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2000 North Central Division Meeting Abstracts

June 18-20, 2000 - Columbus, Ohio

Posted online October 4, 2000


A summary of 1999 growth chamber and field trial laboratory assays and 1998 and 1999 field trial disease control results after lactofen application to soybean. T. G. BEAN (1) and T. L. Graham (2). (1) Field Development Scientist, Valent USA Corporation, Franklin, PA 16323; (2) Dept. of Plant Pathology, Ohio State University, Columbus, OH 43210. Publication no. P-2000-0001-NCA.

Cobra 2 EC plus adjuvant applications were made to soybeans at the V3 and R1 growth stage in 1998 and at the R1 growth stage in 1999. Cobra provided significant Sclerotinia sclerotiorum control in a 1998 field trial and significant Phytophthora sojae control in two 1999 field trials. The R1 growth stage application provided better Sclerotinia sclerotiorum control than did the V3 stage applications. Laboratory cotyledon assay results indicate that lactofen is a strong inducer of soybean isoflavones and glyceollin elicitation competency. Analysis of lactofen field treated trifoliate leaves in 1999 also revealed increased isoflavone levels compared to the control plots.

Effects of inoculum dilutions on wheat streak mosaic tritimovirus disease expression in winter wheat.
C. I. BERGMAN (1), M. A. C. Langham (1), and S. D. Haley (2). (1) Plant Science Dept., South Dakota State University, Brookings, SD 57007; (2) Dept. Soil and Crop Sciences, Colorado State University, Ft. Collins, CO 80523. Publication no. P-2000-0002-NCA.

Ten lines of winter wheat (Triticum aestivum) in four replications were mechanically inoculated in the fall two weeks after emergence with wheat streak mosaic tritimovirus (WSMV). Sap inoculum from infected plants was applied with a high pressure sprayer to each line at dilutions of 1:5, 1:10, or 1:20 (weight:volume). Significant effects were exhibited for plant height, total yield, test weight, and protein. Yields of Arapaho, SD92107, TAM107, Vista, Jagger, SD93267, Sage, and Dawn were all reduced at the 1:20 dilution compared to the 1:5. Yield of KS95-H102 was the lowest at the 1:5 dilution, and higher than the controls at the 1:20. The yield of KS96HW10-1 increased at the 1:5 dilution, was reduced at the 1:10, and was not affected at the 1:20.

New methods for producing, recovering, storing and delivering ascospores of Sclerotinia sclerotiorum and other fungal propagules.
M. G. Boosalis, J. R. STEADMAN, K. Powers, and B. Higgins. Dept. Plant Pathology, University of Nebraska, Lincoln. Publication no. P-2000-0003-NCA.

Ascospores of Sclerotinia sclerotiorum are used to test management strategies of diseases caused by this fungus. In lieu of filter membranes for the deposition of ejected ascospores we use silica sand, calcimate clay mineral particles, petroleum jelly, plastic disks (PD) cut from plastic food bags and aluminum foil disks (AD). Spores on petroleum jelly, silica sand and clay were viable and virulent for over 30 months, whereas viability and virulence was retained for over 6 months on PD and AD. Conidia of Alternaria spp. and Bipolaris sorokinia and chlamydospores of Ustilago tritici stored on petroleum jelly were viable for 24 months while urediospores of wheat leaf rust lost viability after several months. Rinsing ascospores off PD and AD did not inhibit their germination or pathogenicity. Silica sand and clay particles with ascospores intact are used as inoculum. This would retain the gelatinous matrix that is associated with freshly ejected ascospores.

Greenhouse evaluation of commercial and public soybean cultivars for resistance to rhizoctonia root and crown rot.
C. A. Bradley, G. L. Hartman, D. S. Mueller, and W. L. Pedersen. Dept. of Crop Sciences, University of Illinois, Urbana, IL 61801. Publication no. P-2000-0004-NCA.

Rhizoctonia root and crown rot of soybean, caused by Rhizoctonia solani, can be a problem wherever soybean is grown, and there are no known commercial soybean cultivars released for resistance. Seven hundred soybean cultivars were evaluated for resistance to Rhizoctonia solani (root and crown rot) in the greenhouse. Cultivars were planted into trays containing a 2:1 sand:soil mix. Each tray also had cultivars Bedford or Jack (susceptible) or Centennial or Savoy (resistant). Plants were inoculated at their bases with a mycelial suspension of Rhizoctonia solani (AG-2) at the VE growth stage and were rated for disease severity using a 0 to 5 scale after 21 days. One hundred forty cultivars had disease ratings one standard deviation below the mean (<2.5 disease rating) and were reevaluated in a replicated trial. Twenty of the 140 cultivars had disease ratings of <2.5 in this test and were reevaluated in another replicated trial. In this final trial, cultivars Savoy, Asgrow 2601, and Asgrow 3701 had the lower disease ratings and were classified as moderately resistant to infection by Rhizoctonia solani.

Control of Rhizoctonia root and crown rot of sugar beet with azoxystrobin.
J. R. BRANTNER and C. E. Windels. Univ. Minn., NW Research & Outreach Center, Crookston, 56716. Publication no. P-2000-0005-NCA.

Azoxystrobin was tested for control of early- and late-season root and crown rot of sugar beet caused by R. solani AG-2-2. Plants were inoculated with R. solani 5 wk after planting. Azoxystrobin (13.9 g a.i./1000 m row) was applied in an 18-cm band after inoculation and again 2 and 4 wk later. After 2½ wk, inoculated plots treated with azoxystrobin had greater (P=0.05) plant stand than untreated plots (75% vs 30%, respectively); stand in noninoculated, no fungicide plots was 97%. At harvest, inoculated plots treated with azoxystrobin had higher (P=0.05) stand than untreated plots (29% vs 3%, respectively); stand in noninoculated, no fungicide plots was 87%. In a late season trial, plants were inoculated with R. solani at row closure. Azoxystrobin was broadcast-applied (0.168 kg a.i./ha) after inoculation and again 3 wk later. At harvest, plant stand in inoculated plots treated with azoxystrobin and in noninoculated, no fungicide plots (86% and 95%, respectively) was (P=0.05) greater than in inoculated plots with no fungicide (63%). Azoxystrobin is more effective in controlling late- than early-season Rhizoctonia root and crown rot of sugar beet.

