J. J. Hao

Germination of Sclerotinia minor and S. sclerotiorum Sclerotia Under Various Soil Moisture and Temperature Combinations

ABSTRACT Sclerotial germination of three isolates each of Sclerotinia minor and S. sclerotiorum was compared under various soil moisture and temperature combinations in soils from Huron and Salinas, CA. Sclerotia from each isolate in soil disks equilibrated at 0, -0.03, -0.07, -0.1, -0.15, and -0.3 MPa were transferred into petri plates and incubated at 5, 10, 15, 20, 25, and 30 degrees C. Types and levels of germination in the two species were recorded. Petri plates in which apothecia were observed were transferred into a growth chamber at 15 degrees C with a 12-h light-dark regime. All retrievable sclerotia were recovered 3 months later and tested for viability. Soil type did not affect either the type or level of germination of sclerotia. Mycelial germination was the predominant mode in sclerotia of S. minor, and it occurred between -0.03 and -0.3 MPa and 5 and 25 degrees C, with an optimum at -0.1 MPa and 15 degrees C. No germination occurred at 30 degrees C or 0 MPa. Soil temperature, moisture, or soil type did not affect the viability of sclerotia of either species. Carpogenic germination of S. sclerotiorum sclerotia, measured as the number of sclerotia producing stipes and apothecia, was the predominant mode that was affected significantly by soil moisture and temperature. Myceliogenic germination in this species under the experimental conditions was infrequent. The optimum conditions for carpogenic germination were 15 degrees C and -0.03 or -0.07 MPa. To study the effect of sclerotial size on carpogenic germination in both S. minor and S. sclerotiorum, sclerotia of three distinct size classes for each species were placed in soil disks equilibrated at -0.03 MPa and incubated at 15 degrees C. After 6 weeks, number of stipes and apothecia produced by sclerotia were counted. Solitary S. minor sclerotia did not form apothecia, but aggregates of attached sclerotia readily formed apothecia. The number of stipes produced by both S. minor and S. sclerotiorum was highly correlated with sclerotial size. These results suggest there is a threshold of sclerotial size below which apothecia are not produced, and explains, in part, why production of apothecia in S. minor seldom occurs in nature.

Microbial Communities Associated with Potato Common Scab-Suppressive Soil Determined by Pyrosequencing Analyses

Potato common scab, caused by Streptomyces spp., is an annual production problem for potato growers, and not effectively controlled by current methods. A field with naturally occurring common scab suppression has been identified in Michigan, and confirmed to have a biological basis for this disease suppression. This field and an adjacent scab nursery conducive to disease were studied using pyrosequencing to compare the two microbial communities. Total DNA was extracted from both the disease-conducive and -suppressive soils. A phylogenetically taxon-informative region of the 16S rRNA gene was used to establish operational taxonomic units (OTUs) to characterize bacterial community richness and diversity. In total, 1,124 OTUs were detected and 565 OTUs (10% dissimilarity) were identified in disease-conducive soil and 859 in disease-suppressive soil, including 300 shared both between sites. Common phyla based on relative sequence abundance were Acidobacteria, Proteobacteria, and Firmicutes. Sequences of Lysobacter were found in significantly higher numbers in the disease-suppressive soil, as were sequences of group 4 and group 6 Acidobacteria. The relative abundance of sequences identified as the genus Bacillus was significantly higher by an order of magnitude in the disease-conducive soil.

Spatial Patterns of Microsclerotia of Verticillium dahliae in Soil and Verticillium Wilt of Cauliflower

ABSTRACT The spatial patterns of microsclerotia of Verticillium dahliae in soil and wilt symptoms on cauliflower were determined at three sites in each of two fields in 1994 and 1995. Each site was an 8 x 8 grid divided into 64 contiguous quadrats (2 by 2 m each). Soil samples were collected to a depth of 15 cm with a probe (2.5 cm in diameter), and samples from four sites in each quadrat were bulked. Plants in each quadrat were cut transversely, and the number of plants with vascular discoloration and the number without discoloration were recorded. The soil was assayed for microsclerotia by the modified Anderson sampler technique. Lloyds index of patchiness (LIP) was used as an indicator to evaluate the aggregation of microsclerotia in the field. Spatial autocorrelation and geostatistical analyses were also used to assess the autocorrelation of microsclerotia among quadrats. The LIP for microsclerotia was greater than 1, indicating aggregation of propagules; however, the degree of aggregation at most sites was not high. Significant autocorrelation within or across rows was detected in some spatial autocorrelograms of propagules, and anisotropic patterns were also detected in some oriented semivariograms from geostatistical analyses for microsclerotia, indicating the influence of bed preparation in the fields on pathogen distribution. The parameter estimates p and theta in the beta-binomial distribution and the index of dispersion (D) associated with the distribution were used to assess the aggregation of diseased plants at each site. A random pattern of wilt incidence was detected at 7 of 12 sites, and an aggregated pattern was detected at 5 of 12 sites. The degree of aggregation was not high. A regular pattern of wilt severity was detected at all sites. The high disease incidence (77 to 98%) observed at 11 of the 12 sites could be explained by high inoculum density.

