Antibacterial, antibiofilm, and antivirulence potential of the main diterpenes from Copaifera spp. oleoresins against multidrug-resistant bacteria.

To evaluate the antibacterial, antibiofilm and antivirulence potential of the main diterpenes from Copaifera spp. oleoresins against multidrug-resistant (MDR) bacteria. Antimicrobial assays included determination of the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Inhibitory Concentration of Biofilm (MICB50), as well as synergistic and antivirulence assays for eight diterpenes against MDR. The tests revealed that two diterpenes (named 1 and 5) showed the best results, with MIC and MBC between 12.5 and 50 µg/mL against most MDR bacteria. These diterpenes exhibited promising MICB50 in concentration between 3.12-25 µg/mL but showed no synergistic antimicrobial activity. In the assessment of antivirulence activity, diterpenes 1 and 5 inhibited only one of the virulence factors evaluated (Dnase) produced by some strains of S. aureus at subinhibitory concentration (6.25 µg/mL). Results obtained indicated that diterpenes isolated from Copaifera oleoresin plays an important part in the search of new antibacterial and antibiofilm agents that can act against MDR bacteria.

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

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

Cropping System Diversification Reduces Severity and Incidence of Soybean Sudden Death Syndrome Caused by Fusarium virguliforme.

Current management of sudden death syndrome (SDS) of soybean, caused by Fusarium virguliforme, focuses on planting resistant varieties and improving soil drainage; however, these measures are not completely effective. A 6-year study evaluated the effects of cropping system diversification on SDS and soybean yield. SDS, root health, yield, and F. virguliforme density in soil were assessed in a naturally infested field trial comparing a 2-year cropping system consisting of a corn-soybean rotation and synthetic fertilizer applications with 3- and 4-year cropping systems consisting of corn-soybean-oat + red clover and corn-soybean-oat +alfalfa-alfalfa rotations, respectively, with both manure and low synthetic fertilizer rates. In 5 of 6 years, SDS incidence and severity were lower and yield higher in the 3- and 4-year systems than in the 2-year system. SDS severity and incidence were up to 17-fold lower in the diversified systems than in the 2-year system. Incidence and severity of SDS explained 45 to 87% of the variation in yield. Plants in the 2-year system generally showed more severe root rot and lower plant weights than plants in the diversified systems. F. virguliforme density in soil was up to fivefold greater in the 2-year system compared with the 4-year system. The processes responsible for the suppression of SDS and yield protection in the diversified cropping systems still need to be determined.

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

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

Soybean Sudden Death Syndrome Caused by Fusarium virguliforme is Impaired by Prolonged Flooding and Anaerobic Conditions.

High soil moisture usually favors soybean sudden death syndrome (SDS), caused by Fusarium virguliforme (Fv), but the effects of the duration of the flooding period and accompanying anaerobic conditions on the soybean-Fv interaction are not clear. Greenhouse studies were conducted using susceptible and resistant cultivars exposed to the following treatments: 3, 5, or 7 days of continuous flooding, repeated short-term flooding of 8 h/week for 3 weeks, and a no-flood check treatment. At 7, 14, and 21 days after flooding (DAF), seedlings in the no-flood, 3-day, and repeated short-term treatments showed the highest root rot and foliar symptom severity, whereas seedlings in the 7-day treatment showed the lowest severity. Fv inoculum density in soil was lowest in the 7-day flooding treatment. In a hydroponic system, the steady transcript levels of soybean defense genes and Fv candidate virulence genes were measured in response to different oxygen levels using qPCR. Fv-infected roots exposed to 12 h of anaerobic conditions showed down-regulation of the defense-related soybean genes Laccase, PR3, PR10, PAL, and CHS, and the Fv virulence genes pectate lyase (PL), and Fv homolog of the pisatin demethylase (PDA). Our study suggests that short-term flooding tends to increase SDS, while prolonged flooding negatively impacts SDS due to reduction of Fv density in soil. Moreover, anaerobic conditions down-regulate both soybean defense genes and Fv candidate virulence genes.

Usefulness of 10 genomic regions in soybean associated with sudden death syndrome resistance.

