Decayed Apple Fruit Research Articles

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.

First Report of Penicillium expansum Isolates Resistant to Pyrimethanil from Stored Apple Fruit in Pennsylvania

Apples in the United States are stored in low-temperature controlled atmospheres for 9 to 12 months and are highly susceptible to blue mold decay. Penicillium spp. cause significant economic losses worldwide and produce mycotoxins that contaminate processed apple products. Blue mold is managed by a combination of cultural practices and the application of fungicides. In 2004, a new postharvest fungicide, pyrimethanil (Penbotec 400 SC, Janseen PMP, Beerse, Belgium) was registered for use in the United States to control blue mold on pome fruits (1). In this study, 10 blue mold symptomatic 'Red Delicious' apples were collected in May 2011 from wooden bins at a commercial facility located in Pennsylvania. These fruit had been treated with Penbotec prior to controlled atmosphere storage. Ten single-spore Penicillium spp. isolates were analyzed for growth using 96-well microtiter plates containing Richards minimal medium amended with a range of technical grade pyrimethanil from 0 to 500 μg/ml. Conidial suspensions adjusted to 1 × 105 conidia/ml were added to three 96-well plates for each experiment; all experiments were repeated three times. Nine resistant isolates had prolific mycelial growth at 500 μg/ml, which is 1,000 times the discriminatory dose that inhibited baseline sensitive P. expansum isolates from Washington State (1). However, one isolate (R13) had limited conidial germination and no mycelial proliferation at 0.5 μg/ml and was categorized as sensitive. One resistant (R22) and one sensitive (R13) isolate were selected on the basis of their different sensitivities to pyrimethanil. Both isolates were identified as P. expansum via conventional PCR using β-tubulin gene-specific primers according to Sholberg et al. (2). Analysis of the 2X consensus amplicon sequences from R13 and R22 matched perfectly (100% identity and 0.0 E value) with other P. expansum accessions in GenBank including JN872743.1, which was isolated from decayed apple fruit from Washington State. To determine if pyrimethanil applied at the labeled rate of 500 μg/ml would control R13 or R22 in vivo, organic 'Gala' apple fruit were wounded, inoculated with 50 μl of a conidial suspension (1 × 104 conidia/ml) of either isolate, dipped in Penbotec fungicide or sterile water, and stored at 25°C for 7 days. Twenty fruit composed a replicate within a treatment and the experiment was performed twice. Non-inoculated water-only controls were symptomless, while water-dipped inoculated fruit had 100% decay with mean lesion diameters of 36.8 ± 2.68 mm for R22 and 38.5 ± 2.61 mm for R13. The R22 isolate caused 30% decay with 21.6 ± 5.44 mm lesions when inoculated onto Penbotec-treated apples, while the R13 isolate had 7.5% decay incidence with mean lesion diameters of 23.1 ± 3.41 mm. The results from this study demonstrate that P. expansum pyrimethanil-resistant strains are virulent on Penbotec-treated apple fruit and have the potential to manifest in decay during storage. To the best of our knowledge, this is the first report of pyrimethanil resistance in P. expansum from Pennsylvania, a major apple growing region for the United States. Moreover, these results illuminate the need to develop additional chemical, cultural, and biological methods to control this fungus.

Identification of Penicillium expansum causing postharvest blue mold decay of apple fruit

