J. E. Adaskaveg

Effects of continuous 0.3 ppm ozone exposure on decay development and physiological responses of peaches and table grapes in cold storage

Continuous ozone exposure at 0.3 ppm (v/v) (US-OSHA Threshold Limit Value for short term exposure) inhibited aerial mycelial growth and sporulation on ‘Elegant Lady’ peaches wound inoculated with Monilinia fructicola, Botrytis cinerea, Mucor piriformis ,o rPenicillium expansum and stored for 4 weeks at 5 °C and 90% relative humidity (RH). Aerial growth and sporulation, however, resumed afterward in ambient atmospheres. Ozone exposure did not significantly reduce the incidence and severity of decay caused by these fungi with the exception of brown rot. Gray mold nesting among ‘Thompson Seedless’ table grapes was completely inhibited under 0.3 ppm ozone when fruit were stored for 7 weeks at 5 °C. Gray mold incidence, however, was not significantly reduced in spray inoculated fruit. Continuous ozone exposure at 0.3 ppm increased water loss after 5 weeks of storage at 5 °C and 90% RH in ‘Zee Lady’ peaches but not after 4 weeks of storage in ‘Flame Seedless’ grapes. Respiration and ethylene production rates of ‘O’Henry’ peaches were not affected by previous exposure to 0.3 ppm ozone. In every test, no phytotoxic injuries of fruit tissues were observed in ozonated or ambient atmosphere treatments.

Effect of Phosphite on Tomato and Pepper Plants and on Susceptibility of Pepper to Phytophthora Root and Crown Rot in Hydroponic Culture

Tomato and pepper plants were grown hydroponically in a greenhouse using phosphate or technical and commercial formulations of phosphite as sources of phosphorus nutrition to determine the effects on plant development and susceptibility to Phytophthora root and crown rot. Phosphite-treated tomato and pepper plants were deficient of phosphate and developed phosphorus-deficiency symptoms. Growth of plants (leaf area and leaf, stem, and root dry weights) that were fertilized with phosphite was significantly (P < 0.05) reduced compared with phosphate-fertilized plants. In Phytophthora capsici-inoculated pepper plants, incidence of Phytophthora crown rot was significantly reduced in phosphite-treated plants compared with no phosphorus or phosphate-treated plants. Incidence of crown rot in pepper plants treated with 1 mM phosphate plus 0.3 mM phosphite was intermediate between plants treated with only phosphite (1 mM or 0.1 mM) and plants treated with phosphate (1 mM).

Comparative Efficacy of the New Postharvest Fungicides Azoxystrobin, Fludioxonil, and Pyrimethanil for Managing Citrus Green Mold

Three new fungicides (i.e., azoxystrobin, fludioxonil, and pyrimethanil) are currently being introduced for postharvest management of citrus green mold in the United States. The effectiveness of each fungicide was evaluated when applied alone (at 1,000 to 1,200 mg/liter) or in mixtures (at 500 mg/liter each component) to lemon fruit that were wound-inoculated with imazalil/thiabendazole (TBZ)-sensitive or -resistant isolates of Penicillium digitatum. In laboratory studies when aqueous fungicide solutions were applied 9 to 21 h after inoculation, pyrimethanil showed the highest level of green mold control. The efficacy of fludioxonil and azoxystrobin was very high at the early timings, but decreased as time after inoculation increased. Differences in fungicide performance were not due to multiple fungicide resistance, but more likely due to differences in fungicide mobility in fruit tissue. Azoxystrobin-fludioxonil mixtures were significantly more effective when compared to single-fungicide treatments. Mixtures of imazalil with pyrimethanil were the most effective in controlling decay. The efficacy of all fungicides was significantly lower when mixed into a packing fruit coating as compared to aqueous or storage fruit coating applications. In laboratory and packingline studies, the lowest incidence of green mold decay was obtained when azoxystrobin-fludioxonil and imazalil-pyrimethanil were applied as aqueous solutions that were followed by a fruit coating. Among the new fungicides, azoxystrobin and fludioxonil applied in water or storage fruit coating, respectively, provided the best anti-sporulation activity. Storage fruit coating improved the activity of both fungicides. Pyrimethanil was the least effective fungicide in suppressing sporulation of the pathogen on decaying fruit. Overall, among the mixtures, azoxystrobin-fludioxonil and TBZ-fludioxonil had high anti-sporulation activity in aqueous and storage fruit coating applications. New integrated management programs should be based on monitoring of fungicide sensitivities in pathogen populations, rotating mixtures of products with different modes of action, and using appropriate fungicide application strategies.