Evaluation of Burkard cyclonic spore sampler efficiency.
C. BUTTKE and L. Francl. Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105. Publication no. P-2000-0006-NCA.

The Burkard cyclonic sampler takes in air volumetrically, creates a vortex in an aluminum cylinder, and deposits particulates in an eppendorf vial. Sampling efficiency was determined for ascospores of Sordaria fimicola and Gibberella zeae under variable power and with and without a surfactant. Results showed an average of 78% of the S. fimicola ascospores entered the vial, while 22% lodged inside the cylinder. G. zeae ascospores were tested identically, but only 25% entered the vial, while 75% remained lodged inside the cylinder. When testing a liquid silicone product (Sigmacote) on the cylinder wall, approximately 17% of the S. fimicola spores entered the vial. The aluminum collecting cylinder should be rinsed thoroughly when collecting samples. Tests of Sigmacote with G. zeae had 99.7% of the spores lodged inside the cylinder. When the power supply was decreased from 200mA to 140mA, spore counts decreased, indicating that the Burkard needs full power to run efficiently. A 38 amp hour battery maintained greater than 150mA to the Burkard for four days. A solar panel 10W or greater powering a battery should maintain the amperage needed for the Burkard to function properly.

Introduction to the late wilt disease of maize.
L. M. BUTTONOW (1) and A. H. Ellingboe (1). (1) Dept. Plant Pathology, University of Wisconsin, Madison, WI 53706. Publication no. P-2000-0007-NCA.

For the past five decades, late wilt disease, causal agent Cephalosporium maydis, has been an economically important disease of maize in Egypt; it is considered one of the most significant factors reducing maize yield. When one considers the high economic and agricultural importance of this disease, research progress is extremely slow. Maize lines resistant to late wilt disease have been found, however, these lines have been low yielding, resulting in poor acceptance and use by producers. Other means of control in the field have not been found. The literature on late wilt disease contains contradictory reports, a lack of consensus as to inoculation and screening techniques, an incomplete understanding of the field epidemiology and the genetics of the pathogen, and a lack of communication between workers. The focus of this paper is the comparison of four greenhouse-inoculation systems commonly used by workers: root inoculation at planting with grain-grown pathogen, root inoculation at 3 weeks after planting with cut roots, stem injection of conidia, and stem toothpick inoculation at 3 weeks after planting. We found that disease progress was dependent on the inoculation system used. This result may explain part of the contradictions in the literature and argues for increased focus on the basic biology of the pathogen.

Identification of resistance to soybean cyst nematode in Glycine tomentella.
R. J. CAMPBELL, G. L. Hartman, G. R. Noel, and T. Hymowitz. Dept. Crop Sciences, University of Illinois, Urbana, IL 61801. Publication no. P-2000-0008-NCA.

Transfer of soybean cyst nematode (SCN) resistance to soybean from a wild perennial relative of soybean (Glycine tomentella) has been reported. Our research objective is to determine SCN resistance levels in all 328 known accessions of G. tomentella. In our initial study, 12 accessions representing all four chromosome levels (2n=38,40,78,80) within G. tomentella were screened for resistance to SCN races 1, 3, and 4. Resistant, susceptible and intermediate plants were found between and within accessions. Susceptibility appears to be correlated with chromosome number. All 328 accessions will be tested in year 2000. We will begin to study the mode of inheritance of this SCN resistance, and ultimately transfer new sources of genetic resistance to SCN into soybean.

Developing microsatellite markers for the soybean brown stem rot pathogen Phialophora gregata.
W. CHEN and X. Shi. Illinois Natural History Survey, and University of Illinois at Urbana-Champaign, Champaign, 61820. Publication no. P-2000-0009-NCA.

Isolates of P. gregata from soybean were separated into two distinct genotypes (A & B). The two genotypes showed cultivar-preferential infection under field conditions. In order to study gene flow between the two genotypes and variation within genotypes, we have developed several microsatellite markers for P. gregata. Total genomic DNA was digested with two four-basepair recognition enzymes, and the digested DNA fragments ranging from 300 to 600 bps were isolated and used to construct a DNA library. The DNA library was further enriched for microsatellites by producing single stranded DNA followed by selective second strand extension with microsatellite oligonucleotides as primers. After sequencing selected clones and confirming microsatellite sequences, specific primers were designed to flank the microsatellite repeats. Among three microsatellites tested so far, two microsatellites produced monomorphic bands for P. gregata from soybean. One microsatellite consisting of ACGA repeats produced two alleles which correspond to the separation of the genotypes A and B.

Multiple infection of soybean by the brown stem rot pathogen Phialophora gregata.
Y.-C. CHEN (1), C. R. Grau (2), and W. Chen (1). (1) Illinois Natural History Survey, 607 E. Peabody Drive, Champaign, 61820; (2) Dept. of Plant Pathology, University of Wisconsin, Madison, WI 53706. Publication no. P-2000-0010-NCA.

In a previous study we discovered that soybean isolates of P. gregata are characterized as either genotype A or genotype B, and the two genotypes preferentially infect susceptible and resistant cultivars under field conditions, respectively. Most infected plants in field environments are colonized by one pathogen genotype if assayed by PCR using primers specific for the two genotypes. In these same studies, however, a few plants exhibited DNA bands characteristic of both genotypes A and B. Studies were designed to determine whether those plants were infected by a new genotype of the pathogen or the plants were co-infected by both genotypes. Seven isolates of P. gregata were recovered from a single soybean plant that showed DNA bands of both genotypes collected from Arlington, Wisconsin. Six of the seven strains were genotype A, and one strain was genotype B. Results show that the plants exhibited DNA bands of both genotypes were infected by both A and B genotypes and not by a new genotype of P. gregata.

Maize dwarf mosaic virus movement in resistant and susceptible maize.
Z. B. CHEN (1), T. Meulia (2), M. G. Redinbaugh (1,3), S. A. Hogenhout (4), and D. T. Gordon (1). (1) The Department of Plant Pathology; (2) Molecular and Cellular Imaging Center and (4) Department of Entomology, The Ohio State University and (3) USDA, ARS Corn and Soybean Research, OARDC, Wooster, OH 44691. Publication no. P-2000-0011-NCA.

