Brown Rot Of Stone Fruit Research Articles

Culturable bacteria from plum fruit surfaces and their potential for controlling brown rot after harvest

Fruit microflora have been the richest source of antagonists against fruit decays and the active ingredient in all currently available commercial biocontrol products. A comprehensive evaluation of plum bacteria for biocontrol activity against Monilinia fructicola, which causes brown rot of stone fruit, is important for determining their biocontrol potential. Resident culturable bacterial microflora of plums from early fruit development until maturity were characterized. The most dominant genera were Curtobacterium (19.88%), Pseudomonas (15.06%), Microbacterium (13.86%), and Clavibacter (12.65%). These genera occurred at all four isolation times and accounted for 61.45% of all isolates. Microbacterium and Curtobacterium dominated at the early stage of fruit development while Pseudomonas and Clavibacter were dominant at the end of the season. Less prevalent genera were Enterobacter (5.42%), Chrysomonas (4.82%), and Pantoea (4.22%). Most frequently isolated species were Microbacterium lacticum, Clavibacter michiganensis, Curtobacterium flaccumfaciens, Enterobacter intermedius, and Chrysomonas luteola. The seasonal succession of genera was observed in both MANOVA and frequency analysis. Primary and secondary screening of plum-inhabiting bacteria for control of brown rot on wounded fruit resulted in selection of several antagonists among which Pantonea agglomerans and Citrobacter freundii were the most effective. These antagonists grew well in plum wounds and increased by four log units during first three days at 24°C, and two log units after seven days at 4°C. Results indicate that plum microflora are an excellent source of antagonists against brown rot decay originating from wounds after harvest.

Use of Biofungicides for Controlling Plant Diseases to Improve Food Availability

Biological control of fungal plant pathogens can improve global food availability, one of the three pillars of food security, by reducing crop losses, particularly for low-income farmers. However, the interrelationships of many environmental variables can result in multiple interactions among the organisms and their environment, several of which might contribute to effective biological control. Here, we present an advanced survey of the nature and practice of biological control when it is used to control brown rot in stone fruit. Specifically, we describe the population dynamics of Penicillium frequentans and Epicoccum nigrum and their efficacy as biocontrol agents against brown rot disease under field conditions. The size of P. frequentans population after an application of a P. frequentans conidial formulation during the crop season is bigger than that of E. nigrum following the application of an E. nigrum conidial formulation. Moreover, applications of a P. frequentans conidial formulation during the crop season also caused a higher reduction in the number of Monilinia spp. conidia on the fruit surface than that found after applications of an E. nigrum formulation during the growing season.

First Report of Brown Rot Caused by Monilinia fructicola on Stored Apple in Serbia.

Monilinia fructicola (G. Winter) Honey is a causal agent of brown rot of stone fruits, occasionally affecting pome fruits as well. The pathogen is commonly present in North and South America, Oceania, and Asia, but listed as a quarantine organism in Europe (4). After its first discovery in France in 2001, its occurrence has been reported in Germany, Hungary, Italy, Poland, Romania, Slovenia, Spain, Switzerland, Austria, and the Slovak Republic (1). In February 2011, during a survey for fungal postharvest pathogens in cold storage conditions, apple fruits (Malus domestica Borkh.) grown and stored in the Grocka Region, Serbia, were collected. All pathogens from symptomatic fruits were isolated on potato dextrose agar (PDA). One isolate from apple fruit cv. Golden Delicious with brown rot symptoms was identified as M. fructicola based on morphological and molecular characters. Colonies cultivated on PDA at 22°C in darkness were colorless, but later became grayish, developing mass of spores in concentric rings. Colony margins were even. Conidia were one-celled, limoniform, hyaline, measured 12.19 to 17.37 (mean 13.8) × 8.62 to 11.43 μm (mean 9.9), and were produced in branched monilioid chains (3). Morphological identification was confirmed by PCR (2) using genomic DNA extracted from the mycelium of pure culture, and an amplified product of 535 bp, specific for the species M. fructicola, was obtained. Sequence of the ribosomal (internal transcribed spacer) ITS1-5.8S-ITS2 region was obtained using primers ITS1 and ITS4 and deposited in GenBank (Accession No. JN176564). Control fruits were inoculated with sterile PDA plugs. After 3 days of incubation in plastic containers with high humidity at room temperature, typical symptoms of brown rot developed on inoculated fruits, while control fruits remained symptomless. The isolate recovered from symptomatic fruits showed the same morphological and molecular features of the original isolate. To our knowledge, this is the first report of M. fructicola in Serbia. Further studies are necessary for estimation of economic importance and geographic distribution of this quarantine organism in Serbia.

