Comparative Pathogenicity and Virulence of Fusarium Species on Sugar Beet.

In all, 98 isolates of three Fusarium spp. (18 Fusarium oxysporum, 30 F. graminearum, and 50 Fusarium sp. nov.) obtained from sugar beet in Minnesota were characterized for pathogenicity and virulence on sugar beet in the greenhouse by a bare-root inoculation method. Among the 98 isolates tested, 80% of isolates were pathogenic: 83% of the F. oxysporum isolates, 57% of the F. graminearum isolates, and 92% of the Fusarium sp. nov. isolates. Symptoms varied from slight to moderate wilting of the foliage, interveinal chlorosis and necrosis, and vascular discoloration of the taproot without any external root symptoms. Among the pathogenic isolates, 14% were highly virulent and 12% were moderately virulent. Most of the highly virulent isolates (91%) and moderately virulent isolates (89%) were Fusarium sp. nov. All pathogenic isolates of F. graminearum and most pathogenic isolates (87%) of F. oxysporum were less virulent. In general, more-virulent isolates induced first foliar symptoms earlier compared with less-virulent isolates. This study indicates that both F. oxysporum and Fusarium sp. nov. should be used in greenhouse and be present in field studies used for screening and developing sugar beet cultivars resistant to Fusarium yellows complex for Minnesota and North Dakota.

Association of 'Candidatus Liberibacter solanacearum' with Zebra Chip Disease of Potato Established by Graft and Psyllid Transmission, Electron Microscopy, and PCR.

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with 'Candidatus Liberibacter' sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of 'Ca. L. solanacearum' previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of 'Ca. L. solanacearum' with potatoes affected by ZC in the United States, Mexico, and Guatemala.

Genetic relationships among populations of Gibberella zeae from barley, wheat, potato, and sugar beet in the upper Midwest of the United States.

Gibberella zeae, a causal agent of Fusarium head blight (FHB) in wheat and barley, is one of the most economically harmful pathogens of cereals in the United States. In recent years, the known host range of G. zeae has also expanded to noncereal crops. However, there is a lack of information on the population genetic structure of G. zeae associated with noncereal crops and across wheat cultivars. To test the hypothesis that G. zeae populations sampled from barley, wheat, potato, and sugar beet in the Upper Midwest of the United States are not mixtures of species or G. zeae clades, we analyzed sequence data of G. zeae, and confirmed that all populations studied were present in the same clade of G. zeae. Ten variable number tandem repeat (VNTR) markers were used to determine the genetic structure of G. zeae from the four crop populations. To examine the effect of wheat cultivars on the pathogen populations, 227 strains were sampled from 10 subpopulations according to wheat cultivar types. The VNTR markers also were used to analyze the genetic structure of these subpopulations. In all populations, gene (H = 0.453 to 0.612) and genotype diversity (GD = or >0.984) were high. There was little or no indication of linkage disequilibrium (LD) in all G. zeae populations and subpopulations. In addition, high gene flow (Nm) values were observed between cereal and noncereal populations (Nm = 10.69) and between FHB resistant and susceptible wheat cultivar subpopulations (Nm = 16.072), suggesting low population differentiation of G. zeae in this region. Analysis of molecular variance also revealed high genetic variation (>80%) among individuals within populations and subpopulations. However, low genetic variation (<5%) was observed between cereal and noncereal populations and between resistant and susceptible wheat subpopulations. Overall, these results suggest that the populations or subpopulations are likely a single large population of G. zeae affecting crops in the upper Midwest of the United States.

Survival, Dispersal, and Primary Infection Site for Cercospora beticola in Sugar Beet.

