Genetic Diversity of Pseudomonas savastanoi pv. savastanoi in California and Characterization of Epidemiological Factors for Olive Knot Development.

Olive knot, caused by Pseudomonas savastanoi pv. savastanoi, is a limiting disease in the production of table and oil olives in California. The genetic variability among 152 strains from major production areas of California was determined using BOX, ERIC, and REP primers in repetitive element sequence-based polymerase chain reaction. Overall genetic variability was low, and strains shared at least 82% similarity. Phenetic analyses identified several genotypes but most strains belonged to one of two major groups. Three copper-resistant strains had two fingerprints that were distinct from any of the sensitive strains, indicating that they may have been introduced from other production areas or hosts. In inoculations, two copper-resistant strains were mostly equally as virulent as two copper-sensitive strains. Inoculum was exuded at high levels (>108 CFU/g of knot tissue) within 10 min from hydrated olive knots, and concentrations were 2- to 3-log higher than the minimum needed to induce knot formation. Arbequina olive was significantly more susceptible to infection and developed a higher incidence of knots on leaf scar and lateral wounds (59.7 to 80.6% incidence) than Manzanillo (47.4 to 68.2% incidence). In wound-healing studies, both types of wounds were less susceptible to infection ≥10 days after injury, indicating a critical period for infection and application of bactericides during favorable environments.

Efficacy of Copper and New Bactericides for Managing Olive Knot in California.

Baseline sensitivities were established for kasugamycin and oxytetracycline for 147 strains of Pseudomonas savastanoi pv. savastanoi collected from olive knots throughout California. Minimum inhibitory concentrations for ≥95% growth inhibition ranged from 1.86 to 11.52 and 0.13 to 0.40 µg/ml for kasugamycin and oxytetracycline, respectively. In copper sensitivity evaluations, 95.3% of the strains collected grew at concentrations of metallic copper equivalent (MCE) of <20 µg/ml, 2.7% grew at MCE between 20 and 30 µg/ml (moderately sensitive), and 2% grew at MCE of 150 µg/ml (resistant). Copper resistance was never reported previously in the olive knot pathogen, and pathogenicity studies confirmed a high virulence of the copper-resistant strains. In comparative field studies, kasugamycin at 200 µg/ml performed equally to the standard copper hydroxide treatment (MCE of 1,260 µg/ml) for reducing knot development on lateral wounds of Arbequina and Manzanillo olive inoculated with a copper-sensitive strain and was better than copper using a highly copper-resistant strain. Oxytetracycline at 200 µg/ml was not as effective as copper or kasugamycin but significantly reduced the disease as compared with the untreated control. Field studies on application timings of copper, kasugamycin, and copper-kasugamycin mixtures to inoculated wounds indicated that treatments within 24 h of inoculation resulted in higher disease control than applications at later times. In greenhouse trials, copper or copper-kasugamycin applied to wounds 7 days before inoculation persisted and reduced knot incidence by >50%. Our findings indicate that kasugamycin is an effective bactericide for controlling olive knot and that the time of any bactericide application after inoculation is critical in managing the disease.

Identification of Alternaria Species Causing Heart Rot of Pomegranates in California.

