Modeling disease progression of Camellia twig blight using a recurrent event model.

To improve control of camellia twig blight (CTB) using sanitation methods, a more complete epidemiologic understanding of this disease is necessary. Three CTB disease stages were modeled using recurrent event analysis. Wound inoculated stems were observed at regular intervals for appearance of disease symptoms. Survival times (time from inoculation until symptom appearance) for the three disease stages (mild, moderate, and severe) were regressed against stem diameter, monthly mean hours/day within a specified temperature range (15 to 30 degrees C), and season (spring, summer, fall, and winter). For all three CTB disease stages, stem diameter had a protective effect on survival times, while monthly mean hours/day in the specified temperature range and warmer seasons were risk factors. Based upon median ratios, the mild disease stage developed 2 to 3 times faster in spring, summer, and fall than in winter. Similarly, moderate and severe disease stages developed 2 to 2.5 times faster. For all three disease stages, seasonal differences in stage development were smaller among fall, spring, and summer, varying from 1 to 1.6 times faster. Recurrent event modeling of CTB progression provides knowledge concerning developmental expression of this disease, information necessary for creating a comprehensive, integrated disease management program.

Survival Analysis to Determine the Length of the Incubation Period of Camellia Twig Blight Caused by Colletotrichum gloeosporioides.

Camellia twig blight, caused by Colletotrichum gloeosporioides, is a disease common to several Camellia species in the southern United States. To determine the potential seasonal differences in incubation periods, stems of Camellia sasanqua 'Rosea' plants grown in pine bark under ambient conditions were wounded and inoculated monthly with C. gloeosporioides mycelium. The time until appearance of the first symptom of disease (incubation period length) was recorded for all stems. Stems that did not display a disease symptom by the last day of the observation period were recorded as censored observations. Survival analysis using Kaplan-Meier estimates, Cox proportional hazards, and extended Cox models was used to analyze the data. Incubation period length was regressed against stem diameter, monthly mean hours per day in a specified temperature range (15 to 30°C), and a categorical season variable approximating the four host growth stages (winter dormancy, spring leaf and stem growth, summer stem hardening and bud set, fall cessation of leaf and stem growth and opening of flowers) at the time stems were inoculated. Stems of thicker diameter tended to have greater incubation period length, while higher monthly mean hours per day in the specified temperature range decreased incubation period length. In comparison to winter months, spring, summer, and fall months were all associated with significantly higher risks for disease symptom appearance. The median incubation period lengths for the spring, summer, fall, and winter months were 18, 23, 28, and 57 days, respectively.

Microscopic and Macroscopic Studies of the Development of Puccinia hemerocallidis in Resistant and Susceptible Daylily Cultivars.

Infection and colonization of eight daylily cultivars, which varied in resistance to daylily rust, by Puccinia hemerocallidis was studied macroscopically and microscopically. After germination of urediniospores, appressoria formed at the tip of germ tubes and the fungus penetrated the host through stomatal openings 2 days after inoculation (DAI). Under the infection sites, intercellular hyphae aggregated and formed uredia, which released urediniospores 8 DAI. Resistant cultivars, characterized by the development of rapid death of host cells, were separated into three qualitative categories based on absence and presence of necrotic lesions without or with sporulation. In highly resistant cvs. Prairie Blue Eyes and Bertie Ferris, no macroscopic disease symptoms were observed on leaf surfaces although a few collapsed cells were detected microscopically. Both resistant and moderately resistant reactions were characterized by necrotic lesions with many collapsed cells under infection sites. The difference between these two reactions was that uredia and urediniospores were observed in the moderately resistant cv. Chicago Apache, but not in resistant cvs. Buttered Popcorn and Stella De Oro. Susceptible cultivars, characterized by the absence of a hypersensitive response, were separated into two qualitative categories based on restriction of intercellular hyphal growth that delayed development of uredia and formation of urediniospores. Compared to the susceptible cv. Pardon Me, moderately susceptible cvs. Mary Todd and Chorus Line had a delayed latent period and reduced amount of sporulation. The results indicate that hypersensitive cell death is one of the resistance responses to daylily rust. Necrotic lesions on leaf surfaces are associated with the number of collapsed host cells. Delayed latent period and reduced sporulation that resulted from restriction of intercellular hyphal growth could represent another type of resistance response in the daylily-rust pathosystem.

