Spatiotemporal Patterns in the Airborne Dispersal of Spinach Downy Mildew.

Downy mildew is the most devastating disease threatening sustainable spinach production, particularly in the organic sector. The disease is caused by the biotrophic oomycete pathogen Peronospora effusa, and the disease results in yellow lesions that render the crop unmarketable. In this study, the levels of DNA from airborne spores of P. effusa were assessed near a field of susceptible plants in Salinas, CA during the winter months of 2013-14 and 2014/15 using rotating-arm impaction spore-trap samplers that were assessed with a species-specific quantitative polymerase chain reaction (qPCR) assay. Low levels of P. effusa DNA were detectable from December through February in both winters but increased during January in both years, in correlation with observed disease incidence; sharp peaks in P. effusa DNA detection were associated with the onset of disease incidence. The incidence of downy mildew in the susceptible field displayed logistic-like dynamics but with considerable interseason variation. Analysis of the area under the disease progress curves suggested that the 2013-14 epidemic was significantly more severe than the 2014-15 epidemic. Spatial analyses indicated that disease incidence was dependent within an average range of 5.6 m, approximately equivalent to the width of three planted beds in a typical production field. The spatial distribution of spores captured during an active epidemic most closely fit a power-law distribution but could also be fit with an exponential distribution. These studies revealed two important results in the epidemiology of spinach downy mildew in California. First, they demonstrated the potential of impaction spore-trap samplers linked with a qPCR assay for indicating periods of high disease risk, as well as the detection of long-distance dispersal of P. effusa spores. Second, at the scale of individual crops, a high degree of spatial aggregation in disease incidence was revealed.

Development and Deployment of Systems-Based Approaches for the Management of Soilborne Plant Pathogens.

Biological suppression of soilborne diseases with minimal use of outside interventive actions has been difficult to achieve in high input conventional crop production systems due to the inherent risk of pest resurgence. This review examines previous approaches to the management of soilborne disease as precursors to the evolution of a systems-based approach, in which plant disease suppression through natural biological feedback mechanisms in soil is incorporated into the design and operation of cropping systems. Two case studies are provided as examples in which a systems-based approach is being developed and deployed in the production of high value crops: lettuce/strawberry production in the coastal valleys of central California (United States) and sweet basil and other herb crop production in Israel. Considerations for developing and deploying system-based approaches are discussed and operational frameworks and metrics to guide their development are presented with the goal of offering a credible alternative to conventional approaches to soilborne disease management.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence.

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

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.

Clonal expansion of Verticillium dahliae in lettuce.

Few studies in population biology have documented how structure and diversity of pathogens evolve over time at local scales. With the historical samples of Verticillium dahliae available from lettuce, we investigated the structure and diversity of this pathogen in time and space. Three hundred twenty-nine V. dahliae isolates from lettuce fields collected over 18 years were characterized with polymorphic microsatellite markers and polymerase chain reaction tests for race and mating type. Genetic variation within and among commercial lettuce fields in a single season was also investigated using an additional 146 isolates. Sixty-two haplotypes (HTs) were observed among the 329 isolates. A single HT was frequently observed over multiple years and locations (61.40%). Genetic diversity, allelic richness, and private allelic richness suggested a relatively recent clonal expansion. Race 1 (93.63%) and MAT1-2-1 (99.69%) were overwhelmingly represented among the isolates. Linkage disequilibrium was significant (P < 0.001) for all populations, suggesting limited sexual recombination in the sampled populations from lettuce. Populations from 2006, 2009, and 2010 had higher numbers of unique HTs, implying a recent introduction of novel HTs. We conclude that V. dahliae population from lettuce evaluated in this study is expanding clonally, consistent with an asexually reproducing pathogen, and the movement of clonal genotypes locally occurs over time.

Comparative pathogenicity, biocontrol efficacy, and multilocus sequence typing of Verticillium nonalfalfae from the invasive Ailanthus altissima and other hosts.