Cercospora beticola survives as stromata in infected crop residue. Spores produced on these survival structures serve as primary inoculum during the next cropping season. This study was conducted to determine how long C. beticola can survive at different soil depths, the mechanism of inoculum dispersal, and the primary infection site in sugar beet. Longevity of C. beticola was studied over a 3-year period under field conditions at Fargo, ND. C. beticola-infected leaves were placed at depths of 0, 10, and 20 cm and retrieved after 10, 22, and 34 months. Survival of C. beticola inoculum declined with time and soil depth. Inoculum left on the soil surface, 0 cm in depth, survived the longest (22 months) compared with that buried at 10 cm (10 months) and 20 cm (10 months). C. beticola dispersal from the primary source of inoculum was studied in the field for three growing seasons. Sugar beet plants were surrounded with plastic cages with and without ground cover, or exposed with and without ground cover. Significantly higher disease severity was observed on exposed plants than caged plants with or without ground cover, suggesting that wind was the major dispersal factor for C. beticola inoculum. The primary infection site by C. beticola was determined in a greenhouse study. Leaves, roots, and stems of healthy sugar beet plants were inoculated with C. beticola. Cercospora leaf spot symptoms were observed only on plants that were leaf inoculated, suggesting that the leaf was the primary infection site for C. beticola.

First Report of a Defect of Processing Potatoes in Texas and Nebraska Associated with a New Phytoplasma.

An outbreak of a new potato disease occurred in Texas and Nebraska causing a serious defect in potato chips produced from commercial processing potatoes. The defect consists of patchy brown discoloration of chips and can be a cause for rejection of contracted potatoes by the processor. Infected potato plants exhibit symptoms of the purple top wilt syndrome similar to those of the purple top disease in processing potatoes caused by clover proliferation phytoplasma recently found in Washington and Oregon (3). Foliar symptoms include stunting, chlorosis, slight purple coloration of new growth, swollen nodes, proliferated axillary buds, and aerial tubers. Tuber symptoms include mild vascular discoloration and brown flecking of medullary rays. Seed potatoes from affected plants produce hair sprouts. Total nucleic acid was extracted from leaf and stolon tissue of symptomatic plants in the field and from tuber samples exhibiting the defect from commercial storages. Nested polymerase chain reactions (PCR) were performed using phytoplasma-universal 16SrDNA-based primers (P1/P7 and R16F2n/R16R2) (2) to detect the presence of phytoplasmas in these samples. A negative control, devoid of DNA templates in the reaction mix, was included in all PCR assays. In 2004, 13 foliar samples tested positive for phytoplasmas using PCR. None of the apparently symptomless plants or tubers tested positive. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA using enzymes AluI, MseI, HhaI, BfaI, and Tsp509I indicated that four samples are associated with a phytoplasma belonging to subgroup A (16SrI-A) of the "Candidatus Phytoplasma asteris" (aster yellows phytoplasma) group (16SrI), and nine plant samples were associated with a new phytoplasma related to, but distinct from, the stolbur phytoplasma group (16SrXII). Nucleotide sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. DQ174114-DQ174123) confirmed the results on the basis of RFLP analyses. Sequences of cloned 16S rDNAs were analyzed with previously described phytoplasma strains available in GenBank using DNAStar's (Madison, WI) Lasergene software MegAlign program. The new phytoplasma is only distantly related to the stolbur phytoplasma, sharing 96.6% sequence homology. In 2005, 14 defective tuber samples from storage and 16 symptomatic plants from the field tested positive for the new phytoplasma. In Texas and Nebraska, it appears that at least two distinct phytoplasmas seem to be involved in the disease complex contributing to the defects of processed products produced from infected potatoes. Previous reports have suggested a similar defect of chipping potatoes, but the phytoplasma associated with the disease was not identified (1). To our knowledgek, this the first report of this new phytoplasma associated with disease and defects of potato and the first report of this phytoplasma in the United States. References: (1) E. E. Bantarri et al. Trans. ASAE 33:221, 1990. (2) I.-M. Lee et al. Int. J. Sys. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Plant Dis. 88:429, 2004.

First Report of Fusarium graminearum Causing Dry Rot of Potato in North Dakota.

