Verneta L. Gaskins

Draft Genome Sequence of Penicillium expansum Strain R19, Which Causes Postharvest Decay of Apple Fruit.

ABSTRACT Among the species that cause blue mold, isolates of Penicillium expansum are the most prevalent and virulent species, causing more than 50 percent of postharvest decay. We report the draft genome sequence of P. expansum R19 in order to identify fungal virulence factors and to understand the mechanism of infection.

Genome Sequence of Penicillium solitum RS1, Which Causes Postharvest Apple Decay

ABSTRACT Penicillium species cause postharvest decay, commonly known as blue mold, in pome fruits, such as apples and pears. To devise novel strategies to prevent and reduce economic losses during storage, the genome sequence of Penicillium solitum RS1 is reported here for the first time.

First Report of Botryosphaeria dothidea Causing White Rot on Apple Fruit in Maryland

Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not has a worldwide distribution infecting species from over 80 genera of plants (1). Apart from being an important pathogen of apple trees in many countries, B. dothidea can cause pre- and postharvest decay on apple fruit (2). It has been known to cause canker and dieback of forest trees in Serbia (3), but has not been recorded either on apple trees or apple fruit. In December 2010, apple fruit cv. Idared (Malus × domestica Borkh.) with symptoms of white rot were collected from one storage in the area of Svilajnac in Serbia. The incidence of the disease was low but the symptoms were severe. Affected fruit were brown, soft, and almost completely decayed, while the internal decayed tissue appeared watery and brown. A fungus was isolated from symptomatic tissue of one fruit after surface sterilization with 70% ethanol (without rinsing) and aseptic removal of the skin. Small fragments of decayed tissue were placed on potato dextrose agar (PDA) and incubated in a chamber at 22°C under alternating light and dark conditions (12/12 h). Fungal colonies were initially whitish, but started turning dark gray to black after 5 to 6 days. Pycnidia were produced after 20 to 25 days of incubation at 22°C and contained one-celled, elliptical, hyaline conidia. Conidia were 17.19 to 23.74 μm (mean 18.93) × 3.72 to 4.93 μm (mean 4.45) (n = 50). These morphological characteristics are in accordance with those described for the fungus B. dothidea (4). Genomic DNA was isolated from the fungus and internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The nucleotide sequence has been assigned to GenBank Accession No. KC994640. BLAST analysis of the 528-bp segment showed a 100% similarity with several sequences of B. dothidea deposited in NCBI GenBank, which confirmed morphological identification. Pathogenicity was tested by wound inoculation of five surface-sterilized, mature apple fruit cv. Idared with mycelium plugs (5 mm in diameter) of the isolate grown on PDA. Five control fruit were inoculated with sterile PDA plugs. After 5 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22°C, typical symptoms of white rot developed on inoculated fruit, while wounded, uninoculated, control fruit remained symptomless. The isolate recovered from symptomatic fruit showed the same morphological features as original isolate. To the best of our knowledge, this is the first report of B. dothidea on apple fruit in Serbia. Apple is widely grown in Serbia and it is important to further investigate the presence of this pathogen in apple storage, as well as in orchards since B. dothidea may cause rapid disease outbreaks that result in severe losses. References: (1) G. H. Hapting Agriculture Handbook 386, USDA, Forest Service, 1971. (2) A. L. Jones and H. S. Aldwinckle Compendium of Apple and Pear Diseases. APS Press, St. Paul, MN, 1990. (3) D. Karadžic et al. Glasnik Šumarskog Fakulteta 83:87, 2000. (4) B. Slippers et al. Mycologia 96:83, 2004.

Carbon, nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ∼7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.

Characterization of Postharvest Fungicide-Resistant Botrytis cinerea Isolates From Commercially Stored Apple Fruit

Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruit, vegetables, and ornamentals. Fludioxonil-sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had values for effective concentrations for inhibiting 50% of mycelial growth of 0.0004 to 0.0038 μg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sublethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multiresistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multiresistant B. cinerea strain caused decay on apple fruit treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multipostharvest fungicide-resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanisms to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil, which may pose control challenges in the packinghouse environment and during long-term storage.

Penicillium solitum produces a polygalacturonase isozyme in decayed Anjou pear fruit capable of macerating host tissue in vitro

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5–6, with an optimum at pH 4.5. PG activity was detectable 0–70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca2+, Mg2+, Mn2+ and Fe2+ and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.

Dominant Selectable Markers for Penicillium spp. Transformation and Gene Function Studies

Penicillium spp. has been genetically manipulated and gene function studies have utilized single gene deletion strains for phenotypic analysis. Fungal transformation experiments have relied on hygromycin and hygromycin phosphotransferase (hph) as the main dominant selectable marker (DSM) system in Penicillium spp. This poses a limitation on the number of loci that can be analyzed and complemented in reverse genetic studies. Additionally, many economically important Penicillium spp. have not been evaluated to determine the utility of additional chemicals that can serve as DSMs. Therefore, six compounds were examined for 15 blue mold strains and their Minimum Inhibitory Concentrations (MICs) determined. Phleomycin, neomycin and G418 were deemed ineffective, as Penicillium spp. growth was observed on media amended with 1000 μg/ml of each compound. The efficacy of bialophos to inhibit fungal growth was intermediate, with MICs ranging from 250 to 1000 μg/ml and was species-dependent. However, chlorimuron ethyl and benlate had the lowest MIC values and minimal variation in efficacy within and between species. Therefore, benlate and chlorimuron ethyl are good candidates for use as since corresponding fungal resistance genes have been cloned, characterized and are available from a variety of public and academic sources.