Sudden death syndrome (SDS) is an important soybean [Glycine max (L) Merrill] disease caused by the soilborne fungus Fusarium virguliforme. Currently, 14 quantitative trait loci (QTL) had been confirmed associated with resistance or tolerance to SDS. The objective of the study was to evaluate usefulness of 10 of these QTL in controlling disease expression. Six populations were developed providing a total of 321 F2-derived lines for the study. Recombinant inbred lines (RIL) used as parents were obtained from populations of 'Essex' × 'Forrest' (EF), 'Flyer' × 'Hartwig' (FH), and 'Pyramid' × 'Douglas' (PD). Disease resistance was evaluated in the greenhouse at three different planting times, each with four replications, using sorghum infested with F. virguliforme homogeneously mixed in the soil (Luckew et al., Crop Sci 52:2215-2223, 2012). Four disease assessment criteria-foliar disease incidence (DI), foliar leaf scorch disease severity (DS), area under the disease progress curve (AUDPC), and root rot severity-were used. QTL were identified in more than one of the disease assessment criteria, mainly associated with lines in the most resistant categories. Five QTL (qRfs4, qRfs5, qRfs7, qRfs12, and Rfs16) were associated with at least one of the disease assessments across multiple populations. Of the five, qRfs4 was associated with DI, AUDPC, and root rot severity, and Rfs16 with AUDPC and root rot severity. The findings suggest it may be possible for plant breeders to focus on stacking a subset of the previously identified QTL to improve resistance to SDS in soybean.

A ten-year experience and follow-up of three hundred patients fitted with the Biomet/Lorenz Microfixation TMJ replacement system.

The purpose of this paper is to present the postoperative results obtained after full temporomandibular joint (TMJ) reconstruction employing the Biomet/Lorenz Microfixation TMJ replacement system (Jacksonville, FL, USA) in 300 patients (201 unilateral, 99 bilateral). Objective data (maximum inter-incisal opening; MIO) and subjective data (function and speech, diet, and pain) were collected preoperatively and at postoperative evaluations performed over a 10-year period (mean 3.5, standard deviation 2.1 years). The MIO measures were obtained using a calliper rule. Subjective data were evaluated using a visual analogue scale with scores ranging from 0 to 5 for each variable. The results were analyzed with the paired t-test (two-sided, α=5%). Each patient showed significant improvements for all of the variables at evaluation on postoperative day 7. The results for MIO, function and speech, and diet, showed improvements at each postoperative evaluation over a maximum of 3 years, with stabilization of the results from the fourth year. Complaints of pain decreased considerably up to the 1-month postoperative evaluation, and no patient reported severe pain at 6 months after surgery. The results presented show that the reconstruction of the TMJ through the installation of the Biomet/Lorenz system prosthesis is a safe and effective option for proper reestablishment of the joint and stomatognathic system function; significant long-term improvements in mandibular range of motion are promoted and pain levels decrease.

