Effects of Soil Conditions on Root Rot of Soybean Caused by Fusarium graminearum.

Fusarium graminearum is an important soybean pathogen that causes seedling disease, root rot, and pre- and postemergence damping-off. However, effects of soil conditions on the disease are not well understood. The objective of this greenhouse study was to determine the impacts of soil texture, pH, and soil moisture on seedling root rot symptoms and detrimental effects on seedling development caused by F. graminearum. F. graminearum-infested millet was added (10%, vol/vol) to soil with four different textures (sand, loamy sand, sandy loam, and loam). Soil moisture was maintained at saturation, field capacity or permanent wilting point at soil pH levels of 6 or 8. Seedlings were evaluated 4 weeks after planting for root rot, root length, root and shoot dry weights, leaf area, and F. graminearum colonization (by qPCR). There was a significant interaction between soil moisture and soil texture for root rot assessed visually (P < 0.0001). Highest severity (67%) and amount of F. graminearum DNA were observed at pH 6 and permanent wilting point in sandy loam soils. Pot saturation resulted in the lowest levels of disease in sandy loam and loam soils (11.6 and 10.8%, respectively). Reductions in seedling growth parameters, including root length, foliar area, shoot and root dry weights, and root tips, relative to the noninfested control, were significantly greater in sandy loam soils. In contrast, there were no significant growth reductions in sand. This study showed that levels of root rot increased under moisture-limiting conditions, producing detrimental effects on plant development.

Pathogenicity of Fumonisin-producing and Nonproducing Strains of Aspergillus Species in Section Nigri to Maize Ears and Seedlings.

Species of Aspergillus section Nigri are commonly associated with maize kernels, and some strains can produce fumonisin mycotoxins. However, there is little information about the extent to which these fungi contribute to fumonisin contamination in grain, the damage they cause to maize ears, or their effects on maize seed germination and seedling health. We compared fumonisin-producing and nonproducing strains of A. niger, A. welwitschiae, A. phoenicis, A. tubingensis, and A. carbonarius from the United States and Italy in laboratory and field studies to assess their ability to contribute to fumonisin contamination, to cause maize ear rot, and to affect seed germination and seedling growth. In laboratory experiments, some strains of each Aspergillus species reduced germination or seedling growth, but there was high variability among strains within species. There were no consistent differences between fumonisin-producing and nonproducing strains. In field studies in Iowa and Illinois, strains were variable in their ability to cause ear rot symptoms, but this was independent of the ability of the Aspergillus strains to produce fumonisins. Contamination of grain with fumonisins was not consistently increased by inoculation with Aspergillus strains compared with the control, and was much greater in F. verticillioides-inoculated treatments than in Aspergillus-inoculated treatments. However, the ratio of the FB analogs FB2 and FB1 was altered by inoculation with some Aspergillus strains, indicating that FB2 production by Aspergillus strains occurred in the field. These results demonstrate the pathogenic capabilities of strains of Aspergillus in section Nigri, but suggest that their effects on maize ears and seedlings are not related to their ability to produce fumonisins, and that fumonisin contamination of grain caused by Aspergillus spp. is not as significant as that caused by Fusarium spp.

Seed Treatment Effects on Maize Seedlings Coinfected with Rhizoctonia solani and Pratylenchus penetrans.

The roots of maize seedlings typically are attacked by a complex of organisms that includes fungal pathogens and plant-parasitic nematodes but few studies have examined the effects of these organisms in combination. Rhizoctonia solani can be an important component of the seedling disease complex; like other fungi, its effect on the plant may be influenced by the activity of nematodes such as the root-lesion nematode Pratylenchus penetrans. In this study, we assessed the impact of seed treatments, including fungicide-nematicide combinations, on maize seedlings exposed to R. solani and P. penetrans alone or in combination. In growth-chamber and greenhouse experiments, seed treated with various active ingredient combinations were planted in an autoclaved sand-soil mixture with or without inoculum of R. solani. In some treatments, a suspension of P. penetrans adults and juveniles was added to the sand-soil mixture. In the greenhouse experiments, infection by R. solani caused dramatic reductions in root length, volume, surface area, and numbers of root tips and root forks, whereas P. penetrans infestation alone reduced only shoot fresh weight. Statistical interactions between the effects of the two organisms were not significant, although fungal infestation significantly reduced the numbers of nematodes extracted from roots. Seed treatments significantly improved most root development variables, and the combination that included four fungicides, thiamethoxam, and abamectin was the best treatment for most variables. Results were similar in the growth-chamber experiments, where R. solani caused significant reductions in nearly all shoot and root development measurements, and seed treatment with sedaxane, alone or combined with abamectin, consistently provided the best results. R. solani was more damaging to seedlings than P. penetrans, and the combination of the two organisms did not cause more damage than R. solani alone. Seed-treatment active ingredients that specifically targeted R. solani (sedaxane) and P. penetrans (abamectin) had large positive effects on seedling health, causing significant improvements in root and shoot growth and development compared with untreated seedlings exposed to these pathogens.