Alternaria heart rot of pomegranate fruit is generally considered a minor disease. However, the current listing of the causal pathogen in California as "Alternaria sp." or as "A. alternata and other Alternaria spp." restricts trade of the crop to some major export markets where quarantines are in place in an effort to prevent the spread of unknown or undescribed pathogens. Thus, species identification of the pathogen is critical in determining whether infected fruit should be regulated by quarantines. In this study, 86 isolates of Alternaria were collected from pomegranate fruit with Alternaria heart rot symptoms from major production areas in California. An unweighted pair group method with arithmetic means analysis based on amplified fragment length polymorphisms revealed two main clusters, each with a high degree of variability. One of the clusters contained 24 isolates from pomegranate and one reference isolate of Alternaria arborescens. Reference isolates of A. alternata and A. tenuissima were found among pomegranate isolates in the other cluster, and these two species could not be separated. In maximum-parsimony analysis of ribosomal DNA internal transcribed spacer sequence data, representative pomegranate isolates all clustered with reference sequences of species in section Alternaria. Single-nucleotide differences separated A. arborescens, A. gaisen, and two pomegranate isolates from the majority of the remaining isolates. Sequence comparisons of gapdh and OPA10-2 loci indicated that none of the pomegranate isolates were identical to A. gaisen, which is a quarantine pathogen. Due to high genetic similarity and variability of morphological characteristics, revisions of the taxonomy of small-spored Alternaria spp. have been proposed by others. Based on this recent taxonomic work and work herein by us, pomegranate isolates from California can be assigned to A. alternata and A. arborescens. Reassessment of export restrictions for California pomegranate due to the previous pathogen classification is warranted. Fruit injection inoculations with conidia of 12 representative isolates 3 months before harvest caused typical symptoms of Alternaria heart rot, whereas flower inoculations did not result in fruit disease.

Quaternary Ammonium Compounds as New Sanitizers for Reducing the Spread of the Olive Knot Pathogen on Orchard Equipment.

Olive knot, caused by the wound pathogen Pseudomonas savastanoi pv. savastanoi, is a serious bacterial disease that can be disseminated by orchard equipment. Greenhouse studies confirmed that cutting tools contaminated during contact with olive knots are able to spread the pathogen to healthy olive tissue. Quaternary ammonium compounds (QACs) were assessed as sanitizing agents for contaminated equipment as a disease management strategy. In laboratory in vitro tests, QACs exhibited high toxicity against the bacterium over a broad pH range from 6 to 9 using short exposure periods (15 to 60 s) and low concentrations (5 µg/ml). QACs applied to contaminated hard surfaces in the presence of an organic load reduced bacterial recovery by ≥3.6 log10 CFU/ml. In field trials, sanitation of hedging equipment that was contaminated with the pathogen (2 × 107 CFU/ml) and used to prune olives, was successful and sometimes completely prevented new infections from occurring. Application of additional foliar spray treatments of copper or copper-kasugamycin mixtures after hedging significantly improved disease control. In laboratory and field studies, sodium hypochlorite was significantly less effective than QAC compounds in the presence of organic matter. A nonphenolic QAC formulation, however, was ineffective as a preventative treatment when applied prior to inoculation of olive wounds, whereas a copper hydroxide application was highly effective. Based on data from this research, a QAC formulation was registered for field use as a sanitizer for olive equipment in California in 2015.

Postharvest Strategies for Managing Phytophthora Brown Rot of Citrus using Potassium Phosphite in Combination with Heat Treatments.

Phytophthora brown rot, caused by several species of Phytophthora, is an economically important disease of citrus in areas with rainfall during the late stages of fruit development. Recent export restrictions of California orange fruit to China due to the presence of brown rot caused by the quarantine pathogen Phytophthora syringae have mandated more rigorous disease management. We evaluated postharvest applications with the phosphonate fungicide potassium phosphite in combination with heat treatments. In timing studies, potassium phosphite at 1,500 µg/ml was most effective when applied within 18 h after inoculation of orange fruit with P. citrophthora, reducing the incidence of decay by >96% as compared with the control. Potassium phosphite was also highly effective in inoculations with P. syringae. Heated water treatments at 60°C were consistently and highly effective in reducing the incidence of brown rot after inoculation with P. citrophthora, whereas treatments at 55 or 50°C were more variable and generally less effective. Two-stage treatments of fruit were conducted in the laboratory to simulate current packinghouse practices and to evaluate any interaction of the efficacy of potassium phosphite with treatments of two commonly used postharvest fungicides (i.e., imazalil and thiabendazole [TBZ]) or a postharvest carnauba-based fruit coating. In these studies, an aqueous imazalil-potassium phosphite (2,000 µg/ml) dip at ambient temperature that was followed by a spray treatment of imazalil and TBZ prepared in fruit coating significantly reduced the incidence of brown rot from the control. When the aqueous dip was applied at 54°C, brown rot developed in only 1% of the fruit as compared with 76% in the water control. The efficacy of potassium phosphite was also demonstrated in commercial packinghouse treatments. Based on our research, this fungicide was registered for postharvest use against brown rot of citrus and is exempt from tolerance in the United States and many other countries.