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

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

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

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

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

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

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

Fusarium spp. are widespread soilborne pathogens that cause important soybean diseases such as damping-off, root rot, Fusarium wilt, and sudden death syndrome. At least 12 species of Fusarium, including F. proliferatum, have been associated with soybean roots, but their relative aggressiveness as root rot pathogens is not known and pathogenicity has not been established for all reported species (2). In collaboration with 12 Iowa State University extension specialists, soybean roots were arbitrarily sampled from three fields in each of 98 Iowa counties from 2007 to 2009. Ten plants were collected from each field at V2-V3 and R3-R4 growth stages (2). Typical symptoms of Fusarium root rot (2) were observed. Symptomatic and asymptomatic root pieces were superficially sterilized in 0.5% NaOCl for 2 min, rinsed three times in sterile distilled water, and placed onto a Fusarium selective medium. Fusarium colonies were transferred to carnation leaf agar (CLA) and potato dextrose agar and later identified to species based on cultural and morphological characteristics. Of 1,230 Fusarium isolates identified, 50 were recognized as F. proliferatum based on morphological characteristics (3). F. proliferatum isolates produced abundant, aerial, white mycelium and a violet-to-dark purple pigmentation characteristic of Fusarium section Liseola. On CLA, microconidia were abundant, single celled, oval, and in chains on monophialides and polyphialides (3). Species identity was confirmed for two isolates by sequencing of the elongation factor (EF1-α) gene using the ef1 and ef2 primers (1). Identities of the resulting sequences (~680 bp) were confirmed by BLAST analysis and the FUSARIUM-ID database. Analysis resulted in a 99% match for five accessions of F. proliferatum (e.g., FD01389 and FD01858). To complete Koch's postulates, four F. proliferatum isolates were tested for pathogenicity on soybean in a greenhouse. Soybean seeds of cv. AG2306 were planted in cones (150 ml) in autoclaved soil infested with each isolate; Fusarium inoculum was applied by mixing an infested cornmeal/sand mix with soil prior to planting (4). Noninoculated control plants were grown in autoclaved soil amended with a sterile cornmeal/sand mix. Soil temperature was maintained at 18 ± 1°C by placing cones in water baths. The experiment was a completely randomized design with five replicates (single plant in a cone) per isolate and was repeated three times. Root rot severity (visually scored on a percentage scale), shoot dry weight, and root dry weight were assessed at the V3 soybean growth stage. All F. proliferatum isolates tested were pathogenic. Plants inoculated with these isolates were significantly different from the control plants in root rot severity (P = 0.001) and shoot (P = 0.023) and root (P = 0.013) dry weight. Infected plants showed dark brown lesions in the root system as well as decay of the entire taproot. F. proliferatum was reisolated from symptomatic root tissue of infected plants but not from similar tissues of control plants. To our knowledge, this is the first report of F. proliferatum causing root rot on soybean in the United States. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathologic Society, St. Paul, MN, 1999. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (4) G. P. Munkvold and J. K. O'Mara. Plant Dis. 86:143, 2002.

A New TaqMan Real-Time Polymerase Chain Reaction Assay for Quantification of Fusarium virguliforme in Soil.

The quantification of the soilborne pathogen Fusarium virguliforme inoculum in soil is important for epidemiological studies of soybean sudden death syndrome (SDS). Classical dilution plating methods to determine inoculum density in soil have yielded inconsistent results due to slow growth, variable colony morphology of the pathogen, and the presence of other fungi with similar phenotype. A TaqMan real-time polymerase chain reaction assay was developed based on sequences of the FvTox1 gene of F. virguliforme. The gene differed by four single-nucleotide proteins from the other SDS-causing species. Assay specificity was tested on 48 fungal isolates that varied in taxonomic relatedness. Assay sensitivity was appraised on 10-fold serial dilutions of genomic DNA, conidia suspensions, and soil spiked with conidia. Applicability of the assay was evaluated on field and greenhouse soil samples, and on roots of symptomatic plants. The assay detected only DNA sequences specific to F. virguliforme. The detection limit of the assay was 5 pg/µl, 1,000 conidia/ml, and 1,000 conidia/g soil for genomic DNA, conidial suspensions, and soil with conidia, respectively. The assay was specific to F. virguliforme and was used successfully to quantify inoculum density in soil and soybean roots. The assay can be used as a diagnostic tool for rapid screens of field and greenhouse soil, and for symptomatic and asymptomatic plants.

Effect of Soil Temperature and Plant Age at Time of Inoculation on Progress of Root Rot and Foliar Symptoms of Soybean Sudden Death Syndrome.

Sudden death syndrome (SDS) of soybean is favored by planting in cool soil but epidemics can be severe even when planting occurs later in the season into warmer soil. Our objective was to determine how soil temperature affects susceptibility of plants exposed to Fusarium virguliforme at different ages. Soybean plants were grown in rhizotrons in water baths at 17, 23, and 29°C. Subsets of plants were inoculated 0, 3, 7, and 13 days after planting (DAP) by drenching soil with a conidial suspension. Root rot developed in all inoculated plants but severity decreased with increasing temperature and plant age at inoculation. Severity of foliar symptoms also decreased with increasing plant age. Whereas plants inoculated 0 DAP developed severe foliar symptoms at all temperatures, plants inoculated 3 and 7 DAP developed symptoms only at 17 and 23°C, and those inoculated 13 DAP never developed foliar symptoms at any temperature. Root length at inoculation was negatively correlated with disease severity. Our findings suggest that roots are most susceptible to infection during the first days after seed germination and that accelerated root growth in warmer temperatures reduces susceptibility to root infection conducive to foliar symptoms. However, soil temperature may not affect infections that occur as soon as seeds germinate.

