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.