Seed Treatment Effects on Maize Seedlings Coinfected with Fusarium spp. and Pratylenchus penetrans.

Seedling diseases of maize are caused by a complex of organisms, including fungi in the genus Fusarium. Root-lesion nematodes (Pratylenchus spp.) are common in fields where maize is grown, and they are known to interact with Fusarium spp. in several crops. The objectives of this study were to assess the impacts of seed treatment combinations on maize seedlings coinfected with Pratylenchus penetrans and two Fusarium spp. that cause seedling disease symptoms (Fusarium graminearum and F. verticillioides) and to determine whether there were interactions between P. penetrans and the Fusarium spp. Growth-chamber experiments were conducted with fungicide- or nematicide-treated or untreated maize seed planted in a sand-soil mixture infested with inoculum of either F. graminearum or F. verticillioides. A suspension of 4,000 P. penetrans (mixed stages) was added to the pots at the time of planting. After 30 days, shoot length and fresh and dry shoot and root weights were determined. Total root length and fine root length, root volume, numbers of root tips and forks, and root surface area were measured through analysis of digital images of the root systems. After 42 days, P. penetrans nematodes were extracted and quantified from roots and soil. There were significant effects of the treatments on root health with interactions between Fusarium spp. and P. penetrans. F. graminearum caused the greatest reductions in root and shoot growth, and interactions with P. penetrans were more evident for F. verticillioides than for F. graminearum. Image analysis of root system architecture showed that seed treatment significantly improved root system characteristics. Seed treatments containing the nematicide abamectin in combination with fungicides reduced root infection by P. penetrans and provided the healthiest root system when under attack by the Fusarium-Pratylenchus complex.

Real-Time PCR Assay for Detection of Sphacelotheca reiliana Infection in Maize (Zea mays) Seedlings and Evaluation of Seed Treatment Efficacy.

Head smut of maize, caused by the fungus Sphacelotheca reiliana, is an economically important disease in all major maize-producing countries. Although seed treatments are commonly used for management purposes, evaluating these treatments for efficacy is both time consuming and inefficient. Therefore, in order to improve the capacity for evaluating seed treatment fungicides, we developed a real-time PCR-based seedling assay for S. reiliana infection. We optimized growth chamber conditions and inoculation methods to achieve infection incidence of 60 to 80% in inoculated, nontreated controls. The effects of five commercially available fungicidal seed treatment formulations on seedling infection incidence were compared by PCR analysis of root and mesocotyl tissues. Tebuconazole, fludioxonil, sedaxane, and Maxim Quattro (fludioxonil+mefenoxam+azoxystrobin+thiabendazole) reduced the incidence of infection (P < 0.05) compared with the control, but no difference was found between the azoxystrobin treatment and the control. All rates tested for both sedaxane and tebuconazole were equally effective for seeds coated with 106 teliospores∙seed-1. Sedaxane, at a rate of 0.1 mg/kernel, eliminated seedling infection if seeds were infested with a low inoculum concentration (101 teliospores∙seed-1). The assay developed here is a valuable tool not only for the detection of fungal infection at the seedling stage, but also for testing the relative efficacies of seed treatments for reducing incidence of infection.

Transgenic Virus Resistance in Crop-Wild Cucurbita pepo Does Not Prevent Vertical Transmission of Zucchini yellow mosaic virus.