Plant Age Affects Root Infection and Development of Foliar Symptoms of Soybean Sudden Death Syndrome.

Soybean sudden death syndrome is characterized by root rot followed by the development of foliar symptoms. However, it is not known how time of infection affects disease severity. Soybean plants were inoculated at 0, 4, 7, 14, 21, 28, and 35 days after planting (DAP) by drenching potting media with conidia of Fusarium virguliforme, then incubating in growth chambers at 17°C for 7 days followed by 24°C for 31 days. Root rot and severity of foliar symptoms were assessed 18 and 38 days after inoculation (DAI). Root rot developed on plants inoculated at all ages but plants inoculated at seed stage (0 DAP) had the highest (P < 0.01) root rot severity (>90%). At 38 DAI, foliar symptoms were severe (>80%) on plants inoculated at 0 DAP but did not develop on plants inoculated at all other ages. Xylem colonization by F. virguliforme was more frequent in plants inoculated at 0 DAP than on plants inoculated at later stages. The results of this study suggest that soybean roots become less susceptible to xylem colonization and the subsequent development of foliar symptoms as plants mature. Therefore, practices aimed at protecting seed and seedling roots from infection may improve soybean sudden death management.

Strawberry Plant Extracts Stimulate Secondary Conidiation by Colletotrichum acutatum on Symptomless Leaves.

ABSTRACT Conidial suspensions of Colletotrichum acutatum were prepared in 1:27, 1:45, and 1:81 (wt/vol) dilutions of an extract of strawberry (cv. Tristar) flowers or leaves in water. Strawberry leaves and plastic coverslips were sprayed with the conidial suspensions, incubated at 25 degrees C and continuous wetness for 48 h, and the number of conidia and appressoria were counted. In another experiment, leaves and coverslips were sprayed with a conidial suspension in water, incubated for 72 h to establish C. acutatum populations, and placed in a growth chamber under dry conditions for up to 6 weeks. At each sampling time, leaves and coverslips were sprayed with flower extracts, leaf extracts, or water, incubated for 48 h at 25 degrees C and continuous wetness, and the number of conidia and appressoria were counted. Flower extracts significantly (P

Influence of Temperature and Wetness Duration on Conidia and Appressoria of Colletotrichum acutatum on Symptomless Strawberry Leaves.

ABSTRACT Strawberry leaves (cv. Tristar) inoculated with Colletotrichum acuta-tum conidia were incubated at 10, 15, 20, 25, 30, and 35 degrees C under continuous wetness, and at 25 degrees C under six intermittent wetness regimes. The number of conidia and appressoria was quantified on excised leaf disks. In order to assess pathogen survival, inoculated leaves were frozen and incubated to induce acervular development. Germination, secondary3 conidiation, and appressorial development were significantly (P /= 0.95) related to appressorial populations prior to this treatment and was greatest following periods of continuous wetness. Production of secondary conidia and appressoria of C. acutatum on symptomless strawberry leaves under a range of environmental conditions suggests that these processes also occur under field conditions and contribute to inoculum availability during the growing season.

Germination and Sporulation of Colletotrichum acutatum on Symptomless Strawberry Leaves.

ABSTRACT The germination and sporulation of Colletotrichum acutatum were characterized over time on strawberry leaves (cv. Tristar) and plastic coverslips incubated at 26 degrees C under continuous wetness. Conidia germinated within 3 h after inoculation and formed melanized appressoria with pores by 9 h after inoculation. Host penetration was not observed up to 7 days after inoculation. Production of secondary conidia on conidial and hyphal phialides began within 6 h after inoculation. Secondary conidiation was responsible for up to a threefold increase in the total number of conidia within 7 days after inoculation. Primary conidia and hyphae began to collapse 48 h after inoculation, whereas melanized appressoria remained intact. These findings suggest that appressoria and secondary conidia of C. acutatum produced on symptomless strawberry foliage may be significant sources of inoculum for fruit infections.