Maize dwarf mosaic potyvirus (MDMV) causes an important disease in corn worldwide. The disease is controlled primarily through use of resistant hybrids. The maize inbred, Pa405 has a high level resistance to MDMV conferred by a single dominant gene on chromosome 6. To develop an understanding of the resistance mechanism, the distribution of MDMV-A coat protein and RNA in vascular puncture inoculated Pa405 and a susceptible inbred (Oh28) were compared. After inoculation, the viral coat protein and RNA were found in the scutellum and embryo of both inbreds, demonstrating that MDMV-A entered and replicated in Pa405 cells. However, the virus was more invasive in Oh28 suggesting that virus movement was limited in the resistant line. To further characterize limitations to virus movement in Pa405, the MDMV-A genome was cloned, and a full-length cDNA construct was inserted downstream of a T3 promoter. Sequence analysis of the genome revealed a polyprotein sequence with similarity to other potyviruses. Transcripts, synthesized from the clone, are being tested for their infectivity in maize. To identify maize proteins involved in the resistance response, a "virus overlay" assay was used to identify several MDMV-binding proteins in both Pa405 and Oh28. Work to identify, characterize and isolate these maize proteins is ongoing.

Petiole inoculation as a tool to screen soybean germplasm for resistance to Sclerotinia sclerotiorum.
L. DEL RIO, N. C. Kurtzweil, and C. R. Grau. Dept. Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706. Publication no. P-2000-0012-NCA.

A petiole inoculation technique was used to evaluate soybean germplasm for resistance to Sclerotinia sclerotiorum in a controlled environment. Plastic drinking straws (2.5 cm length) were used to cut and retain mycelium plugs from the leading edge of two-day-old colonies of S. sclerotiorum. The petiole of the first trifoliate leaf was cut 2.5 cm from the stem when the second leaf was fully open and capped with the straw containing inoculum. Wilt and plant mortality were rated multiple times during the incubation period and were used to calculate wilt and resistance indices. A negative correlation was detected between the resistance index and disease incidence in field experiments for nine (r=-0.55, P<0.08), and six (r=-0.64, P<0.06) cultivars. Soybean PIs 548-625, 547-799, 547-803 (MG I), and 507-803 (MG 0) expressed a higher resistance index (P<0.05) than the commercial cultivar S19-90.

Occurrence of soybean viruses and association with green stem of soybean in Wisconsin.
D. M. DOUGHTY (1), M. E. Lee (1), N. C. Kurtzweil (1), C. M. Boerboom (2), and C. R. Grau (1). Department of Plant Pathology; (2) Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706. Publication no. P-2000-0013-NCA.

Green stem syndrome (GSS) of soybeans has been observed for years and while incidence of GSS is usually 1-5%, in recent years fields have been observed with 50-75% incidence. Plants retain green stems, leaves and petioles well past the point of normal maturity and may have symptoms including leaf distortion similar to herbicide injury, mosaic patterns, leaf rugosity, and mottled seed. Because soybean viruses are often implicated as causal agents of GSS, a virus survey was initiated in 1999 to determine incidence. Soybean cultivars common to 13 statewide evaluation trials were sampled by collecting a leaf from five plants per replicate plot, and were assayed using ELISA. Percent incidence was based on the number of plots positive for a specific virus. Statewide incidence for alfalfa mosaic virus was 28%, soybean mosaic virus 27%, tobacco streak virus 17%, bean yellow mosaic virus 6%, tobacco ringspot virus 6% and bean pod mottle virus 2%. Tobacco ringspot virus, soybean mosaic virus and bean pod mottle virus were associated with plants expressing GSS.

Managing Fusarium head blight (scab) with fungicides.
M. A. DRAPER. Plant Science Dept., South Dakota State University, Brookings, SD 57007. Publication no. P-2000-0014-NCA.

Fusarium head blight (scab) has been a problem in South Dakota wheat since 1993, particularly on spring wheat in the northeastern quarter of the state. Fungicides have been a proven element in effective scab management. Ten treatments were evaluated during 1999 in South Dakota as a part of a thirteen-state, uniform regional trial to evaluate fungicide suppression of scab under different environments. Scab suppression can be achieved with several fungicide products, but Folicur has provided the best return on yield. Folicur treatment resulted in about a 60% reduction in leaf disease and a 30-60% reduction in scab in 1999, but no significant reduction in vomitoxin levels. New strobilurin chemistry fungicides, Quadris, Stratego, and BAS 500, showed increased levels of vomitoxin and are being monitored closely. All three of the new fungicides showed great promise in suppressing leaf diseases and scab. BAS 500 was similar to Folicur in both disease suppression and increased yield. Overall, Folicur continues to provide the best performance against scab and the greatest potential for full Section 3 labeling from EPA in the near future.

Relative titer of PVY in three inoculated potato cultivars over time.
M. A. DRAPER (1) and N. C. Gudmestad (2). (1) Plant Science Dept., South Dakota State University, Brookings, SD 57007; (2) Dept. Plant Pathology, North Dakota State University, Fargo, ND 58103. Publication no. P-2000-0015-NCA.

Mosaic, caused by potato virus Y (PVY), has been a problem in US-grown potatoes since the mid-1980s. Cultivars Shepody and Russet Norkotah, express mosaic symptoms poorly when infected by PVY. These cultivars and Red La Soda, a cultivar that expresses mosaic, were grown from single eye seed pieces and inoculated with a local strain of PVY(^O), transmitted to the plants by green peach aphids (Myzus persicae). The aphids acquired PVY from detached PVY-infected potato leaves and were transfered to the first leaves, soon after emergence. PVY replicated at similar rates in the three potato cultivars. Over a four-week period of time, there was no significant difference in rate or distance of virus movement with all cultivars. No cultivar showed impaired PVY movement. Following inoculation of a basal leaf, PVY was detected by ELISA in upper, middle, or basal leaves within two weeks of inoculation. The relative virus titer, as measured by ELISA, was not significantly different among the three cultivars. These factors together indicate a susceptible response in all three cultivars.

Risk factors associated with Stewart’s disease of corn.
P. D. ESKER and F. W. Nutter, Jr. Department of Plant Pathology, Iowa State University, Ames, IA 50011. Publication no. P-2000-0016-NCA.