Verticillium wilt, caused by Verticillium nonalfalfae, is currently killing tens of thousands of highly invasive Ailanthus altissima trees within the forests in Pennsylvania, Ohio, and Virginia and is being considered as a biological control agent of Ailanthus. However, little is known about the pathogenicity and virulence of V. nonalfalfae isolates from other hosts on Ailanthus, or the genetic diversity among V. nonalfalfae from confirmed Ailanthus wilt epicenters and from locations and hosts not associated with Ailanthus wilt. Here, we compared the pathogenicity and virulence of several V. nonalfalfae and V. alfalfae isolates, evaluated the efficacy of the virulent V. nonalfalfae isolate VnAa140 as a biocontrol agent of Ailanthus in Pennsylvania, and performed multilocus sequence typing of V. nonalfalfae and V. alfalfae. Inoculations of seven V. nonalfalfae and V. alfalfae isolates from six plant hosts on healthy Ailanthus seedlings revealed that V. nonalfalfae isolates from hosts other than Ailanthus were not pathogenic on Ailanthus. In the field, 100 canopy Ailanthus trees were inoculated across 12 stands with VnAa140 from 2006 to 2009. By 2011, natural spread of the fungus had resulted in the mortality of >14,000 additional canopy Ailanthus trees, 10,000 to 15,000 Ailanthus sprouts, and nearly complete eradication of Ailanthus from several smaller inoculated stands, with the exception of a few scattered vegetative sprouts that persisted in the understory for several years before succumbing. All V. nonalfalfae isolates associated with the lethal wilt of Ailanthus, along with 18 additional isolates from 10 hosts, shared the same multilocus sequence type (MLST), MLST 1, whereas three V. nonalfalfae isolates from kiwifruit shared a second sequence type, MLST 2. All V. alfalfae isolates included in the study shared the same MLST and included the first example of V. alfalfae infecting a non-lucerne host. Our results indicate that V. nonalfalfae is host adapted and highly efficacious against Ailanthus and, thus, is a strong candidate for use as a biocontrol agent.

Distribution of Lettuce Big-Vein Incidence Under Three Irrigation Systems.

Spatial patterns of lettuce big-vein (LBV) incidence under furrow, sprinkler, and subsurface drip irrigation systems were determined. Because LBV pathogen is a virus and is vectored by the soilborne chytrid Olpidium brassicae, different irrigation systems likely affect the movement of the vector and were hypothesized to result in different distribution patterns and levels of the disease. Lettuce plants were mapped by recording the location of each LBV-infected or healthy plant in arbitrarily selected plots of sizes 16 by 30, 20 by 30, and 18 by 50 m in Salinas, Gonzales, and Santa Maria in California. Data were arrayed into different quadrat sizes by rearrangement, and disease incidence was calculated for each quadrat. Frequency distribution analysis and spatial autocorrelation analyses were performed on this data. LBV incidence was aggregated in all furrow-irrigated fields, four of five subsurface drip-irrigated fields, and two of three sprinkler-irrigated fields. The remaining fields had a random distribution. As the quadrat size increased, index of aggregation decreased, and vice versa. In fields under sprinkler irrigation, regardless of whether the spatial pattern of LBV was random or aggregated, no directional orientation occurred. However, under furrow or subsurface drip irrigation, the aggregation mostly occurred across the rows. Although irrigation type influenced LBV distribution pattern and incidence in lettuce fields, the differential effects of irrigation type on vector O. brassicae could not be discerned in this study. The sprinkler irrigation practiced in lettuce production until thinning may influence the vector distribution and the subsequent irrigation methods adapted for the remainder of the season in individual fields may play a significant role in disease incidence.

Verticillium Wilt of Spineless Safflower Caused by Verticillium dahliae in California.

