed by colony and spore morphology. Sensitivity of A. solani isolates to boscalid was determined by the spiral gradient endpoint method (2). For all isolates, the effective concentration for 50% reduction in growth was outside the range of the spiral plate dilution series (i.e., isolates were either completely insensitive or completely sensitive to boscalid). In total, 46 isolates (20 collected in 2009 and 26 collected in 2010) were tested against boscalid. Experiments were carried out twice with 2009 isolates using mycelial strips and conidial suspensions. Experiments with 2010 isolates were carried out three times using only conidial suspensions. Of the isolates from 2009, 15% were insensitive. There was no difference between the use of mycelial strips or conidial suspensions. In 2010, 62% of isolates were insensitive. By location, 72% of isolates from Parma, 73% from Rupert, 63% from Aberdeen, and 44% from Bonners Ferry were insensitive. Resistance to boscalid has been reported in A. alternata isolates from pistachio (1,3). However, to our knowledge, this is the first report of resistance to boscalid in isolates of A. solani on potato. These data suggest that resistance to boscalid is widespread in Idaho, even in areas like Bonners Ferry where potato cultivation is limited. Boscalid insensitivity in vitro may not translate directly to commercial production and currently there is no evidence to suggest that boscalid has failed to control early blight in Idaho. However, the discovery of insensitive isolates suggests that boscalid should be considered at high risk of resistance development. References: (1) H. Avenot et al. Plant Dis. 91:1345, 2007. (2) H. Förster et al. Phytopathology 94:163, 2004. (3) N. Rosenzweig et al. (Abstr.) Phytopathology 93(suppl.):S75, 2003.

First Report of Rhizoctonia solani AG4 HG-II Infecting Potato Stems in Idaho.

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.

First Report of Rhizoctonia solani AG2-2IIIB Infecting Potato Stems and Stolons in the United States.

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.

Optimizing Fungicide Timing for the Control of Rhizoctonia Crown and Root Rot of Sugar Beet Using Soil Temperature and Plant Growth Stages.

Azoxystrobin is applied early in the sugar beet growing season in north-central United States for control of Rhizoctonia damping-off and Rhizoctonia crown and root rot caused by Rhizoctonia solani anastomoses groups (AGs) 4 and 2-2, respectively. Fungicide application timings based on crop growth stage and soil temperature thresholds were evaluated in inoculated small-scale trials and in commercial fields with a history of Rhizoctonia crown and root rot. Soil temperature thresholds of 10, 15, and 20°C were selected for fungicide application timings and used to test whether soil temperature could be used to better time applications of azoxystrobin. In both small- and large-plot trials, timing applications after attainment of specific soil temperature thresholds did not improve efficacy of azoxystrobin in controlling damping-off or Rhizoctonia crown and root rot compared with application timings based on either planting date, seedling development, or leaf stage in a susceptible (E-17) and a resistant (RH-5) cultivar. Application rate and split application timings of azoxystrobin had no significant effect on severity of crown and root rot. Other environmental factors such as soil moisture may interact with soil temperature to influence disease development. Cv. RH-5 had higher sugar yield attributes than the susceptible cultivar (E-17) in seasons conducive and nonconducive to crown and root rot development. All isolates recovered from both small- and large-plot trials in all years were AG 2-2. R. solani AG 4 was not identified in any samples from any year.

First Report of Potato Tuber Sprout Rot Caused by Fusarium sambucinum in Michigan.

