L. J. du Toit

Sequence diversity of the nucleoprotein gene of iris yellow spot virus (genus Tospovirus, family Bunyaviridae) isolates from the western region of the United States.

Summary.Iris yellow spot virus (IYSV), a tentative virus species in the genus Tospovirus and family Bunyaviridae, is considered a rapidly emerging threat to onion production in the western United States (US). The present study was undertaken to determine the sequence diversity of IYSV isolates from infected onion plants grown in California, Colorado, Idaho, Oregon, Utah and Washington. Using primers derived from the small RNA of IYSV, the complete sequence of the nucleoprotein (NP) gene of each isolate was determined and the sequences compared. In addition, a shallot isolate of IYSV from Washington was included in the study. The US isolates of IYSV shared a high degree of sequence identity (95 to 99%) with one another and to previously reported isolates. Phylogenetic analyses showed that with the exception of one isolate from central Oregon and one isolate from California, all the onion and shallot isolates from the western US clustered together. This cluster also included onion and lisianthus isolates from Japan. A second distinct cluster consisted of isolates from Australia (onion), Brazil (onion), Israel (lisianthus), Japan (alstroemeria), the Netherlands (iris) and Slovenia (leek). The IYSV isolates evaluated in this study appear to represent two distinct groups, one of which largely represents isolates from the western US. Understanding of the population structure of IYSV would potentially provide insights into the molecular epidemiology of this virus.

Fusarium proliferatum Pathogenic on Onion Bulbs in Washington

Fusarium oxysporum f. sp. cepae and an unidentified Fusarium species have been reported to cause bulb rot of onion (Allium cepa L.) in Washington (1). In August and September 2002, a salmon-pink discoloration was observed on the outer three to four layers of dry scales of approximately 20% of white onion bulbs of cv. Cometa F1, in each of two 20-acre fields in the Columbia Basin of central Washington. Isolations from the discolored areas of the dry scales onto water agar and potato dextrose agar (PDA) yielded fungal colonies characteristic of F. proliferatum (3). The isolates formed long, V-shaped chains of microconidia on polyphialides. Pathogenicity of the isolates of F. proliferatum was tested on white onion bulbs purchased at a local grocery store. The outermost dry scales of each bulb were removed, and the bulb was inoculated by one of three methods: (i) a 5-mm3 section of the fleshy scales was removed using a scalpel, the wound was filled with a 3-mm2 plug of PDA colonized by F. proliferatum, the plug was covered with the section of scale that had been removed, and the inoculation site was covered with Parafilm; (ii) the basal plate of the bulb was dipped into a suspension of 106 microconidia per ml; or (iii) the basal plate was dipped into the spore suspension after wounding by inserting a dissecting needle into the bulb to a depth of 1 cm. A noninoculated bulb provided a control treatment. Bulbs were incubated in a moist chamber at 13°C and examined for discoloration of the outer scales and development of bulb rot. After 2 weeks, salmon-pink discoloration of the outer scales was observed at the inoculation site for both methods of dip inoculation, but not for the plug inoculation method. After 3 weeks, water-soaked, tan to golden, shrunken, soft tissue was observed on the remainder of each dip inoculated bulb, but symptoms of basal rot did not develop. Symptoms were similar to those reported in Idaho for a bulb rot of stored onions caused by F. proliferatum (2). One of the nonwounded inoculated bulbs did not develop a bulb rot, but pinkish discoloration was observed beneath the outer scales and in the neck. F. proliferatum was reisolated from the inoculated bulb tissues. The discoloration observed on the white onions raised concern about the potential for infection to develop into bulb rot in storage. However, thorough curing of the bulbs immediately upon storage restricted infection to the outer dry scales. Similar symptoms were observed at harvest on the bulbs of other white onions in a cultivar trial located near Quincy, WA, although symptoms were not observed on yellow or red cultivars in the trial. The same symptoms were later observed on approximately 70% of bulbs harvested from a 32-acre fresh-market crop of the cv. Sterling in the Columbia Basin. These symptomatic bulbs were rejected for the fresh market. To our knowledge, this is the first report of infection of onion bulbs by F. proliferatum in Washington, which in 2001, had the third largest acreage of onions in the United States after California and Oregon (USDA National Agricultural Statistics Service). References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (2) S. K. Mohan et al. (Abstr.) Phytopathology 87:S67, 1997. (3) P. E. Nelson et al. Fusarium species: An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983.