Identification and Pathogenicity of Fungal Pathogens Associated with Stem-End Rot of Avocado in California.

Stem-end rot of harvested avocado fruit commonly occurs wherever the crop is cultivated. Multiple fungal species have been described as causal agents. To determine the causal pathogens of stem-end rot in California, fungal isolations were conducted from symptomatic fruit, and fungi were identified by morphological and molecular techniques. In 2010 and 2011, a total of 177 isolates were recovered from 290 avocado fruit collected from seven orchards in one of the major avocado growing areas in Southern California. The majority of isolates was identified as Neofusicoccum luteum (65%), with the remainder either as Colletotrichum gloeosporioides (33%) or Phomopsis sp. (2%). In a pathogenicity test, N. luteum caused significantly (P < 0.05) more severe stem-end rot than either C. gloeosporioides or Phomopsis sp. No significant (P > 0.05) differences in stem-end rot severity were observed between inoculations with N. luteum isolated from fruit stem-end rot and N. luteum or N. parvum isolated from branch cankers. This confirms that stem-end rot of avocado can be initiated by fungi causing branch cankers. Although low humidity and rainfall during much of the growing and harvest seasons in California are considered unfavorable conditions for the development of avocado stem-end rot, the identification of the causal pathogens is of value when decays have to be managed during outbreaks, and it stresses the importance of managing branch cankers.

QoI Resistance in Fusicladium carpophilum Populations from Almond in California and Evaluation of Molecular Resistance Mechanisms.

Disease management failures have been reported in California for almond scab caused by Fusicladium carpophilum following quinone outside inhibitor (QoI) applications. Resistance in the pathogen populations was found to be common and at high incidence in the major almond-growing regions beginning in 2003, 4 years after registration of azoxystrobin on this crop. Two levels of azoxystrobin resistance, moderate and high, were identified with 50% effective concentration (EC50) values between 0.15 and 10 µg/ml or >40 µg/ml, respectively. Reference isolates collected before resistance was detected had EC50 values <0.05 µg/ml. High-resistance was associated with a G143A mutation in the mitochondrial cytochrome b gene. For the less commonly found moderately resistant isolates, no mutations in the gene were detected between codons 122 and 212. Using primers targeting the G143A mutation or the cytochrome b gene of all F. carpophilum isolates in quantitative polymerase chain reaction (qPCR) analyses, the frequency of highly resistant isolates was accurately determined in mixtures of conidia with selected ratios of sensitive and resistant isolates. The frequency of high resistance in bulked samples of scab lesions, however, was generally underestimated compared with in vitro testing of fungicide sensitivity of fungal isolates from the same lesions. Competition experiments using conidial suspensions demonstrated stability of the highly resistant genotype in the presence of different amounts of sensitive and moderately resistant genotypes. Analysis of covariance of linear regressions of cycle threshold values on DNA concentrations derived from qPCR amplifications using two primer pairs for cytochrome b alleles with and without the G143 mutation showed that several isolates differed in their slopes and midpoints. Thus, heteroplasmy of mitochondrial-inherited QoI resistance is suggested as a likely cause for incongruence in estimating resistance frequencies using the two methods.

Distinguishing Galactomyces citri-aurantii from G. geotrichum and characterizing population structure of the two postharvest sour rot pathogens of fruit crops in California.

