C. L. Xiao

Development of PCR Assays for Diagnosis and Detection of the Pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in Apple Fruit

Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple. Infection by these two pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to those of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important during the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is an important step to implement relevant pre- and postharvest measures for disease control. The aim of this study was to develop polymerase chain reaction (PCR) assays for diagnosis and early detection of latent infections of apple fruit by P. washingtonensis and S. pyriputrescens. Species-specific primers based on the ribosomal DNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit inoculated with P. washingtonensis, S. pyriputrescens, or B. cinerea and compared with identifications using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 h of processing time, compared with 5 to 6 days using culture-based methods. Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.

Occurrence and Phenotypes of Pyrimethanil Resistance in Penicillium expansum from Apple in Washington State

Penicillium expansum is the cause of blue mold in stored apple fruit. In 2010-11, 779 isolates of P. expansum were collected from decayed apple fruit from five packinghouses, tested for resistance to the postharvest fungicide pyrimethanil, and phenotyped based on the level of resistance. In 2010, 85 and 7% of the isolates were resistant to pyrimethanil in packinghouse A and B, respectively, where pyrimethanil had been used for four to five consecutive years. In 2011, pyrimethanil or fludioxonil was used in packinghouse A, and 96% of the isolates from the fruit treated with pyrimethanil were resistant but only 4% of the isolates from the fruit treated with fludioxonil were resistant to pyrimethanil, suggesting that fungicide rotation substantially reduced the frequency of pyrimethanil resistance. No pyrimethanil-resistant isolates were detected in 2010 in the three other packinghouses where the fungicide had been used recently on a small scale. However 1.8% of the isolates from one of the three packinghouses in 2011 were resistant to pyrimethanil. A significantly higher percentage of thiabendazole-resistant than thiabendazole-sensitive isolates were resistant to pyrimethanil. Of the pyrimethanil-resistant isolates, 37 to 52, 4 to 5, and 44 to 58% were phenotyped as having low, moderate, and high resistance to pyrimethanil, respectively. Fludioxonil effectively controlled pyrimethanil-resistant phenotypes on apple fruit but pyrimethanil failed to control phenotypes with moderate or high resistance to pyrimethanil and only partially controlled the low-resistance phenotype.

Occurrence of Fludioxonil Resistance in Penicillium digitatum from Citrus in California

Penicillium digitatum is the causal agent of green mold, one of the most important postharvest diseases of citrus (Citrus spp.). Fludioxonil can be used alone or in combination with azoxystrobin for the control of green mold and other postharvest diseases on citrus. Baseline sensitivity to fludioxonil in P. digitatum populations from California citrus packinghouses has been previously established (Kanetis et al. 2008). To monitor resistance to fludioxonil in P. digitatum, 20 Penicillium spp. isolates were obtained from decayed oranges by isolation from decayed tissue and 22 isolates were recovered from potato dextrose agar plates amended with 0.5 mg/liter fludioxonil (Kanetis et al. 2006) and exposed to packinghouse air for 5 min each sampling time during March to July 2013 in two California citrus packinghouses. All isolates were single-spore cultured and identified as P. digitatum based on morphological characters (Pitt 1979). The sequence analysis of the internal transcribed spacer (ITS) region, using the primers ITS1/ITS4, was conducted to confirm the species-level identification. A MegaBLAST search showed that the sequences of all isolates had 99% homology (E-value = 0.0) with that of P. digitatum deposited at GenBank (Accession No. AF033471.1). In a mycelial growth assay, 15 of the 42 P. digitatum isolates were able to grow at 0.5 μg/ml, a previously established discriminatory concentration (Kanetic et al. 2006). EC50 values (the effective concentration that inhibits fungal growth by 50% relative to the control) of fludioxonil for the resistant isolates were >100 mg/liter following a method described by Li and Xiao (2008). To assess whether fludioxonil at the label rate was able to control fludioxonilresistant isolates, ‘Eureka’ lemons were wounded with a stainless steel rod (1 × 2 mm) and inoculated with 10 μl of conidial suspensions (6 × 10 conidia/ml) of a representative sensitive or a resistant isolate. After 4 h, inoculated fruit were dipped for 30 s in either a formulated product of fludioxonil, Graduate (50% active ingredient; Syngenta Crop Protection, Research Triangle Park, Raleigh, NC) at 1,198 mg/liter, or water as a control, and then stored at 20°C in air for 7 days. There were four 20-fruit replicates for each treatment and the experiment was performed twice. All inoculated water-dipped fruit were decayed, while fludioxonil completely controlled green mold on fruit inoculated with the fludioxonil-sensitive isolate. However, 100% fruit inoculated with the fludioxonil-resistant isolate and treated with fludioxonil were decayed. Fludioxonil-insensitive isolates have been reported to occur in natural populations of P. digitatum before its commercial use, but at very low frequency (1.4 to 2.5 × 10) (Kanetis et al. 2006). This is the first report of fludioxonil resistance in P. digitatum collected from commercial citrus packinghouses after the introduction of the fungicide on the market. These fludioxonil-resistant isolates were obtained from packinghouses where the fungicide had been used during citrus packing for 2 consecutive years, approximately for a 3-month period in each year, indicating that fludioxonil-resistant individuals emerged quickly in P. digitatum populations. Our results show that fludioxonil failed to control green mold incited by the fludioxonil-resistant isolate, suggesting that appropriate fungicide resistance management practices need to be implemented to ensure adequate control.