Evaluation of Onion Cultivars for Resistance to Enterobacter cloacae in Storage

Sixty-nine storage onion (Allium cepa) cultivars (seven white, five red, and 57 yellow cultivars) were evaluated in the Washington State University Onion Cultivar Trials in the semiarid Columbia Basin of central Washington in 2007-08 and/or 2008-09. Each cultivar was inoculated with Enterobacter cloacae, cured, stored under commercial storage conditions, and evaluated for bacterial storage rot symptoms approximately 4.5 months after storage. Noninoculated bulbs of each cultivar served as a control treatment in each experiment. In addition, bulbs injected with water served as a second control treatment in the 2008-09 experiment. Inoculation of onion bulbs with E. cloacae resulted in significantly higher incidence and severity of Enterobacter bulb decay compared to noninoculated bulbs and bulbs injected with sterile water. For bulbs inoculated with E. cloacae, mean severity of bacterial storage rot per cultivar ranged from 5 to 19% of the cross-section evaluated for each onion bulb in 2007-08 and from 9 to 29% in 2008-09. For noninoculated bulbs, mean severity ranged from 0 to 1% in 2007-08 and 0 to 3% in 2008-09. For bulbs injected with water in the 2008-09 experiment, severity of bulb rot ranged from 0 to 10% per cultivar, with four cultivars (OLYX05-26, RE-E, Redwing, and Talon) displaying bulb rot ratings significantly greater than 0%. For the 33 cultivars included in both experiments, a significant correlation in bulb rot severity ratings was detected for the 2007-08 versus 2008-09 experiments (r = 0.43 at P = 0.013). Redwing, Red Bull, T-433, Centerstone, and Salsa had low severity ratings in both experiments; whereas Montero, OLYS05N5, Caveat, and Granero had severe bulb rot ratings in both experiments. The results demonstrate that it should be possible to select for increased resistance to Enterobacter bulb decay in storage onion cultivars.

Disease Severity and Yield of Sweet Corn Hybrids with Resistance to Northern Leaf Blight

Reactions of supersweet (sh2) sweet corn to northern leaf blight (NLB) and associated yields were evaluated in Belle Glade, Florida and Urbana, Illinois in yield-loss trials, hybrid evaluations, and evaluations of breeding materials. Hybrids differed significantly for NLB in all trials. Severity of NLB ranged from 0 to 66% on 35 sh2 hybrids in yield-loss trials, and from 0 to 60% on 80 sh2 hybrids in hybrid evaluations. NLB ratings ranged from 1 to 9 (approximately 0 to 80% severity) on 375 hybrids and 186 inbred lines in evaluations of breeding materials. Various methods of rating NLB and ratings from multiple dates were highly correlated, with correlation coefficients ranging from 0.76 to 0.98. Yield, measured as weight of ears and number of marketable ears from inoculated plots as a percentage of that from control plots, decreased as disease severity increased. Linear or quadratic regression models explained 31 to 70% of the variation in percent yield as a function of disease severity at harvest. The effects of NLB on yield were limited by NLB-resistance in several hybrids, including CCO 3268, Chieftain, Crisp N Sweet 710A, Day Star, Envy, Forever, GSS 1526, Jupiter, Midship, Prime Plus, Sch 5005, and SummerSweet 7630. Although high levels of partial resistance to NLB were prevalent among 375 new experimental sh2 hybrids and 186 sh2 inbred lines evaluated in 1995, use of the gene HtN may increase in the near future as breeders are incorporating this resistance into new inbreds and hybrids. Breeders and plant pathologists would be wise to continue improving partial resistance to NLB without using the gene HtN in genotypes with adequate levels of partial resistance, because the widespread use of the gene HtN will select for virulent races of Exserohilum turcicum which occur in Florida, or for races with new combinations of virulence.

First Report of Enterobacter cloacae Causing Onion Bulb Rot in the Columbia Basin of Washington State