Culture-Based Assessment of Microbial Communities in Soil Suppressive to Potato Common Scab

A field in East Lansing, MI, showed a decline of potato common scab compared with an adjacent potato field. To confirm that the decline was due to biological factors, the soil was assayed. In the greenhouse, putative common-scab-suppressive soil (SS) was either treated with various temperatures or mixed with autoclaved SS at various ratios. Pathogenic Streptomyces scabies was incorporated into the treated soil at 106 CFU/cm3 of soil, followed by planting of either potato or radish. Disease severity was negatively correlated with the percentage of SS in the mixture and positively correlated with temperature above 60°C. The soil was screened for four groups of potential antagonists (general bacteria, streptomycetes, fluorescent pseudomonads, and bacilli) pairing in culture with S. scabies. The frequency of antagonistic bacteria in SS was higher than common-scab-conducive soil (CS) in all four groups but only pseudomonads and streptomycetes were significantly higher. The population of pathogenic Streptomyces spp. in the rhizosphere of CS was significantly higher than SS. Dilution plating of CS and SS samples showed no clear trends or differences in populations of total fungi, total bacteria, streptomycetes, fluorescent pseudomonads, and bacilli but terminal restriction fragment polymorphism analysis revealed two distinct microbial communities were present in SS and CS.

Effects of Broccoli Rotation on Lettuce Drop Caused by Sclerotinia minor and on the Population Density of Sclerotia in Soil

Field experiments were conducted at Spence Road site and at the Hartnell College East Campus site in Salinas, CA, to determine the effects of crop rotation with broccoli or a fallow period on lettuce drop caused by Sclerotinia minor and the density of pathogen sclerotia in the soil. Treatments at the Spence Road site with low inoculum density (<7 sclerotia per 100 cm3 of soil) distributed randomly included: successive crops of lettuce (LLL), lettuce rotated with broccoli (LBL), and lettuce followed by a fallow period (LFL). Treatments at the Hartnell site with high inoculum density (>7 sclerotia per 100 cm3 of soil) distributed uniformly included: continuous lettuce (LLLL), broccoli-lettuce-broccoli-lettuce (BLBL), broccoli-broccoli-lettuce-lettuce (BBLL), and fallow-lettuce-fallow-lettuce (FLFL). At the Spence Road site, continuous lettuce did not increase lettuce drop incidence for at least 2 years, although an increase in soilborne sclerotia was observed annually but was below the threshold at which a correlation between inoculum density and disease incidence is observed. Rotation with broccoli resulted in small reductions in disease incidence only in the first year. The density of sclerotia was lowest in the LFL treatment, and the highest in the LLL. At the Hartnell site, rotation with broccoli significantly reduced both sclerotia and lettuce drop incidence. The number of broccoli crops rather than the sequence of lettuce rotations with broccoli was critical for reducing the numbers of S. minor sclerotia in soil. Fallowing after a lettuce crop resulted in marginal reductions in sclerotia and lettuce drop incidence. Viability of recovered sclerotia was not significantly different between treatments, although differences between seasons were detected. Results suggest that rotations with broccoli can be a practical lettuce drop management strategy.

Comparative analyses of lettuce drop epidemics caused by Sclerotinia minor and S. sclerotiorum

Temporal and spatial patterns of lettuce (Lactuca sativa) drop caused by Sclerotinia minor and S. sclerotiorum were determined in lettuce fields in the Salinas, Santa Maria, and San Joaquin Valleys in California during 1995 to 1998. Of the 25 commercial fields assessed, 14 had predominantly S. minor, 9 had predominantly S. sclerotiorum, and 2 had varying levels of both species. Sclerotinia infections were classified based on symptoms: those caused directly by eruptive germination of sclerotia (type I) and those caused by the airborne ascospores (type II). The precise location of diseased and healthy plants was mapped and lettuce drop progress was determined at different crop growth stages. Spatial patterns of disease incidence were analyzed using 1-by-1-, 2-by-2-, 3-by-3-, and 4-by-4-m quadrat sizes. Regardless of the analytical method employed, disease incidence with type I infection showed an aggregated pattern in a majority of the fields evaluated and random patterns in fields where incidence was low. In all fields with type I infection, disease progress followed the monomolecular model, typical of soilborne diseases. For fields with aggregated distribution, spatial dependence was observed up to 10 m and was either isotropic or random in direction, suggesting the potential influence of tillage operations on inoculum distribution and disease incidence. Lettuce drop incidence in fields with type II infection was erratic in time and peaked within a very short time. However, disease incidence showed an aggregated pattern in all fields evaluated. Spatial dependence of quadrats generally was detected in two adjacent directions, suggesting a directional gradient perhaps caused by wind direction during ascospore dissemination. Increasing quadrat sizes usually increased the degree of aggregation of lettuce drop, but not the distribution pattern itself. These results demonstrate that the source of inoculum and the type of infections they cause are most likely to determine spatial patterns of lettuce drop in the field.