Penicillium expansum (Link) Thom. is one of the most important postharvest pathogens of apple fruit worldwide. It causes blue mold, a decay that can lead to significant economic losses during storage, which can also impact fruit destined for processing due to the production of carcinogenic mycotoxin patulin. Apple fruit cvs. Idared, Golden Delicious and Braeburn with blue mold symptoms were collected from five storage facilities in Serbia and nine fungal isolates were obtained. Pathogenicity of the isolates was tested and proven by artificial inoculation of healthy apples cv. Idared. In order to identify the causal agents of decay, morphological and molecular methods were used. Colony morphology and microscopic features were observed on differential media, and isolates were tested for the production of cyclopiazonic acid. Molecular analysis included PCR amplification with species specific primers for P. expansum based on polygalacturonase gene (Pepg1), universal primers for internal transcribed spacer rDNA region and primers based on ?-tubulin gene. All isolates formed compact blue green colonies with characteristic earthy odor. Conidiophores were terverticillate with smooth septate stipes and conidia were smooth, globose to subglobose, born in colums. The average size of conidia was 3.38 ? 0.49 (SD) x 3 ? 0.36 (SD) ?m. Using species specific primers PEF/PER the texpected amplicons of ~404 bp were obtained in all nine tested isolates and PCR conducted with the Bt-LEVUp4/ Bt-LEV-Lo1 and universal ITS1/ITS4 primer pairs generated amplicons of the expected sizes of ~800 bp and ~600 bp, respectively. MegaBlast analyses of the 2X consensus of nucleotide sequences of the isolate JP1 partial ?-tubulin gene and ITS region showed 99-100% and 100% similarity with several P. expansum sequences of corresponding regions of this species deposited in GenBank. Based on morphological and molecular features, the isolates obtained from decayed apple fruit collected in several storage facilities in Serbia were identified as P. expansum.

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

Botryosphaeria dothidea (Moug.:Fr.) Ces. De Not. causes perennial cankers on apple trees and causes white rot on apple fruit in the field and during storage (1). Prolonged periods of warm wet weather favor rapid disease outbreaks that result in severe losses, which range from 25 to 50% for the southeastern United States (3). A B. dothidea isolate was obtained from decayed 'Fuji' apple fruit exhibiting white rot symptoms from a local farm market in Beltsville, MD, in May 2010. The fruit had characteristic large dark brown lesions with irregular margins and decay expanded unevenly toward the core and the tissue was soft. The pathogen was isolated from symptomatic tissue by spraying the lesion surface with 70% ethanol. The skin with aseptically removed with a scalpel and small pieces of tissue were placed on potato dextrose agar (PDA) and incubated at 20°C. Once fungal growth was evident, the cultures were hyphal-tip transferred to individual PDA plates and incubated at 20°C. The B. dothidea isolate produced black aerial mycelium with a white margin on PDA and had a black reverse. Conidiomata were evident after 10 to 14 days at 20°C only on oatmeal agar. Conidia were hyaline, smooth and straight, fusiform with an subobtuse apex and a truncate base 20 to 26 (24.33) × 4 to 7 (5) μm (n = 50). Genomic DNA was isolated from the fungus and amplified with gene specific primers (ITS 4 and 5) for the ribosomal DNA internal transcribed spacer region ITSI-5.8S-ITS2 as described by White et al. (4). Both forward and reverse strands of the 542-bp amplicon were sequenced and assembled into a contig. The nucleotide sequence (GenBank Accession No. KC473852) indicated 99% identity to B. dothidea isolate CMM3938 (JX513645.1) and to voucher specimens CMW 25686, 25696, and 25222 (FM955381.1, FM955379.1, and FM955377,1). Koch's postulates were conducted using three 'Golden Delicious' apple fruit that were wound-inoculated with 50 μl of a mycelial suspension of the fungus, obtained from aseptically scraping a 7-day-old PDA culture, and was also repeated using 'Fuji' apple fruit. Large, brown, slightly sunken, soft lesions with undefined edges developed 5 days after inoculation at 20°C and water-only inoculated fruit were symptomless. The fungus was reisolated from infected tissue and was morphologically identical to the original isolate from decayed apple fruit. To determine if the B. dothidea isolate was resistant to postharvest fungicides, the minimum inhibitory concentration (MIC) was conducted using the 96 well plate method with a mycelial suspension of the fungus as described by Pianzzola et al. (2). The MIC for the isolate was >1 ppm for Mertect and Scholar and 50 ppm for Penbotec, which are well below the labeled rates for these postharvest fungicides and the experiment was repeated. To our knowledge, this is the first report of B. dothidea causing white rot on apple fruit in Maryland.