Genetic Characterization of Binucleate Rhizoctonia Species Causing Web Blight on Azalea in Mississippi and Alabama.

Web blight on containerized azalea is an annual problem for commercial nurseries during summer months in the southern United States. Losses to web blight are associated with the cost of fungicide applications, delayed marketing of diseased plants, and plant death. Two hundred and eleven isolates of binucleate Rhizoctonia were recovered from azalea leaves with web blight symptoms from two nurseries in Mississippi and Alabama over 3 years. The internal transcribed spacer region (ITS) of the ribosomal DNA (rDNA) was sequenced from all isolates to determine genetic identity. A single anastomosis group (AG) of binucleate Rhizoctonia represented 92% of the samples collected from infected leaves. Genetic data and hyphal fusion experiments confirmed that these isolates belong to AG-U, which was recently identified from root and stem infections on miniature rose in Japan. Isolates of binucleate Rhizoctonia belonging to anastomosis groups AG-R, CAG-7 (=AG-S), and AG-G were also identified in the sample in low frequency. This is the first report of the occurrence of binucleate Rhizoctonia AG-U in the United States.

Spore Germination, Infection Structure Formation, and Colony Development of Erysiphe pulchra on Dogwood Leaves and Glass Slides.

Spore germination, infection structure formation, and colony development of Erysiphe pulchra on glass slides and leaf disks of a susceptible flowering dogwood line were examined using light and scanning electron microscopes. On both glass slides and leaf disks, germination of conidia started within 2 h after inoculation (hai). One to four germ tubes grew from two poles of a conidium, one or two of the germ tubes formed initial appressoria, and only one of the germ tubes with initial appressoria formed secondary appressoria. However, formation of initial and secondary appressoria was delayed on glass slides (48 and 72 hai, respectively) compared with that on dogwood leaf disks (3 and 24 hai, respectively). Branching hyphae did not grow from germinated conidia on glass slides. However, on dogwood leaf disks, branched hyphae were observed 48 hai. In epidermal cells, the fungus formed compact and globose haustoria. Conidia formation on conidiophores started on leaf disks 7 days after inoculation.

Effect of Temperature on Sporulation of Botryosphaeria dothidea, B. obtusa, and B. rhodina.

Three Botryosphaeria spp. were grown on autoclaved apple and peach stems in cotton-plugged tubes with constant moisture at 6, 12, 18, 24, and 30°C to determine the effect of temperature on sporulation. Number of conidia per pycnidium was determined weekly from 4 to 10 weeks after inoculation. The experiment was repeated three times. Maximum sporulation occurred at 24°C with B. dothidea and at 18 and 24°C with B. obtusa. Spore production of both fungi showed a quadratic curvilinear response to temperature. Pycnidia were erumpent, typical of their habit in nature. Maximum sporulation of B. rhodina occurred at 12, 24, and 30°C instead of at a distinctive peak. Of the three fungi, B. rhodina produced the greatest number of conidia per pycnidium at all temperatures. Mycelia and pycnidia of B. rhodina grew on top of the bark, which is atypical of their habit in nature. For spore production by B. dothidea, there was a significant interaction between temperature and time. Maximum sporulation over the 10-week period occurred in week 4 and/or 6 for B. dothidea at 12, 18, and 24°C, with a linear response at 12 and 24°C (P ≤0.05). Conidial maturation of B. obtusa and B. rhodina had a quadratic curvilinear response due to temperature, with a maximum maturation at 12, 18, and 24°C with B. obtusa and at 24°C with B. rhodina. Spore maturation would affect longevity of conidial viability. Maximum spore production over time and percent pigmented spores over time by B. obtusa, and spore maturation over time by B. rhodina occurred in weeks 8, 9, and 10 with a significant linear response (P ≤ 0.05). All three Botryosphaeria spp. produced conidia over the 6 to 30°C range and over the 7-week period (weeks 4 to 10), with maximum sporulation or spore maturation at 18 to 24°C.

Activity of Chlorine Dioxide in a Solution of Ions and pH Against Thielaviopsis basicola and Fusarium oxysporum.