Zucchini yellow mosaic virus (ZYMV) is an economically important pathogen of cucurbits that is transmitted both horizontally and vertically. Although ZYMV is seed-transmitted in Cucurbita pepo, the potential for seed transmission in virus-resistant transgenic cultivars is not known. We crossed and backcrossed a transgenic squash cultivar with wild C. pepo, and determined whether seed-to-seedling transmission of ZYMV was possible in seeds harvested from transgenic backcrossed C. pepo. We then compared these transmission rates to those of non-transgenic (backcrossed and wild) C. pepo. The overall seed-to-seedling transmission rate in ZYMV was similar to those found in previous studies (1.37%), with no significant difference between transgenic backcrossed (2.48%) and non-transgenic (1.03%) backcrossed and wild squash. Fewer transgenic backcrossed plants had symptom development (7%) in comparison with all non-transgenic plants (26%) and may be instrumental in preventing yield reduction due to ZYMV. Our study shows that ZYMV is seed transmitted in transgenic backcrossed squash, which may affect the spread of ZYMV via the movement of ZYMV-infected seeds. Deep genome sequencing of the seed-transmitted viral populations revealed that 23% of the variants found in this study were present in other vertically transmitted ZYMV populations, suggesting that these variants may be necessary for seed transmission or are distributed geographically via seeds.

Trichothecene Genotype of Fusarium graminearum Isolates from Soybean (Glycine max) Seedling and Root Diseases in the United States.

Fusarium graminearum is an economically important pathogen that causes Fusarium head blight of wheat, barley, and oat, and Gibberella ear and stalk rot of maize. More recently, F. graminearum was reported as a soybean seedling and root pathogen in North America (1,5), causing seed decay, damping-off, and brown to reddish-brown root rot symptoms. Type B trichothecene mycotoxins are commonly produced by F. graminearum, which can be categorized into three trichothecene genotypes; those that produce 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), or nivalenol (NIV). The 15-ADON genotype is dominant in populations from small grains and maize in North America (4), but the 3-ADON genotype has recently been found (4). F. graminearum was known as a pathogen of wheat and maize in North America for over a century before it was reported as a soybean pathogen. Therefore, we hypothesized that recent reports on soybean could be associated with the appearance of the 3-ADON genotype. The objective of this research was to determine the trichothecene genotype of F. graminearum isolates from soybean in the United States. Thirty-eight isolates from soybean were evaluated. Twenty-seven isolates came from a 3-year survey for Fusarium root rot from 2007 to 2009 in Iowa. Other isolates (Ahmad Fakhoury, Southern Illinois University, Carbondale) were collected from soybean seedlings during a multi-state survey in 2012, and included three isolates from Illinois, three from Indiana, and five from Nebraska. Species identification and lineage of F. graminearum were confirmed by sequencing the translation elongation factor gene (EF1-α) using EF-1H and EF-2T primers. A maximum likelihood analysis of the EF1-α, including voucher strains from nine lineages of F. graminearum (2), placed all 38 isolates into lineage 7, F. graminearum sensu stricto (representative GenBank accessions KJ415349 to KJ415352). To determine the trichothecene genotype of each isolate we used three multiplex PCR assays. The first two assays targeted a portion of trichothecene biosynthesis genes Tri3 and Tri12 (4), while the third assay targeted portions of the Tri3, Tri5, and Tri7 genes (3). The PCR for the first two assays was conducted as described by Ward et al. (4) using four sets of primers: 3CON, 3NA, 3D15A, and 3D3A; and 12CON, 12NF, 12-15F, and 12-3F for the Tri3 and Tri12 genes, respectively. The PCR for the third assay was conducted as described by Quarta et al. (3) using the following primers: Tri3F971, Tri3F1325, Tri3R1679, Tri7F340, Tri7R965, 3551H, and 4056H. The amplification products were analyzed by gel electrophoresis. All 38 isolates produced amplicons consistent with the 15-ADON genotype; ~610 and 670 bp for the Tri3 and Tri12 genes, respectively (4), and two amplicons of ~708 and 525 bp for the Tri3/Tri5 genes (3). Our results indicated that the dominant trichothecene genotype among isolates of F. graminearum from soybean is 15-ADON, and the introduction of 3-ADON isolates does not explain the recent host shift of F. graminearum to soybean in North America. To our knowledge, this is the first assessment of trichothecene genotypes in F. graminearum populations from soybean from the United States. References: (1) K. E. Broders et al. Plant Dis. 91:1155, 2007. (2) K. O'Donnell et al. Fungal Gen. Biol. 41:600, 2004. (3) A. Quarta et al. FEMS Microbiol. Lett. 259:7, 2006. (4) T. D. Ward et al. Fungal Gen. Biol. 45:473, 2008. (5) A. G. Zue et al. Can. J. Plant Pathol. 29:35, 2007.

Distribution and Frequency of Fusarium Species Associated with Soybean Roots in Iowa.

A 3-year survey was conducted in Iowa to characterize the distribution and frequency of species of Fusarium associated with soybean roots. Ten plants were collected from each of 40 to 57 fields each year at V2 to V5 and R3 to R4 soybean growth stages. Fusarium colonies were isolated from symptomatic and symptomless roots and identified to species based on cultural and morphological characteristics. Species identification was confirmed by amplification and sequencing of the translation elongation factor (EF1-α) gene. Fifteen species were identified; Fusarium oxysporum was isolated most frequently, accounting for more than 30% of all isolates. F. acuminatum, F. graminearum, and F. solani were also among the most frequent and widespread species. Eleven other species were recovered from few fields, accounting for less than 10% of all isolates in a given year. No consistent trends were observed in geographic distribution of species. Variability in species frequency was found between soybean growth stages. Fusarium oxysporum was recovered at higher frequency during vegetative stages (40%) than reproductive stages (22%). Conversely, species such as F. acuminatum, F. graminearum, and F. solani were recovered more often from reproductive-stage plants. No significant differences in species composition were observed among fields differing in tillage practices and row spacing.

First Report of Fusarium commune Causing Damping-off, Seed Rot, and Seedling Root Rot on Soybean (Glycine max) in the United States.

During 2007 to 2009, symptomatic and asymptomatic soybean plants were collected from fields in 18 Iowa counties. Fusarium isolates were recovered from surface-sterilized root tissue on peptone PCNB agar (2). Single-spore isolates were transferred to synthetic low nutrient agar (SNA) overlain with pieces (1 × 2 cm) of sterile filter paper, and to potato dextrose agar (PDA), and placed in the dark for 10 to 14 days for morphological identification (4). Twenty-three isolates were identified as Fusarium commune K. Skovg., O'Donnell & Nirenberg, previously in the F. oxysporum species complex (4). Colonies on PDA had white, fluffy, aerial mycelium with magenta to violet pigmentation in the medium. On SNA, macroconidia, chlamydospores, and microconidia on monophialides and polyphialides were consistent with the species description (4). Identification of all 23 isolates was confirmed by DNA sequencing of the translation elongation factor (EF1-α) gene, using ef1 and ef2 primers, and the mitochondrial small subunit (mtSSU), using primers MS1 and MS2 (4) [GenBank accessions for two representative isolates: EF1-α (JX289892 and JX289893), and mtSSU (JX289894 and, JX289895)]. Pathogenicity of two representative isolates of F. commune was tested on soybean (cv. AG2403) in a greenhouse, in water baths set at 18°C, using autoclaved soil mixed with infested sand-cornmeal inoculum (3). The experiment entailed a completely randomized design (CRD) with five replications (single plant/150 ml cone) per treatment, and was conducted three times. Dry root and shoot weights, and root rot severity (visual estimate of percent root rot on the entire root system) were evaluated after 6 weeks. Mean seedling emergence in soil infested with F. commune was 47 and 40% for the two isolates; in contrast, non-inoculated control plants had 100% emergence. There were significant differences in root (P < 0.0001) and shoot (P < 0.0001) weights, and root rot severity (P < 0.0001), between inoculated and non-inoculated plants. Seedlings that emerged were severely stunted and had dark brown lesions. F. commune was reisolated from infected roots of inoculated plants, but not from non-inoculated plants. Pathogenicity of both isolates to soybean (cv. MN1805) was also tested using a petri dish assay, in which eight seeds were placed on a plate with a 4-day-old culture growing on 2% water agar (1). Plates were rated 7 days later for seed germination, seed rot, and lesion development, using an ordinal scale (1). The experiment entailed a CRD with three replicate plates/treatment, and was conducted three times. Germination of inoculated seeds ranged from 37.5 to 75.0%, and germinated seedlings had dark brown lesions on the taproots. There was a significant difference between isolates in the petri dish assay (P = 0.0030); one isolate was less aggressive, but both isolates resulted in significantly more disease than on the non-inoculated control plants, which had 100% germination and no symptoms (P < 0.0001). F. oxysporum is a known soybean pathogen (1), but isolates of F. commune may have been misidentified as F. oxysporum in previous studies. To our knowledge, this is the first report of F. commune as a pathogen on soybean in the U.S.A. References: (1) K. E. Broders et al. Plant Dis. 91:727, 2007. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (3) G. P. Munkvold and J. K. O'Mara. Plant Dis. 86:143, 2002. (4) K. Skovgaard et al. Mycologia. 94:630, 2003.