Fusarium dry rot of potato can be caused by several species of Fusarium, but F. sambucinum is considered the primary cause in stored potatoes in North America and Europe (2). Potato tubers of cvs. Shepody and Russet Burbank with severe dry rot were collected from a commercial processing storage facility in central North Dakota during 2003-2004. Pathogen isolations were made from infected tubers on one-half strength acidified potato dextrose agar (APDA). Only F. graminearum was isolated from all rotted tubers used. Identification was based on colony morphology and conidial and perithecial characteristics, which included a carmine coloration of the underside of the agar and white fluffy mycelium on APDA, the presence of black perithecia on carnation leaf agar, and large distinctive macroconidia (1). The identity was confirmed by the Fusarium Research Institute at Pennsylvania State University. Pathogenicity was tested in potato tubers and greenhouse-grown potato plants cv. Atlantic. Nine tubers were wounded by removal of a plug of tissue with a cork borer, 3 mm in diameter and 5 mm deep, and inoculated by placing either 100 µl of a conidial suspension (5 × 104 conidia per ml) from a 7-day-old culture or a mycelial plug, 3 mm in diameter, from a 7-day-old culture in the wound. Nine tubers wounded and treated with either sterile distilled water or one-half strength APDA served as controls. Plant inoculations were performed by cutting a slit in the lower stem with a sterile scalpel and placing a cotton collar saturated with a conidial suspension (5 × 104 conidia per ml) around the wound and held in place with a clothespin. Four plants were inoculated with a conidial suspension, and four plants were treated with sterile distilled water. All tubers inoculated with either Fusarium treatment developed typical potato dry rot symptoms consisting of a brown, dry decay with mycelium lined cavities, and F. graminearum was reisolated from all symptomatic tubers. The control tubers did not develop symptoms. No symptoms developed in any of the greenhouse inoculated plants. Fifteen isolates were tested for sensitivity to thiabendazole, and all were sensitive with EC50 (50% effective concentration) values ranging from 0.8 to 3.7 µl/ml. The results indicate that F. graminearum can cause dry rot of potato, and to our knowledge, this is the first report of F. graminearum as a cause of potato dry rot. These results have epidemiological implications in the persistence, spread, and management of F. graminearum in cereals and potatoes, since potato is often used in rotation with other hosts of F. graminearum, including wheat, barley, and corn. References: (1) 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. (2) 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, R. Loria, G. D. Franc, and D. P. Weingartner, eds. The American Phytopathological Society, St. Paul, MN, 2001.

Organization and transcription of the gene encoding potato UDP-glucose pyrophosphorylase.

The organization of the gene encoding potato UDP-glucose pyrophosphorylase, one of the key enzymes of carbohydrate metabolic pathway is presented. The gene cloned from cultivar (cv.) Lemhi consists of a 6.6-kb structural and a 1-kb regulatory region. The structural region contains 20 exons and 19 introns. The coding sequence with exception of three bases is identical with the UGPase cDNA previously cloned from Danshaku-Imo cv. [Katsube et al. (1990) UDP-Glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing. J. Biochem. 108, 321-326]. The largest intron contains a tandem repeat consisting of 50 nt core units. A putative polyadenylation site is situated 79 bp downstream of the translation stop codon. A transcription start point (tsp) and a putative TATA-box were located 84 bp and 141 bp upstream of the translation start, respectively. The regulatory region contained general enhancer, suppressor, and regions responsible for tissue specificity of UGPase expression.

Variability in Virulence Among Asexual Progenies of Phytophthora infestans.

ABSTRACT One hundred two single zoospore isolates of Phytophthora infestans, derived asexually from four parental isolates of US-8 genotype and one isolate of US-1 genotype, were characterized for their virulence phenotypes to determine changes in virulence during asexual reproduction. Potato differentials, each containing a major gene for resistance to P. infestans (R1 to R11), were used to characterize the virulence patterns. Isolates were also characterized for mating type, glucose-6-phosphate isomerase (Gpi) banding pattern, and DNA fingerprints using probe RG57 to determine any genotypic changes in the single zoospore isolates. A subset of these single zoospore isolates was tested for response to mefenoxam to determine any shifts in sensitivity. Results showed that single zoospore isolates derived from parent PI-1 (US-8, 11 isolates) were identical to their parental virulence. Isolates derived from parent PI-191 (US-8, 29 isolates) showed some differences in virulence, mainly toward R8 and R9. Isolates derived from parent PI-126 (US-8, 14 isolates) demonstrated a higher level of virulence diversity. Isolates derived from parents PI-52 (US-1, 28 isolates) and PI-105 (US-8, 20 isolates) showed the highest level of virulence variability among the single zoospore isolates. Mating type, Gpi banding pattern, and DNA fingerprints for the single zoospore isolates were, in most cases, identical to the parental isolates. Single zoospore isolates showed different levels of sensitivity to mefenoxam. Virulence and other genetic changes during asexual reproduction are likely to play a major role in changing the race structure of P. infestans populations. This continuous change in the race structure is a serious problem and now poses a new challenge for utilization of race-specific resistance to manage late blight of potato.