Fusarium dry rot is one of the most important diseases of potato (Solanum tuberosum L.), affecting tubers in storage and whole seed or seed pieces after planting (2). Fusarium sambucinum Fuckel (teleomorph Giberella pulicaris) is the most common pathogen causing dry rot of stored tubers in North America. (4). Cut seed potato tubers of cvs. FL1879 and Pike with severe sprout rot were collected in Michigan during May 2006. As well as having rotted sprouts, all diseased tubers had dry rot. When diseased sprouts were cut in half, brown, necrotic lesions could be seen spreading down the center of the sprout in vascular tissue and at the base of the sprout in tuber tissue. Pathogen isolations were made from both infected tuber tissue and diseased sprouts on potato dextrose agar (PDA). In both cases, only F. sambucinum was isolated from diseased sprout and tuber tissue. Identification of the pathogen was based on colony and conidial morphology. This included white, fluffy mycelium on the surface and crimson coloration of the colonies viewed from the underside of PDA plates and large distinctive macroconidia (3). Identification was confirmed by comparison of ITS (internal transcribed spacer) sequence data with reference isolates. The ITS region of rDNA was amplified by polymerase chain reaction (PCR) with primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the sequence obtained showed a 100% homology with F. sambucinum Fuckel. For inoculum production, isolates were grown on PDA at 8°C for 14 days prior to inoculation. Pathogenicity was tested in potato tubers of cv. FL1879 with a single isolate collected from diseased sprouts. Whole seed tubers with 4 mm long sprouts were cut in half longitudinally with a sterile knife to ensure that seed pieces had viable sprouts. The cut surfaces of seed pieces were spray inoculated with 200 ml of conidial suspension (1 × 104 conidia ml-1) over the entire cut surface to give a final dosage of approximately 1 ml per seed piece. Care was taken to limit inoculum spray to the cut surface so that sprouts were not inoculated. Seed pieces (40 per replicate × 4 replicates) were then placed in plastic boxes (30 × 15 × 10 cm) and incubated in the dark at 18°C and 95% relative humidity for 30 days in a controlled environment chamber. As a control, cut seed pieces were spayed with sterile distilled water and incubated as above. All tubers inoculated with the pathogen developed typical Fusarium dry rot symptoms consisting of a brown, dry decay of tuber tissue with mycelial lined cavities. Sprouts on inoculated tubers developed symptoms that were observed in the initially collected seed pieces, and F. sambucinum was reisolated from all infected sprouts. The noninoculated control tubers did not develop any symptoms of dry rot. The results of the pathogenicity tests indicate that F. sambucinum caused sprout rot on potato seed pieces. Since only the cut surfaces of tubers were inoculated, it is assumed that infection of sprouts is systemic through the tuber. To our knowledge, this is the first report of F. sambucinum causing a sprout rot of developing sprouts on seed tubers in the United States. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) L. E. Hanson et al. Phytopathology 86:378, 1996. (3) P. E. Nelson et al. Pages 118-119 in: Fusarium Species: An Illustrated Manual for Identification. The Pennsylvania State University, University Park and London, 1983. (4) G. A. Secor and B. Salas. Fusarium dry rot and Fusarium wilt. Pages 23-25 in: Compendium of Potato Diseases. 2nd ed. W. R. Stevenson et al., eds. The American Phytopathological Society, St. Paul, MN, 2001.

Assessment of immunoglobulin-M immunosorbent agglutination assay (ISAGA) for detecting toxoplasma specific IgM.

An immunoglobulin-M immunosorbent agglutination assay (ISAGA) was introduced to detect toxoplasma specific IgM. This assay incorporates mu chain capture and use of entire toxoplasma trophozoites as an antigen source. The performance of the ISAGA was compared with that of a double sandwich enzyme linked immunosorbent assay (DS-ELISA) currently used in the Public Health Laboratory Service Toxoplasma Reference Laboratories. The ISAGA was found to be more sensitive than DS-ELISA but there was no demonstrable difference in the specificity or reproducibility between the two assays. The ISAGA is suitable for the diagnosis of acute toxoplasmosis in immunocompetent patients and as a screening test for recent infection in pregnant women. The persistence of ISAGA reactivity, however, is such that additional serological assessment is required to define the risk of congenital infection.

Ultrastructure of the Infection of Sorghum bicolor by Colletotrichum sublineolum.