A growth assay in lemon juice and polymerase chain reaction amplifications using newly designed species-specific primers from endopolygalacturonase and ß-tubulin genes rapidly differentiated isolates of the morphologically similar fruit sour rot pathogens Galactomyces citriaurantii and G. geotrichum. Isolates of both species were collected from agricultural soils and decaying fruit at locations within and outside California, including worldwide locations, and were used in population genetic studies based on amplified fragment length polymorphic (AFLP) DNA markers. For all four geographically defined subpopulations (three counties of California and locations outside California) among 97 isolates of G. citri-aurantii and for the two subpopulations (origin within or outside California) among 35 isolates of G. geotrichum, the proportion of polymorphic loci and haplotypic diversity was high. In total, 82 unique haplotypes were identified for G. citri-aurantii for the four subpopulations and, of these, 80 haplotypes were unique among subpopulations. For G. geotrichum, 25 unique haplotypes were identified among the two subpopulations and no haplotype was shared. Indices of genetic differences (F(ST)) between subpopulations within each species were all low (e.g., 0.038 for G. geotrichum and 0.085 to 0.226 for G. citriaurantii), indicating a low level of genetic differentiation. Following clone correction, mating type segregation ratios for G. citri-aurantii did not significantly (P > 0.1) deviate from a 1:1 ratio for all four subpopulations or the entire population. Tests of the index of association (I(A)) and parsimony tree-length permutation tests (PTLPT) supported a random mating structure for clone-corrected data for the Kern, Tulare, and Ventura County subpopulations and the null hypothesis of random mating could not be rejected. Additionally, PTLPT also supported random mating for the "outside of California" population. For G. geotrichum, random mating was only tested using I(A) and PTLPT and the null hypothesis of random mating was not rejected (P > 0.05) using clone-corrected data. Further evidence that sexual recombination likely occurs in both species of Galactomyces was the lack of grouping consistency in the unweighted pair-group method with arithmetic mean clustering of AFLP data. A high confidence based on bootstrap values was obtained for only a few of the nodes in each of the two trees. A mixed reproduction system with an out-crossing sexual mating system and a prolific asexual phase is proposed for both species.

Efficacy and Application Strategies for Propiconazole as a New Postharvest Fungicide for Managing Sour Rot and Green Mold of Citrus Fruit.

Few postharvest treatments are available for managing sour rot of citrus caused by Galactomyces citri-aurantii and they are generally not very effective. The demethylation-inhibiting (DMI) triazole fungicides propiconazole and cyproconazole were found to be highly effective and more efficacious than other DMIs evaluated, such as metconazole and tebuconazole, in reducing postharvest sour rot of citrus. Additional studies were conducted with propiconazole as a postharvest treatment because it has favorable toxicological characteristics for food crop registration in the United States and the registrant supports a worldwide registration. Regression and covariance analyses were performed to determine optimal time of application after inoculation and fungicide rate. In laboratory studies, decay incidence increased when propiconazole applications were delayed from 8 to 24 h (lemon) or 18 to 42 h (grapefruit) after inoculation. Effective rates of the fungicide were 64 to 512 µg/ml and were dependent on inoculum concentration of the sour rot pathogen and on the type of citrus fruit. Propiconazole was found to be compatible with sodium hypochlorite at 100 µg/ml and 1 to 3% sodium bicarbonate without loss of efficacy for decay control on lemon. The addition of hydrogen peroxide/peroxyacetic acid at 80 µg/ml slightly decreased the effectiveness of propiconazole. Heated (48°C) solutions of propiconazole did not significantly improve the efficacy compared with solutions at 22°C. In experimental packing-line studies, aqueous in-line drenches applied alone or followed by applications of the fungicide in storage or packing fruit coatings were highly effective, reducing sour rot to between 0 and 1.2% compared with 83.8% decay incidence in the control when treatments were made up to 16 h after inoculation. When the fungicide was applied in either fruit coating, decay was only reduced to 49.1 to 57.1% incidence. Tank mixtures of propiconazole with the citrus postharvest fungicides fludioxonil and azoxystrobin were highly effective in reducing green mold caused by isolates of Penicillium digitatum sensitive or moderately resistant to imazalil and sour rot. Propiconazole will be an important postharvest fungicide for managing sour rot of citrus and potentially can be integrated into current management practices to reduce postharvest crop losses caused by DMI-sensitive isolates of P. digitatum.

Toxicity and Resistance Potential of Selected Fungicides to Galactomyces and Penicillium spp. Causing Postharvest Fruit Decays of Citrus and Other Crops.

A diverse collection of isolates of Galactomyces citri-aurantii and G. geotrichum, the causal pathogens of sour rots of citrus and other fruit crops, respectively, was evaluated for sensitivity to demethylation-inhibiting (DMI) fungicides of the triazole group. Propiconazole was found to be highly effective in reducing mycelial growth of both species in vitro. For 139 isolates of G. citri-aurantii, a mean effective concentration for 50% reduction of mycelial growth (EC50 value) of 0.34 µg/ml was determined; whereas, for 33 isolates of G. geotrichum, this value was 0.14 µg/ml. In a comparison of additional DMI fungicides, mean EC50 values for 60 isolates of G. citri-aurantii and 20 isolates of G. geotrichum, were 0.27 and 0.17 µg/ml for cyproconazole, 0.25 and 0.14 µg/ml for metconazole, and 1.16 and 0.73 µg/ml for tebuconazole, respectively. Propiconazole was also highly active against mycelial growth of imazalil-sensitive isolates of Penicillium digitatum, the pathogen that causes green mold of citrus, with a mean EC50 value of 0.008 µg/ml for 63 isolates. Imazalil-resistant isolates of this fungus were cross-resistant to propiconazole. When G. citri-aurantii and P. digitatum were grown at selected pH values between 3 and 9, inhibition by propiconazole occurred over the entire pH range. The fungicide was most effective at pH 5 when compared with the non-fungicide-amended control grown at the same pH. In laboratory mass platings of single-spore isolates sensitive to propiconazole onto selective media, isolates with an up to 81.6-fold decrease in sensitivity to the fungicide were recovered for P. digitatum. For G. geotrichum, isolates with an approximately twofold decrease in sensitivity were obtained. No isolates with reduced sensitivity were recovered for G. citri-aurantii. Propiconazole is currently being registered for postharvest use on citrus and other crops, and the information provided will be valuable in monitoring of fungicide resistance and in designing effective fungicide application strategies.

Effect of wetness duration and temperature on the development of anthracnose on selected almond tissues and comparison of cultivar susceptibility.

Blossoms, leaves, fruit, and woody tissues of almond can be affected by anthracnose caused by Colletotrichum acutatum. Because the disease occurs throughout rainy spring seasons, the effect of temperature and wetness duration on disease development was evaluated in controlled studies. The lowest inoculum concentration where disease developed on leaves was 10(4) conidia/ml. Longer wetness durations were needed for leaves than for blossoms and disease increased linearly with increasing wetness durations. Inoculation temperature mainly affected final disease levels. Temperature during incubation affected the rate of disease development, while final disease levels were very similar at 10, 15, or 20°C. An analysis of covariance was performed to compare regressions of the effects of wetness and temperature on disease development for several almond cultivars. For blossom inoculations at 15°C in growth-chamber studies, a common slope model was statistically sufficient to describe all four cultivars. Cultivar Nonpareil (NP) had a significantly (P<0.05) lower adjusted means at the midpoint than cultivars Carmel (CA), NePlus Ultra (NU), and Wood Colony (WC). For blossom inoculations at 20°C and for leaf inoculations at all temperatures evaluated, an unequal slope model was statistically justified for comparing regression lines. For blossoms, the slopes were significantly different (P<0.05) for pair-wise comparisons of CA-NU, NU-WC, and NP-WC. For leaves, most of the cultivars responded differently to infection at different temperatures. Two of the pair-wise comparisons demonstrated unequal slopes at all three temperatures evaluated (i.e., NU-NP and NU-WC). Overall, for blossoms and leaves, NP was the least susceptible, NU was the most susceptible, and WC and CA showed an intermediate susceptibility. In field blossom and fruit studies, a common slope model was statistically sufficient to describe all four cultivars. NP had a significantly lower midpoint (i.e., was less susceptible) than CA or WC, whereas no significant difference (P > 0.1) occurred in comparisons between CA and WC.

Effectiveness of Kasugamycin Against Erwinia amylovora and its Potential Use for Managing Fire Blight of Pear.

Comparative field studies on the management of fire blight were conducted in California on Asian and Bartlett pear using single-bactericide, mixture, and rotation treatments of selected compounds. Treatment efficacy was evaluated based on the natural occurrence of the disease or after inoculation with Erwinia amylovora. Kasugamycin at 100 mg/liter demonstrated similar or higher pre- and post-infection activity than the industry standards, streptomycin and oxytetracycline. Phytotoxicity caused by kasugamycin was observed only when five or six sequential weekly applications were done. In a six-spray rotation program including three bactericides (copper, kasugamycin, and oxytetracycline), with each being used twice, phytotoxicity was minor. Baseline sensitivity concentrations for kasugamycin were established for growth of 376 isolates of E. amylovora from California. Values for the lowest concentration where a reduction in growth on nutrient agar was observed ranged from 3.5 to 18.3 mg/liter, with a mean value of 8.7 mg/liter. Values for ≥95% inhibition of growth ranged from 6.9 to 46.7 mg/liter, with a mean value of 18.5 mg/liter. These inhibitory values for kasugamycin were higher than those for streptomycin or oxytetracycline. The in vitro activity of all three compounds was highly dependent on the agar medium used in the sensitivity assay. The activity of kasugamycin was also highly dependent on the pH of the medium and was significantly higher at pH 5.1 than pH 7.3. With the planned registration in the United States, kasugamycin represents the first new, highly effective bactericide for managing fire blight in over 40 years.

Determination of natural resistance frequencies in Penicillium digitatum using a new air-sampling method and characterization of fludioxonil- and pyrimethanil-resistant isolates.

ABSTRACT Fungicide resistance was identified in natural populations of Penicillium digitatum, the causal agent of green mold of citrus, to two of three new postharvest fungicides before their commercial use. Using a new air-sampling method where large populations of the pathogen in citrus packinghouses were exposed to agar plates with a continuous, wide-range fungicide concentration gradient, isolates with reduced sensitivity to fludioxonil or pyrimethanil were obtained. Resistance frequencies to fludioxonil and pyrimethanil were calculated as 9.5 x 10(-7) to 1.5 x 10(-5) and 7.3 x 10(-6) to 6.2 x 10(-5), respectively. No isolates resistant to azoxystrobin were detected. Isolates with reduced sensitivity to fludioxonil or pyrimethanil were also obtained in laboratory selection studies, where high concentrations of conidial mixtures of isolates sensitive to the three fungicides were plated onto agar amended with each fungicide at 10 microg/ml. Isolates obtained from fludioxonil selection plates in laboratory and packinghouse experiments were placed into two categories based on mycelial growth: moderately resistant isolates had 50% effective concentration (EC(50)) values of 0.1 to 0.82 microg/ml and highly resistant isolates had EC(50) values > 1.5 microg/ml. Isolates resistant to pyrimethanil all had EC(50) values >8 microg/ml. Representative isolates of the two categories with reduced sensitivity to fludioxonil varied widely in their virulence and sporulation capacity as measured by the incidence of decay and degree of sporulation on inoculated fruit, respectively, whereas pyrimethanil-resistant isolates were mostly similar to the wild-type isolate. Fungicide sensitivity characteristics for isolates from fludioxonil and pyrimethanil selection plates remained stable after passages on nonamended agar, and disease could not be controlled after treatment with the respective fungicides. Types of fungicide resistance were visualized on thiabendazole- (TBZ) and imazalil-amended selection plates that were exposed in packinghouses where resistance to these fungicides was known to occur. The qualitative, single-site resistance to the benzimidazole TBZ was visualized by two distinct subpopulations in regard to fungicide sensitivity, whereas the quantitative, multi-site resistance to the demethylation inhibitor imazalil was apparent as a continuous density gradient of colonies along the fungicide concentration gradient. Types of resistance could not be assigned to fludioxonil or pyrimethanil because a limited number of resistant colonies was obtained on each plate. Thus, with this new method, we were able to estimate fungicide resistance frequencies as well as characterize and visualize types of resistance within populations of a fungal species. This information will be used to design resistance management strategies for previous and newly registered postharvest fungicides of citrus.