Characterizing Fenbuconazole and Propiconazole Sensitivity and Prevalence of 'Mona' in Isolates of Monilinia fructicola from New York.

Demethylation inhibitor (DMI) resistant populations of Monilinia fructicola, the causal agent of brown rot of stone fruit, and the presence of the genetic DMI resistance determinant 'Mona' have been reported throughout the eastern United States. In this study, we endeavored to conduct a comprehensive investigation of DMI sensitivity, the prevalence of 'Mona', and implications of DMI use for M. fructicola populations from New York and Pennsylvania. Of the 18 orchards surveyed, only 9 were primarily composed of isolates with either resistance or reduced sensitivity to fenbuconazole and propiconazole. The DMI resistance determinant 'Mona' was only found in 5 orchards, present in isolates with a range of sensitivity phenotypes, and not always present in resistant isolates. These results suggested that 'Mona' only contributes to a portion of the quantitative resistance response to DMI fungicides. On detached blossoms and fruit, protective applications of fenbuconazole (Indar 2F) against isolates with resistance or reduced sensitivity resulted in significantly (P < 0.05) lower brown rot incidence compared to applications of propiconazole (Orbit 3.6EC) and water controls. By comparison, therapeutic applications of fenbuconazole and propiconazole against isolates with resistance or reduced sensitivity provided little to no reduction in brown rot incidence on blossoms and fruit.

Selection of a Suitable Medium to Determine Sensitivity of Monilinia fructicola Mycelium to SDHI Fungicides

Succinate dehydrogenase inhibitor (SDHI) fungicides are important tools to control preharvest and postharvest brown rot of stone fruits. In this study we determined the mycelial growth rate of Monilinia fructicola isolates on various media and picked the three that allowed the fastest growth to assess the sensitivity of mycelium to SDHI fungicides boscalid, fluopyram, and penthiopyrad. Minimal medium (MM) supported mycelial growth the best and yielded lowest EC50 values for three SDHI fungicides. EC50 values corresponded with disease incidence data obtained from detached fruit assays. Penthiopyrad had significantly greater intrinsic activity in vitro compared to fluopyram at the α = 0.05 level and compared to boscalid at the α = 0.1 level. However, detached fruit assays revealed that this ‘advantage’ did not carry over in vivo. In conclusion, MM appears to be the best medium currently available to assess the sensitivity of M. fructicola mycelium in vitro.

First Report of Brown Rot of Apple Caused by Monilinia fructicola in Germany.

Monilinia fructicola (G. Wint.) Honey is a causal agent of brown rot of stone fruits but may also affect pome fruits. M. fructicola is common in North America, Oceania, and South America as well as in Asia, but it is listed as a quarantine pathogen in Europe (3). Since its first discovery in Europe in 2001 (France), it has been reported in Spain, Slovenia, Italy, and Switzerland. Recently, the fungus was also detected in orchards of blackberries and plums in the State of Baden-Württemberg, Germany (4). In July 2010, apples (Malus domestica Borkh.) of the cultivar Jonagold were found in a residential backyard in Fronhausen an der Lahn located in the State of Hessen, Germany with symptoms resembling brown rot caused by Monilinia species. Affected apples were at or near maturity with brown decay that had spread throughout the fruits. On the surface of the decaying apples was tan to white zones of sporulation. Upon isolation, the mycelium grew at a linear rate of 9.2 mm per day at 22°C on potato dextrose agar forming branched, monilioid chains of grayish colonies with concentric rings and little sporulation. The lemon-shaped spores had an average size of 14 × 9 μm, a shape and size consistent with M. fructicola. The ribosomal ITS1-5.8S-ITS2 region was PCR-amplified from genomic DNA obtained from mycelium using primers ITS1 and ITS4. A BLAST search in GenBank revealed highest similarity (99%) to M. fructicola sequences from isolates collected in China, Italy, and Slovenia (GenBank Accession Nos. FJ515894.1, FJ411109.1, GU967379.1). The M. fructicola sequence from the apple isolate was submitted to GenBank (Accession No. JF325841). The pathogen was also identified to the species level and confirmed to be M. fructicola using two novel PCR techniques based on cytochrome b sequences (1,2). Pathogenicity was confirmed by inoculating three surface-sterilized, mature apples cv. Gala with a conidial suspension (105 spores/ml) of the apple isolate. Fruit were stab inoculated at three equidistant points to a depth of 10 mm using a sterile needle. A 30-μl droplet was placed on each wound; control fruit received sterile water without conidia. After 5 days of incubation at room temperature in air-tight plastic bags, the inoculated fruits developed typical brown rot symptoms with sporulating areas (as described above). The developing spores on inoculated fruit were confirmed to be M. fructicola. All control fruits remained healthy. To our knowledge, this is the first report of M. fructicola on apple in Germany and more indication of further geographical spread of the quarantine disease in Germany.