Accurate prediction of the risk for Stewart’s disease of corn would help seed corn producers to select low-disease-risk sites and use insecticide sprays in a cost-effective manner. Factors important in estimating risks for this disease include: (i) mean monthly temperatures during Dec, Jan, and Feb, (ii) prevalence of the disease the previous season, (iii) population dynamics of the corn flea beetle (Chaetocnema pulicaria) (vector), and (iv) the proportion of beetles infested with Pantoea stewartii. In 1999, Stewart’s disease occurred in 58% of 1317 Iowa fields inspected, and large populations of the corn flea beetle were found in the southern 2/3 of Iowa. ELISA tests found that 50 to 70% of the beetles sampled from mid- to late season were infested with P. stewartii. The Stewart’s disease risk for Iowa in 2000 is predicted to be moderate- to high based on geospatially referenced mean monthly air temperatures. However, considering all four risk factors, we predict that Stewart’s disease will be high throughout Iowa. Thus, seed and foliar applications of insecticide will be needed to reduce disease risk in 2000.

Susceptibility of soybeans and dry beans to root rot pathogens in Minnesota.
C. ESTEVEZ DE JENSEN, M. ElAraby, J. A. Percich, and J. E. Kurle. Dept. of Plant Pathology, University of MN, St. Paul, MN 55108. Publication no. P-2000-0017-NCA.

Susceptibilities of dry bean (Phaseolus vulgaris) ‘Montcalm’ and soybean (Glycine max) ‘Sturdy’ to infection by Fusarium solani f. sp phaseoli (Fsp) F. solani (Fs), and Rhizoctonia solani (Rs) were determined in growth chamber studies. Five-day-old seedlings germinated from disinfected seed of both crops were established in growth pouches. They were then inoculated with isolates of Fsp or Fs (1 × 10(^6) macroconidia / ml) or with 1 cm disks of PDA supporting actively growing cultures of Rs. Fusarium solani f. sp. phaseoli was pathogenic to bean and soybean resulting in root disease severity (DS) of 7.5 (dry bean) and 3.2 (soybean) (1-9 scale). Fusarium solani infected both dry bean and soybean resulting in a DS of 2.5 and 3.2 respectively. Plant biomass was decreased for bean (65%) and soybean (4%) when infected with Fsp when compared with noninfected plants. Rhizoctonia solani caused severe root rot and decreased plant biomass of infected dry bean (42%) and soybean (19%). The results suggest that crop sequences containing either of these crops may increase the risk of these root rots in Minnesota.

Monitoring Fusarium head blight temporal progress. L. J. FRANCL. Dept. of Plant Pathol., North Dak. State Univ., Fargo, ND 58105. Publication no. P-2000-0018-NCA.

A method was developed to measure Fusarium head blight (FHB) progression in field-grown spring wheat. Wheat susceptible to FHB was planted in one-year old wheat stubble in replicated blocks on three dates and soil was mulched with plastic to accelerate maturation of the first planting. A weather station and Burkard air sampler were placed on-site. Every day from heading until the milk stage, culms were cut at the first node and collected. Five heads per replicate were shaken in water to dislodge inoculum, which was assayed on selective medium. Twelve culms per replicate were inserted directly into a floral preservative solution, while 12 others were first misted for 24 h to promote disease expression. Culms then were kept in a growth chamber at 22 C with a 12 h photoperiod for 14 days. FHB intensity was evaluated before heads senesced. Under these conditions, FHB severity in 1999 ranged from 0 to 100% and sporodochia could sometimes be seen. This data series can be related to time, environment, spore dispersal, inoculum level, and plant growth stage. Once it is conducted in multiple environments, this approach should lead to a greater understanding of FHB epidemiology and development of a forecasting model.

Foliar-applied calcium sulfate increased yield and reduced white mold in dry bean.
P. L. GROSS, J. R. Venette, and R. S. Lamppa. Dept. Plant Pathology, North Dakota State University, Fargo, ND 58105-5012. Publication no. P-2000-0019-NCA.

Dry bean Phaseolus vulgaris L. is an important crop in North Dakota and white mold Sclerotinia sclerotiorum is the worst disease problem. Five grower fields in three North Dakota counties were chosen for white mold strip trials in 1999. Five treatments (Thiophanate methyl or benomyl at 1.7 and 0.56 kg/ha, calcium sulfate liquid at 9.4 L/ha plus thiophanate methyl or benomyl at 0.56 kg/ha, calcium sulfate liquid at 9.4 L/ha) were arranged so that each treated strip was adjacent to an untreated strip. White mold incidence and severity was determined at 8 paired sites 80 m apart in each treated and untreated strip. Incidence was reduced by 13% and 15% with fungicides alone at 0.56 kg/ha and 1.7 kg/ha respectively, by 26% with fungicide plus calcium sulfate, and by 2.5% with calcium sulfate alone, compared to the untreated control. Severity was reduced by 15% with fungicide treatment applied at 1.7 kg/ha and by 14% with calcium sulfate. Yields were increased by all treatments. Calcium sulfate used alone had the highest yield increase of 446 kg/ha compared to the untreated check.

Fungicide application timing effects on field severity of Fusarium head blight in wheat and barley.
S. Halley (1), M. P. MCMULLEN (1), J. Pederson (1), and J. Moos (2). (1) Dept. Plant Pathology; (2) Dept. Agricultural Systems and Engineering, North Dakota State University, Fargo, ND 58105. Publication no. P-2000-0020-NCA.

Optimum timing of fungicide application for control of Fusarium head blight (FHB) in spring wheat, durum, and barley was evaluated in a greenhouse environment. For each crop, 20 plants were planted in each of five replicate trays per treatment and grown to heading stage. Folicur (tebuconazole) fungicide (4 fl oz/acre in 9 gpa) was applied at three separate Feekes growth stages, 10.3, 10.51, or 10.54., using a track sprayer fitted with forward/backward XR8001 flat fan nozzles. Plants were inoculated with 5000 spores/ml of Fusarium graminearum at Feekes 10.51 using atomization. FHB field severity (incidence × head severity) was measured 21 days after inoculation. For each crop, FHB field severity was lowest when Folicur was applied at Feekes 10.51 and highest when treatment was delayed to Feekes 10.54. An additional trial on barley indicated that fungicide application on the day of, or 2 days after, inoculation to the head gave significantly lower FHB severities compared to 3 or 4 days after inoculation.

Effect of preharvest benomyl spray on winter storage of carrots.
J. M. HANSEN (1), D. J. Tobias (1), N. F. Balbyshev (1), R. W. Stack (1), and C. W. Lee (2). (1) Dept. Plant Pathology; (2) Dept. Plant Science, North Dakota State University, Fargo, ND 58105. Publication no. P-2000-0021-NCA.