First Report of a New Leaf Blight Caused by Phacidiopycnis washingtonensis on Pacific Madrone in Western Washington and Oregon

In recent years, a leaf blight disease, consisting of browned, desiccated leaves occurring mainly in the lower parts of the canopy, has been observed during wet springs on Pacific madrone (Arbutus menziesii) in western Washington and Oregon. In May 2009 and 2011, severe outbreaks occurred and symptomatic leaves from madrones growing in the region were sampled to determine the causal agent. Two symptoms, leaf necrosis or blotching along the edges and tips of the leaves, and leaf spot, were observed. Small segments of diseased tissue were cut from the leaves, surface-disinfected, rinsed, and plated on malt extract agar. Fifty percent of the leaf blotch and 30% of leaf spot samples yielded a fungus that was fast-growing (20 mm diameter in 4 days at 25°C) and produced colonies that were a pale gray with dark gray reverse and a felty texture. On potato dextrose agar (PDA), pycnidia formed and exuded conidia in peach-colored droplets after 2 weeks under room temperature and light conditions. Pycnidia were spherical and 12.5 to 39.8 μm, average 24.2 μm in diameter. Conidia were hyaline, ovoid, and 5.8 to 8.5 × 3.1 to 4.7 μm (average 7.0 × 3.7 μm). The fungus was identified as Phacidiopycnis washingtonensis based on its morphology (1). To confirm the identity, the internal transcribed spacer (ITS) region of the rDNA was amplified with ITS1/ITS4 primers (2) and sequenced (GenBank Accession Nos. JQ743784 to 86). BLAST analysis showed 100% nucleotide identity with those of P. washingtonensis in GenBank (AY608648). The fungus was also isolated from lesions on green shoots and the petiole and leaf blade of dead attached leaves. To test pathogenicity, 3-year-old Pacific madrone seedlings (three for each isolate) were inoculated with five isolates of the fungus and maintained in the greenhouse (25°C); the experiment was conducted twice. Five leaves from each tree were cold injured (-50°C) at a marked 5 × 5 mm2 area with a commercial aerosol tissue freezing product prior to inoculation and five leaves were not cold injured. A 5-mm-diameter mycelial plug cut from the margin of 6-day-old PDA culture was applied to the marked areas on the upper leaf surface. The inoculated area was covered with moist cheese cloth and wrapped with Parafilm. Leaves treated with blank PDA plugs served as control. Leaves were enclosed in plastic bags to maintain moisture for the first 15 h post inoculation and cheese cloths were removed after 15 days. All cold-injured inoculated leaves showed symptoms of blight starting at 2 weeks after inoculation, and no symptoms appeared on the controls. On non-cold injured inoculated leaves, only one isolate caused symptoms (80% of all leaves). The fungus was re-isolated from diseased leaves. These results suggest that P. washingtonensis is able to cause foliar blight on Pacific madrone when leaves are subjected to cold stress. Increased disease severity on madrone observed in spring 2011 in Washington and Oregon may have been due to predisposition of foliage to extreme cold in November 2010 and February 2011. This fungus has previously been reported to cause a postharvest fruit rot disease on apple fruit and a canker and twig dieback disease of apple and crabapple trees in WA (1). To our knowledge, this is the first report of P. washingtonensis causing a leaf blight disease on Pacific madrone in North America. References: (1) C. L. Xiao et al. Mycologia 97:464, 2005. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Control of bull’s-eye rot of apple caused by Neofabraea perennans and Neofabraea kienholzii using pre- and postharvest fungicides

Bulls-eye rot is a major postharvest disease of apple caused by several fungi belonging to the Neofabraea and Phlyctema genera. Chemical control of these fungi is a crucial component of disease management for apples that are conventionally grown. The efficacy of several preharvest and postharvest applied fungicides were evaluated to identify effective chemistries that can control bulls-eye rot incited by Neofabraea perennans and N. kienholzii on apples. In general, the preharvest fungicide thiophanate-methyl was found to be effective at reducing disease caused by N. perennans and N. kienholzii. Two postharvest fungicides, thiabendazole and pyrimethanil, also provided disease control that was far superior to other chemical compounds evaluated in this study. The efficacy of thiabendazole and pyrimethanil was unaffected by application method (fungicide dip compared with thermofog). Despite providing satisfactory control of bulls-eye rot, integration of these three chemicals into disease management programs should proceed judiciously with consideration of their impact on the development of fungicide resistance and influence on diversity in populations of apple postharvest pathogens.