Chlorine dioxide (ClO2) is a disinfestant used to control pathogens in water. To determine if interactions between inorganic ions and pH levels effect ClO2 activity in vitro, concentrations of ClO2 (0, 1, 3, 5, 7, 9, 22, 24, 46, 58, and 70 mg/liter) were mixed for 10 min in solutions containing a nitrogen and hard water solution with equal concentrations of ammonium, nitrate, and synthetic hard water (0 and 100 mg/liter) and a divalent metal ion solution with equal concentrations of copper, iron, manganese, and zinc (0, 1, 3, and 5 mg/liter) at pH 5 and 8. Macro- and microconidia of Fusarium oxysporum f. sp. narcissi or conidia and aleuriospores of Thielaviopsis basicola were injected into each suspension for 30 s, captured on filter paper disks that were flushed with water, and plated on 50% potato dextrose agar. Spore germination was quantified after 1 day. ClO2 activity had a similar effect on both fungal species and all types of propagules with interactions among the divalent metal ion solution, nitrogen and hard water solution, and pH treatments. A higher concentration of ClO2 was required at pH 8 than at pH 5 to achieve a lethal dose resulting in 50% mortality of spores (LD50). The addition of the divalent metal ion solution required an increase in ClO2 concentration to maintain a LD50. When combined with the nitrogen and hard water solution, the divalent metal ion solution placed a higher demand on ClO2 at pH 5 and a lower demand on ClO2 at pH 8, thus requiring an increase and decrease in a ClO2 concentration, respectively, to achieve a LD50. Chlorine dioxide doses resulting in 50% mortality ranged from 0.5 to 7.0 mg/liter for conidia of F. oxysporum, 0.5 to 11.9 mg/liter for conidia of T. basicola, and 15.0 to 45.5 mg/liter for aleuriospores of T. basicola.

Dose Curves of Disinfestants Applied to Plant Production Surfaces to Control Botrytis cinerea.

Lethal dose curves were calculated using probit analysis for six disinfestants (chlorazene hydrosol, hydrogen dioxide, hydrogen peroxide, iodine, quaternary ammonium chloride, sodium hypochlorite) when applied on seven substrates (galvanized metal, stainless steel, polyethylene ground fabric, polyethylene pot plastic, pressure-treated pine, exterior latex-painted pine, raw pine) that had been inoculated with Botrytis cinerea conidia. Mortality was determined by percentage of ungerminated versus germinated conidia that had been rubbed off of a substrate onto half-strength potato dextrose agar (hPDA) 16 to 24 h previously. Based on overlapping confidence limits (95% CL) of the lethal doses resulting in 90 and 50% mortality (LD90 and LD50, respectively) and significance of slopes, differences occurred between substrates with all six disinfestants. LD90 values ranged from 0.21 to 4.54 g a.i./liter for chlorazene hydrosol, 4.99 to 40.3 g a.i./liter for hydrogen dioxide, 63.0 to 233.1 g a.i./liter for hydrogen peroxide, 0.42 to 2.45 g a.i./liter for iodine, 0.64 to 6.46 g a.i./liter for quaternary ammonium chloride, and 0.87 to 6.84 g a.i./liter for sodium hypochlorite. For hydrogen dioxide, quaternary ammonium chloride, and sodium hypochlorite, a binomial lethal dose (LDb) was calculated by plating the inverted inoculated substrates on hPDA, then recording the presence or absence of B. cinerea mycelial growth over 7 days. Lethal doses resulting in the absence of mycelial growth (LDb100) were equal to or greater than the LD90 values for most disinfestants and substrates. Results demonstrate for the six disinfestants that dose should be selected based on the substrate being disinfested of B. cinerea conidia.

Sequential Sampling to Assess the Incidence of Infection by Monilinia vaccinii-corymbosi in Mechanically Harvested Rabbiteye Blueberry Fruit.

ABSTRACT Blueberry fruit infected by Monilinia vaccinii-corymbosi, the causal agent of mummy berry disease, are unsuitable for use in processed food products. Fruit shipments that exceed a disease incidence threshold of 0.5% are redirected to alternative markets with substantial reductions in economic return to the producer. Because of this low tolerance, a sampling procedure with defined statistical properties is needed to determine disease incidence in the packinghouse. In this study, a sequential sampling plan was developed based on counts and dispersion patterns of infected fruit in 23 loads of mechanically harvested rabbiteye blueberries. Each load was sampled 20 to 100 times, with each sample containing 550 cm(3) of fruit. Various dispersion statistics (k of the negative binomial distribution, Lloyd' index of patchiness, and Iwao' b) were computed, all of which suggested aggregation of infected fruit. Because k was variable across loads, Iwao' regression procedure, which does not assume a single frequency distribution with fixed parameters describing the counts of infected fruit, was used to develop upper and lower stop lines for sequential sampling. For alpha = 0.05 and assuming a total of 250 fruit per 550-cm(3) sample, the resulting sampling plan would require only one sample to conclude that a load exceeds the threshold if the number of infected fruit in that sample is greater than four. A minimum of six samples would be needed to conclude that disease incidence in a load is below the threshold if the cumulative total of infected fruit in these samples is zero. Resampling analysis showed that most fruit loads could be classified reliably with <10 samples per load; for loads with a disease incidence very close to the 0.5% threshold, <50 samples were needed on average. Stop lines for sequential sampling for different fruit size classes are presented.