A major consideration for carrot production in northern regions is the necessity to store the crop over the winter. In 1995 and 1996, growers in North Dakota and Minnesota suffered severe losses of stored carrots due primarily to white mold (WM) (Sclerotinia sclerotiorum (Lib) deBary). In 1997 trials, a preharvest application of benomyl reduced WM storage rot by 50%, but these results could not be repeated in 1998. In 1999, two experiments were done to test the effectiveness of preharvest benomyl application on the incidence of WM in stored carrots. In trials, a benomyl spray 10 days prior to harvest resulted in lower WM incidence. After 4 months of cold storage at 4 C, 9.7% of benomyl-sprayed carrots were decayed due to WM compared to 18.5% decay in unsprayed checks in one trial. In the other trial, the WM was 5.2% in the benomyl-sprayed versus 37.6% in the check. It appears that properly timed preharvest application of benomyl will substantially reduce losses to WM of carrots in storage.

Reaction of soybean germplasm with soybean cyst nematode resistance to brown stem rot development.
T. J. HUGHES, N. C. Kurtzweil, and C. R. Grau. Dept. of Plant Pathology, University of Wisconsin, Madison, WI 53706. Publication no. P-2000-0022-NCA.

Field and greenhouse experiments indicate a strong relationship between some sources of soybean cyst nematode (SCN) resistance and resistance to Phialophora gregata, causal agent of brown stem rot (BSR). To determine which sources of SCN resistance conferred resistance to BSR, 10 plant introductions (PI) with different sources of SCN resistance and 12 varieties representing six of these sources, were root-dip inoculated in greenhouse assays with isolate H96-5. BSR severity was calculated as the percentage of symptomatic leaf, stem, and root tissue. PI 209332 was the only SCN-resistant source with resistance (3%) to BSR development. Reaction of Peking (50%) and PI 88788 (62%) were higher than two BSR susceptible checks, Sturdy (19%) and Williams 82 (32%). SCN-resistant varieties derived from PI 88788 showed varying levels of symptom severity. Dwight was similar to PI 88788 while Freeborn, Fayette, Jack, and Bell were similar to varieties with the Rbs 1, 2, and 3 (2-10%) genes for BSR resistance. Studies are underway to determine the source of BSR resistance observed in PI 88788 derived soybean varieties.

Genetics of maize resistance to Maize chlorotic dwarf waikivirus.
M. W. JONES (1,2), R. Louie (1,2), R. A. Anderson (1,3), and M. G. Redinbaugh (1,2). (1) USDA-ARS Corn and Soybean Research, OARDC, Wooster, OH 44691; (2) Department of Plant Pathology; (3) Department of Entomology, The Ohio State University. Publication no. P-2000-0023-NCA.

Ineffective screening methods and low levels of disease resistance have hampered genetic analysis of maize resistance to maize chlorotic dwarf. This disease is caused by Maize chlorotic dwarf waikivirus, which is vectored by the leafhopper, Graminella nigrifrons. Germplasm identified as resistant by one or more screening methods (natural infection, single inoculation of 14-day old plants, and multiple inoculation of 3-4 day old seedlings) was re-evaluated with the highly effective multiple inoculation screening method. In this screen, inbreds Oh1VI, Mp705, and inbred lines derived from Compuesto Cuba 551 (CC551) and Dominican Republic (DR) were highly resistant. In contrast, T232, Oh1EP, Pa11, and CI21, which were resistant to MCDV in the natural infection and/or single inoculation screens, were susceptible in the multiple inoculation screen. Tests of F(1) crosses (resistant inbred X susceptible inbred Va35) and subsequent F(2) populations indicated the MCDV resistance was quantitative, and AUDPC scores for the F(1) crosses suggested the inheritance of resistance was polygenic. QTL analyses and ANOVA of 300 F(2) progeny of Oh1VI x Va35 indicated two independent QTLs of equal strength located on chromosomes three and ten. Experiments to map loci controlling resistance in Mp705, CC551 and DR are still underway.

Increased susceptibility to Sclerotinia stem rot of soybeans associated with increased planting density.
J. E. Kurle and M. ElAraby. Dept. of Plant Pathology, University of Minnesota, St. Paul, MN 55108. Publication no. P-2000-0024-NCA.

The incidence of Sclerotinia stem rot (SSR) or white mold of soybean Glycine max caused by Sclerotinia sclerotiorum increases when soybeans are planted in narrow rows or at high plant populations. The increase is attributed to high humidity and low temperatures in the crop canopy. Soybean (cvs. Sturdy and S19-90) was planted at five planting rates: 38, 61, 84, 106, and 129 plants/m(^2). At growth stage V4, plants were inoculated with S. sclerotiorum mycelium by placing plugs of PDA cut from the edge of growing S. sclerotiorum colonies over the cut ends of main stems. Susceptibility to SSR was indicated by the length of the lesion developing from the point of inoculation. Increasing plant density was correlated with increasing susceptiblity to SSR (r = 0.39, p < 0.001). The result indicates that increased incidence of SSR associated with high planting rates or narrow rows may be a result of physiological or structural changes caused by crowding.

Plant population and row spacing effects on Sclerotinia stem rot severity in soybean.
J. E. Kurle (1) and S. Naeve (2). (1) Dept. of Plant Pathology, University of Minnesota, St. Paul, MN 55108; (2) Dept. of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108. Publication no. P-2000-0025-NCA.

Sclerotinia stem rot (SSR) or white mold of soybean (Glycine max) caused by Sclerotinia sclerotiorum is an important disease in the North Central states. Increased prevalence of SSR in soybean has been attributed to more frequent use of narrow row spacing and high plant populations. We examined the effect of three row spacings on SSR incidence (25 cm, 50 cm and 76 cm) and three plant populations (100,000; 175,000 , and 225,000 plants ha(^-1)) planted in a factorial design at seven locations with a history of SSR occurrence. In 1999 SSR developed at four locations. Incidence of SSR was affected by the interaction of row spacing and plant population at one location (P=9.7), by plant population at two locations (P<=0.01, P=1.5), and was present only in plots planted at 225,000 plants ha(^-1) at a fourth location where average incidence was < 5%. The results indicate that plant population has a greater influence on white mold incidence than row spacing.