First Report of Fusarium armeniacum Causing Seed Rot and Root Rot on Soybean (Glycine max) in the United States.

In a survey for Fusarium root rot, soybean plants were sampled from eight counties across Iowa in 2008 to 2009. Fusarium isolates were recovered from surface-sterilized symptomatic and asymptomatic root tissue by culturing on peptone PCNB agar (2). Single-spore isolates were transferred to carnation leaf agar (CLA) and potato dextrose agar (PDA) for morphological identification; 11 isolates were identified as F. armeniacum (Forbes, Windels, and Burgess) Burgess and Summerell (previously F. acuminatum ssp. armeniacum) (2). Colonies on PDA produced white aerial mycelium, red to apricot pigment in agar, and bright orange sporodochia in the center of the culture. Some isolates produced a pionnotal form of slow-growing colonies with little aerial mycelium and abundant orange sporodochia. On CLA, macroconidia in orange sporodochia on carnation leaves and chlamydospores formed abundantly, but microconidia were absent (2). Species identity for the 11 isolates was confirmed by sequencing of the elongation factor gene (EF1-α) using ef1 and ef2 primers (4) (reference sequences deposited in GenBank JX101763 and JX101764). Pathogenicity of seven F. armeniacum isolates was tested using surface-sterilized soybean seed, cv. AG2403, in a petri dish assay with 3-day-old cultures on 2% water agar (1). Germination, seed rot, and lesion development were scored 7 dai using an ordinal scale (1). The experiment was a completely randomized design (CRD), had three replicate plates per isolate, and was conducted twice. All seven isolates were pathogenic on soybean, though variation in aggressiveness was observed among isolates (P < 0.0001) related to colony morphology on PDA. Seed germination was 0 to 40% when inoculated with four isolates showing white fluffy aerial mycelium on PDA. Seedlings were severely stunted with dark brown lesions covering a majority of the root system. When inoculated with three isolates showing the pionnotal form of slow-growing mycelium, germination was 70 to 100%, with few small brown lesions (~5 to 10 mm) on the roots. Noninoculated controls showed 100% germination and no symptoms. Pathogenicity was also tested in a growth chamber assay at 18°C using autoclaved soil mixed with an infested sand-cornmeal inoculum (3). Data for dry root and shoot weights and root rot severity (visually scored on a % scale) were collected at 6 weeks. The CRD experiment had five replications (single plant in a cone containing 150 ml infested soil), and was conducted twice. Root symptoms and similar variation in aggressiveness among isolates (based on colony morphology) was observed in inoculated plants. Isolates differed significantly for effects on root weight (P = 0.0125), shoot weight (P = 0.0035), and root rot severity (P = 0.0158). F. armeniacum was reisolated from infected root tissue, but not from noninoculated controls. Recovered isolates maintained their original colony morphology. F. armeniacum was previously reported in Minnesota on symptomless corn (2), but it has not been reported on soybean and its pathogenicity has not been established on any crop. To our knowledge, this is the first report of F. armeniacum as a pathogen on soybean in the United States. References: (1) K. E. Broders et al. Plant Dis. 91:727, 2007. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (3) G. P. Munkvold and J. K. O'Mara. Plant Dis. 86:143, 2002. (4) K. O'Donnell et al. Proc. Natl. Acad. Sci. 95:2044, 1998.

Meta-analysis of yield response of hybrid field corn to foliar fungicides in the U.S. Corn Belt.