Functions of Lipopeptides Bacillomycin D and Fengycin in Antagonism of Bacillus amyloliquefaciens C06 towards Monilinia fructicola

In previous studies, Bacillus amyloliquefaciens C06 has been proven to be effective in controlling brown rot of stone fruit caused by Monilinia fructicola. When tested in vitro, cell-free filtrate of B. amyloliquefaciens C06 significantly inhibited mycelial growth and conidial germination of the fungal pathogen. This study aimed to determine the role of the antifungal compound(s) in the cell-free filtrate of B. amyloliquefaciens C06 by an approach combining a DNA-based suppression subtractive hybridization (SSH) method with MALDI-TOF-MS analysis. It was demonstrated that B. amyloliquefaciens C06 harbored two genes, bmyC and fenD, involved in biosynthesis of bacillomycin D and fengycin, two lipopeptides belonging to the iturin and fengycin family, respectively. To determine the roles of bacillomycin D and fengycin of B. amyloliquefaciens C06 in suppressing M. fructicola, the mutants of B. amyloliquefaciens C06 deficient in producing bacillomy- cin D, fengycin or both were constructed, and evaluated in vitro together with the wild-type B. amyloliquefaciens C06. The results indicated that bacillomycin D and fengycin jointly contributed to the inhibition of conidial germination of M. fructicola, and fengycin played a major role in suppressing mycelial growth of the fungal pathogen.

First Report of the β-tubulin E198A Allele for Fungicide Resistance in Monilinia fructicola from South Carolina.

Resistance to methyl benzimidazole carbamates (MBCs) in Monilinia fructicola, the causal agent of brown rot of stone fruits, is known to be present in South Carolina peach orchards, but the molecular mechanism of resistance has not been investigated. Nine isolates were collected from peach in five counties in South Carolina and examined in petri dish assays on potato dextrose agar (PDA) for resistance to the MBC fungicide thiophanate-methyl (Topsin-M 70WP; Ceraxagri, King of Prussia, PA) at the discriminatory dose of 50 μg/ml. Isolates that grew on the fungicide-amended medium were considered highly resistant (HR). The β-tubulin gene from four sensitive (S) and five HR M. fructicola isolates was PCR-amplified with primer pair TubA and TubR1 as described previously (1). Sequence analysis revealed several silent mutations in introns and exons in S and HR isolates and the presence of the previously described E198A allele in HR but not S isolates (1). Nucleotide sequences of the β-tubulin gene from three S (BS, S2, MfEgpc1) and two HR isolates (MfPdt6 and BR2) were submitted to GenBank under accession numbers HM051379, HM051380, HM051381, HM051382, and HM051383, respectively. To our knowledge, this is the first report of the E198A in M. fructicola isolates from South Carolina and the East Coast. This allele is responsible for high levels of MBC resistance in M. fructicola (1). A previously reported PCR-based method using primers HRF+HRR designed to detect the E198A mutation in M. fructicola HR isolates (1) was improved by adding primer TR739 (5'-TCA CGA CGA ACA ACA TCA AGA-3') to the PCR cocktail. This additional internal primer amplified a 222-bp fragment from all S and HR isolates and therefore provided a useful, additional control. The confirmation of the E198A allele in M. fructicola isolates provides another useful tool to detect MBC resistance in commercial peach orchards in South Carolina. Reference: (1) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003.

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self-splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5'-3') 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above-mentioned 1166 bp intron, a self-splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron.

First Report of Brown Rot Caused by Monilinia fructicola Affecting Peach Orchards in Slovenia.

Monilinia fructicola, the causal agent of brown rot, is a destructive fungal pathogen that affects mainly stone fruits (Prunoideae). It causes fruit rot, blossom wilt, twig blight, and canker formation and is common in North and South America, Australia, and New Zealand. M. fructicola is listed as a quarantine pathogen in the European Union and was absent from this region until 2001 when it was detected in France. In August 2009, mature peaches (Prunus persica cv. Royal Glory) with brown rot were found in a 5-year-old orchard in Goriška, western Slovenia. Symptoms included fruit lesions and mummified fruits. Lesions were brown, round, rapidly extending, and covered with abundant gray-to-buff conidial tufts. The pathogen was isolated in pure culture and identified based on morphological and molecular characters. Colonies on potato dextrose agar (PDA) incubated at 25°C in darkness had an average daily growth rate of 7.7 mm. They were initially colorless and later they were light gray with black stromatal plates and dense, hazel sporogenous mycelium. Colony margins were even. Sporulation was abundant and usually developed in distinct concentric zones. Limoniform conidia, produced in branched chains, measured 10.1 to 17.7 μm (mean = 12.1 μm) × 6.2 to 8.6 μm (mean = 7.3 μm) on PDA. Germinating conidia produced single germ tubes whose mean length ranged from 251 to 415 μm. Microconidia were abundant, globose, and 3 μm in diameter. Morphological characters resembled those described for M. fructicola (1). Morphological identification was confirmed by amplifying genomic DNA of isolates with M. fructicola species-specific primers (2-4). Sequence of the internal transcribed spacer (ITS) region (spanning ITS1 and ITS 2 plus 5.8 rDNA) of a representative isolate was generated using primers ITS1 and ITS4 and deposited in GenBank (Accession No. GU967379). BLAST analysis of the 516-bp PCR product revealed 100% identity with several sequences deposited for M. fructicola in NCBI GenBank. Pathogenicity was tested by inoculating five mature surface-sterilized peaches with 10 μl of a conidial suspension (104 conidia ml-1) obtained from one representative isolate. Sterile distilled water was used as a control. Peaches were wounded prior to inoculation. After 5 days of incubation at room temperature and 100% relative humidity, typical brown rot symptoms developed around the inoculation point, while controls showed no symptoms. M. fructicola was reisolated from lesion margins. Peach and nectarine orchards in a 5-km radius from the outbreak site were surveyed in September 2009 and M. fructicola was confirmed on mummified fruits from seven orchards. The pathogen was not detected in orchards from other regions of the country, where only the two endemic species M. laxa and M. fructigena were present. To our knowledge, this is the first report of M. fructicola associated with brown rot of stone fruits in Slovenia.

γ-Polyglutamic acid (γ-PGA) produced by Bacillus amyloliquefaciens C06 promoting its colonization on fruit surface

Bacillus amyloliquefaciens C06, an effective biological agent in controlling brown rot of stone fruit caused by Monilinia fructicola, was also found to produce extra-cellular mucilage and form mucoid colonies on semi-solid surfaces. This study aimed to characterize the extra-cellular mucilage produced by B. amyloliquefaciens C06 using transposon mutagenesis and biochemical and physical analyses. The mucilage production in B. amyloliquefaciens C06 was demonstrated to be associated with ywsC gene expression and characterized to be of high molecular weight, consisted of only glutamic acid and linked with non-peptide bonds, thus identified as γ-polyglutamic acid (γ-PGA). Compared with wild type B. amyloliquefaciens C06, its mutants deficient in producing γ-PGA, e.g. M106 and C06Δ ywsC showed less efficiency in biofilm formation, surface adhesion and swarming ability. It was also demonstrated that γ-PGA was not essential for C06 to form colony on semi-solid surfaces, but was able to improve its colony structure. In vivo evaluation showed that disruption of γ-PGA production in C06Δ ywsC impaired its efficiency of colonizing apple surfaces.

Reduced Sensitivity in Monilinia fructicola Field Isolates from South Carolina and Georgia to Respiration Inhibitor Fungicides.

Quinone outside inhibitor (QoI) and succinate dehydrogenase inhibitor (SdhI) fungicides are respiration inhibitors (RIs) used for preharvest control of brown rot of stone fruit. Both chemical classes are site-specific and, thus, prone to resistance development. Between 2006 and 2008, 157 isolates of Monilinia fructicola collected from multiple peach and nectarine orchards with or without RI spray history in South Carolina and Georgia were characterized based upon conidial germination and mycelial growth inhibition for their sensitivity to QoI fungicides azoxystrobin and pyraclostrobin, SdhI fungicide boscalid, and a mixture of pyraclostrobin + boscalid. There was no significant difference (P = 0.05) between EC50 values for inhibition of conidial germination versus mycelial growth. The mean EC50 values based upon mycelial growth tests for 25 isolates from an orchard without RI-spray history were 0.15, 0.06, 2.23, and 0.09 μg/ml for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. The respective mean EC50 values for 76 isolates from RI-sprayed orchards in South Carolina were 0.9, 0.1, 10.7, and 0.13 μg/ml and for 56 isolates from RI-sprayed orchards in Georgia were 1.2, 0.1, 8.91, and 0.17 μg/ml. Overall, mean EC50 values of populations from RI-sprayed orchards increased three-, two-, five-, and twofold between 2006 and 2008 for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. A subset of 10 M. fructicola isolates representing low and high EC50 values for azoxystrobin, boscalid, and boscalid + pyraclostrobin was selected for a detached fruit assay to determine disease incidence and severity following protective treatments of formulated RI fungicides at label rates. Brown rot incidence was greater than 50% when fruit were inoculated with isolates having EC50 values of 2, 4, and 0.6 μg/ml for azoxystrobin, boscalid, and pyraclostrobin + boscalid, respectively. Pyraclostrobin failed to control any of the isolates tested in detached fruit assays. Based on minimum inhibitory concentration and brown rot incidence data, we recommend using 3 and 0.75 μg/ml as discriminatory doses to distinguish between sensitive isolates and those with reduced sensitivity to azoxystrobin and pyraclostrobin + boscalid, respectively. Results from our in vitro and in vivo assays indicate a shift toward reduced sensitivity in M. fructicola from the southeastern United States. No cross-resistance was observed between the QoI and the SdhI fungicides, which implies that rotation or tank mixtures of these two chemical classes can be used as a resistance management strategy.

Control of brown rot of stone fruits by brief heated water immersion treatments

The effectiveness of brief (30 or 60 s) immersion in water at 24, 50, 55, 60, 65, or 70 °C was evaluated for the control of brown rot, caused by Monilinia fructicola, on California-grown peaches, nectarines, and plums. Inoculated fruits were treated and either stored at 20 °C for 5 days or at 0 °C and 95% RH for 30 days followed by 5 days at 20 °C to simulate commercial marketing conditions. Immersion in water at 55 °C for 60 s or at 60 °C for 30 or 60 s significantly reduced both decay incidence and severity among the remaining wounds that developed the disease. Water temperatures of 65 °C or higher were phytotoxic and caused moderate to severe surface injuries. Immersion in water at 60 °C for 60 s was effective for plums and it reduced the incidence of brown rot from more than 80% among control fruit to less than 2%. In nectarines, this treatment reduced decay incidence from 100 to less than 5% on fruit stored at 20 °C and from 73 to 28% on cold-stored fruit. Therefore, brief immersion in heated water can be an effective approach to manage postharvest brown rot of stone fruits, especially for the organic fruit industry.