ABSTRACT Ultrastructural studies of the infection of susceptible and resistant cultivars of Sorghum bicolor by Colletotrichum sublineolum were conducted. Initial penetration events were the same on both susceptible and resistant cultivars. Germ tubes originating from germinated conidia formed globose, melanized appressoria, that penetrated host epidermal cells directly. Appressoria did not produce appressorial cones, but each penetration pore was surrounded by an annular wall thickening. Inward deformation of the cuticle and localized changes in staining properties of the host cell wall around the infection peg suggests that penetration involves both mechanical force and enzymic dissolution. In compatible interactions, penetration was followed by formation of biotrophic globular infection vesicles in epidermal cells. Filamentous primary hyphae developed from the vesicles and went on to colonize many other host cells as an intracellular mycelium. Host cells initially survived penetration. The host plasma membrane invaginated around infection vesicles and primary hyphae and was appressed tightly to the fungal cell wall, with no detectable matrix layer at the interface. Necrotrophic secondary hyphae appeared after 66 h and ramified through host tissue both intercellularly and intracellularly, forming hypostromatic acervuli by 114 h. Production of secondary hyphae was accompanied by the appearance of electron-opaque material within infected cells. This was thought to represent the host phytoalexin response. In incompatible interactions, infection vesicles and primary hyphae were formed in epidermal cells by 42 h. However, they were encrusted with electron-opaque material and appeared dead. These observations are discussed in relation to the infection processes of other Colletotrichum spp. and the host phytoalexin response.

Nutrition in adolescence.

This review discusses five questions concerning nutrition in adolescence. Do adolescents have special nutritional needs? What are the recommended dietary intakes? What do adolescent schoolchildren eat? What nutritional disorders occur at this age? The prevalence of iron deficiency anaemia, rickets, coeliac disease, iodine deficiency, dental caries, weight disorders, zinc deficiency and pellagra are discussed. What is the role of school meals or other food supplementation programmes?

Human pinch-force discrimination.

Pinch-sustaining tasks such as holding a pencil, fork, or key require the exertion of different levels of force. There is little information concerning normal subjects' ability to discriminate differences in their pinching force, so the purpose of this study was to evaluate the ability of 24 normal young women to discriminate differences in their self-generated isometric tip and lateral pinching force. Resistance forces of 10, 25, 50, and 75% of known normal maximum pinching force were selected as standards. Subjects were presented a series of paired resistance settings of which the first resistance in each pair was the standard and the second resistance a comparator of some greater amount. This procedure of paired comparisons was continued until subjects' threshold of discrimination between two pinching forces was established. The results indicated that subjects' pinch-force discrimination at the standard of 50% of reported maximum pinching force was significantly better for the tip condition than for the lateral condition. This study has described an instrumentation and the methodology for assessing individuals' ability to discriminate differences in their pinching force at submaximal levels.

Prevalence of cleft uvula among school children in kindergarten through grade five.

The purpose of this study was to determine the prevalence of bifid uvula among elementary school children in grades K through 5. Three examiners performed independent intraoral examinations of 709 children enrolled in a Jacksonville, Florida metropolitan public school. The structure of the uvula was evaluated as normal or bifid. If bifid, the cleft was classified as: (a) notched, (b) extending up to 1/4 the length of the uvula, (c) extending from 1/4 to 3/4 of the length of the uvula, or (d) extending the full length of the uvula. Prevalence of some form of uvular cleft was observed among 16 (2.26%) of the children while full length uvular cleft was found in only two (0.3%) of the children. These findings are congruent with percentages reported in other studies. It is recommended that speech-language pathologists identify children with bifid uvulas and provide appropriate counseling and follow-up study.

The assessment and manipulation of personal diet: an undergraduate practical in human nutrition.

The Department of Paediatrics at King Saud University, Riyadh, Saudi Arabia offers to medical undergraduates an optional course in nutrition and growth. This paper describes the practical experiences offered in dietary assessment and manipulation which was one component of the course, together with an assessment of its value by the students and the authors. Generally, it seemed that the component had been successful but three changes might be considered: although hand analysis of crude dietary data to determine nutrient intakes is a useful exercise, computer analysis should also be included; the principles of dietary manipulation (in effect, the principles of therapeutic dietetics) will require more emphasis; the students should make a detailed analysis of a patient's diet as well as their own. Regarding the nutrition and growth course as a whole: assessment of clinical skills would probably benefit from the same structured approach as used in the dietetic assessment; a preliminary assessment of the students' nutritional knowledge at the start of the course should be incorporated.

Sorrento Asian food tables: food tables, recipes and customs of mothers attending Sorrento Maternity Hospital, Birmingham, England.

Information on food customs and recipes, collected from Indian, Pakistani and Bangladeshi immigrant women attending the Sorrento Maternity Hospital, in Birmingham, UK is presented. The calculated nutrient composition of these recipes is given, together with the composition of a number of Asian foods and spices taken from various food tables.

Nutrient intake of pregnant Asian women at Sorrento Maternity Hospital, Birmingham.

The dietary intake of pregnant Asian women (that is originating from the Indian subcontinent) attending Sorrento Maternity Hospital in Birmingham was determined, using the weighed and recall techniques, at five-weekly intervals from 18 to 38 weeks of pregnancy. Mean energy intake of the group was 7.1 MJ (1700 kcal)/d. The intakes of most nutrients were substantially below those consumed by pregnant European women in Britain, a little below those of expectant Pakistani mothers in Islamabad, and about the same as those of expectant East London mothers. Intakes of vitamin D, total folate, vitamin B6, zinc and magnesium were particularly low. These observations suggest that a number of Asian women in Birmingham are likely to experience nutritional stress in pregnancy, and there is some anthropometric and biochemical evidence from Sorrento, published elsewhere (Bissenden et al. 1981), to support this. A possibly beneficial feature of the diet was a low sodium intake (2 g/d). Previous work at this hospital has noted a lower prevalence of hypertension in pregnant Asian women (Wharton et al. 1980; Bissenden et al. 1981).

Subethnic variation in the diets of Moslem, Sikh and Hindu pregnant women at Sorrento Maternity Hospital, Birmingham.

The previous paper (Eaton et al. 1984) described the nutrient intake of pregnant Asian women attending Sorrento Maternity Hospital, Birmingham using the weighed and recall methods. The present paper describes the subethnic variation in nutrient intake by comparing the results from Pakistanis, Sikhs, Hindus and Bangladeshis and also describes food eaten by the pregnant women. Generally, Sikhs had the highest intake of most nutrients (mean energy 7.5 MJ (1800 kcal)/d) and the greatest variety of foods; they ate chapatti and paratha but few ate meat. Hindus had a very similar diet but more ate meat, chicken and rice. Pakistanis had an energy intake about 10% below that of the Sikhs and Hindus; meat was eaten, and intake of fruit, and therefore vitamin C, was quite large. Bangladeshis were the smallest women; they had the lowest intake of energy (mean energy 6.5 MJ (1555 kcal)/d) and most nutrients, except for protein, so that 15% of energy was provided by protein. Fish, rice and a low-fat intake were other features of their diet. From a nutritional standpoint, peoples coming from the Asian subcontinent should be divided into subethnic groups; the collective term 'Asian' is insufficient. It is not clear whether these differences have any effect on the life and health of the individuals. Comparison of groups does not suggest an obvious relationship between dietary intake and fetal growth; however, there is other evidence to implicate the possible role of deficiencies of protein, energy, zinc and pyridoxine. The results provide some support for the community nutritional policies of (a) offering vitamin D supplements to all pregnant Asian women and (b) fortifying bread with calcium, thiamin and nicotinic acid. There is probably no need to offer vitamin A and C supplements but they are harmless. Indications for iron supplementation are no different from those for white English women.

Placental zinc in normal and intra-uterine growth-retarded pregnancies.

The zinc concentration of placental tissue and cord blood in sixteen mothers who gave birth to normal babies was measured. The blood volume of each placenta was estimated from measurements of haemoglobin concentration of placental homogenate and cord blood, and, by deduction, the Zn content of blood-free placental tissue was calculated. Results were compared with eleven mothers whose fetuses showed a low biparietal diameter velocity between 17 and 28 weeks gestation and with ten mothers who gave birth to intra-uterine growth-retarded (IUGR) babies. As expected, placental weight was significantly correlated with infant birth weight. Blood-free placental tissue contained about four times more Zn (approximately 10 micrograms Zn/g) than cord blood (approximately 2.5 micrograms Zn/ml). Concentrations of Zn in blood-free placental tissue were similar in all three groups, but the cord blood Zn of mothers producing IUGR babies was significantly lower than that of the other two groups. Results of the present study suggested that fetal growth retardation in the mothers studied could not be explained by differences in blood-free placental Zn concentration, but that there may be some association between lower cord blood Zn levels and intra-uterine growth retardation.