Ultrastructure of the Penetration and Infection of Pansy Roots by Thielaviopsis basicola.

ABSTRACT Transmission electron microscopy was used to study the penetration and infection of pansy roots by Thielaviopsis basicola. Events observed in 7- to 10-day-old roots produced on moist filter paper differed slightly from those in roots from 4-week-old plants washed free of potting media prior to inoculation. By 3 h postinoculation (PI), epidermal cells of roots produced on filter paper exhibited aggregated cytoplasm and papilla formation in response to germ tube tips. The presence of callose in papillae was demonstrated using immunogold labeling. Papilla formation was not effective in preventing host cell penetration. A slender infection hypha emerged from a germ tube tip and grew through a papilla. Its tip then expanded to form a globose infection vesicle. By 6 h PI, infection hyphae emerged from infection vesicles, and invaded host cells showed signs of necrosis. By 8 h PI, infection hyphae had grown into cortical cells in spite of papilla formation in these cells. By 24 h PI, distinctive intracellular hyphae were present in necrotic cortical cells. In washed roots, most epidermal cells failed to respond to invasion. Hyphae simply grew through these cells and contacted cortical cells that exhibited aggregated cytoplasm and papillae formation. Infection structures similar to those produced in epidermal cells from roots grown on filter paper then formed in cortical cells of washed roots. The fact that T. basicola formed infection structures only in cells that responded to invasion suggests that T. basicola has a more complex relationship with its host than would be expected in a nectrotrophic pathogen. We believe that T. basicola is best described as a necrotrophic hemibiotroph.

Evaluation of Methods to Detect Fruit Infected by Monilinia vaccinii-corymbosi in Mechanically Harvested Rabbiteye Blueberry.

Blueberry fruit infected by Monilinia vaccinii-corymbosi (causal agent of mummy berry disease) are unfit for processing because of the formation of hardened structures (pseudosclerotia) within them. In commercial packinghouses in Georgia, fruit loads exceeding the tolerance level for mummy berry are appraised at lower quality grades, resulting in severe economic penalties to producers. Two methods to detect and enumerate mummy berry in blueberry loads were evaluated in the laboratory using fruit samples with known numbers of infected fruit. The first method involved destructive processing of the samples in a blender. The resulting blueberry puree was passed through a series of screens and the number of pseudosclerotia of M. vaccinii-corymbosi retained on the screens assessed tactilely. The second method consisted of visual symptom assessment of intact fruit. Bias and coefficients of variation of the blender method in five experiments ranged from -63.0 to 152.4% and 6.9 to 44.1%, respectively, indicating that the method was inaccurate and imprecise. Several factors probably contributed to its poor performance, including the formation of multiple fragments from single pseudosclerotia during blending and subjectivity in the tactile assessment of pseudosclerotia. Bias and coefficients of variation of the visual assessment method in four experiments ranged from -3.41 to 1.97% and 1.16 to 5.17%, respectively. Thus, the visual method was considerably more accurate and more precise than the blender method. Visual assessment was further evaluated under commercial packinghouse conditions, with >70,000 fruit assessed individually for symptoms of mummy berry and other abnormalities. Bias ranged from -11.1 to 33.3%, indicating that visual assessment was less accurate under packinghouse conditions than under laboratory conditions. This was due to the low number of infected fruit encountered in most of the loads, which resulted in large relative errors if only a single fruit was misidentified. In a two-year packinghouse survey, a high incidence of partial infection, together with successional variations in discoloration of infected portions of the fruit as the harvest season progressed, resulted in a greater variation of mummy berry symptoms than previously described.