Characterization of genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in field isolates of Penicillium digitatum.

Genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in isolates of Penicillium digitatum were evaluated and compared to those characterized in other fungi. Resistant isolates were naturally occurring in packinghouses and were not associated with crop losses. For the phenylpyrrole fludioxonil, EC50 values were 0.02 to 0.04 microg/ml for sensitive, 0.08 to 0.65 microg/ml for moderately resistant (MR), and > 40 microg/ml for highly resistant (HR) isolates. Two fludioxonil-sensitive isolates evaluated were also significantly more sensitive to the unrelated dicarboximide fungicide iprodione, that also disrupts osmotic regulation, than the MR and HR isolates. There was no consistent relationship, however, between the HR and MR isolates and their sensitivity to iprodione or osmotic stress. Although, two nucleotide substitutions were found in a sequence analysis of the N-terminal amino acid repeat region of the os-1-related histidine kinase gene among isolates of P. digitatum, these were not correlated with fludioxonil resistance. In mycelia not exposed to fludioxonil, the amount of phosphorylated OS-2-related protein (PdOS-2) was higher in fludioxonil-sensitive isolates and lowest in the HR isolate. An increase in PdOS-2 was observed for sensitive and resistant isolates after exposure to fludioxonil. In addition, glycerol content in untreated mycelia of the fludioxonil-sensitive isolate was significantly higher than in resistant isolates. After exposure to fludioxonil, glycerol concentrations significantly increased in the sensitive and MR isolates, but not in the HR isolate. Thus, our studies indicate that the mode of action of fludioxonil in P. digitatum is probably the mitogen-activated protein kinase pathway that stimulates glycerol synthesis in sensitive and MR isolates. The general suppression of this pathway in resistant isolates was supported by the fact that growth and sporulation of MR and HR isolates were significantly reduced from that of sensitive isolates. In studies on the mode of action of anilinopyrimidines (AP), EC50 values for mycelial growth of P. digitatum and the previously characterized Botrytis cinerea were determined for cyprodinil and pyrimethanil using a defined culture medium without and with the addition of selected amino acids and homocysteine. The addition of amino acids resulted in a reduced toxicity of the two AP fungicides in both fungi, but the effect of each additive was significantly lower for P. digitatum than for B. cinerea. This suggests that methionine biosynthesis is not the primary target site of APs in P. digitatum.

Spiral gradient dilution, a rapid method for determining growth responses and 50% effective concentration values in fungus-fungicide interactions.

ABSTRACT A new technique, the spiral gradient dilution method, was evaluated for determining 50% effective concentrations (EC(50) values) of fungicides for the inhibition of mycelial growth and conidial germination of various fungi. In this method, an agar medium is plated with a fungicide solution by means of a spiral plater, which applies the fungicide in a 2.5-log dilution in a continuous radial concentration gradient. Fungal inoculum is then placed along radial lines across the gradient. After incubation of the plates, distinct growth shapes were observed in different fungus-fungicide interactions. Mycelial growth responses to increasing fungicide concentrations ranged from abrupt to gradual transitions. Conidial germination responses were similar; in addition, distinct zones of confluent growth, nonconfluent growth, and outlier colonies were also identified, depending on the fungus-fungicide interaction. The fungicide concentration at the radial distance at which 50% reduction of growth or spore germination occurred, compared with growth or germination on unamended media, was calculated by a computer program. EC(50) values were obtained for mycelial growth in 22 fungus-fungicide interactions and for conidial germination in five interactions. The fungi evaluated were members of the Zygomycota, Ascomycota, Basidiomycota, and Deuteromycota. Nine fungicides, belonging to six different chemical classes, were tested. EC(50) values were compared with those obtained by the traditional agar dilution method. In linear regression analyses of the two methods, the models were highly significant (P < 0.01), and coefficients of determination (r(2)) were 0.92 for the mycelial growth assays and 0.94 for the conidial germination assays. Regression slopes were not significantly different from 1 (P > 0.05) with optimal program settings in the software. Estimated bias, coefficients of variation, and actual confidence intervals for the regression slope were 13.5%, 6.5%, and 1.14 +/- 0.14 for the mycelial growth assays and 7.5%, 14.5%, and 1.08 +/- 0.37 for the conidial germination assays. These analyses indicate that the spiral gradient dilution method is accurate and precise compared with the agar dilution method for calculating EC(50) values of fungicides in continuous growth responses to fungicide concentration gradients.