First Report of Potato Powdery Scab, Caused by Spongospora subterranea f. sp. subterranea, in North Dakota.

Powdery scab is a serious disease of potatoes (Solanum tuberosum L.) that can cause extensive surface defects on susceptible potato cultivars. The pathogen, Spongospora subterranea (Wallr.) Lagerh. f. sp. subterranea J. A. Tomlinson, is an important concern in seed potato production because current seed certification standards in North Dakota have a zero tolerance for this pathogen. Powdery scab had not previously been identified in North Dakota. Until recently this disease was not thought to be present in U.S. commercial potato-producing areas. Powdery scab has now been shown to be more widely distributed than previously thought (1), having been identified in several locations in eastern and western North America. However, environmental conditions in North Dakota, including high soil pH, low rainfall, and high temperatures, are not considered favorable for powdery scab development. Diseased potatoes were found in a field from Griggs County, ND, in the fall of 1994 and characteristic cystosori were present in erumpent lesions on infected tubers. The field was an irrigated circle planted to three different cultivars. The cultivars Goldrush and Red Norland were symptomless, while cv. Red La-Soda was severely affected. Up to 30% of the tuber surfaces were diseased and disease incidence approached 25%. The infected crop was destroyed. Pathogenicity was confirmed by planting pieces of diseased tuber tissue adjacent to healthy seed pieces of Russet Burbank. Transmission of the obligate parasite occurred naturally by simulating conditions that were favorable for infection, acidic potting mix (Jiffy-Mix) maintained at a high water potential. Progeny plants were found to be infected on both tubers and roots after 60 days. Control plants that were not exposed to diseased tuber pieces were unaffected. North Dakota, a major seed-potato-producing state, ships seed to every other potato-producing area in the U.S. and much of Canada. Infected seed shipped to areas with a favorable environment for disease development may result in crops becoming diseased and unmarketable. The cooler temperatures and higher than normal rainfall during the 1992 to 1995 seasons may account for powdery scab development in North Dakota. Recent increases in irrigated agriculture and potato production in North Dakota could lead to the infestation of new areas, restricting the value of that land for potato production. Reference: (1) B. Christ et al. Am. Potato J. 65:583, 1988.

The Effect of Wounding, Temperature, and Inoculum on the Development of Pink Rot of Potatoes Caused by Phytophthora erythroseptica.

The effect of wounding, temperature, and inoculum on the development of pink rot caused by Phytophthora erythroseptica, was studied for its potential impact on postharvest infection. Tissue plugs cut from pink rot infected tubers and plugs of similar size from laboratory cultures of the pathogen were highly effective inoculum sources on wounded tubers. Severe wounding, temperatures of 15 to 25°C, and high inoculum density affected the infection risk. Regardless of source or amount of inoculum, any degree of wounding greatly increased incidence of infection of tubers by P. erythroseptica. Infections in unwounded tubers started at 15°C, whereas in wounded tubers infection started at 10°C. Incidence of pink rot was high when two or three of the factors (severe wounding, high temperature, high inoculum level) were favorable. Incidence of pink rot was intermediate when only one factor was favorable. Incidence of pink rot was low or absent without a favorable factor (no wounding, low temperature, and low inoculum), Since infected tuber tissue may serve as potential inoculum source for postharvest infection of tubers by P. erythroseptica, the removal of pink rot infected tubers at harvest is desirable. Avoidance of wounding and rapid cooling of storage bins to 10°C may also help control pink rot.

Sensitivity of North American Isolates of Phytophthora erythroseptica and Pythium ultimum to Mefenoxam (Metalaxyl).