First Report of Neofusicoccum ribis Causing Postharvest Decay of Apple Fruit from Cold Storage in Pennsylvania.

Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.), previously known as Botryosphaeria ribis (Grossenb. & Duggar), is an aggressive fungal plant pathogen that is part of the N. ribis/N. parvum species complex that causes stem cankers on a variety of woody plant species (2). An isolate of N. ribis was obtained from decayed 'Honeycrisp' apple fruit from a commercial cold storage facility located in Pennsylvania in October of 2011. The decayed apple fruit sample had a brownish lesion that was soft, dry, and leathery on the surface while sporulation was not evident. To conduct Koch's postulates, three 'Golden Delicious' apple fruits were wound-inoculated with a 50-μl mycelial suspension, obtained from aseptically scraping a 7-day-old potato dextrose agar (PDA) culture of the fungus, and was repeated using 'Fuji' apple fruit. The inoculated fruit developed lesions, while water-inoculated fruit were symptomless after 5 days at 20°C. N. ribis was reisolated from infected tissue and was morphologically identical to the original isolate. Genomic DNA was isolated, a portion of the β-tubulin gene was amplified with the gene specific primers, and the amplicon was sequenced and analyzed using BLAST (1). The nucleotide sequence (GenBank Accession No. KC47853) had 99% identity with N. ribis SEGA8 isolate (JN607146.1). The N. ribis isolate produced a grayish-white mycelium with abundant aerial hyphae on PDA and had an olive-colored reverse. Microscopic investigation revealed septate mycelia with right angle branching and conidiomata were not evident on PDA, V8, oatmeal agar (OMA), or water agar (WA). Growth on WA was sparse and transparent, and aerial mycelial growth was not produced. Growth rate analyses were conducted on PDA, V8, and OMA and were 10.1 (±1.39), 20.4 (±1.15), and 17.6 (±0.70) mm/day at 20°C and the experiment was repeated. The minimum inhibitory concentrations (MIC) for the N. ribis isolate was carried out for three postharvest fungicides as described by Pianzzola et al. (3). Briefly, 96 well plates were filled with PDA alone (0 ppm) and PDA amended with 10 fungicide concentrations ranging from 1 to 1,200 ppm for thiabendazole (Mertect), and 1 to 1,000 ppm for fludioxonil (Scholar) and pyrimethanil (Penbotec). A mycelial suspension of the fungus was obtained from pure culture, 50 μl of the mycelial suspension was pipetted into each well, and allowed to grow for 72 h at 25°C. The experiment was conducted twice. The N. ribis isolate displayed MIC values of >1 ppm thiabendazole (Mertect), >1 ppm fludioxonil (Scholar), and 50 ppm pyrimethanil (Penbotec), which are all well below the labeled application rates for these postharvest fungicides. To our knowledge, this is the first report of N. ribis causing postharvest decay on apple fruit obtained from a commercial storage facility in Pennsylvania.

Molecular characterization, fitness and mycotoxin production of benzimidazole-resistant isolates of Penicillium expansum