Relationship of soil pH and brown stem rot of soybean.
N. C. KURTZWEIL and C. R. Grau. Department of Plant Pathology, University of Wisconsin, Madison, WI 53706. Publication no. P-2000-0026-NCA.

Brown stem rot (BSR), caused by Phialophora gregata, is commonly found in Midwest soybean fields, occurring over a range of soil types and soil pH. While incidence of BSR has increased with shortened crop rotations and decreased tillage, fields are encountered that remain relatively free of BSR symptoms despite pathogen presence and management practices that should favor the pathogen and disease. Although climate, soil moisture, and cultivar influence BSR severity, results from several years of small plot and large on-farm trials suggest that symptom severity is greatest as soil pH approaches 6.0 and severity declines as soil approaches pH 7.0. A yield difference of 16-19 bu/a between BSR resistant and BSR susceptible cultivars was obtained at soil pH below 6.0, compared to a 5 bu/a yield difference for resistant and susceptible cultivars at soil pH above 6.5. Symptom severity of BSR susceptible cultivars was inversely correlated with soil pH (r=-0.41, p=0.0001), but not for BSR resistant cultivars (r=0.08 p=NS). Population density of P. gregata in host tissue decreased as soil pH increased. Knowledge of soil pH can be used to tailor BSR management systems for specific fields.

Evaluation of winter wheat cultivars and lines for wheat streak mosaic tritimovirus resistance or tolerance.
M. A. C. LANGHAM (1), S. D. Haley (2), D. C. Doxtader (1), S. Kalsbeck (1), and R. S. Little (1). (1) Plant Science Department, South Dakota State University, Brookings, SD 57007; (2) Soil & Crop Sciences, Colorado State University, Fort Collins, CO 80523. Publication no. P-2000-0027-NCA.

Ninety-six winter wheat (Triticum aestivum) cultivars and lines in three replications were evaluated for response to wheat streak mosaic tritimovirus (WSMV) in a field study. Two rows of a four-row plot were inoculated with infected sap extract using high-pressure spray. The remaining rows served as uninoculated controls. Disease severity, height, total yield, test weight, and heading date were recorded. Average disease severity (scale of 0-5) ranged from 1.9-4.1. Heights ranged from a 30% increase to 22.7% decrease. The hard red winter wheat cultivars, Alliance, Crimson, Scout 66, and Tandem had less than 10% yield loss. Yield losses up to 41.6% were recorded among the remaining cultivars. Among hard red wheat experimental lines, 12 lines had yield losses under 10%, but other lines had losses up to 61%. Five hard white winter wheat lines had yield losses under 10%, and maximum yield loss among these was 63.3%.

Characterization of phytoplasmas detected in Wisconsin.
M. E. LEE and C. R. Grau. University of Wisconsin, Madison, WI 53706. Publication no. P-2000-0028-NCA.

Phytoplasmas are commonly associated with diverse plant hosts in Wisconsin. No recent work has been done to characterize the phytoplasmas present in Wisconsin using molecular methods. We investigated the presence of phytoplasmas in soybeans and carrots and found aster yellows phytoplasma subgroups A and B (16SrI-A and 16SrI-B), according to Ing-Ming Lee’s RFLP classification system. Phylogenetic analysis of the 16S rDNA gene sequence suggests the phytoplasmas in carrot and soybean differ from each other. Of 121 soybean leaf samples collected from throughout Wisconsin, 2 tested positive. These positive samples were located at the West Madison Agricultural Research Station next to an alfalfa field. The positive soybean plants could not be distinguished based on symptoms from the surrounding plants. Of 37 aster yellows symptomatic and asymptomatic carrots sampled from areas near Hancock and Plover, Wisconsin, only one carrot plant did not contain phytoplasma in young leaves or root tips. Phytoplasmas were most frequently detected in the roots (97%) versus the leaves (73%) of carrots.

Relationships among environmental variables, ascospore dispersal, and inoculum of Gibberella zeae.
S. MARKELL and L. Francl. Dept. of Plant Pathology, North Dakota State University, Fargo, ND 58105. Publication no. P-2000-0029-NCA.

Ascospores of Gibberella zeae, thought to be the primary inoculum for Fusarium head blight, are borne on infested plant residues. A Burkard 7-day spore sampler was placed in one-year-old wheat stubble left unplowed and fallow. Greenhouse grown, potted wheat plants were exposed daily in the stubble field for a 24 h period. Four heads per pot were cut, shaken in a solution of distilled water and Tween20, and 0.5 ml was spread on Komada’s medium to determine colony forming units per head per day. A Campbell weather station was also on the site. Environmental variables were correlated with spores collected in the sampler and inoculum recovered from the heads. Six of seven major spore dispersal events occurred within 2 h of rain. Daily inoculum levels peaked when rainfall occurred within the previous 24 or 48 h. Time of day, relative humidity, and wind speed affected spore dispersal. Further research and analysis may provide a prediction model of inoculum level based on environmental variables.

Effect of Sclerotinia stem rot on seed yield of two soybean cultivars inoculated at two growth stages.
G. A. MARS (1), B. D. Nelson (1), and T. C. Helms (2). (1) Depts. Plant Pathology and (2) Plant Sciences, North Dakota State University, Fargo, 58105. Publication no. P-2000-0030-NCA.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is an important soybean disease in the Northern Plains. Seed yield loss (measured as total seed weight) due to Sclerotinia stem rot was evaluated in two cultivars, Traill (less susceptible) and M301 (highly susceptible), inoculated at two growth stages (R-3 and R-5; 15 days apart) in the field using two inoculation methods. One method was infested tissue paper appressed to the stem and the other was infested ground wheat grain sprinkled over the top of the plants. Following inoculation, an irrigation system maintained high humidity for 30 days. Both types of inoculations at R-3 resulted in significant seed weight reductions on individual plants of both cultivars. Although plants inoculated at the R-5 stage were diseased, there was no significant reduction in seed weight. The mean seed weight reduction per diseased plant was 50% for the tissue paper method and 42% and 45% for Traill and M301, respectively, for the grain inoculum method. The two cultivars did not significantly differ in seed weights, irrespective of the inoculation method.

Preferential infection of soybean cultivars by genotypes of the brown stem rot pathogen Phialophora gregata.
X. Q. MENG (1), C. R. Grau (2), and W. Chen (1). (1) Dept. of Crop Sciences, Univ. of Illinois, and Ill. Nat. Hist. Surv., Champaign; (2) Dept. of Plant Pathology, Univ. of Wisconsin-Madison. Publication no. P-2000-0031-NCA.