The use of foliar fungicides on field corn has increased greatly over the past 5 years in the United States in an attempt to increase yields, despite limited evidence that use of the fungicides is consistently profitable. To assess the value of using fungicides in grain corn production, random-effects meta-analyses were performed on results from foliar fungicide experiments conducted during 2002 to 2009 in 14 states across the United States to determine the mean yield response to the fungicides azoxystrobin, pyraclostrobin, propiconazole + trifloxystrobin, and propiconazole + azoxystrobin. For all fungicides, the yield difference between treated and nontreated plots was highly variable among studies. All four fungicides resulted in a significant mean yield increase relative to the nontreated plots (P < 0.05). Mean yield difference was highest for propiconazole + trifloxystrobin (390 kg/ha), followed by propiconazole + azoxystrobin (331 kg/ha) and pyraclostrobin (256 kg/ha), and lowest for azoxystrobin (230 kg/ha). Baseline yield (mean yield in the nontreated plots) had a significant effect on yield for propiconazole + azoxystrobin (P < 0.05), whereas baseline foliar disease severity (mean severity in the nontreated plots) significantly affected the yield response to pyraclostrobin, propiconazole + trifloxystrobin, and propiconazole + azoxystrobin but not to azoxystrobin. Mean yield difference was generally higher in the lowest yield and higher disease severity categories than in the highest yield and lower disease categories. The probability of failing to recover the fungicide application cost (p(loss)) also was estimated for a range of grain corn prices and application costs. At the 10-year average corn grain price of $0.12/kg ($2.97/bushel) and application costs of $40 to 95/ha, p(loss) for disease severity <5% was 0.55 to 0.98 for pyraclostrobin, 0.62 to 0.93 for propiconazole + trifloxystrobin, 0.58 to 0.89 for propiconazole + azoxystrobin, and 0.91 to 0.99 for azoxystrobin. When disease severity was >5%, the corresponding probabilities were 0.36 to 95, 0.25 to 0.69, 0.25 to 0.64, and 0.37 to 0.98 for the four fungicides. In conclusion, the high p(loss) values found in most scenarios suggest that the use of these foliar fungicides is unlikely to be profitable when foliar disease severity is low and yield expectation is high.

Effects of Virus Infection on Susceptibility of Soybean Plants to Phomopsis longicolla.

Infection of soybean by Bean pod mottle virus (BPMV) or Soybean mosaic virus (SMV) has been reported to increase susceptibility to seed infection by Phomopsis spp., but the mechanism is unclear. Effects of virus infection on susceptibility to Phomopsis longicolla were studied in greenhouse experiments. Three soybean cultivars were inoculated with BPMV at growth stage V2 to V3, and with P. longicolla at R3, R5, or R7. Inoculation with BPMV did not increase the incidence of stem infection by P. longicolla, but it increased susceptibility to seed infection of cultivars Spansoy 201 at R5, and Pioneer brand 92M02 at R3, R5, and R7. A delay in maturity was observed only in 92M02. Thus, BPMV predisposed soybean plants to seed infection by P. longicolla, but this predisposition was not due solely to prolonging maturation. In separate experiments, two soybean cultivars were inoculated with SMV (V2 to V3) and P. longicolla (R3 and R5). Inoculation with SMV did not increase the incidence of stem or seed infection by P. longicolla. The SMV-Phomopsis spp. relationship may be cultivar and strain dependent. Results suggest that the risk of soybean seed infection by P. longicolla may be higher when BPMV vector populations are high and BPMV infection is widespread.

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

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.

Systemic Infection by Fusarium verticillioides in Maize Plants Grown Under Three Temperature Regimes.

Fusarium verticillioides causes seedling decay, stalk rot, ear rot, and mycotoxin contamination (primarily fumonisins) in maize. Systemic infection of maize plants by F. verticillioides can lead to kernel infection, but the frequency of this phenomenon has varied widely among experiments. Variation in the incidence of systemic infection has been attributed to environmental factors. In order to better understand the influence of environment, we investigated the effect of temperature on systemic development of F. verticillioides during vegetative and reproductive stages of plant development. Maize seeds were inoculated with a green fluorescent protein-expressing strain of F. verticillioides, and grown in growth chambers under three different temperature regimes. In the vegetative-stage and reproductive-stage experiments, plants were evaluated at tasseling (VT stage), and at physiological maturity (R6 stage), respectively. Independently of the temperature treatment, F. verticillioides was reisolated from nearly 100% of belowground plant tissues. Frequency of reisolation of the inoculated strain declined acropetally in aboveground internodes at all temperature regimes. At VT, the high-temperature treatment had the highest systemic development of F. verticillioides in aboveground tissues. At R6, incidence of systemic infection was greater at both the high- and low-temperature regimes than at the average-temperature regime. F. verticillioides was isolated from higher internodes in plants at R6, compared to stage VT. The seed-inoculated strain was recovered from kernels of mature plants, although incidence of kernel infection did not differ significantly among treatments. During the vegetative growth stages, temperature had a significant effect on systemic development of F. verticillioides in stalks. At R6, the fungus reached higher internodes in the high-temperature treatment, but temperature did not have an effect on the incidence of kernels (either symptomatic or asymptomatic) or ear peduncles infected with the inoculated strain. These results support the role of high temperatures in promoting systemic infection of maize by F. verticillioides, but plant-to-seed transmission may be limited by other environmental factors that interact with temperature during the reproductive stages.

