DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple.

The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.

Methodology for Determining Relationships Between Inoculum Concentration of Botrytis cinerea and Penicillium expansum and Stem End Decay of Pear Fruit.

The objective of this research was to determine quantitative relationships between incidence of stem end decay of pear fruit and inoculum concentration of Botrytis cinerea and Penicillium expansum using dry conidia applied to pear fruit in a settling tower. Five concentrations of conidia were applied to pear fruit, fruit were stored at -1°C for 8 months, and stem end decay was evaluated. In addition, conidia were washed from the surface of inoculated fruit, and DNA was extracted and quantified with real-time polymerase chain reaction (PCR). The linear regression relationships between percent stem end gray mold and B. cinerea conidia per liter of air or per square centimeter of fruit surface were significant (P = 0.01). At the highest inoculum dose introduced into the settling tower, conidia per liter of air, conidia per square centimeter, and percent stem end gray mold at 8 months after inoculation were 12, 31, and 39, respectively for 2000 and 6, 33, and 67, respectively for 2001. Similarly, the linear regression relationships between percent stem end blue mold and P. expansum conidia per liter of air or per square centimeter of fruit surface were significant (P = 0.01 and 0.05, respectively). At the highest inoculum dose introduced into the settling tower, conidia per square centimeter and percent stem end blue mold at 8 months after inoculation were 39 and 26, respectively for 2000 and 66 and 23, respectively for 2003. Real-time PCR provided a rapid, quantitative measure of B. cinerea and P. expansum DNA on pear fruit surfaces. Because of possible year-to-year shifts in susceptibility of fruit to decay, disease incidence:inoculum dose relationships may be of most value compared within years rather than across years. This would facilitate comparison of decay risk among orchards in order to determine which fruit is most suitable for long-term storage.

Biological control of apple blue mold with Pseudomonas fluorescens.

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.

Effect of Preharvest Application of Cyprodinil on Postharvest Decay of Apples Caused by Botrytis cinerea.

Dry-eye rot and gray mold of apple are important diseases caused by Botrytis cinerea. Fungicides available for their control are lacking, and this study was conducted to determine if cyprodinil (Vangard) could be used for this purpose. The mean EC50 value of cyprodinil for 32 Botrytis spp. isolates (27 from apple) was 0.02 µg ml-l, indicating that apple isolates are generally very sensitive. Some of the isolates (19%) were less sensitive and had EC50 values greater than 0.03 µg ml-l, and one isolate from 'Gala' apple was considerably less sensitive at 0.095 µg ml-l. Bloom sprays of cyprodinil alone in 1998 and 1999 or in combination with myclobutanil or metiram in 1998 reduced Botrytis spp. infection on developing fruit. Postharvest application of cyprodinil in 1998 indicated that cyprodinil protected apples from gray mold for 3 months. Cyprodinil applied 2 to 3 weeks before harvest in 1999 reduced lesion diameters 68 and 62% on 'Jonagold' and 'Gala' apples, respectively, that had been wounded and inoculated with B. cinerea after storage at 1°C for 6 months. In similar trials on 'Gala' apples in 2000 and 2001, preharvest applications of cyprodinil consistently reduced gray mold incidence and lesion diameter on inoculated apples stored for 6 months. New preharvest use patterns for cyprodinil are discussed for control of postharvest diseases caused by B. cinerea.

Antimicrobial Activity of Gaseous Allyl Isothiocyanate.

A simple model system was constructed to evaluate the microbistatic and microbicidal properties of gaseous allyl isothiocyanate (AIT) against bacterial cells and fungal conidia deposited on agar surfaces. Salmonella typhimurium , Listeria monocytogenes Scott A, and Escherichia coli O157:H7 were inhibited when exposed to 1,000 µg AIT per liter. Pseudomonas corrugata , a Cytophaga species, and a fluorescent pseudomonad failed to grow in the presence of 500 µg AIT per liter. Germination and growth of Penicillium expansum , Aspergillus flavus , and Botrytis cinerea conidia was inhibited in the presence of 100 µg AIT per liter. Bactericidal and sporicidal activities varied with strain and increased with time of exposure, AIT concentration, and temperature. E. coli O157:H7 was the most resistant bacterial species tested.