Isolates of P. gregata from soybean were separated into two distinct genotypes (A and B). The two genotypes showed cultivar-preferential infection in field environments. An experiment was designed to study the cultivar-preferential infection in a controlled environment. Four isolates each of the two genotypes were used individually or in combination to inoculate resistant (Bell) and susceptible (Sturdy) cultivars. When the isolates were used individually, all isolates of both genotypes infected both cultivars. Genotype A isolates caused both stem discoloration and defoliation on cv. Sturdy, whereas genotype B isolates caused stem discoloration only. Bell expressed mild symptoms to most of the isolates. When the two genotypes were mixed in 1:1 ratio, genotype A isolates preferentially infected cv. Sturdy, whereas genotype B isolates preferentially infected cv. Bell. The results confirm field observations and also support the concept that P. gregata is composed of defoliating and non-defoliating strains.

A computer program to quantify the "just noticeable difference" when assessing disease severity.
F W. NUTTER, Jr. (1), P. D. Esker (1), J. Guan (1), and C. Conover. (1) Dept. Plant Pathology, Iowa State University, Ames, IA 50011. Publication no. P-2000-0032-NCA.

Psychophysical laws (e.g., Weber’s Law and Fechner’s Law) that have been proposed as the basis for the development and use of nonlinear disease rating scales (e.g., Horsfall-Barrett scale) have recently been called into question (Can. J. Plant Pathology 17:174-185). A new computer program "Disease Comparison" was developed to measure the "just noticeable difference" (JND) in disease severity when raters were asked to compare images of diseased leaves (grape downy mildew) to the level of a known standard (e.g., 25, 37, and 50%). Preliminary studies were performed with nine raters, who recorded whether each of 150 images was < , >, or = to the comparison stimulus (image). All raters could correctly identify that images were not equal with a JND of + 5 to 6%. The JND was not significantly (P <= 0.05) affected by the disease severity of the comparison levels (25, 37, 50%). The results of this study suggest that a linear disease rating scale is more appropriate than the Horsfall-Barrett scale which apparently has no psychophysical basis for assessing the severity of at least some plant diseases.

Mapping the temporal and spatial spread of gray leaf spot of corn in Iowa using GIS.
F.W. NUTTER, Jr. (1), P. D. Esker (1), and R. Rubsam (1). (1) Dept. Plant Pathology, Iowa State University, Ames, IA 50011. Publication no. P-2000-0033-NCA.

Geographic Information Systems (GIS) is a powerful tool that can be used to geospatially reference disease and/or pathogen information over time to assess the risk for diseases in specific years and fields. We geospatially referenced the spread of Cercospora zeae-maydis (casual agent of gray leaf spot) in Iowa from 1981 to 1997 by obtaining the original hard copies of corn inspection data from the Iowa Dept. of Ag. and Land Stewardship, Des Moines, IA. A GIS database was created using ArcView GIS software (ESRI Inc., Redlands, CA, USA). Prevalence data was mapped by county and year for C. zeae-maydis, using ArcView. In 1981, gray leaf spot was reported in 4 out of 28 counties inspected. By 1997, however, gray leaf spot was reported in 44 out of 46 counties inspected. Prevalence based on the percentage of fields with gray leaf spot disease increased from 8% in 1981 to 95% in 1997. The disease prevalence maps for gray leaf spot clearly indicate that farmers should place greater emphasis through the use of resistant hybrids and/or the application of fungicides and not rely solely on practices that reduce initial inoculum.

Quantifying the effect of prescribed burning on disease dynamics in tallgrass prairies.
R. J. PERCIFIELD (1), T. Rosburg (2), and F. W. Nutter, Jr. (1). (1) Dept. Plant Pathology, Iowa State University, Ames, IA 50011; (2) Dept. Biology, Drake University, Des Moines, IA 50311. Publication no. P-2000-0034-NCA.

The management program most utilized to maintain the health and diversity of prairie "remnants" is prescribed burning. However, optimal seasonal timing and frequency of burning for managing plant diseases is unknown. The objective of this study was to quantify the impact of spring burning on disease dynamics in 3 Iowa tallgrass prairies. Disease incidence and severity were visually assessed at Cayler, Doolittle, and Sheeder Prairies in Iowa from sections burned in April 1999 and from sections burned in prior years. Four prairie species (Helianthus rigidus, Desmodium canadense, Andropogon gerardii, Sorghastrum nutans) were assessed for disease incidence and severity at 2-wk intervals from late May to mid-September. Predominant pathogens were: Puccinia andropogonis on A. gerardii, Stagnospora simplicior on S. nutans, and Puccinia helianthi on H. rigidus. Prescribed burning significantly reduced initial inoculum, which delayed the onset of disease epidemics at all 3 locations by up to 29 days, but had no effect on the rate of epidemics.

Fusarium head blight reaction in F2 and F3 generations of a spring wheat recombinant population.
R. W. Stack (1), R. C. Frohberg (2), J. Mitchell-Fetch (1), and J. M. HANSEN (1). (1) Dept. of Plant Pathology; (2) Dept. of Plant Sciences, North Dakota State Univ., Fargo, ND 58105. Publication no. P-2000-0035-NCA.

From the spring wheat cross ND2709/ND688, 107 individual F2 plants and F3 families were screened for Fusarium head blight (FHB) reaction in greenhouse tests. ND2709 is resistant and ND688 is susceptible to FHB. On each individual F2 plant 3-5 heads were inoculated at anthesis by spikelet injection with F. graminearum. Seed was saved from non-inoculated heads of the F2 plants. Fifteen F2:3 seeds were planted in three replicates. Wheat heads were inoculated at anthesis as before. In both experiments, individual heads were scored for FHB symptoms on a 0-100% scale at 3.5 weeks postinoculation. Both the F2 and F3 generations showed highly significant differences in FHB, but the correlation between generations was low (r = 0.37). Selecting the "best" and "worst" of the F2 plants did result in population shifts in the F3, but F2 selection would have neither retained the most resistant F3 lines nor discarded the most susceptible ones. Phenotypic selection for FHB resistance in spring wheat should be deferred until at least the F3 generation.

Internal stem browning can be an unreliable indicator of colonization of soybean stems by Phialophora gregata.
G. M. TABOR, G. L. Tylka, and C. R. Bronson. Dept. of Plant Pathology, Iowa State University, Ames, Iowa 50011. Publication no. P-2000-0036-NCA.