Spineless selections of Carthamus tinctorius (safflower) are grown as commercial field grown cutflower crops in coastal California. In 2010, field plantings of spineless safflower in Santa Clara County developed symptoms of a wilt disease. Affected plants were stunted and slow to develop. As plants developed flower buds, lower leaves turned yellow and wilted. As disease developed, lower leaves turned tan and desiccated; in extreme cases, the entire plant wilted and died. Examination of the taproot and stem vascular tissue revealed a tan to light brown, longitudinal, vascular discoloration. Disease distribution was patchy but in some plantings up to 50% of the plants were unharvestable due to loss of quality or plant death. Isolation from symptomatic vascular tissue consistently resulted in the recovery of a fungus with white aerial mycelium, verticillate conidiophores, single-celled, ovoid to ellipsoid, hyaline conidia, and solitary black microsclerotia that were rounded to elongated or irregular in shape. Three single conidial isolates were chosen for species and race identification after DNA extraction using the FastDNASPIN Kit (MP Biomedicals, Solon, OH). Verticillium species-specific PCR amplified a 500-bp amplicon that is specific to Verticillium dahliae from all three isolates. All three isolates also amplified the race 2-specific 270-bp band in PCR. No amplification was observed in race 1-specific PCR. Based on morphological and molecular data, the fungus was identified as V. dahliae (1,3). Pathogenicity of two isolates was tested individually by soil drench inoculations using 10 ml of conidial suspensions (7 × 106 conidia/ml) for each of 10 containerized plants grown in a peat moss mix in 7.6 cm diameter pots. Five safflower selections were inoculated and maintained in a greenhouse. After 6 weeks, as plants began to form flowers, inoculated plants showed lower leaf dieback and plant wilting. Vascular discoloration was observed when plants were dissected. V. dahliae was consistently recovered from symptomatic tissue. Control plants that only received water did not develop symptoms. The experiment was repeated and the results were consistent. To our knowledge, this is the first report of Verticillium wilt of ornamental, spineless safflower. This disease has been reported previously on agronomic safflower grown as an oilseed crop (2). This finding has significance for coastal crop rotation decisions as ornamental safflower is yet another host that could augment V. dahliae soil inoculum levels for crops such as strawberry and vegetables. References: (1) P. Inderbitzin et al. PLoS One 6: e28341, 2011. (2) J. M. Klisiewicz. Plant Dis. 65:237, 1981. (3) Maruthachalam et al. Phytopathology 100:1222, 2010.

Impact of consumer-driven changes to crop production practices on lettuce drop caused by Sclerotinia sclerotiorum and S. minor.

Increasing demands for value-added salad products have triggered revolutionary changes in the production practices of vegetable salad crops in recent years. One of the pivotal changes is the adaptation of 2-m-wide beds for increased vegetable biomass per unit area. The move away from the traditional 1-m-wide raised beds in cool-season vegetable production and the associated irrigation practices potentially can have a major influence on diseases affecting cool-season vegetables. To assess the potential impacts of this shift on lettuce drop caused by Sclerotinia minor and S. sclerotiorum, the two bed widths and different irrigation frequencies within each were compared in two separate field experiments over four lettuce crops in 2 years. Treatments included 1- and 2-m bed widths with twice-weekly, weekly and biweekly drip irrigation serving as subplot treatments that were begun immediately following thinning. Incidence of lettuce drop was evaluated weekly thereafter until maturity. For S. sclerotiorum, 36 half-liter soil samples were also collected once each season and assayed for the number of sclerotia, and apothecia were counted weekly in a 10-m(2) area for each plot. Regardless of the species, the effects of bed width and irrigation frequency were both significant. Twice-weekly irrigation and 2-m bed width resulted in higher lettuce drop incidence than other treatments. For S. sclerotiorum, twice-weekly irrigation and 2-m bed width also significantly increased the number of apothecia per unit area and the accumulation of soilborne sclerotia over multiple cropping seasons. Results demonstrated that the 2-m bed width combined with the practiced frequency of irrigations can result in higher lettuce drop caused by S. minor and increased incidence of airborne infection by S. sclerotiorum in the Salinas Valley where, historically, it has not been a serious threat. Increased incidence of S. sclerotiorum infection in commercial lettuce fields in the Salinas Valley between 2001 and 2006 validates these experimental results. These relatively new crop production practices can alter the balance of the two Sclerotinia spp. that has long existed in California.