Etiology and Management of a Mandarin Rind Disorder in California.

Recent reports of a preharvest rind disorder of Satsuma mandarin fruit resulted in severe crop losses in some growing areas of California (Butte, Fresno, Tulare, and Kern Counties). Symptoms were more frequently observed on fruit on the outer perimeter of the tree and on the exposed side of the fruit. Fungal isolations from affected fruit were inconsistent among orchard sites and growing seasons. Isolations resulted in species of Alternaria, Fusarium, Macrophomina, Ulocladium, and Cladosporium. In laboratory studies, mandarin fruit that were soaked in water for 6 h at 15 to 35°C, air dried, and incubated at 20°C for 3 days developed lesions similar to those observed in the field. When fruit were treated with an agricultural summer spray oil or the antitranspirant di-1-p-menthene prior to water soaking, the incidence and severity of the disorder were significantly reduced. Similar results were obtained in 2 years of field trials at two sites using one to three preharvest applications with the agricultural antitranspirant or summer oil starting after fruit color break. In most trials, a single application of these treatments at 1 or 2 weeks after color break was highly effective. Fungicides were inconsistent in their efficacy, whereas trees that were physically protected from rain did not develop the disorder. These data suggest that this mandarin rind disorder is a physiological, abiotic disorder rather than a pathological problem caused by fungi and can be economically managed with water-repellant treatments.

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.

Visualization of localized pathogen-Induced pH modulation in almond tissues infected by Colletotrichum acutatum using confocal scanning laser microscopy.

Modulation of pH within the host during infection of almond by the anthracnose pathogen Colletotrichum acutatum was studied using confocal scanning laser microscopy and the dual emission fluorescence indicator SNARF-1. This highly sensitive method allowed visualization of the spatial distribution of localized pathogen-induced pH modulation within and in proximity to fungal infection structures in host tissue at the cellular level. Ratiometric measurement of fluorescence at two emission wavelengths and in situ calibration allowed the quantification of pH ranges. After incubation of leaf epidermal tissue with SNARF-1, distinct alkaline (pH 8 to > or =9), red-spectrum (650 nm wave length) fluorescent zones developed as partial or complete halos around many fungal appressoria and in infection vesicles at 24 to 36 h after inoculation. In samples taken after 48 to 72 h, colonizing hyphae in the biotrophic phase and subsequently in the necrotrophic phase were also emitting the red fluorescence that extended into the surrounding host tissue, as also verified by depth analyses. Host epidermal cells were intact and apparently alive during the fungal alkalization process, with no visible disruption of cell structure. Generally, the pH of epidermal cells in noninoculated samples or in areas away from the infection in inoculated samples was lower than pH 7 with green (i.e., 500 to 550 nm wave length) fluorescence detected. Using standard electrodes, a significant increase in pH and ammonia concentration in leaf and fruit tissue was also measured but only at advanced stages of disease. In contrast, hyphae of the pathogen Alternaria alternata were mostly acidic and no change in fluorescence was found inside invaded host cells. The sequence of events in the C. acutatum-almond interaction includes penetration, production of ammonia by C. acutatum, and subsequent pH modulation within almond epidermal tissue to an alkaline environment that leads to further colonization of the host.

Comparison of Molecular Procedures for Detection and Identification of Guignardia citricarpa and G. mangiferae.

Citrus black spot, caused by Guignardia citricarpa, is a serious fruit spot disease and is widely distributed in Asia, southern Africa, and South America, but does not occur in North America or the Mediterranean region. A nonpathogenic species, G. mangiferae, is cosmopolitan with a wide host range and can colonize citrus fruit and leaves saprophytically. Detection and identification of Guignardia spp. on citrus fruit is necessary for epidemiological, management, and regulatory purposes. In this study, we compared published and unpublished polymerase chain reaction primer sets for their specificity and sensitivity in the detection and differentiation of the two Guignardia spp. All primers evaluated successfully identified the two species using purified DNA from fungal cultures or mycelia as source materials. However, some primer sets were not highly effective in detecting G. citricarpa when DNA was extracted directly from single characteristic black spot lesions on fruit. Thus, new primer pairs for both species were designed from the internal transcribed spacer region that were highly sensitive and specific for detection of G. citricarpa using DNA recovered from single lesions on fruit by a rapid DNA extraction procedure.