A 4-year study (1997 to 2000) was conducted to determine the sensitivities of the potato tuber rot pathogens, Phytophthora erythroseptica and Pythium ultimum, to mefenoxam (metalaxyl). A total of 2,277 tubers showing symptoms of "water rot" were collected from 16 states and 2 Canadian provinces. From these, 849 isolates of P. erythroseptica and 213 isolates of P. ultimum were obtained, and 805 and 190 isolates, respectively, were tested for their ability to grow on V8 medium amended at 0.01 to 100 µg/ml with fungicide. Isolates ranged widely in their responses to mefenoxam. The presence of resistant isolates (EC50 > 100 µg ml-1) of P. erythroseptica in the potato producing areas of Maine was confirmed. The presence of P. erythroseptica isolates in Idaho and Minnesota resistant to mefenoxam is reported for the first time. The proportion of P. erythroseptica isolates resistant to mefenoxam varied from 2.9 to 36.2% between 1997 and 2000. The proportion of resistant P. ultimum isolates represented only a small proportion of the isolates tested (3.7%). A single resistant P. ultimum isolate was recovered from Washington, whereas most of the resistant isolates obtained (5 of 7) were collected in Minnesota during the final year of the study. This is the first report of resistance in P. ultimum pathogenic to potato tubers. These observations suggest that pink rot and leak could become significant problems in the future, particularly in those areas where resistance has been detected. Our results have implications for the effective management of water rot. Monitoring the sensitivity of the pathogen population to mefenoxam in all production areas should be considered and integrated as a part of the overall disease management strategy.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Potato Purple Top Disease in Washington and Oregon.

An epidemic of purple top disease of potato (Solanum tuberosum) occurred in the Columbia Basin Region of Washington and Oregon in 2002 and 2003, causing great economic loss in the potato industry (1). Symptoms of potato purple top (PPT) were characterized by upright terminal shoots, upward leaf rolling, chlorosis, red or purplish discoloration of new leaves, proliferation of axillary shoots with basal swelling, and the formation of aerial tubers. Preliminary studies on PPT disease suggested phytoplasma as a possible cause (1). In this study, 78 potato samples (including five asymptomatic) were collected from five fields throughout the region. A nested polymerase chain reaction (PCR) with primer pair P1/P7 in the first amplification followed with primer pair R16F2n/R16R2 was performed to detect the presence of phytoplasmas in infected plants (2). Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Eighty-four percent (63% in the first amplification) of the symptomatic samples and 60% (0% in the first amplification) of the asymptomatic samples tested positive. Low phytoplasma titers and the presence of PCR inhibitors accounts for the low detection rate in the first PCR amplifications. RFLP analyses of 16S rDNA with enzymes MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma associated with PPT belonged to the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) (2). 16SrVI-A currently consists of three members, CP (GenBank Accession No. AY500130), potato witches'-broom (GenBank Accession No. AY500818), and vinca virescence (VR) (GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (2). The taxonomic affiliation of PPT phytoplasma was confirmed by phylogenetic analysis of cloned 16S rDNA (GenBank Accession Nos. PPT4, AY496004; PPT8, AY496005). The 16S rDNA sequences of the PPT strains were closely related to VR with 99.7% sequence homology compared with 99.2% with CP. A high correlation between the symptoms and the presence of 16SrVI-A phytoplasmas in the potato plants suggests that these phytoplasmas play an etiological role in PPT disease. To gain further evidence, a modified test of Koch's postulates was conducted. Infected tissues from four phytoplasma-positive potato samples (including PPT4 and PPT8) were grafted onto healthy potato seedlings. Within 60 days after grafting, the potato seedlings developed symptoms similar to those in the original diseased samples. The newly infected plants were maintained through cuttings. RFLP analysis of 16S rDNA indicated that the phytoplasmas detected in each of the seedlings and cuttings were identical to those in the scions. These results confirmed the probable etiological role of CP group, subgroup 16SrVI-A phytoplasma strains in PPT disease in Washington and Oregon. There are two other confirmed cases of phytoplasmas (BLTVA and aster yellows phytoplasma) associated with PPT disease in Utah (4) and Mexico (3). References: (1) P. B. Hamm et al. Potato Prog. Vol. 3, No. 1, 2003. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) N. E. Leyva-Lopez et al. Can. J. Microbiol. 48:1062, 2002. (4) C. D. Smart et al. Phytopathology 83:1399, 1993.

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot.

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.