Interactions between Coniothyrium minitans and Sclerotinia minor affect biocontrol efficacy of C. minitans.

Coniothyrium minitans, marketed as Contans, has become a standard management tool against Sclerotinia sclerotiorum in a variety of crops, including winter lettuce. However, it has been ineffective against lettuce drop caused by S. minor. The interactions between C. minitans and S minor were investigated to determine the most susceptible stage in culture to attack by C. minitans, and to determine its consistency on S minor isolates belonging to four major mycelial compatibility groups (MCGs). Four isolates of S. minor MCG 1 and 5 each from MCGs 2 and 3 and one from MCG 4 were treated in culture at purely mycelial, a few immature sclerotial, and fully mature sclerotial phases with a conidial suspension of C. minitans. Sclerotia from all treatments were harvested after 4 weeks, air dried, weighed, and plated on potato dextrose agar for recovery of C. minitans. S. minor formed the fewest sclerotia in plates that received C. minitans at the mycelial stage; C. minitans was recovered from nearly all sclerotia from this treatment and sclerotial mortality was total. However, the response of MCGs was inconsistent and variable. Field experiments to determine the efficacy of C. minitans relative to the registered fungicide, Endura, on lettuce drop incidence and soil inoculum dynamics were conducted from 2006 to 2009. All Contans treatments had significantly lower numbers of sclerotia than Endura and unsprayed control treatments, and drop incidence was as low as in Endura-treated plots (P > 0.05). Although the lower levels of lettuce drop in Contans treatments were correlated with significantly lower levels of sclerotia, the lower levels of lettuce drop, despite the presence of higher inoculum in the Endura treatment, was attributable to the prevention of infection by S. minor. A useful approach to sustained lettuce drop management is to employ Contans to lower the number of sclerotia in soil and to apply Endura to prevent S. minor infection within a cropping season.

Phenological and phytochemical changes correlate with differential interactions of Verticillium dahliae with broccoli and cauliflower.

Cauliflower (Brassica oleracea var. botrytis subvar. cauliflora) is susceptible to wilt caused by Verticillium dahliae but broccoli (B. oleracea var. italica subvar. cyamosa) is not. Infection of broccoli and cauliflower by a green fluorescent protein-expressing isolate of V. dahliae was examined using epifluorescence and confocal laser-scanning microscopy to follow infection and colonization in relation to plant phenology. Plant glucosinolate, phenolic, and lignin contents were also assayed at 0, 4, 14, and 28 days postinoculation. V. dahliae consistently infected and colonized the vascular tissues of all cauliflower plants regardless of age at inoculation, with the pathogen ultimately appearing in the developing seed; however, colonization decreased with plant age. In broccoli, V. dahliae infected and colonized root and stem xylem tissues of plants inoculated at 1, 2, or 3 weeks postemergence. However, V. dahliae colonized only the root xylem and the epidermal and cortical tissues of broccoli plants inoculated at 4, 5, and 6 weeks postemergence. The frequency of reisolation of V. dahliae from the stems (4 to 22%) and roots (10 to 40%) of mature broccoli plants was lower than for cauliflower stems (25 to 64%) and roots (31 to 71%). The mean level of aliphatic glucosinolates in broccoli roots was 6.18 times higher than in the shoots and did not vary with age, whereas it was 3.65 times higher in cauliflower shoots than in the roots and there was a proportional increase with age. Indole glucosinolate content was identical in both cauliflower and broccoli, and both indole and aromatic glucosinolates did not vary with plant age in either crop. Qualitative differences in characterized glucosinolates were observed between broccoli and cauliflower but no differences were observed between inoculated and noninoculated plants for either broccoli or cauliflower. However, the phenolic and lignin contents were significantly higher in broccoli following inoculation than in noninoculated broccoli or inoculated cauliflower plants. The increased resistance of broccoli to V. dahliae infection was related to the increase in phenolic and lignin contents. Significant differential accumulation of glucosinolates associated with plant phenology may also contribute to the resistant and susceptible reactions of broccoli and cauliflower, respectively, against V. dahliae.

