Fruit Cuticle Research Articles

Fruit-Surface Flavonoid Accumulation in Tomato Is Controlled by a SlMYB12-Regulated Transcriptional Network

The cuticle covering plants' aerial surfaces is a unique structure that plays a key role in organ development and protection against diverse stress conditions. A detailed analysis of the tomato colorless-peel y mutant was carried out in the framework of studying the outer surface of reproductive organs. The y mutant peel lacks the yellow flavonoid pigment naringenin chalcone, which has been suggested to influence the characteristics and function of the cuticular layer. Large-scale metabolic and transcript profiling revealed broad effects on both primary and secondary metabolism, related mostly to the biosynthesis of phenylpropanoids, particularly flavonoids. These were not restricted to the fruit or to a specific stage of its development and indicated that the y mutant phenotype is due to a mutation in a regulatory gene. Indeed, expression analyses specified three R2R3-MYB–type transcription factors that were significantly down-regulated in the y mutant fruit peel. One of these, SlMYB12, was mapped to the genomic region on tomato chromosome 1 previously shown to harbor the y mutation. Identification of an additional mutant allele that co-segregates with the colorless-peel trait, specific down-regulation of SlMYB12 and rescue of the y phenotype by overexpression of SlMYB12 on the mutant background, confirmed that a lesion in this regulator underlies the y phenotype. Hence, this work provides novel insight to the study of fleshy fruit cuticular structure and paves the way for the elucidation of the regulatory network that controls flavonoid accumulation in tomato fruit cuticle.

A new species of Zygophiala associated with the flyspeck complex on apple from China

Zygophiala qianensis is described as a new fungal species associated with the cuticle of apple fruit sampled from an orchard in Shaanxi Province, China. Conidiophores were separate, arising from superficial hyphae, erect, scattered, subcylindrical, irregularly flexuous, consisting of four parts: a hyaline supporting cell that gives rise to a smooth, dark brown stipe, terminating in a finely verruculose, medium brown apical cell that gives rise to (1–)2(–3) medium brown, finely verruculose, doliiform to ellipsoidal, polyblastic conidiogenous cells, with 1–2 prominent scars, apical and lateral, darkened, thickened. Conidia were solitary, fusiform to obclavate, hyaline, smooth and thick-walled, transversely (0–)1(–7)-septate, mostly 1–2-septate, prominently constricted at the septum; apex obtuse, base subtruncate, with a darkened, thickened hilum. Zygophiala qianensis is compared morphologically to other species of Zygophiala, and a phylogenetic analysis of their DNA sequence data is presented.

Phototransformation of imidacloprid on isolated tomato fruit cuticles and on tomato fruits

Imidacloprid, a neonicotinoid insecticide, is widely used in plant protection to prevent crop losses. The objective of this study was to show the photochemical fate of imidacloprid on plant surfaces by irradiation experiments on isolated tomato fruit cuticles and tomato fruits ( Lycopersicon esculentum Mill.). Imidacloprid spiked samples were irradiated both under sunlight simulator and natural sunlight conditions for 24 h, which resulted in recoveries of 23% and 24%, respectively, if isolated cuticles were studied. On whole tomato fruits, recoveries were 33% and 71%, respectively. Similar results were obtained on cuticles spiked with the formulation Confidor and irradiated under natural sunlight. However, on tomato fruits the application of Confidor resulted in a higher loss (56%) of imidacloprid. During all irradiation experiments both on cuticles and whole fruits, 1-[(6-chloropyridin-3-yl)methyl]imidazolidin-2-imine was generally formed at 10–14 mol%, but different other photoproducts were also detected in low amounts, whereas N-nitrosoimidacloprid was only detected under natural sunlight conditions. Rapid photodegradation of imidacloprid could be demonstrated in all experiments. The identified photoproducts, 1-[(6-chloropyridin-3-yl)methyl]imidazolidin-2-imine and N-nitrosoimidacloprid, are possible reaction partners for plant cuticle constituents to form cuticle bound residues.

Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95-98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.

Three‐dimensional imaging of plant cuticle architecture using confocal scanning laser microscopy

Full appreciation of the roles of the plant cuticle in numerous aspects of physiology and development requires a comprehensive understanding of its biosynthesis and deposition; however, much is still not known about cuticle structure, trafficking and assembly. To date, assessment of cuticle organization has been dominated by 2D imaging, using histochemical stains in conjunction with light and fluorescence microscopy. This strategy, while providing valuable information, has limitations because it attempts to describe a complex 3D structure in 2D. An imaging technique that could accurately resolve 3D architecture would provide valuable additions to the growing body of information on cuticle molecular biology and biochemistry. We present a novel application of 3D confocal scanning laser microscopy for visualizing the architecture, deposition patterns and micro-structure of plant cuticles, using the fluorescent stain auramine O. We demonstrate the utility of this technique by contrasting the fruit cuticle of wild-type tomato (Solanum lycopersicum cv. M82) with those of cutin-deficient mutants. We also introduce 3D cuticle modeling based on reconstruction of serial optical sections, and describe its use in identification of several previously unreported features of the tomato fruit cuticle.

Spatially resolved quantification of agrochemicals on plant surfaces using energy dispersive X‐ray microanalysis

In the present study the principle of energy dispersive X-ray microanalysis (EDX), i.e. the detection of elements based on their characteristic X-rays, was used to localise and quantify organic and inorganic pesticides on enzymatically isolated fruit cuticles. Pesticides could be discriminated from the plant surface because of their distinctive elemental composition. Findings confirm the close relation between net intensity (NI) and area covered by the active ingredient (AI area). Using wide and narrow concentration ranges of glyphosate and glufosinate, respectively, results showed that quantification of AI requires the selection of appropriate regression equations while considering NI, peak-to-background (P/B) ratio, and AI area. The use of selected internal standards (ISs) such as Ca(NO(3))(2) improved the accuracy of the quantification slightly but led to the formation of particular, non-typical microstructured deposits. The suitability of SEM-EDX as a general technique to quantify pesticides was evaluated additionally on 14 agrochemicals applied at diluted or regular concentration. Among the pesticides tested, spatial localisation and quantification of AI amount could be done for inorganic copper and sulfur as well for the organic agrochemicals glyphosate, glufosinate, bromoxynil and mancozeb.

Sorption and Interaction of the Flavonoid Naringenin on Tomato Fruit Cuticles

The flavonoid naringenin accumulates in tomato fruit epidermis during ripening. The sorption of this flavonoid to enzymatically isolated cuticles of Solanum lycopersicum was studied as a function of the temperature and naringenin concentration at two stages of fruit growth. The selected stages were mature green, without flavonoids in the cuticle, and ripe tomato, with significant amounts of flavonoids in the cuticle. Sorption isotherms showed different behaviors that could be explained in terms of different affinities of the sorbed flavonoid for the cuticular matrix. The partition coefficient of naringenin in the system cuticle/water solution was a function of temperature and concentration. Changes in the free energy, enthalpy, and entropy for the phase transfer of naringenin to cuticle were also calculated, indicating the existence of naringenin-naringenin interactions replacing naringenin-cuticular matrix interactions at high concentrations with the final result of solid precipitations in the form of clusters within the cutin matrix.

Morphological characteristics of leaf epidermis and size variation of leaf, flower and fruit in different ploidy levels in Buddleja macrostachya (Buddlejaceae)

Abstract Buddleja macrostachya (Buddlejaceae) is a widespread shrub native to the Sino‐Himalayan mountains and beyond. It has been found to occur at two ploidy levels, hexaploid, 2n=6x=114 and dodecaploid, 2n=12x=228. To determine if morphological characters might be used as indicators of ploidy levels, we measured floral and fruit length, relative and absolute leaf size, trichome density on both leaf surfaces, and stomatal density and length in different populations of B. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length increased with an increase at ploidy level (P<0.01), whereas adaxial cell and stomatal density decreased with an increase at ploidy level (P<0.01). We found no conspicuous differences in relative leaf size (P>0.05) in different populations. Other characters studied such as trichome type, cuticular membrane and ornamentation of stomata, cell and stomatal shape, and anticlinal wall pattern were quite constant in this species. Thus it appears that flower and fruit length, absolute leaf size, and stomatal frequency and length can be used to distinguish hexaploid from dodecaploid cytotypes either in the field or in herbarium specimens.

Surfactant Effects on the Affinity of Plant Cuticles with Organic Pollutants

To precisely predict organics accumulation and crop safety, the affinity of fruit cuticles for naphthalene and 1-naphthol was investigated with the presence of three surfactants below and above the critical micelle concentration (CMC), including anionic sodium dodecylbenzene sulfonate (SDBS), cationic cetyltrimethylammonium bromide (CTMAB), and nonionic polyoxyethylene (20) sorbitan monolaurate (Tween 20). Tomato and apple cuticles with distinct compositions were selected. With increasing SDBS concentrations, apparent sorption coefficients (K(d)*) of 1-naphthol by both cuticles first increased a bit and then decreased slightly. The K(d)* of naphthalene by tomato cuticle is sensitive to SDBS concentration with a sharp increase and then decrease, whereas SDBS has little effect on naphthalene K(d)* by apple cuticle. For CTMAB with lower CMC, the naphthalene K(d)* decreased more quickly. Tween 20 seems to be ineffective on naphthalene sorption by both cuticles. Nevertheless, the intrinsic sorption coefficients (K(d)) were almost promoted by the coexisting surfactants, resulting from the cuticle-sorbed surfactant's plasticizing effect.

Biomechanics of isolated tomato (Solanum lycopersicum) fruit cuticles during ripening: the role of flavonoids.

Flavonoids accumulate in tomato (Solanum lycopersicum L.) fruit cuticles during ripening. Their quantitative contribution to the biomechanical properties of the cuticle is studied in six tomato genotypes which show presence or absence of these compounds at the red ripe stage of fruit development. Tomato cuticles with flavonoids at red ripe showed a dramatic increase of these compounds between mature green and red ripe stages together with a significant increase in the elastic modulus. On the other hand, cuticles without flavonoids displayed a similar biomechanical behaviour at mature green and red ripe stages. The absence of flavonoids could also be related with a predominance of the viscoelastic performance of the cuticle. Thus, the increase of phenolics in tomato fruit cuticles during ripening is correlated with a more rigid cutin network that reinforces the mechanical function of polysaccharides which tend to diminish at this stage due to cell wall disassembly. A role of phenolics as biomechanical modulators of the cuticle behaviour is proposed.

Application of GC-MS for the detection of lipophilic compounds in diverse plant tissues.

BackgroundThe concept of metabolite profiling has been around for decades and technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. However, studies are generally confined to polar compounds alone. Here we describe a simple method for lipophilic compounds analysis in various plant tissues.ResultsWe choose the same preparative and instrumental platform for lipophilic profiling as that we routinely use for polar metabolites measurements. The method was validated in terms of linearity, carryover, reproducibility and recovery rates, as well as using various plant tissues.As a first case study we present metabolic profiling of Arabidopsis root and shoot tissue of wild type (C24) and mutant (rsr4-1) plants deficient on vitamin B6. We found significant alterations in lipid constituent contents, especially in the roots, which were characterised by dramatic increases in several fatty acids, thus providing further hint for the role of pyridoxine in oxidative stress and lipid peroxidation.The second example is the lipophilic profiling of red and green tomato fruit cuticles of wild type (Alisa Craig) and the DFD (delayed fruit deterioration) mutant, which we compared and contrasted with the more focused wax analysis of these plants reported before.ConclusionWe can rapidly and reliably detect and quantify over 40 lipophilic metabolites including fatty acids, fatty alcohols, alkanes, sterols and tocopherols. The method presented here affords a simple and rapid, yet robust complement to previously validated methods of polar metabolite profiling by gas-chromatography mass-spectrometry.

Phenanthrene Sorption by Fruit Cuticles and Potato Periderm with Different Compositional Characteristics

Phenanthrene sorption by fruit cuticles (tomato, apple, and grape), potato tuber periderm, and their fractions were investigated to elucidate effects of compositional characteristics on affinity of plant cuticle (above-ground) and periderm (below-ground) with hydrophobic organic contaminants (HOCs). The distinct roles of the extractable lipids (waxes), the depolymerizable lipids (cutin/suberin), the nonsaponifiable lipids (cutan/suberan) and polysaccharide are discussed. The cutin/suberin rather than waxes dominates the sorption properties of bulk cuticle/periderm, but the sorption coefficient (K(d)) is linearly correlated with the total lipid contents. Polysaccharide plays a negative role in HOC sorption due to its obviously poor affinity with HOCs and restriction effect on the other powerful sorptive regions in cuticle/periderm. The significantly lower sorption of periderm than cuticle is attributed to the former having higher polysaccharide and lower depolymerizable lipids. The linear correlation of K(d) of bulk cuticle/periderm with polysaccharide content is observed for a potential prediction of plant uptake.

Cuticular calcium penetration is directly related to the area covered by calcium within droplet spread area

Calcium penetration from salt solutions of calcium chloride and calcium acetate was measured through isolated tomato fruit ( Solanum lycopersicum L., cultivar ‘Panovy’) cuticles after 4, 24, and 48 h. Droplet spread area (volume = 1 μL) and the area ultimately covered with calcium were determined with a scanning electron microscope which has an integrated energy dispersive X-ray microanalysis system. An ethoxylated rapeseed oil surfactant (RSO 5) was used for inducing different degrees of droplet spread and together with different calcium concentrations of the solutions to vary the concentration gradient in the penetration experiments. As a rule, penetration of calcium from CaCl 2 was higher than that of Ca(CH 3COO) 2. The addition of RSO 5 to salt solutions resulted in enhanced calcium penetration in comparison to unformulated solutions. However, extending penetration time from 4 to 48 h predominantly did not enhance the penetrated calcium amount significantly. Droplet spread area was enhanced up to 4-fold after adding RSO 5, whereas the area ultimately covered with calcium did not exceed twice as much. This resulted in lower percentage area covered by calcium in droplet footprints from formulated solutions in comparison to solutions without addition of RSO 5. Correlation analyses showed that penetration was not related to droplet spread area but significantly correlated with the area covered by calcium. These observations were true for unformulated calcium salts as well as for calcium solutions with addition of RSO 5. Therefore, the area ultimately covered with calcium or even other xenobiotics within a droplet footprint should be considered in developing mathematical models to simulate active ingredient (a.i.) penetration.

만수 품종 배의 과피 갈변 원인 구명

This research was carried out to elucidate the causes of fruit skin browning in 'Mansu' pear for the last 2 years. It was observed that skin browning was induced even by the small mechanical injury produced during grading and packing for the market supplies after harvest on the fruits of 'Mansu' pear. The incidences of fruit skin browning in pears treated with artificial mechanical injuries were investigated between 'Niitaka' and 'Mansu' pears. The results showed that fruits of 'Mansu' are more susceptible to skin browning than those of 'Niitaka', We also found that the epidermis of fruits in 'Mansu' pear was thinner than that of 'Niitaka', and that there was lower incidence of fruit browning in epidermis of pears with high chlorophyll content than those with low chlorophyll content. The skin browning in fruits could be considerably reduced by sorting and grading them wrapped with paper bags for the fruits of 'Mansu' pear.

Applicability of the energy dispersive x‐ray microanalysis for quantification of irregular calcium deposits on fruit and leaf cuticles

In our studies, we evaluated the relation between CaCl(2) concentration and the scanning electron microscope-energy dispersive x-ray microanalysis parameters, net intensity, peak/background and standardized percentage of atoms and percentage of weight after application of single microdroplets on enzymatically isolated tomato fruit cuticles and apple seedling leaves. After analysis, the Ca/Cl ratio was calculated and the area ultimately covered with Ca and Cl within the droplet spread area determined. According to our findings, all evaluated parameters were influenced by both droplet volume and calcium chloride concentration, whereas Pearson's analysis revealed a strong correlation between net intensity and area ultimately covered by Ca or Cl, respectively. Simple linear regressions for net intensity, peak/background and Ca/Cl ratio showed variable determination coefficients (R(2)) ranging between 0.49 and 0.79. Multiple regression equations comprising net intensity, Ca/Cl ratio and deposit area were established to estimate the amount of calcium present on the cuticles. Equation slopes depended on droplet volumes, with determination coefficients of 0.89 and 0.81 for 0.5 and 1.0-microL droplets, respectively. The importance of the physicochemical properties of the spray solution was exploited in another study where a hydrophobic or a hydrophilic adjuvant was added to a 10 g L(-1) CaCl(2) solution, which was applied to apple seedling leaves. The addition of adjuvants increased values of net intensity and peak/background, which correlated significantly with the area covered by calcium. The importance of the methodology for studies on the interaction between leaf applied fertilizers (also extended to agrochemicals) and the characteristics of target surfaces is discussed.

NMR characterization of hydration and thermal stress in tomato fruit cuticles

In its natural environment, the plant cuticle, which is composed of the biopolymer cutin and a mixture of surface and embedded cuticular waxes, experiences a wide variety of temperatures and hydration states. Consequently, a complete understanding of cuticular function requires study of its thermal and mechanical properties as a function of hydration. Herein, we report the results of a comprehensive 13C nuclear magnetic resonance (NMR) relaxation study of hydrated tomato fruit cuticle. Cross-polarization and direct-polarization experiments serve to measure the solid-like and liquid-like components, respectively, of hydrated cuticle. Localized, high-frequency motions are probed by T 1(C) spin relaxation measurements, whereas T 1ρ(H) and T 1ρ(C) experiments reflect low-frequency, lower amplitude polymer-chain motions. In addition, variable-temperature measurements of T 1(C) and T 1ρ(C) for dry tomato cuticles are used to evaluate the impact of temperature stress. Results of these experiments are interpreted in terms of changes occurring in individual polymer motions of the cutin/wax components of tomato cuticle and in the interaction of these components within intact cuticle, both of which are expected to influence the functional integrity of this protective plant covering.

Polyphenol oxidase and its relationship with oleuropein concentration in fruits and leaves of olive (Olea europaea) cv. 'Picual' trees during fruit ripening

Oleuropein, the main phenolic compound of olive fruit, has important antioxidant properties that are responsible for some of the nutritional properties of fruits and the defence mechanism of leaves. Polyphenol oxidase (PPO) activity changes during fruit ripening in many plants. We studied the kinetics and molecular properties of PPO in fruits and leaves of olive (Olea europaea L.) cv. 'Picual' trees and the relationship between PPO and oleuropein concentration during fruit ripening. Polyphenol oxidase showed hyperbolic kinetics in fruits and leaves. Significant increases in PPO specific activity, V(max), K(m )and catalytic efficiency occurred during fruit ripening. Based on SDS-PAGE under partially denaturing conditions and in-gel staining with DL-3,4-dihydroxyphenylalanine, PPO activity was found in one major protein of 55 and 50 kDA in fruits and leaves, respectively. During the last stages of fruit maturation, a second 36 kDa protein was observed in fruits but not in leaves, indicating that this protein could serve as a marker of the final phase of fruit maturation. Under fully denaturing conditions, only one 27.7 kDa immunoreactive band was detected in fruits. Both the amount of PPO activity and the amount of PPO protein increased significantly during fruit maturation. Immunohistochemical studies indicated that PPO is located in the epidermis, parenchyma and companion vascular cells of leaves as well as in the epidermis of fruit. During fruit maturation, oleuropein concentration measured by HPLC significantly decreased in fruits and increased in leaves.

Influência do plástico branco, poda verde e amino quelant®-K na qualidade de pêssegos 'Santa Áurea'

A qualidade das frutas é determinada por fatores relacionados ao clima, solo, cultivar, as práticas culturais adotadas, época e forma de colheita e processos de manipulação da fruta na pós-colheita. Uma das formas de melhorar esta qualidade é a utilização de práticas de manejo na pré-colheita. Assim, este trabalho teve por objetivo avaliar o efeito do plástico branco, da poda verde e do nutriente aminoquelant®-K, associado ou não ao tratamento com plástico branco e poda verde, na qualidade de pêssegos 'Santa Áurea'. Os atributos de qualidade avaliados foram: percentagem visual de vermelho, intensidade de vermelho na epiderme, determinada pelo sistema CIEL*a*b*, massa, diâmetro, firmeza de polpa, sólidos solúveis totais (SST), acidez total titulável (ATT) e relação SST/ATT. As práticas utilizadas causaram o aumento do percentual de vermelho na epiderme das frutas; o aminoquelant®-K isolado proporcionou maior acidez das frutas; a melhor relação SST/ATT foi obtida com o uso do plástico branco sob a copa e pela combinação do plástico com o nutriente aminoquelant®-K.