In August of 2006, onion plants of cv. Redwing exhibiting premature dieback and bulb rot were obtained from a commercial onion crop under center pivot irrigation in the Columbia Basin of Washington State. High temperatures during the summer were similar to those in 2004, which preceded significant outbreaks of Enterobacter rot of onion bulbs in storage. Fungal pathogens of onion were not observed. Bacteria from infected bulb tissue were isolated and purified on nutrient broth yeast extract (NBY) agar, and 537 isolates were evaluated for the ability to ferment glucose anaerobically. Of the facultative anaerobes (~50% of all isolates), 48 isolates were arginine dihydrolase positive, indole negative, and unable to degrade pectin, i.e., characteristics typical of the genus Enterobacter (2), which includes Enterobacter cloacae, a bacterial pathogen reported to cause onion bulb rot in California and Colorado (1,3). Sixteen of the putative Enterobacter isolates, along with four strains of E. cloacae known to be pathogenic on onion (1) (ATCC 23355 and ATCC 13047, 310 (H. F. Schwartz, Colorado State University), and E6 (J. Loper, USDA ARS), were tested for pathogenicity on onion bulbs (8 to 10 cm in diameter; cv. Tamara). The isolates were grown overnight in NBY broth at 28°C, harvested by centrifugation and resuspended to an OD600 = 0.3 (~108 CFU/ml) in sterile distilled water. After the outermost fleshy scale of each bulb was removed, each bulb was surface disinfected in 0.6% NaOCl for 2 min, dipped in sterile distilled water, and then dipped in 95% ethanol. Each bulb was air dried before a 0.5-ml aliquot of bacterial suspension was injected into the shoulder of the bulb with a 20-gauge needle. Three bulbs were inoculated for each isolate, placed in individual plastic bags, sealed, and incubated at 30°C in the dark. Three bulbs injected with water and three noninjected bulbs served as controls. After 14 days, each bulb was sliced through the center and rated for rot. Thirteen isolates induced rot symptoms on the inner fleshy scales of all inoculated bulbs. Of these, seven also caused tan-to-brown discoloration of the inner fleshy scales; similar symptoms were caused by the four pathogenic reference strains of E. cloacae (1). No symptoms were observed in any of the controls. Symptoms were not observed when the bacteria, prepared as described above, were infiltrated into onion leaves. Bacteria were reisolated from the symptomatic inoculated bulb tissue and confirmed to be Enterobacter spp. by the above physiological tests. In addition, an isolate designated ECWSU2 and the corresponding strain recovered from one of the inoculated symptomatic bulbs, along with the four reference strains, were evaluated for anaerobic growth on a variety of carbon sources by using API 50 CHE test strips (bio Mérieux Vitek, Inc., Hazlewood, MO). The physiological test data along with sequence analysis of a portion of the 16S rRNA gene of each isolate confirmed all of these isolates to be E. cloacae (4; Ribosomal Database Project [ http://rdp.cme.msu.edu/ ]). To our knowledge, this is the first report of E. cloacae causing a bulb rot of onion in Washington State. References: (1) A. L. Bishop and R. M. Davis. Plant Dis. 74:692, 1990. (2) J. G. Holt et al. Bergeys Manual of Determinative Bacteriology. Williams and Wilkins, Baltimore, MD, 1994. (3) H. F. Schwartz and K. Otto. Plant Dis. 84:808, 2000. (4) L. Verdonck et al. Int. J. Syst. Bacteriol. 37:4, 1987.

Effects of Silk Maturity and Pollination on Infection of Maize Ears by Ustilago maydis

Host resistance is the most efficient method of controlling common smut of maize (Zea mays), caused by Ustilago maydis. Precise timing of ear inoculations with U. maydis relative to silk maturity and pollination may improve the ability to screen maize germ plasm for resistance. The objectives of this study were to determine the length of time maize kernels can be infected by U. maydis through silks, and to examine the effects of pollination on infection through silks. Two field studies were done in 1995, 1996, and 1997 at the University of Illinois South Farms. In the date-of-inoculation study, ears were inoculated at 2- to 3-day intervals from early silk emergence until 16 days after silk emergence. In the date-of-planting study, hybrids were planted on four dates and ears were inoculated on the same day for all planting dates. In each study, ear shoots were covered with shoot bags prior to silk emergence to prevent pollination, or ear shoots were left uncovered to allow silks to be pollinated normally. Maize ears were susceptible to infection by U. maydis from silk emergence until 8 to 14 days after silk emergence. During this period of susceptibility, incidence of ears with galls decreased as silks aged. Incidence of ears with galls on plants inoculated 7 days apart differed by as much as 70%. The period that maize ears were susceptible to infection by U. maydis was shorter and incidence of ears with galls decreased more rapidly when silks were exposed to pollen than when silks were not exposed. The silk channel method of inoculating for common smut does not appear to be practical for large-scale evaluations of numerous lines. The method is practical for evaluating a limited number of lines or for inducing ear galls for commercial production of huitlacoche (smut galls eaten at an immature stage).

Variation associated with silk channel inoculation for common smut of sweet corn.

Efforts at breeding for resistance to common smut of maize (Zea mays), caused by Ustilago maydis, are hampered by the lack of a reliable and efficient method of inoculation. Silk channel injection is one of the most efficient methods of inoculating for ear galls but is less consistent than acceptable for accurate assessment of the response of genotypes. The objective of this study was to examine how the silk channel inoculation method can be modified to reduce variation while maintaining efficiency for large-scale field inoculations. Variation associated with inoculum concentration and variation among people inoculating were examined. Incidence and severity of symptomatic ears increased with inoculum concentration. Concentrations between 105 and 106 sporidia/ml are recommended. Variation among people was greater than variation among inoculum concentrations. Incidence and severity ratings were lower for people inexperienced at inoculating with U. maydis than for experienced people. Variation among people inoculating can be controlled by appropriate experimental design.