Molecular variation among isolates of Verticillium dahliae and polymerase chain reaction-based differentiation of races.

Verticillium dahliae is a soilborne fungal pathogen that causes vascular wilt in a variety of economically important crops worldwide. There are two races of V. dahliae that infect tomato and lettuce. Although race-1-specific resistance has been identified in both tomato and lettuce, no resistant sources are available for race 2. Molecular analyses were employed to characterize the genetic variability and race structure of 101 isolates of V. dahliae from a variety of hosts, mainly from central and coastal California, and 10 isolates exotic to this area. Analyses of the 16 simple sequence repeat (SSR) markers illustrated that tomato subpopulations from central California were distinct relative to the marigold subpopulations. In contrast, cotton and olive isolates showed admixture with tomato isolates. Analyses of both the ribosomal DNA intergenic spacer regions and SSR markers revealed high genetic variability among isolates but were unable to delineate races of V. dahliae. However, a polymerase chain reaction (PCR) assay was applied to amplify a race-1-specific amplicon from the isolates in many hosts from different geographic areas, and was coupled with virulence assays for validation of the data. Results of the PCR assay showed 100% concordance with the virulence assay to differentiate race 1 from race 2 of 48 isolates from tomato. The results indicate that the PCR assay can be applied to differentiate the two races to support our related aim of breeding host resistance, and further reveal insights into the distribution of races in tomato and lettuce cropping systems in California.

Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae.

Interactions between lettuce and a green fluorescent protein (GFP)-expressing, race 1 isolate of Verticillium dahliae, were studied to determine infection and colonization of lettuce cultivars resistant and susceptible to Verticillium wilt. The roots of lettuce seedlings were inoculated with a conidial suspension of the GFP-expressing isolate. Colonization was studied with the aid of laser scanning confocal and epi-fluorescence microscopes. Few differences in the initial infection and colonization of lateral roots were observed between resistant and susceptible cultivars. Hyphal colonies formed on root tips and within the root elongation zones by 5 days, leading to the colonization of cortical tissues and penetration of vascular elements regardless of the lettuce cultivar by 2 weeks. By 8 to 10 weeks after inoculation, vascular discoloration developed within the taproot and crown regions of susceptible cultivars well in advance of V. dahliae colonization. Actual foliar wilt coincided with the colonization of the taproot and crown areas and the eruption of mycelia into surrounding cortical tissues. Advance colonization of stems, pedicels, and inflorescence, including developing capitula and mature achenes was observed. Seedborne infection was limited to the maternal tissues of the achene, including the pappus, pericarp, integument, and endosperm; but the embryo was never compromised. Resistant lettuce cultivars remained free of disease symptoms. Furthermore, V. dahliae colonization never progressed beyond infected lateral roots of resistant cultivars. Results indicated that resistance in lettuce may lie with the plant's ability to shed infected lateral roots or to inhibit the systemic progress of the fungus through vascular tissues into the taproot.

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.

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.

Biocontrol of Lettuce Drop Caused by Sclerotinia sclerotiorum and S. minor in Desert Agroecosystems.

Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to determine the efficacy of several biocontrol agents for the management of lettuce drop caused by Sclerotinia spp. Commercial formulations of Trichoderma harzianum (Plantshield, Supersivit), Gliocladium virens (Soilgard), Coniothyrium minitans (Contans), and Bacillus subtilis (Companion) were evaluated and compared with the chemical fungicide iprodione (Rovral) against Sclerotinia sclerotiorum and S. minor. A single application of biocontrol products or of Rovral did not reduce lettuce drop caused by either Sclerotinia species. However, two applications of Contans, one at planting and one at post-thinning, significantly reduced the incidence of lettuce drop caused by S. sclerotiorum and increased yield but had no effect on S. minor at both locations in both years. Two applications of other biocontrol products did not significantly reduce disease incidence despite medium to high recovery following application. In contrast, Contans was only sporadically recovered following application. In vitro fungicide sensitivity evaluation revealed that both Trichoderma and Gliocladium species were tolerant to iprodione, dicloran (Botran), and vinclozolin (Ronilan) up to 1,000 ppm a.i., whereas both Sclerotinia spp. and C. minitans were sensitive to all three fungicides above 1 ppm. In summary, Contans was the most effective treatment for the control of lettuce drop caused by S. sclerotiorum, but no treatment was effective against S. minor in the desert lettuce production systems.