Development of fruit cuticle in cherry tomato (Solanum lycopersicum).

The cuticle of a plant plays an important role in many physiological events of fruit development and ripening. Despite this, little is known about cuticle formation and development. We include a detailed morphological study at the microscopic level of cuticle during fruit growth and ripening using tomato as a fruit model. In addition, a study of the differences in cuticle thickness and composition during development is included. The four genotypes studied in this work showed a similar timing of the main morphological events: initiation of epidermal differentiation, changes in the distribution of the lipid, pectin and cellulose material within the cuticle, appearance of pegs, beginning of cuticle invaginations, maximum thickness and loss of polysaccharidic material. Fruit growth, measured by fruit diameter, showed a positive correlation with the increase of cuticle thickness and the amount of cuticle and their cutin and polysaccharide components per fruit unit during development. By contrast, cuticle waxes showed a different behaviour. Two important characteristics of cuticle growth were observed during tomato fruit development. First, the amount of cuticle per surface area reached its maximum in the first 15 days after anthesis and remained more or less constant until ripening. Second, there was a significant loss of polysaccharidic material from the beginning of ripening (breaker stage) to full red ripe.

First Report of Five Sooty Blotch and Flyspeck Fungi on Prunus americana in the United States

The sooty blotch and flyspeck (SBFS) complex includes more than 30 fungi that blemish the cuticle of apple fruit, causing economic losses in humid regions worldwide (1). In August 2005, we sampled SBFS-infested wild plum (Prunus americana) fruit growing in hedgerows in Iowa. Colonies were categorized according to mycelial type (1), and isolates were made from representative colonies onto acidified water agar (AWA). Plum skins with SBFS signs were excised, pressed, and photographed. DNA was obtained from purified isolates and also from mycelium and fruiting bodies scraped directly from plum fruit skins. Extracted DNA was amplified using primer pair ITS1-F/Myc1-R (ACTCGTCGAAGGAGCTACG) and PCR products were sequenced using primer pair ITS-1F/ITS4. Six sequences were obtained from pure cultures and seven from colonies on plum fruit skin. BLAST analysis of the 470-bp sequences showed 100% homology to five known species in the SBFS complex: Zygophiala cryptogama, Zygophiala wisconsinensis, Pseudocercosporella sp. RH1, and Stomiopeltis spp. RS1 and RS2 (GenBank Accession Nos. AY598854, AY598853, AY5988645, AY598882, and AY598883, respectively). Observations of colony and fruiting structure morphology from cultures on potato dextrose agar (PDA) and colonies on plums confirmed species identity. A modified version of Koch's postulates was conducted to verify that these fungi caused the signs observed on plum and could also infest apple fruit. In June 2006, 1-month-old cultures on PDA were pulverized in a blender with sterile distilled water, passed through four layers of sterile cheesecloth, and transferred to sterile jars. Each isolate was inoculated onto 20 fruit on plum trees (P. americana) on the Iowa State University (ISU) campus and 20 fruit on cv. Golden Delicious apple trees at the ISU Research Station, Gilbert, IA. Each fruit was disinfested with 70% ethanol, air dried, swabbed with inoculum, and covered with a Fuji bag. At harvest, fungal colonies on fruit were reisolated onto AWA. DNA was extracted from pure cultures; when isolations on agar were unsuccessful, DNA was extracted directly from colonies on fruit. PCR was conducted using ITS1-F/Myc1-R, and PCR products were sequenced using ITS1-F/ITS4. All five species were reisolated and sequenced from apple. Pseudocercosporella sp. RH1 and Stomiopeltis sp. RS1 were sequenced from inoculated plums. Although flyspeck, presumably caused by Schizothyrium pomi, was reported on Japanese plum (P. salicina) in Japan (2) and black cherry (P. serotina) in the United States (3), to our knowledge this is the first report of SBFS fungi on plum in the United States and the first confirmation that fungi from plum can produce SBFS signs on apple fruit. Wild plum may therefore act as a reservoir host, providing inoculum for SBFS infestations on apple.