Penicillium expansum field-strains resistant to benzimidazole fungicides were isolated in high frequency from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, N. Greece. In vitro fungitoxicity tests resulted in the identification of two different resistant phenotypes: highly (BEN-HR) and moderately (BEN-MR) carbendazim-resistant. Thirty seven percent of the isolated P. expansum strains belonged to the BEN-HR phenotype, carried no apparent fitness penalties and exhibited resistance levels higher than 60 based on EC50 values. Cross resistance studies with other benzimidazole fungicides showed that all BEN-HR and BEN-MR isolates were also less sensitive to benomyl and thiabendazole. Fungitoxicity tests on the response of BEN-HR isolates to fungicides belonging to other chemical classes revealed no cross-resistance relationships between benzimidazoles and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the anilinopyrimidine cyprodinil, the QoI pyraclostrobin, the imidazole imazalil and the triazole tebuconazole, indicating that a target-site modification is probably responsible for the BEN-HR phenotype observed. Contrary to the above, some BEN-MR isolates exhibited an increased sensitivity to cyprodinil compared to benzimidazole-sensitive ones. BEN-MR isolates had fitness parameters similar to the benzimidazole-sensitive isolates except for conidia production which appeared significantly decreased. Analysis of mycotoxin production (patulin and citrinin) showed that all benzimidazole-resistant isolates produced mycotoxins at concentrations significantly higher than sensitive isolates both on culture medium and on artificially inoculated apple fruit. Comparison of the β-tubulin gene DNA sequence between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in most but not all HR isolates tested. Molecular analysis of the β-tubulin gene in moderately resistant isolates did not reveal any amino acid substitution. This is the first report on the existence and distribution of highly mycotoxigenic field isolates of P. expansum resistant to the benzimidazoles indicating a high potential risk of increased mycotoxin contamination of pome fruit and by-products.

Molecular Characterization of Pyraclostrobin Resistance and Structural Diversity of the Cytochrome b Gene in Botrytis cinerea from Apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.

Molecular Characterization of Boscalid Resistance in Field Isolates of Botrytis cinerea from Apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.

First Report of Occurrence of Pyrimethanil Resistance in Penicillium expansum from Stored Apples in Washington State.

Blue mold caused by Penicillium expansum is a major postharvest fruit rot disease of apples (Malus domestica) worldwide. Pyrimethanil was registered in late 2004 in the United States for postharvest use on apples. Since then, pyrimethanil has been increasingly used in Washington State as a postharvest drench treatment for control of blue mold and other postharvest diseases in apples. Baseline sensitivity to pyrimethanil in P. expansum populations from apples in Washington State has been established and all isolates in the baseline population were sensitive to pyrimethanil (1). To monitor resistance to pyrimethanil in P. expansum populations, blue mold-like decayed apple fruit were sampled from May to August 2009 from the fruit that had been drenched with pyrimethanil prior to storage from fruit packinghouses. Isolation of Penicillium species from decayed fruit was attempted. Isolates of Penicillium species were identified to species according to the descriptions by Pitt (2). In total, 186 P. expansum isolates were collected and tested for resistance to pyrimethanil in a conidial germination assay on an agar medium amended with pyrimethanil at the discriminatory concentration of 0.5 μg ml-1 (1). Isolates that were able to germinate were considered resistant to pyrimethanil. Of the 186 isolates tested, one was resistant to pyrimethanil. EC50 (the effective concentration that inhibits fungal growth by 50% relative to the control) of pyrimethanil for the resistant isolate was determined according to a method described previously (1) and the test was done twice. EC50 values of pyrimethanil on mycelial growth and conidial germination for the resistant isolate were 9.9 and 3.1 μg/ml, respectively, which were 7.4-fold and 16.5-fold higher than the means of the baseline population (1). To evaluate whether pyrimethanil at label rate is still able to control this resistant isolate, 'Fuji' apples were wounded, inoculated with conidial suspensions (1 × 104 conidia ml-1) of either the resistant isolate or a pyrimethanil-sensitive isolate, treated with either pyrimethanil or sterile water as controls, and stored at 20°C for 10 days following a method described previously (1). There were four 20-fruit replicates for each treatment. The experiment was performed twice. All inoculated fruit in the nontreated controls were decayed. Pyrimethanil applied at label rate completely controlled blue mold incited by a pyrimethanil-sensitive isolate, but 75% of the fruit that were inoculated with the resistant isolate and treated with pyrimethanil developed blue mold. To our knowledge, this is the first report of pyrimethanil resistance in P. expansum from decayed apple fruit collected from commercial packing houses. The pyrimethanil-resistant isolate was obtained from a packing house in which pyrimethanil had been used as a postharvest drench treatment in each of four consecutive years, suggesting that pyrimethanil-resistant individuals are emerging in P. expansum populations in Washington State after repeated use of pyrimethanil. Our results also indicate that pyrimethanil resistance in P. expansum reported in this study can result in failure of blue mold control in apples with pyrimethanil.

Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione, fludioxonil and cyprodinil and characterization of fitness parameters and patulin production

A total of 236 Penicillium expansum field isolates from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, Northern Greece were tested for their sensitivity to tebuconazole, fludioxonil, iprodione and cyprodinil. Preliminary fungitoxicity tests on the response of the isolates showed several phenotypes, distinguished according to their sensitivity to fungicides tested. The EC 50 values ranged from 0.64 to 5 (average = 0.98) μg/ml for iprodione, 0.9 to 7.3 (average = 2.66) μg/ml for tebuconazole, 0.008 to 1.28 (average = 0.55) μg/ml for cyprodinil and from 0.013 to 0.47 (average = 0.08) μg/ml for fludioxonil. A bimodal distribution of the EC 50 values of isolates with distinct sensitive and resistant populations to fludioxonil and tebuconazole were observed. In the case of cyprodinil, a much broader, hundred-fold, range of sensitivity was found, probably indicating that some isolates are relatively insensitive to cyprodinil compared to the most sensitive ones. Isolates exhibiting simultaneously reduced sensitivity to tebuconazole and fludioxonil or tebuconazole and iprodione or to tebuconazole and cyprodinil were also observed at low frequencies. A small portion of the population (7.5%) showed multiple resistance to tebuconazole, fludioxonil and iprodione. Study of fitness determining parameters showed that the resistance to tebuconazole, fludioxonil and iprodione had a significant adverse effect on mycelial growth rate and pathogenicity. Contrary to that, these fitness parameters were not affected in the isolates showing reduced sensitivity to cyprodinil. Analysis of patulin production on YES-agar growth medium and on artificially inoculated apple fruit showed that all isolates were mycotoxigenic. Most of the cyprodinil-insensitive isolates produced patulin at concentrations similar to or relatively higher (up to 1.5-fold on growth medium) than the sensitive ones. In contrast, a significant reduction (up to 98% of multiple resistant isolates) in patulin production was observed in all other phenotypes, indicating an adverse effect of fitness penalties on the mycotoxigenic ability of resistant isolates. The above mentioned data clearly show a considerable risk for the selection of P. expansum isolates resistant to fludioxonil, iprodione, tebuconazole and cyprodinil. The potential risk of increased patulin contamination of apples and their byproducts by the appearance and predominance of highly mycotoxigenic isolates of P. expansum resistant to the anilinopyrimidines is discussed.

First Morphomolecular Identification of Penicillium griseofulvum and Penicillium aurantiogriseum Toxicogenic Isolates Associated with Blue Mold on Apple

Postharvest blue mold decay caused by Penicillium spp. is the most important disease of fresh apple fruit in the world, which extend from the field to the store. Two new Penicillium spp. responsible for apple fruit decay were recovered. The morphological and molecular features of Penicillium griseofulvum and Penicillium aurantiogriseum isolated from apple fruits were characterized morphologically and molecularly. Pathogenicity test exhibited that both P. griseofulvum and P. aurantiogriseum were responsible for blue mold decay in storage apple fruits. Lesion diameter indicated that P. aurantiogriseum was more aggressive than P. griseofulvum. All tested isolates were able to synthesize citrinin in addition to patulin. Not all of the isolates belonging to the same species showed the same profile of secondary metabolites. Microsatellite-primed polymerase chain reaction was able to differentiate these isolates at the species level and divided the analyzed isolates into two genetically different groups. Little intraspecific variability was evident. Microsatellite-primed polymerase chain reaction analysis proved to be an objective, rapid, and reliable tool to identify Penicillium spp. involved in blue mold of apple. This is the first report of occurrence of P. griseofulvum and P. aurantiogriseum on imported apple fruits in Saudi Arabia.