Growth chamber experiments were conducted to assess the relationship between internal stem browning and colonization of soybean stems by Phialophora gregata, the causal agent of soybean brown stem rot (BSR). P. gregata (10(^8) spores/plant) was added to soil at the base of 2-week-old susceptible (Sturdy) and resistant (BSR101) cultivars. Internal stem browning and presence of P. gregata were assessed over 12 weeks. Stems of Sturdy were colonized earlier than stems of BSR101; however, the fungus had colonized the full length of the stems of all infected plants by 12 weeks. All Sturdy and 75% of BSR101 plants were infected; yet, 59% of infected Sturdy plants and 67% of infected BSR101 plants had no internal stem browning. These results indicate that both resistant and susceptible varieties can be colonized extensively by P. gregata without detectable stem browning. The possibility of asymptomatic P. gregata infections should be considered when breeding and selecting for BSR resistance.

Soybean cyst nematode increases colonization of soybean stems by Phialophora gregata.
G. M. TABOR, G. L. Tylka, and C. R. Bronson. Dept. of Plant Pathology, Iowa State University, Ames, Iowa 50011. Publication no. P-2000-0037-NCA.

Two growth chamber experiments were conducted to test if Heterodera glycines, the soybean cyst nematode, increases susceptibility of soybeans to Phialophora gregata, causal agent of brown stem rot (BSR) of soybeans. Four cultivars with various combinations of resistance or susceptibility to both pathogens were planted in soil mix with four pathogen treatment combinations. H. glycines was applied as eggs (1,600/plant), and P. gregata was applied as artificially infested soybean straw (7.5 × 10(^8) cfu/plant). After 10 weeks, presence of P. gregata was assessed by plating stem pieces on a semi-selective medium. In the presence of H. glycines, incidence of stem colonization by P. gregata was 38 to 79% for BSR-resistant cultivars which was significantly greater than the 13 to 33% in the absence of H. glycines. For these cultivars, stem length colonized was 22 to 39% with H. glycines, versus 6 to 13% without H. glycines. Incidence and colonization were 90 to 100% with or without H. glycines for a BSR-susceptible cultivar. Our findings suggest that H. glycines infection increases colonization of soybean stems by P. gregata.

Genetic variation of Monilinia vaccinii-corymbosi populations determined with amplified fragment-length polymorphic DNA markers.
T. R. Thompson (1), J. S. LEHMAN (1), L. A. Wasilwa (2), and P. V. Oudemans (2). (1) Otterbein College, Westerville, OH 43081; (2) Rutgers University, Chatsworth, NJ 08019. Publication no. P-2000-0038-NCA.

Genetic variation of Monilinia vaccinii-corymbosi (Mvc) populations was examined with AFLPs. Isolates of Mvc were collected from Nova Scotia (NS) and New Jersey (NJ). Variation in allelic frequencies was calculated for AFLP loci in 16 isolates from each population. Allelic variation was partitioned into within- and between-population components (Nei’s Gst). Linkage disequilibrium was calculated among all pairs of loci. Nine bands were identified. Seven of the bands were found in both populations while two were found only in the NS population. A Gst value of 0.10 indicated that 10% of the allelic variation was due to differentiation between populations, while 90% was due to within-population variation. Linkage disequilibrium was present to a greater extent in the NS population. We conclude that NJ and NS populations share the same alleles and exhibit little differentiation between populations. Based on disequilibrium values, NJ and NS populations may differentially emphasize sexual and asexual reproduction.


Soil moisture and air temperature effects on Rhizoctonia solani infection of soybean. N. T. TUTTLE (1), A. E. Dorrance (1), and M. Kleinhenz (2). (1) Dept. of Plant Pathology, and (2) Dept. of Hort. and Crop Science, The Ohio State University OARDC, Wooster, OH 44691. Publication no. P-2000-0039-NCA.

Reports on environmental conditions favoring Rhizoctonia solani damping-off, root and stem rot of soybean are conflicting. To determine soil moisture and air temperature limits for infection, inoculum of sterile oat kernels colonized by R. solani isolate 91-1 AG (UNK) was used. A 1:1 v/v mixture of Wooster silt loam and silica sand and vermiculite only were used for soil moisture and air temperature studies respectively. Soil moisture was maintained at 25, 50, 75, or 100 percent (%) of moisture holding capacity (MHC) in a greenhouse. Air temperature studies were in growth chambers maintained at 20, 25, 30, or 35°C. Emergence, plant height, top weight, disease incidence and severity, and root health were recorded on the soybean cultivar Resnik. Disease incidence and severity were greatest at 75(%)MHC and 25°C for this isolate. Disease incidence tended to increase with increases in soil moisture and was greatest at air temperatures of 25°C. Infections occurred, albeit at a lower incidence, at 25 and 100(%) MHC as well as 20 and 35°C.

Osmolytes enhance room temperature shelf-life of freeze-dried, cells of the biocontrol agent Enterobacter cloacae S11:T:07( NRRL 21050).
J. E. VanCauwenberge, D. A. Schisler, and P. J. Slininger. USDA-ARS, NCAUR, Peoria, IL 61604. Publication no. P-2000-0040-NCA.

Enterobacter cloacae
S11:T:07 (NRRL 21050) has been identified as an antagonist effective against the causative agent of potato dry rot (Gibberella pulicaris) during tuber storage. In an effort to improve the shelf life of cells stored at room temperature, 15 osmolytes at 4 concentrations were added to individual E. cloacae cultures at initial inoculation or after 72 hours of incubation. Cultures were harvested at 96 hours incubation, freeze dried, stored at room temperature, and tested for culture viability over 21 days. Five osmolytes were identified that improved the shelf life of E. cloacae. Cell counts for control samples decreased from 10(^10) to 10(^1) CFU/ml by day 5 and approached 0 by day 7. In comparison, day 7 cultures which had turanose or arginine added at initial inoculation were at 10(^8) CFU/ml and 10(^5) CFU/ml, respectively. Day seven cultures which had proline, maltose or sucrose added at 72 hours were at 10(^5) CFU/ml, 10(^4) CFU/ml, or 10(^6) CFU/ml, respectively. Further experimentation will be conducted to optimize product shelf life and determine the influence of osmolytes on cell efficacy.