Optimizing Efficacy of New Postharvest Fungicides and Evaluation of Sanitizing Agents for Managing Citrus Green Mold

Three new fungicides, azoxystrobin, fludioxonil, and pyrimethanil, that belong to different chemical classes are highly effective in managing citrus green mold and are being registered for postharvest use in the United States. Recirculating in-line drenches provided a significantly improved efficacy compared with standard low-volume spray applications. To prevent pathogen contamination of drench solutions, two oxidizing disinfectants, sodium hypochlorite and hydrogen peroxide/peroxyacetic acid (HPPA) solutions, were evaluated. Inhibition of conidial germination of Penicillium digitatum was dependent on the pH of the solution and the exposure time for each sanitizing agent. Chlorine (50 mg/liter) and HPPA (2,700 mg/liter) effectively inhibited germination in 40- and 240-s exposures, respectively, at pH 7. All fungicides tested were compatible and effective with HPPA, whereas fludioxonil, azoxystrobin, and thiabendazole, but not imazalil and pyrimethanil, were compatible with chlorine. In laboratory studies, sodium bicarbonate (SBC, 3%) significantly increased the efficacy of the three fungicides (250 mg/liter) and had no adverse effect on their stability in aqueous solutions. Fludioxonil (300 mg/liter)-SBC mixtures were still highly effective when applied 24 h after fruit inoculation. In experimental packingline studies, SBC or SBC-chlorine improved the efficacy of fludioxonil, whereas azoxystrobin was effective with and without these additives. Heating of drench solutions of fludioxonil (300 mg/liter) to 50°C did not improve decay control. In conclusion, in-line recirculating drench applications and fungicide-sanitizer-SBC mixtures significantly increased fungicide efficacy and provide an integrated approach for optimizing fungicide efficacy. These strategies also should minimize the selection for resistant isolates of the pathogen.

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond.

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.

Identification and etiology of visible quiescent infections of Monilinia fructicola and Botrytis cinerea in sweet cherry fruit.

Visible quiescent infections were detected as small (<1 mm) necrotic flecks on green cv. Bing cherry fruit and as reddish halos surrounding tannish spots (1 to 2 mm) on immature, yellow-pink cv. Rainier cherry fruit in commercial orchards in California. Monilinia fructicola or Botrytis cinerea, the fungal pathogens causing brown rot and gray mold of cherry fruit, respectively, were isolated from most of the viable infections. M. fructicola was isolated more frequently from quiescent infections than B. cinerea in two years of the study, whereas similar isolation frequencies for both fungi were obtained in the other two years of sampling from one commercial Rainier cherry orchard. Using immature-pink Bing fruit that were inoculated in the laboratory, significantly more visible quiescent infections than active decay were reproduced in 6-, 9-, or 12-h wetness-period treatments after inoculation as compared to 18- or 24-h wetness periods where more active decay developed. Non-visible quiescent infections of M. fructicola or B. cinerea of immature Bing and Rainier fruit collected 2 weeks before harvest were identified on surface-sterilized, paraquat-treated fruit. In both years of the study, significantly more brown rot and gray mold occurred on the surface-sterilized, paraquat-treated fruit than on the non-treated or surface-sterilized fruit, indicating the presence of non-visible quiescent infections by these fungi in cherry fruit. Thus, for the first time, we demonstrated the presence of visible quiescent infections caused by M. fructicola and B. cinerea and we confirmed the occurrence of non-visible quiescent infections in sweet cherry fruit in California.