Characterizing a novel strain of Bacillus amyloliquefaciens BAC03 for potential biological control application.

To identify and characterize a bacterial strain BAC03, evaluate its biological control activity against potato common scab (Streptomyces spp.) and characterize an antimicrobial substance produced by BAC03.

Baseline sensitivity of natural population and resistance of mutants in Phytophthora capsici to zoxamide.

Laboratory experiments were conducted to determine the baseline sensitivity of Phytophthora capsici and its risk for developing resistance to zoxamide. In total, 158 P. capsici isolates were collected from China. All 158 isolates were sensitive to zoxamide, with effective concentrations for 50% inhibition of mycelial growth of 0.023 to 0.383 μg/ml and a mean of 0.114 μg/ml, which showed a skewed unimodal distribution. Zoxamide-resistant mutants of P. capsici were obtained by either treating mycelial culture and zoospores with ultraviolet irradiation or adapting a culture on zoxamide-amended plates. The frequency of resistance selection averaged 1.8 × 10(-7). Resistant isolates were also derived by selfing or crossing two sexually compatible isolates, resulting in a mean selection frequency of 0.47. The resistance factor (RF) for zoxamide was 25 to 100 in P. capsici mutants. Through 10 culture transfers, the mutants maintained high levels of RF (between 14 and 134) and had almost equal fitness as their wild-type parents in mycelial growth, sporulation, and virulence. There was no cross resistance between zoxamide and either flumorph, metalaxyl, azoxystrobin, or etridiazole. Based on the results above, P. capsici can develop resistance to zoxamide, and the risk is predicted to be moderate in nature.

Baseline sensitivity and resistance-risk assessment of Phytophthora capsici to iprovalicarb.

Iprovalicarb has been used to control Phytophthora capsici, a devastating pathogen of many economically important crops. To evaluate the risk of fungicide resistance, 158 isolates of P. capsici were examined for sensitivity to iprovalicarb by measuring mycelial growth. Values of effective concentrations for 50% mycelial growth inhibition varied from 0.2042 to 0.5540 μg/ml and averaged 0.3923 (±0.0552) μg/ml, with a unimodal distribution. This is the first report of P. capsici isolates highly resistant to iprovalicarb (resistance factor >100). Resistance of the isolates was stable through 10 transfers on iprovalicarb-free medium, and most resistant isolates had the same level of fitness (mycelial growth, zoospore production, and virulence) as their corresponding parents, indicating that iprovalicarb resistance was independent from other general growth characters. There was cross-resistance among all tested carboxylic acid amide (CAA) fungicides, including iprovalicarb, flumorph, dimethomorph, and mandipropamid, but not with non-CAA fungicides, including azoxystrobin, chlorothalonil, cymoxanil, etridiazole, metalaxyl, and zoxamide. Based on the present results, resistance risk of P. capsici to CAAs could be moderate and resistance management should be considered.

Characterization of a New Streptomyces Strain, DS3024, That Causes Potato Common Scab

A novel strain of Streptomyces (named DS3024) was isolated from a potato field in Michigan in 2006. The taxonomy of the organism was determined by morphology, biochemistry, and genetic analysis. Analysis of the 16S ribosomal RNA gene sequence indicated that the organism was most similar to an isolate of Streptomyces sp., ME02-6979.3a, which is not pathogenic to potato tubers but is distinct from other known pathogenic Streptomyces spp. Strain DS3024 has genes that encode thaxtomin synthetase (txtAB), which is required for pathogenicity and virulence, and tomatinase (tomA), which is a common marker for many pathogenic Streptomyces spp. However, the nec1 gene (associated with virulence in most pathogenic Streptomyces spp.) was not detected. The new strain was capable of growth at pH 4.5, caused necrosis on potato tuber slices, and produced thaxtomin A. In greenhouse experiments, DS3024 caused scab symptoms on potato tubers similar to those caused by Streptomyces scabies on tubers of potato cv. Atlantic, which is scab susceptible. We propose that DS3024 is a new strain of Streptomyces capable of causing common scab on potato tubers. The prevalence of this strain of Streptomyces in potato-producing areas in the north-central United States has not been determined.