Postharvest Decay Management for Stone Fruit Crops in California Using the "Reduced-Risk" Fungicides Fludioxonil and Fenhexamid.

In laboratory studies and experimental packingline trials that closely simulated fungicide treatments under commercial conditions, the "reduced-risk" fungicides fenhexamid and fludioxonil were highly effective in managing postharvest brown rot and gray mold decays of peach, nectarine, and plum caused by Monilinia fructicola and Botrytis cinerea, respectively. Tebuconazole was more effective against brown rot than against gray mold. Additionally, fludioxonil and tebuconazole significantly reduced the incidence of Rhizopus rot caused by Rhizopus stolonifer. In laboratory studies, the QoI fungicide azoxystrobin was not consistent in its decay control activity. The high efficacy of fenhexamid and fludioxonil against brown rot and gray mold was substantiated by low effective concentrations necessary (≤0.063 mg/liter)for 50% inhibition of mycelial growth in vitro. In general, fungicides applied 14 to 16 h after wound inoculation were significantly more effective than those applied before inoculation. These results indicate that the fungicides act mainly as protectants that do not penetrate deeply enough into the fruit to prevent decay from wounds that extend below the fruit epidermis. Laboratory treatments of plum fruit generally were less effective than those of peach or nectarine fruit. Comparative studies using either low-volume spray or high-volume, in-line drench applications over a roller bed demonstrated that drench applications were significantly more effective in reducing postharvest decaysof inoculated plum fruit. Decay incidence using fenhexamid or fludioxonil was ≤1.1%, whereas incidence after the spray applications was between 25.2 and 40.4% for brown rot, between 12.0 and 24.3% for gray mold, or 62.6% for Rhizopus rot (fludioxonil only). This research identified effective replacements for iprodione, a fungicide voluntarily canceled by the manufacturer in 1996.

Effect of Fungicides and Storage Conditions on Postharvest Development of Citrus Black Spot and Survival of Guignardia citricarpa in Fruit Tissues.

Citrus black spot (CBS) is caused by Guignardia citricarpa, which incites lesions on citrus fruit and can induce fruit drop. Quiescent infections occur during the spring and summer, and symptoms appear at fruit maturity or after harvest. Thus, fruit from citrus areas affected by CBS represent a risk for introduction of this pathogen into new areas. The effects of preventive field fungicide programs, postharvest fungicide drenches, packinghouse fungicide applications, and storage temperatures on postharvest symptom development and viability of G. citricarpa in lesions were evaluated in five experiments on Murcott tangor, Valencia oranges, and lemons. Preventive field treatments and fruit storage at 8°C consistently reduced postharvest CBS development, whereas a postharvest fungicide drench or packinghouse treatment with fungicides had no effect on postharvest symptom development. In a separate experiment, postharvest appearance of symptoms was related to the percentage of fruit with symptoms at harvest. The preventive field fungicide program also consistently reduced the percentage of isolation of G. citricarpa from affected fruit, whereas storage temperature and packinghouse fungicide treatment gave variable results. The viability of the fungus declined with storage time of fruit after harvest, but G. citricarpa could still be readily isolated regardless of treatment. In another experiment, the viability of the fungus in detached fruit or peel was minimally affected by temperature or moisture during storage. The frequency of successful isolation declined with time, but G. citricarpa was still recovered frequently from symptomatic tissue at later times. The most effective means to reduce postharvest development of symptoms is through preventive application of fungicides during the fruit growing season and storage of harvested fruit at cold temperatures. None of the measures evaluated substantially reduced viability of G. citricarpa, and the pathogen would likely be introduced on symptomatic fruit from citrus areas with CBS.

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.