Reactions of Open-Pollinated Sweet Corn Cultivars to Stewart's Wilt, Common Rust, Northern Leaf Blight, and Southern Leaf Blight

Over 800 open-pollinated (OP) varieties of sweet corn were grown and named in the century prior to the development of hybrids, but only a few of the historically important OP cultivars exist today. Alleles that could improve disease resistance of modern sweet corn may be present in the OP cultivars still in existence. The objectives of this research were to compare 36 OP sweet corn cultivars to modern commercial hybrids for reactions to Stewarts wilt, common rust, northern leaf blight (NLB), and southern leaf blight (SLB), and to classify the OP cultivars based on phenotypic reactions to these four diseases. Plants were inoculated in 1994, 1995, and 1996 with Erwinia stewartii, Puccinia sorghi, Exserohilum turcicum, or Bipolaris maydis. Symptoms were rated on a whole-plot basis, and ratings were analyzed by analysis of variance (ANOVA). Means were separated by Bayesian least significant difference values. Some of the OP cultivars had phenotypes that were intermediate to moderately resistant to Stewarts wilt, common rust, NLB, or SLB, but none of the cultivars were more resistant than the best commercial hybrids. Distributions of ratings for rust, NLB, and SLB were less disperse for the OP cultivars than for commercial hybrids. Hence, the resistance of modern sweet corn germ plasm to Stewarts wilt, rust, and NLB appears to be greater than that of the OP cultivars. OP cultivars and four standard hybrids were placed into groups based on a hierarchical cluster analysis of disease reactions. The seven groups formed from the cluster analysis of disease ratings were considerably different than those formed from isozyme variation and morphological characteristics. The partial resistance of some cultivars, e.g., Golden Sunshine, Country Gentleman, Stowells Evergreen, and Red, may be relatively diverse since these cultivars were placed in different groups based on isozyme and morphological variation. OP cultivars with moderate levels of resistance may be sources of resistance alleles not present in commercial hybrids.

Effects of Postharvest Onion Curing Parameters on Enterobacter Bulb Decay in Storage

Enterobacter bulb decay is a recently described storage disease of onion (Allium cepa) bulbs caused by Enterobacter cloacae. The disease is generally considered minor but, on occasion, can cause significant losses for onion producers. The impact of postharvest curing temperature and duration on Enterobacter bulb decay of onion was evaluated by inoculating bulbs of the cultivars Redwing and Vaquero with E. cloacae after harvest, curing the bulbs at 25, 30, 35, or 40°C for 2 or 14 days, and storing the bulbs at 5°C for 1, 2, or 3 months. Noninoculated bulbs and bulbs injected with sterile water served as control treatments. The trial was completed using bulbs harvested from commercial onion crops grown in the semi-arid Columbia Basin of central Washington in each of 2008-09 (center-pivot irrigated crop) and 2009-10 (drip irrigated crop). Severity of bulb rot was assessed by cutting each bulb down the center from the neck to the basal plate, and rating the percentage of cut surface area with bacterial rot symptoms. Bulb rot severity was negligible for noninoculated bulbs (mean of 0.3% in the 2008-09 storage trial and 1.0% in the 2009-10 storage trial) and bulbs injected with water (0.8% in the 2008-09 trial and 1.3% in the 2009-10 trial) compared to bulbs inoculated with E. cloacae (15.3% in 2008-09 and 23.3% in 2009-10). Severity of Enterobacter bulb decay was affected significantly (P < 0.05) by season (trial), cultivar, curing temperature, curing duration, and storage duration, with significant interactions among these factors. Enterobacter bulb decay was significantly more severe for bulbs cured at 40°C than for bulbs cured at 25, 30, or 35°C. This effect was even greater when bulbs were cured for 14 days versus 2 days prior to cold storage, and in bulbs stored for 2 or 3 months after curing compared to bulbs stored for 1 month. The increase in bulb rot severity caused by curing bulbs at 40°C for 14 days compared to lower temperatures and shorter durations was greater for Vaquero than Redwing, particularly in the 2008-09 trial. The results suggest that curing temperatures ≤35°C should significantly reduce the risk of Enterobacter bulb decay in storage for these cultivars. If higher curing temperatures are used in order to dry onion necks for long-term storage and reduce the risk of fungal diseases such as neck rot (caused by Botrytis spp.), a shorter curing duration may be necessary to minimize the risk of Enterobacter bulb decay in storage.

Multiplex real‐time PCR assays for detection of four seedborne spinach pathogens

To develop multiplex TaqMan real‐time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach.