B. M. Wu

Characterization of Verticillium dahliae Isolates and Wilt Epidemics of Pepper

Epidemics of Verticillium wilt in pepper fields of the central coast of California and isolates of Verticillium dahliae associated with these epidemics were characterized. The mean incidence of wilted plants per field ranged from 6.3 to 97.8% in fields with Anaheim, jalapeno, paprika, or bell peppers. In general, incidence of wilt in jalapeno and bell pepper crops was lower than in crops of other types of pepper. Inoculum density of V. dahliae in the surveyed pepper fields ranged from 2.7 to 66.6 microsclerotia g-1 dry soil, and the correlation between disease incidence and density of microsclerotia was high (r = 0.81, P < 0.01). Distribution of Verticillium wilt was aggregated in a majority of the pepper fields surveyed, but the degree of aggregation varied. Vegetative compatibility group (VCG) characterization of 67 isolates of V. dahliae indicated that 67% belonged to VCG 2, 22% to VCG 4, and 11% to a new group, designated VCG 6. The pathogenicity of isolates of V. dahliae from bell pepper and tomato plants was tested by inoculating 1-month-old bell pepper (cv. Cal Wonder) and tomato (cv. EP 7) seedlings and incubating the inoculated plants in the greenhouse. Seedlings of bell pepper were susceptible only to the isolates of V. dahliae from pepper, whereas seedlings of tomato were susceptible to both pepper and tomato isolates. Pepper isolates belonging to VCG 2, VCG 4, and VCG 6 were highly pathogenic to bell pepper and chili pepper. Temperatures between 15 and 25°C were optimal for mycelial growth of a majority of isolates of V. dahliae. Molecular characterization of pepper isolates of V. dahliae using a polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD) technique revealed minor variation among these isolates, but unique polymorphic banding patterns were observed for isolates belonging to VCG 6. Verticillium wilt of pepper is a major production constraint in the central coast of California. More aggressive isolates of V. dahliae may have been selected in this region as a result of intensive cropping practices.

Effects of Soil Temperature, Moisture, and Burial Depths on Carpogenic Germination of Sclerotinia sclerotiorum and S. minor

Extensive studies have been conducted on the carpogenic germination of Sclerotinia sclerotiorum, but carpogenic germination in S. minor has not been studied adequately. It remains unclear why apothecia of this pathogen have seldom been observed in nature. In this study, a new method was developed to produce apothecia in the absence of soil or sand, and carpogenic germination without preconditioning was recorded for 95 of the 96 S. sclerotiorum isolates tested. Carpogenic germination of the two species was compared under a variety of temperature, soil moisture, burial depths, and short periods of high temperature and low soil moisture. The optimal temperatures for rapid germination and for maximum germination rates were both lower for S. minor than for S. sclerotiorum. The temperature range for carpogenic germination was also narrower for S. minor than for S. sclerotiorum. A 5-day period at 30 degrees C, either starting on the 10th or 20th day of incubation, did not significantly affect carpogenic germination of S. sclerotiorum. For both S. minor and S. sclerotiorum, the percentage of carpogenically germinated sclerotia increased as soil water potential increased from -0.3 to -0.01 MPa. In the greenhouse, a 10- or 20-day dry period completely arrested carpogenic germination of S. sclerotiorum, and new apothecia appeared after an interval of 35 days following rewetting, similar to the initial carpogenic germination regardless of when the dry period was imposed. In naturally infested fields, the number of sclerotia in 100 cc of soil decreased as depth increased from 0 to 10 cm before tillage, but became uniform between 0 and 10 cm after conventional tillage for both species. Most apothecia of S. minor were, however, produced from sclerotia located at a depth shallower than 0.5 cm while some apothecia of S. sclerotiorum were produced from sclerotia located as deep as 4 to 5 cm. These results provide the much needed information to assess the epidemiological roles of inoculum from sexual reproduction in diseases caused by the two Sclerotinia species in different geographical regions. However, more studies on effects of shorter and incompletely dry periods are still needed to predict production of apothecia of S. sclerotiorum in commercial fields under fluctuating soil temperature and moisture.

Factors affecting the survival of Bremia lactucae sporangia deposited on lettuce leaves

ABSTRACT Experiments to identify the factors affecting survival of Bremia lactucae sporangia after deposition on lettuce leaves were conducted in growth chambers and outdoors under ambient conditions. Lettuce seedlings at the four-leaf stage were inoculated with B. lactucae sporangia under dry conditions. Sporangia deposited on lettuce seedlings were incubated at different temperature and relative humidity (RH) combinations, exposed to 100, 50, 25, and 0% sunlight in the second experiment, and exposed to different artificial lights in wavelength ranges of UVA (315 to 400 nm), UVB (280 to 315 nm), or fluorescent light in the third experiment. After exposure for 0 to 48 h in the first experiment and 0 to 12 h in the second and third experiments, seedlings in two pots were sampled for each treatment, and sporangia were washed from 15 leaves excised from the sampled seedlings. Germination of sporangia was determined in water after incubation in the dark at 15 degrees C for 24 h. The sampled seedlings with remaining leaves were first transferred to optimal conditions for infection (24 h), for the development of downy mildew, and then assessed for disease after 9 days. Sporangia survived much longer at 23 degrees C (>12 h) than at 31 degrees C (2 to 5 h), regardless of RH (33 to 76%). Germination percentage was significantly reduced after exposure to 50 and 100% sunlight. UVB significantly reduced sporangium viability, while fluorescent light and UVA had no effect relative to incubation in the dark. Infection of seedlings followed a pattern similar to germination of sporangia. Solar radiation is the dominant factor determining survival of B. lactucae sporangia, while temperature and RH have small, insignificant effects in coastal areas of California. This suggests that infections by sporangia that survived a day are probable only on cloudy days or on leaves that are highly shaded.

The Sclerotinia sclerotiorum Mating Type Locus (MAT) Contains a 3.6-kb Region That Is Inverted in Every Meiotic Generation

Sclerotinia sclerotiorum is a fungal plant pathogen and the causal agent of lettuce drop, an economically important disease of California lettuce. The structure of the S. sclerotiorum mating type locus MAT has previously been reported and consists of two idiomorphs that are fused end-to-end as in other homothallics. We investigated the diversity of S. sclerotiorum MAT using a total of 283 isolates from multiple hosts and locations, and identified a novel MAT allele that differed by a 3.6-kb inversion and was designated Inv+, as opposed to the previously known S. sclerotiorum MAT that lacked the inversion and was Inv-. The inversion affected three of the four MAT genes: MAT1-2-1 and MAT1-2-4 were inverted and MAT1-1-1 was truncated at the 3’-end. Expression of MAT genes differed between Inv+ and Inv- isolates. In Inv+ isolates, only one of the three MAT1-2-1 transcript variants of Inv- isolates was detected, and the alpha1 domain of Inv+ MAT1-1-1 transcripts was truncated. Both Inv- and Inv+ isolates were self-fertile, and the inversion segregated in a 1∶1 ratio regardless of whether the parent was Inv- or Inv+. This suggested the involvement of a highly regulated process in maintaining equal proportions of Inv- and Inv+, likely associated with the sexual state. The MAT inversion region, defined as the 3.6-kb MAT inversion in Inv+ isolates and the homologous region of Inv- isolates, was flanked by a 250-bp inverted repeat on either side. The 250-bp inverted repeat was a partial MAT1-1-1 that through mediation of loop formation and crossing over, may be involved in the inversion process. Inv+ isolates were widespread, and in California and Nebraska constituted half of the isolates examined. We speculate that a similar inversion region may be involved in mating type switching in the filamentous ascomycetes Chromocrea spinulosa, Sclerotinia trifoliorum and in certain Ceratocystis species.

Analyses of Lettuce Drop Incidence and Population Structure of Sclerotinia sclerotiorum and S. minor

ABSTRACT To understand the geographical distribution of lettuce drop incidence and the structure of Sclerotinia minor and S. sclerotiorum populations, commercial lettuce fields were surveyed in the Salinas, San Joaquin, and Santa Maria Valleys in California. Lettuce drop incidence, pathogen species, and mycelial compatibility groups (MCGs) were determined and analyzed using geostatistic and geographical information system tools. Lettuce drop incidence was lowest in the San Joaquin Valley, and not significantly different between the other two valleys. Semivariogram analysis revealed that lettuce drop incidence was not spatially correlated between different fields in the Salinas Valley, suggesting negligible field-to-field spread or influence of inoculum in one field on other fields. Lettuce drop incidence was significantly lower in fields with a surface drip system than in fields with furrow or sprinkler irrigation systems, suggesting that the surface drip system can be a potential management measure for reducing lettuce drop. In the San Joaquin Valley, S. sclerotiorum was the prevalent species, causing drop in 63.5% of the fields, whereas S. minor also was identified in 25.4% of the fields. In contrast, in the Salinas Valley, S. minor was the dominant species (76.1%) whereas S sclerotiorum only observed in only 13.6% fields, in which only a few plants were infected by S. sclerotiorum. In the Santa Maria Valley, both species frequently were identified, with S. minor being slightly more common. Although many MCGs were identified in S. minor, most of them consisted of only one or two isolates. In all, approximately 91.4% of the isolates belonged to four MCGs. Among them, MCG-1 was the most prevalent group in all three valleys, accounting for 49.8% of total isolates. It was distributed all over the surveyed areas, whereas other MCGs were distributed more or less locally. Populations of S. sclerotiorum exhibited greater diversity, with 89 isolates collected from the Salinas and San Joaquin Valleys belonging to 37 different MCGs. Among them, the most recurrent MCG-A contained 16 isolates, and 30 MCGs contained only 1 isolate each. Many MCGs occurred within only one or a part of the two valleys. Potential reasons for this abundant diversity are discussed.

Effects of Irrigation and Tillage on Temporal and Spatial Dynamics of Sclerotinia minor Sclerotia and Lettuce Drop Incidence.

ABSTRACT The temporal and spatial dynamics of Sclerotinia minor sclerotia and the resulting incidence of lettuce drop were studied under furrow irrigation with conventional tillage and subsurface-drip irrigation with minimum tillage during 1993-95. Lettuce crops were grown each year during the spring and fall seasons. All plants were inoculated immediately after thinning in the spring of 1993. Grids of 24 contiguous quadrats (1 by 1 m(2)) were demarcated in the centers of each 150-m(2) plot. Lettuce drop incidence in each quadrat was evaluated each season prior to harvest. One soil sample (100 cm(3)) was collected from each quadrat at harvest and after tillage prior to planting of the next crop for both spring and fall crops and assayed for S. minor sclerotia using wet sieving. Lloyds index of patchiness, the beta-binomial distribution, and variance of moving window averages were used to evaluate the spatial patterns of sclerotia and lettuce drop incidence under the two irrigation systems and associated tillage treatments. Disease incidence remained significantly higher under furrow irrigation than under subsurface-drip irrigation throughout the study period, and was significantly higher on fall crops than on spring crops. Under furrow irrigation, the number of sclerotia at the end of a crop season increased significantly over that at the beginning of the season, but no significant changes were detected over years. In contrast, the number of sclerotia within a single season did not increase significantly under subsurface drip irrigation, nor was year-to-year accumulation of sclerotia statistically significant. The degree of aggregation of sclerotia increased significantly during a cropping season under furrow irrigation, but not under subsurface drip irrigation. The conventional tillage after harvest under furrow irrigation decreased the degree of aggregation of sclerotia after each season, but the distribution pattern of sclerotia under subsurface-drip irrigation changed little by the associated minimum tillage. Spatial pattern analyses suggested that the aggregation of S. minor sclerotia occurred at a scale of no more than 1 m, and distribution of diseased lettuce plants was random at a scale larger than 1 m. The combination of fewer sclerotia produced by each crop and its unaltered distribution under subsurface drip irrigation and associated minimum tillage makes it a valuable cultural practice for lettuce drop management.

