Methodology for Determining Relationships Between Inoculum Concentration ofBotrytis cinereaandPenicillium expansumand Stem End Decay of Pear Fruit

Abstract

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.

Detection of Botrytis cinerea by loop-mediated isothermal amplification

“…However, assays targeting β‐tubulin and cutinase A have been observed or predicted to cross‐react with B. fabae (Suarez et al. 2005; Spotts et al. 2008).…”

“…2009). Knowledge of pathogen levels can help to inform production and storage decisions (Spotts et al. 2008), and quantitative methods also allow periods of active colonization to be distinguished from periods of quiescence (Cadle‐Davidson 2008).…”

A real-time PCR assay for detection and quantification of Botrytis cinerea in Pelargonium x hortorum plants, and its use for evaluation of plant resistance

“…A number of nucleic-acid-based methods for the detection of B. cinerea in plants are considered highly specific, although they have not been screened for their specificity against nontarget Botrytis species (Brouwer et al 2003;Gachon and Saindrenan 2004;Mehli et al 2005;CadleDavidson 2008;Celik et al 2009;Sanzani et al 2012). In addition, none of the methods described that have considered nontarget Botrytis species discriminated B. cinerea from the phylogenetically related species B. pelargonii or B. fabae, which cause leaf-base necrosis in Pelargonium and chocolate spot in broad bean, respectively (Røed 1949;Suarez et al 2005;Spotts et al 2008;Tomlinson et al 2010). As B. fabae does not attack pelargonium plants (data not shown) and B. pelargonii has never been reported to attack pelargoniums since the description of Røed (1949), we believe that it would be unlikely to detect these species in pelargonium samples.…”

“…Furthermore, the accuracy of the qPCR assay developed is due to the normalization of the fungal DNA with the plant DNA abundance, as revealed through the amplification of the β-actin gene. When the fungal DNA is expressed on the basis of the plant surface area or fresh weight (Brouwer et al 2003;Spotts et al 2008), the validity of the assay can be strongly affected by extensive host tissue damage and variable DNA extraction yields among samples. Leaf cell destruction caused by necrotrophic colonization of B. cinerea might lead to an overestimation of the fungal biomass if these data are not normalized to the plant DNA.…”

Gray Mold on Carrot Caused by Botrytis cinerea in Korea

“…To identify the isolates, the β-tubulin gene was amplified with the Bcin-366r and BT-2M-up primers (Spotts et al, 2008) and directly sequenced. The sequence was compared with data in GenBank (Accession No.…”