Isolate-Cultivar Interactions, In Vitro Growth, and Fungicide Sensitivity of Fusarium oxysporum Isolates Causing Seedling Disease on Soybean.

Fusarium oxysporum is frequently associated with soybean root rot in the United States. Information about pathogenicity and other phenotypic characteristics of F. oxysporum populations is limited. The objective of the research described herein was to assess phenotypic characteristics of F. oxysporum isolates from soybean, including the interaction between isolates and soybean cultivars, fungal growth characteristics in culture, and sensitivity to fungicides commonly used as seed treatment products. The pathogenicity of 14 isolates was evaluated in rolled-towel and Petri-dish assays using 11 soybean cultivars. In the rolled-towel assay, seed were inoculated with a conidial suspension and disease severity was observed. In the Petri-dish assay, F. oxysporum isolates were grown on 2% water agar and seed were placed on the F. oxysporum colony to observe the symptoms that developed. Cultivars differed in susceptibility to F. oxysporum, and significant (P = 0.0140) isolate-cultivar interactions were observed. F. oxysporum isolates differed in radial growth on potato dextrose agar at 25°C. Pyraclostrobin and trifloxystrobin reduced conidial germination with average 50% effective concentration (EC50) of 0.15 and 0.20 µg active ingredient (a.i.)/ml, respectively. Ipconazole reduced fungal growth with average EC50 of 0.23 µg a.i./ml, whereas fludioxonil was ineffective. Our results illustrate soybean F. oxysporum isolate variability and the potential for their management through cultivar selection or seed treatment.

Effects of Tillage Practices on Dissemination and Spatial Patterns of Heterodera glycines and Soybean Yield.

Two field experiments were conducted in central Iowa to assess the effects of tillage on Heterodera glycines dissemination and reproduction and soybean (Glycine max) yield. Plots in both experiments were artificially infested with equivalent numbers of H. glycines cysts. In one experiment, plots were left noninfested or received aggregated or uniform infestation, and a susceptible soybean cultivar was grown for 3 years. By the end of the first growing season and through the second, H. glycines population densities were consistently greater (P ≤ 0.05) in uniformly infested plots than in plots with aggregated infestations. No differences in soybean yield among the treatments were detected. In a second experiment, a 1-m2 area of each plot was infested with H. glycines cysts, susceptible soybeans were grown for four seasons, and crop residue was managed with either ridge-, conventional-, reduced-, or no-tillage. After 1 year, nematode population densities were significantly (P ≤ 0.05) greater in conventional- and reduced-tillage treatments than in no- and ridge-tillage treatments. After 2 years, H. glycines had been disseminated 6.9 m from the infestation site in conventional- and reduced-tillage treatments but only 0.5 and 1.4 m for no-tillage and ridge-tillage treatments, respectively. After 3 years, H. glycines population densities were 10 times greater in conventional- and reduced-tillage treatments than in the no-tillage treatment; conventional-tillage was the only treatment with yield significantly lower (P ≤ 0.05) than the noninfested control. Aggregation of H. glycines eggs was greater (P ≤ 0.05) in no- and ridge-tillage treatments than in conventional- and reduced-tillage treatments. Results indicate tillage can quickly disseminate H. glycines in newly infested fields, facilitating more rapid nematode reproduction and subsequent yield loss.

Seed Transmission of Fusarium verticillioides in Maize Plants Grown Under Three Different Temperature Regimes.

Fusarium verticillioides can be seed transmitted and cause systemic infection of maize; however, the frequency of these phenomena has varied widely among and within individual studies. In order to better understand this variability, we evaluated the effect of temperature on the first step in the systemic infection process, the transmission of F. verticillioides from seed to seedling. Seed of a commercial maize hybrid were inoculated with a strain of F. verticillioides that had been transformed with a gene for green fluorescent protein (GFP). The seed were planted in a greenhouse potting mix and incubated in growth chambers. Plants were incubated at one of three temperature regimes designed to simulate average and extreme temperatures occurring in Iowa during the weeks following planting. Root, mesocotyl, and stem tissues were sampled at growth stages V2 and V6, surface disinfested, and cultured on a semiselective medium. At V2, >90% of root and mesocotyl tissues was infected by the GFP-expressing strain at all three temperature regimes. Also at V2, infection was detected in 68 to 75% of stems. At V6, infection of root and mesocotyl tissues persisted and was detected in 97 to 100% of plants at all three temperature regimes. Plants also had symptomless systemic infection of belowground and aboveground internodes at V6. Infection of the three basal aboveground internodes was 24, 6, and 3% for the low-temperature regime; 35, 9, and 0% for the average-temperature regime; and 46, 24, and 9% for the high-temperature regime. Seed transmission and systemic infection occurred at all temperatures and did not differ significantly among treatments. These results indicate that, if maize seed is infected with F. verticillioides, seed transmission is common and symptomless systemic infection can be initiated under a broad range of temperature conditions.