Comparative Survival of Sclerotia of Sclerotinia minor and S. sclerotiorum

Survival of sclerotia of Sclerotinia minor and S. sclerotiorum was compared in irrigated fields during the summer in two major lettuce production areas in California. More than 50% sclerotia of S. sclerotiorum compared with 4 and 35% of S. minor remained viable after 24 weeks of burial at 15 and 5 cm depths, respectively, in the San Joaquin Valley while >80% of sclerotia survived in the Salinas Valley for both species. The results explain in part the lower infections from S. minor in the San Joaquin Valley. To identify factors that contribute to the rapid decline in the viability of sclerotia, the effects of soil moisture, temperature, and oxygen levels were studied in laboratory. More than 90% of sclerotia of both species survived for at least 3 months in sterilized dry soils at temperatures between 15 and 40 degrees C. Soil moisture did not affect survival at 15 and 25 degrees C. At 35 degrees C, however, survival rates were significantly lower at high (-0.3 to -0.01 MPa) water potential than at low (<-1.0 MPa) water potential. Incubation under ultralow oxygen concentration (0.01%) significantly reduced survival of sclerotia in nonautoclaved moist soils at 25 degrees C, with less than 2% sclerotia surviving over 4 weeks compared with about 45% sclerotia surviving at the ambient oxygen level (21%). The combination of high temperature, high soil moisture, and reduced oxygen in irrigated fields contribute to the lower survival of both Sclerotinia species and the responses of the two species to these conditions shape their relative geographical distribution.

Nonlinear colony extension of Sclerotinia minor and S. sclerotiorum

Fungal colonies initially extend exponentially and reach a constant linear extension rate determined solely by their growth in the peripheral zone. However the radial extension rates of Sclerotinia sclerotiorum and S. minor accelerate over time on PDA. Experiments were conducted to analyze the variable extension rates of the two Sclerotinia species and compare them with those of Verticillium dahliae and Cladosporium sp. In addition, the effects of starter disk size, disk position in the parent colony, the age of the parent colony, the concentration of potato dextrose broth and of incubation temperature also were determined. While the growth of Cladosporium sp. and V. dahliae followed established linear trends, the radial extension of S. sclerotiorum and S. minor colonies continuously accelerated over time until they reached the edge of the (150 mm diam) Petri dish. A polynomial model fitted the radial extension of colonies of Sclerotinia spp. Furthermore the accelerating colony extension rate was partly due to increasing colony radius. The rates of extension from mycelial disks transferred from the parental colony were positively correlated with the radius of the mycelial disks transferred. The rates of extension also were dependent on where the transferred disks were taken from parent colonies and the age and radius of the parent colony. On potato dextrose agar medium the extension rates of colonies of S. sclerotiorum and S. minor also were affected by broth concentration and temperature. With increasing nutrient concentration colony extension rates increased and were highest at 25 C. This study revealed a novel pattern of radial growth for Sclerotinia spp. that diverged from the established growth patterns of fungal colonies. Knowledge of the differences in growth behavior may be exploited in the laboratory studies on fungal competition and hyperparasitism and potentially in disease control strategies.

Dynamics of Verticillium Species Microsclerotia in Field Soils in Response to Fumigation, Cropping Patterns, and Flooding

Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum.

A Model for Multiseasonal Spread of Verticillium Wilt of Lettuce

Verticillium wilt, caused by Verticillium dahliae, is a destructive disease in lettuce, and the pathogen is seedborne. Even though maximum seed infestation rates of <5% have been detected in commercial lettuce seed lots, it is necessary to establish acceptable contamination thresholds to prevent introduction and establishment of the pathogen in lettuce production fields. However, introduction of inoculum into lettuce fields for experimental purposes to determine its long term effects is undesirable. Therefore, we constructed a simulation model to study the spread of Verticillium wilt following pathogen introduction from seed. The model consists of four components: the first for simulating infection of host plants, the second for simulating reproduction of microsclerotia on diseased plants, the third for simulating the survival of microsclerotia, and the fourth for simulating the dispersal of microsclerotia. The simulation results demonstrated that the inoculum density-disease incidence curve parameters and the dispersal gradients affect disease spread in the field. Although a steep dispersal gradient facilitated the establishment of the disease in a new field with a low inoculum density, a long-tail gradient allowed microsclerotia to be dispersed over greater distances, promoting the disease spread in fields with high inoculum density. The simulation results also revealed the importance of avoiding successive lettuce crops in the same field, reducing survival rate of microsclerotia between crops, and the need for breeding resistance against V. dahliae in lettuce cultivars to lower the number of microsclerotia formed on each diseased plant. The simulation results, however, suggested that, even with a low seed infestation rate, the pathogen would eventually become established if susceptible lettuce cultivars were grown consecutively in the same field for many years. A threshold for seed infestation can be established only when two of the three drivers of the disease-(i) low microsclerotia production per diseased plant, (ii) long-tail dispersal gradient, and (iii) low microsclerotia survival between lettuce crops-are present.