Resistance to Pyraclostrobin and Boscalid in Populations of Botrytis cinerea from Stored Apples in Washington State

Gray mold caused by Botrytis cinerea is a major postharvest disease of apple. Pristine, a formulated mixture of pyraclostrobin and boscalid, was recently registered for use on apple. Pristine applied within 2 weeks before harvest is effective in controlling gray mold in stored apple fruit. To determine the baseline sensitivity of B. cinerea populations to these fungicides, 40 isolates from organic and 80 from conventional apple orchards where Pristine had not been used were tested for mycelial growth or conidial germination on fungicide-amended media. To monitor fungicide resistance, gray-mold-decayed apple fruit originating from orchards in which Pristine had been used were sampled from a fruit packinghouse. Isolates of B. cinerea recovered from the fruit were tested for resistance to the two fungicides. In the in vivo study in the orchards, Pristine was applied to fruit 1 day before harvest. Fruit were then harvested, wounded, and inoculated with isolates exhibiting different fungicide-resistance phenotypes. Fruit were stored at 0°C for 8 weeks for decay development. The effective concentration that inhibits mycelial growth by 50% relative to the control (EC50) values for sensitive isolates ranged from 0.008 to 0.132 μg/ml (mean = 0.043, n = 116) for pyraclostrobin and from 0.003 to 0.183 μg/ml (mean = 0.075, n = 117) for Pristine in a mycelial growth assay on potato dextrose agar. The EC50 values of boscalid for sensitive isolates ranged from 0.065 to 1.538 μg/ml (mean = 0.631, n = 29) in a conidial germination assay on water agar. Four isolates were resistant to pyraclostrobin, with resistance factors (RFs) ranging from 12 to 4,193. Of the four pyraclostrobin-resistant isolates, one also was resistant to boscalid (RF = 14) and Pristine (RF = 373), and two exhibited reduced sensitivity to Pristine (RF = 16 and 17). The minimum inhibitory concentration for conidial germination (for boscalid) or mycelial growth (for pyraclostrobin and Pristine) of sensitive isolates was 5 μg/ml, which is thus recommended as a discriminatory concentration for phenotyping isolates for resistance to these fungicides. Of the 56 isolates obtained from decayed apple fruit that had been exposed to Pristine, 11 (approximately 20%) were resistant to both pyraclostrobin and boscalid and 1 was resistant only to pyraclostrobin. Of the additional 43 isolates obtained from decayed apple fruit originating from an organic orchard, 3 were resistant only to pyraclostrobin, 2 were resistant only to boscalid, and 2 were resistant to both fungicides. It appeared that there was no cross resistance between pyraclostrobin and boscalid because of the existence of isolates resistant only to either pyraclostrobin or boscalid. Pristine applied at label rate in the orchard failed to control gray mold on apple fruit inoculated with the Pristine-resistant isolates. This is the first report of multiple resistance to pyraclostrobin, boscalid, and Pristine in field populations of B. cinerea. Our results suggest that the development of dual resistance to pyraclostrobin and boscalid in B. cinerea populations could result in the failure to control gray mold with Pristine.

Purification and Biochemical Characterization of Polygalacturonase Produced by Penicillium expansum During Postharvest Decay of ‘Anjou’ Pear