Incubation of excised apothecia enhances ascus maturation of Sclerotinia sclerotiorum.

Synchronized maturation of ascospores of Sclerotinia sclerotiorum is desirable for establishing a transformation system, conducting genetic analyses of the pathogen, defining the precise epidemiological roles of ascospores and screening plant germplasm for resistance. In general, fresh apothecia collected from germinated sclerotia contained primarily immature or discharged asci. This study was undertaken to investigate whether maturation of asci and ascospores could be enhanced by incubation of excised apothecia and to determine the effects of factors such as temperature, excision time, light and ventilation on maturation of asci and ascospores in excised apothecia. Maturation of asci was compared between intact and excised apothecia that were incubated under similar conditions. Results demonstrated that temperature was an important factor affecting ascus maturation of S. sclerotiorum during incubation of excised apothecia, and the optimum temperature was around 21 C. After incubation at 21 C for 30 h, the percentage of undischarged mature asci in excised apothecia increased up to 70-80%. This increase was accompanied by a significant increase in ascospore production of up to 5 x 10(5) ascospores per apothecium. Detailed time course studies indicated that mature asci peaked at 30-36 h of postexcision incubation. Mature asci and the number of ascospores were higher in open incubation than in closed incubation, suggesting that accumulation of volatile substances was not required for ascus/ascospore maturation during postexcision incubation and ventilation could enhance the maturation process. Light also did not affect the maturation of asci during the incubation of excised apothecia. Germination rates for ascospores from excised apothecia under various treatments were similar to those from untreated apothecia but declined slightly with time postexcision. The incubation of excised apothecia promoted ascus maturation compared with intact apothecia.

Management of Soilborne Diseases in Strawberry Using Vegetable Rotations.

The influence of crop rotation on soilborne diseases and yield of strawberry (Fragaria × ananassa) was determined at a site infested with Verticillium dahliae microsclerotia and at another with no known history of V. dahliae infestation during 1997 to 2000. The rotations studied at the V. dahliae-infested site were (i) broccoli-broccoli-strawberry, (ii) Brussels sprouts-strawberry, and (iii) lettuce-lettuce-strawberry; the treatments at the site with no history of V. dahliae were (i) broccoli-broccoli-strawberry, (ii) cauliflower-cauliflower-strawberry, and (iii) lettuce-lettuce-strawberry. The effects of rotation on V. dahliae and Pythium populations, strawberry vigor, Verticillium wilt severity, and strawberry fruit yield were compared with a standard methyl bromide + chloropicrin fumigated control treatment at both sites. Rotations did not alter total population levels of Pythium spp. at either study site. However, V. dahliae microsclerotia were significantly reduced with br occoli and Brussels sprouts rotations compared with lettuce rotations at the V. dahliae-infested site. Reduced propagules led to lower Verticillium wilt severity on strawberry plants in the broccoli and Brussels sprouts rotations than in lettuce-rotated plots. Strawberry vigor and fruit yield were significantly lower in lettuce-rotated plots than in broccoli- and Brussels sprouts-rotated plots. Despite no detectable microsclerotia at the other site, strawberry vigor and fruit yield were greatest in plots rotated with broccoli, intermediate with cauliflower, and lowest with lettuce. None of the rotation treatments were better than the fumigated control for all variables measured. In the absence of fumigation, rotation with broccoli and Brussels sprouts is an effective cultural practice for managing Verticillium wilt in strawberry production; whereas, in fields with no detectable V. dahliae, broccoli is also a feasible rotational crop that enhances strawberry growth and yield. According to a cost-benefit analysis, the broccoli-strawberry rotation system could be an economically viable option provided growers are able to alternate years for strawberry cultivation.