Regression and artificial neural network modeling for the prediction of gray leaf spot of maize.

ABSTRACT Regression and artificial neural network (ANN) modeling approaches were combined to develop models to predict the severity of gray leaf spot of maize, caused by Cercospora zeae-maydis. In all, 329 cases consisting of environmental, cultural, and location-specific variables were collected for field plots in Iowa between 1998 and 2002. Disease severity on the ear leaf at the dough to dent plant growth stage was used as the response variable. Correlation and regression analyses were performed to select potentially useful predictor variables. Predictors from the best 9 of 80 regression models were used to develop ANN models. A random sample of 60% of the cases was used to train the networks, and 20% each for testing and validation. Model performance was evaluated based on coefficient of determination (R(2)) and mean square error (MSE) for the validation data set. The best models had R(2) ranging from 0.70 to 0.75 and MSE ranging from 174.7 to 202.8. The most useful predictor variables were hours of daily temperatures between 22 and 30 degrees C (85.50 to 230.50 h) and hours of nightly relative humidity >/=90% (122 to 330 h) for the period between growth stages V4 and V12, mean nightly temperature (65.26 to 76.56 degrees C) for the period between growth stages V12 and R2, longitude (90.08 to 95.14 degrees W), maize residue on the soil surface (0 to 100%), planting date (in day of the year; 112 to 182), and gray leaf spot resistance rating (2 to 7; based on a 1-to-9 scale, where 1 = most susceptible to 9 = most resistant).

Influence of Temperature and Relative Humidity on Sporulation of Cercospora zeae-maydis and Expansion of Gray Leaf Spot Lesions on Maize Leaves.

Controlled environment studies were conducted to determine the effects of temperature on the expansion of lesions of gray leaf spot, and the effects of temperature and relative humidity on the sporulation of Cercospora zeae-maydis on maize (Zea mays). For the lesion expansion experiment, potted maize plants were spray inoculated at growth stage V6, bagged, and incubated at 25 to 28°C and 100% relative humidity for 36 to 40 h. Symptomatic plants were transferred to growth chambers and exposed to constant temperatures of 25, 30, and 35°C. Lesion area (length by width) was measured at 4-day intervals for 17 days. For sporulation studies, lesions were excised from naturally infected maize leaves, measured, and incubated at constant temperature (20, 25, 30, or 35°C) and relative humidity (70, 80, 90, or 100%) for 72 h. Sporulation was estimated as the number of conidia per square centimeter of diseased leaf tissue. A quadratic function was used to model the relationship between log-transformed conidia per square centimeter at 100% relative humidity and temperature. Temperature had a significant effect on lesion expansion (P ≤ 0.05). At 25 and 30°C, the rate of lesion expansion was significantly higher than at 35°C (P ≤ 0.05). The largest lesions and the highest mean rate of lesion expansion were observed at 30°C; however, the mean lesion expansion rate at this temperature was not significantly different from that at 25°C. The interaction effect of temperature and relative humidity on the log of conidia per square centimeter of diseased tissue was significant (P ≤ 0.05). At 100% relative humidity, the effect of temperature on sporulation was significant (P ≤ 0.05), with maximum spore production occurring at 25 and 30°C. The quadratic model explained between 49 and 80% of the variation in the log of conidia per square centimeter at 100% with variation in temperature. These results suggest that the rapid increase in gray leaf spot severity generally observed during mid- and late summer may be due to favorable conditions for lesion expansion during this period. When relative humidity is >95%, expanding lesions may serve as a source of inoculum for secondary infections.

FUM1--a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests.

We have analyzed the role of fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for fumonisin biosynthesis. Maize ear rot, ear infection, and fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and fumonisin production. Two independent fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.