A polygalacturonase (PG) was extracted and purified from decayed tissue of 'Anjou' pear fruit inoculated with Penicillium expansum. Ammonium sulfate precipitation, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. PG enzyme activity from healthy and wounded pear tissue was undetectable, which supports the claim that the purified PG is of fungal origin. The purified enzyme had a molecular mass of 41 kDa and a pI of 7.8. Activity of the PG was not associated with a glycosylated protein. The enzyme was active over a broad pH range from 3 to 6, with optimal activity at 4.5 in sodium citrate and sodium acetate buffers. The optimal temperature for activity was 37 degrees C but the enzyme was also active at 0, 5, 10, 20, and 50 degrees C. Thin-layer chromatographic analysis of PG hydrolysis products showed that the enzyme exhibits endo- and exo-activity. The purified enzyme macerated tissue in vitro causing approximately 30% reduction in mass of pear plugs compared with approximately 17% reduction for apple. Additionally, it produced 1.5-fold more soluble polyuronides on pear than apple tissue. This work shows for the first time the production of a PG by P. expansum during postharvest decay of pear fruit is different from the previously described PG produced in decayed apple fruit by the same pathogen.

Isolation, Purification, and Characterization of a Polygalacturonase Produced in Penicillium solitum-Decayed ‘Golden Delicious’ Apple Fruit

Polygalacturonase (PG) was extracted and purified from decayed 'Golden Delicious' apple fruit inoculated with Penicillium solitum. Ammonium sulfate, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. The purified PG most likely originates from the fungus because PG activity from healthy and wounded apple tissue was undetectable. Analysis of cation exchange-purified material using sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 50-kDa band. The enzyme was active over a broad pH range (3 to 7), with optimal activity between pH 4 and 5. PG was highly active at 20 and 37 degrees C but was also detectable at 2, 50, and 75 degrees C. Divalent cations affected PG enzyme activity; Mg and Fe increased, whereas Ca and Mn reduced activity in vitro. Thin-layer chromatographic separation of hydrolysis products and data from a PG plate activity assay based on staining with ruthenium red showed that the enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit.

Suppression of postharvest blue mould of apple fruit by Cryptococcus laurentii and N6‐benzyladenine

AbstractBACKGROUND: Cryptococcus laurentii is a well‐known postharvest yeast antagonist. N6‐benzyladenine (6‐BA), a cytokinin plant hormone, has a role in retarding ripening and senescence of harvested produce. This study was conducted to evaluate the efficacy of C. laurentii and 6‐BA in reducing the blue mould disease of apple fruit.RESULTS: The combination of C. laurentii with 6‐BA (20 µg mL−1) was more effective in suppressing the Penicillium expansum infection in apple fruit wounds than C. laurentii alone, although 6‐BA (20 µg mL−1) alone neither affected the growth of C. laurentii nor reduced the incidence of the blue mould disease in vivo. Moreover, treatment of apple fruit with C. laurentii and 6‐BA (20 µg mL−1) resulted in stimulation of superoxide dismutase activity but in inhibition of the increase in peroxidase activity.CONCLUSION: 6‐BA (20 µg mL−1) could enhance the efficacy of C. laurentii in reducing the postharvest blue mould disease of apple fruit, which offered great potential in minimizing the postharvest decay of apple fruit in an integrated pest management strategy. Copyright © 2008 Society of Chemical Industry

Improving biocontrol using antagonist mixtures with heat and/or sodium bicarbonate to control postharvest decay of apple fruit

‘Golden Delicious’ apples were wound-inoculated with either Colletotrichum acutatumor Penicillium expansumand then treated with various combinations of heat (38 ◦ C) for 4 days, 2% sodium bicarbonate, and two biocontrol agents alone or combined. The fruit were stored for 4 months at 1 ◦ C and then at 20 ◦ C for 2 weeks. Either heat or the antagonists reduced decay caused by C. acutatum, but a combination of the two was required to completely eliminate decay caused by this pathogen in most cases. Sodium bicarbonate alone or in combination with the antagonists had little effect on C. acutatum. The antagonists alone reduced decay caused by P. expansum but tended to be more effective when combined. Sodium bicarbonate increased the effectiveness of decay control by each antagonist alone or in combination. All of the treatments that included heat virtually eliminated decay caused by this pathogen. The proper combination of alternative control measures can provide an effective strategy to reduce postharvest decay of apple fruit. Published by Elsevier B.V.