Efficacy of sodium tetrathiocarbonate and propiconazole in managing Armillaria root rot of almond on peach rootstock

The efficacy of sodium tetrathiocarbonate (STTC or Enzone 31.8%, a liquid formulation that releases carbon disulfide) and the demethylation inhibiting (DMI) fungicide propiconazole (Alamo 1.1EC) was evaluated for management of Armillaria root rot of almond grown on Lovell peach rootstock. After 12 months, pre-/post-plant STTC (189 liters of 3,850 and 500 mg/liter/3 m2 treatment site, respectively) or tarped pre-plant methyl bromide (Dowfume 98%, 454 g a.i./3 m2) soil fumigation treatments significantly (P < 0.05) reduced the recovery of Armillaria mellea from naturally infected root segments at 0.3 and 1.2 m soil depths, compared with control sites. Tarped methyl bromide treatments eradicated the fungus from infested root segments at both depths; however, nontarped sites significantly reduced the recovery only at the 1.2 m depth. Pre-plant STTC (189 liters of 3,850 mg/liter/3 m2) reduced the recovery of the fungus but was not as effective as the pre-/post-plant STTC treatment. Recovery of the fungus in post-plant treatments with STTC (189 liters of 500 mg/liter/3 m2) was not significantly (P > 0.05) different from control sites. Additionally, mortality of almond tree replants from phytotoxicity was significantly higher in post-plant applications of STTC, compared with the other treatments or with the control trees. A gel formulation of 31.8% STTC (1,800 ml of 318,000 mg of STTC per liter per tree stump) applied in wells that were drilled into tree stumps eradicated the fungus from trunk and primary roots but not secondary or tertiary roots, whereas liquid formulations of STTC and metam-sodium (Vapam - 32.7% sodium N-methyldithiocarbamate) eradicated the fungus from only trunks but not roots. Treatments with STTC (189 liters of 500 mg/liter/3 m2) were not effective in preventing mortality of A. mellea-infected, mature, 7- to 8-year-old almond trees. Propiconazole (Alamo 1.1EC) was shown to be toxic to mycelial growth of A. mellea grown on potato dextrose agar with an EC50 value of 0.15 mg/liter. Therapeutic, passive injections of propiconazole into 7- to 8-year-old almond trees (Lovell peach rootstock) were successful in reducing mortality of infected trees during two growing seasons, compared with infected control trees treated with water.

Baseline Sensitivities for New Postharvest Fungicides Against Penicillium spp. on Citrus and Multiple Resistance Evaluations in P. digitatum

For the first time in over 25 years, three new fungicides (azoxystrobin, fludioxonil, and pyrimethanil), all belonging to different chemical classes, are being registered for postharvest use against Penicillium decays of citrus fruit in the United States. Baseline sensitivities of Penicillium digitatum and P. italicum were developed using isolates collected before the commercial use of these new fungicides. In a comparison of methods, EC50 values obtained using the spiral gradient dilution method were very similar to those obtained using traditional agar dilutions of fungicides. For azoxystrobin, the addition of salicylhydroxamic acid (SHAM) did not significantly affect EC50 values for mycelial growth of both species. In additional studies on conidial germination of P. digitatum, SHAM significantly reduced EC50 values for azoxystrobin. For pyrimethanil, the mean EC50 value for mycelial growth obtained using a minimum growth medium for anilinopyrimidine fungicides was significantly lower but comparable to values obtained when using potato dextrose agar . For mycelial growth of P. digitatum, mean EC50 values were 0.014, 0.025, and 0.313 μg/ml, whereas for conidial germination, they were 0.074, 0.163, and 1.195 μg/ml for azoxystrobin, fludioxonil, and pyrimethanil, respectively. For P. italicum, mean EC50 values for mycelial growth for fludioxonil and pyrimethanil were 0.005 and 0.040 μg/ml, respectively. For azoxystrobin, the mean EC50 value for mycelial growth for 33 isolates was 0.029 μg/ml. Four isolates had EC50 values ≥0.772 μg/ml and were considered part of a resistant subpopulation. Multiple resistance between the older and new postharvest fungicide classes on citrus was not detected in P. digitatum, and all isolates that were sensitive or resistant to imazalil or thiabendazole were sensitive to the new compounds. This information is important for monitoring populations of P. digitatum, where resistance against the older fungicides has commonly developed.