Control of Postharvest Decay of Apple Fruit with Candida saitoana and Induction of Defense Responses

ABSTRACT The ability of Candida saitoana to induce systemic resistance in apple fruit against Botrytis cinerea was investigated. To separate the antagonistic activity of C. saitoana from its ability to induce resistance, the antagonist and the pathogen were applied in spatially separated wounds. In fresh apples, C. saitoana applied 0 or 24 h before inoculation with B. cinerea showed no effect on lesion development caused by B. cinerea. When applied 48 to 72 h preinoculation with B. cinerea, however, C. saitoana reduced lesion diameter by more than 50 and 70%, respectively, compared with wounding. C. saitoana had no effect on lesion development on stored apples, regardless of the lag period between yeast treatment and inoculation with B. cinerea. In addition to inducing systemic resistance, C. saitoana increased chitinase and beta-1,3-glucanase activities with a higher accumulation in fresh than in stored apples. In fresh apples, the onset of systemic resistance to B. cinerea coincided with the increase in chitinase and beta-1,3-glucanase activity in systemically protected tissue. These studies show that C. saitoana is capable of inducing systemic resistance in apple fruit and indirectly suggest that antifungal hydrolases are involved in the observed systemic protection.

Butyl acetate and yeasts interact in adhesion and germination of Botrytis cinerea conidia in vitro and in fungal decay of golden delicious apple.

Butyl acetate is a volatile aroma and flavor compound in apple. Conidia of three strains of Botrytis cinerea, a fungus that causes decay of apple fruit in postharvest storage, had greater adhesion to and greater germination on polycarbonate membrane filters on water inside sealed 500 cc glass jars that were injected with 4 microliters butyl acetate than conidia not so exposed. Conidial germination was highly correlated with conidial adhesion. The yeasts Sporobolomyces roseus and Cryptococcus laurentii, but not Saccharomyces cerevisiae, reduced the adhesion and germination promoting effect of butyl acetate. Conidia did not readily utilize butyl acetate as a food source, as shown by lack of tetrazolium violet reduction, whereas S. roseus and C. laurentii, but not S. cerevisiae did. Butyl acetate added to suspensions of conidia increased the electrical conductivity of the suspensions and increased the loss of 14C from 14C-labeled conidia compared to conidia unexposed to butyl acetate. Uptake of [14C]glucose by conidia was not increased by butyl acetate. Wounds of Golden Delicious apples inoculated with conidia (strain F-J-4) in a dilute solution of butyl acetate had greater decay than unexposed wounds. S. roseus and C. laurentii, but not S. cerevisiae, added with the conidia decreased the incidence or size of decay. Results indicated that butyl acetate increased conidial adhesion, stimulating conidial germination, and some yeasts can reduce this effect.

Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses

The biocontrol activity of Aureobasidium pullulans on decay of apple fruit caused by Botrytis cinerea and Penicillium expansum, and its ability to induce biochemical defense responses in apple tissue, were investigated. In apple wounds, A. pullulans multiplied rapidly and controlled decay caused by either B. cinerea or P. expansum .A t the end of the storage period, A. pullulans reduced the incidence of gray and blue mold of apple by 89 and 67%, respectively, compared to the water-treated control. In addition to controlling decay, A. pullulans caused a transient increase in b-1,3-glucanase, chitinase, and peroxidase activities starting 24 h after treatment and reaching maximum levels 48 and 96 h after treatment. An increase in b-1,3-glucanase, chitinase, and peroxidase activity was also triggered by wounding, although, the level of increase was markedly lower than that detected in treated fruit. The ability of A. pullulans to increase activities of b-1,3-glucanase, chitinase, and peroxidase in addition to its known capacity to out-compete pathogen for nutrients and space, may be the basis of its biocontrol activity. © 2000 Elsevier Science B.V. All rights reserved.

A Storage Decay of Apple Fruit Caused by Aureobasidium pullulans.

A Storage Decay of Apple Fruit Caused by Aureobasidium pullulans.