RESOLVING THE STATUS OF MONILINIA spp. IN SOUTH AFRICAN STONE FRUIT ORCHARDS

Geographical distribution records of pathogens and pests are the basis for phytosanitary decision-making. Monilinia fructicola, one of the three Monilinia species responsible for brown rot of stone fruit, is listed as a regulated pest for South Africa. Many disputes about the justification of this classification have arisen during the past years as records have been published regarding the presence of this pathogen in South Africa. Results of several surveys conducted from 1985 in stone fruit orchards, revealed that M. laxa is the only causal agent of brown rot in South Africa. However, another notification of non-compliance (interception of regulated pests) was received by the National Plant Protection Organisation of South Africa in 2003 stating that M. fructicola was detected on consignments of South African Prunus fruits (P. domestica) in the United Kingdom. A detection survey according to inspections and sampling relevant to the biology of Monilinia species were conducted in the identified orchards at various stages throughout the year. Molecular techniques with species-specific primers for M. fructicola, M. laxa and M. fructigena based on the EPPO Diagnostic Protocol for M. fructicola were used for the identification of presumptive positive Monilinia isolates. The absence of M. fructicola from South African stone fruit orchards was again confirmed by this survey and its status in South Africa can be reported as: absent, not known to occur, confirmed by a detection survey. The regulated status of M. fructicola in South Africa is therefore scientifically justified by the results from this survey.

First Report of Brown Rot Caused by Monilinia fructicola in Peach Orchards in Ebro Valley, Spain.

Monilinia fructicola causes brown rot of stone fruit in India, Japan, the Republic of Korea, Oceania, and North and South America and is in the A2 list of quarantine organisms for Europe. M. fructicola was found in peach orchards for the first time in Europe in 2001 in France (4) and later in the Czech Republic (2). M. fructicola was not detected among 428 isolates of Monilinia spp. collected from Spanish peach orchards from 1998 to 2005. In March of 2006, M. fructicola was detected to be overwintering in three mummified peach fruit (cv. Autumn Free) trees in an orchard located in Sudanell (Lleida, Spain). Morphological and molecular identification of isolates were performed according to protocols previously described (1,3). The characteristics of these isolates were: i) colonies were entire and showing concentric rings of spores when grown on potato dextrose agar (PDA); ii) sporogenous tissues were gray to buff; iii) single and nearly straight germ tubes were at least 220 μm long before branching; and iv) growth rates on PDA under long-wave UV/darkness were as much as 20 × 10 mm2. Isolates were further identified by a PCR test using primers developed with sequence-characterized amplification region markers obtained by random amplified polymorphic DNA for M. fructicola: IColaS (GAGACGCACACAGAGTCAG) and IColaAS (GAGACGCACATAGCATTGG) (3). The expected PCR product of 386 bp was produced only in M. fructicola isolates. Koch's postulates were fulfilled with the three isolates by inoculating five healthy fruit with a conidial suspension of each isolate (104 conidia ml-1). Symptoms similar to those observed in the field were small brown spots, which rapidly showed brown rot. Noninoculated control fruit did not show symptoms. The fungus was reisolated on PDA from inoculated fruit after 4 days of incubation at 22°C, 80 to 100% relative humidity, and 16 h under fluorescent lighting, 100 μE·m-2·s-1. To our knowledge, this is the first report of M. fructicola in peach orchards in Spain.

First Report of Brown Rot of Stone Fruit Caused by Monilinia fructicola in Italy

Monilinia fructicola, causal agent of brown rot, is one of the most important fungal pathogens of stone fruit. M. fructicola is a quarantined pathogen in Europe. During the summer of 2008 in 15 orchards located in Piedmont (northern Italy), 12,500 stone fruits (cherries, apricots, peaches, nectarines, and plums) were stored in cold chambers at 4 and 6°C and monitored for 8 weeks for the presence of Monilinia spp. M. fructicola was detected on 0.5% of nectarines (cvs. Sweet Red and Orion) that originated from two orchards in Lagnasco. Symptoms appeared on the fruit during storage, starting 3 weeks after harvest. Fruit rot lesions were brown, sunken, and covered with grayish tufts. The majority of infected fruit became dry and mummified. Brown rot symptoms were similar to those caused by endemic M. fructigena and M. laxa. Symptoms began with a small, circular, brown spot, and the rot spread rapidly. At the same time, numerous, small, grayish stromata developed. Finally, the whole surface of the fruit was covered by conidial tufts. Tissues were excised from diseased stone fruits and cultured on potato dextrose agar (PDA) amended with 25 μg of streptomycin per liter. The isolates produced abundant mycelium on PDA at 20 ± 2°C. Colonies were initially gray, but after sporulation, they became hazel, showing concentric rings (sporulation is sparse in M. laxa or M. fructigena). Conidia were one-celled, ellipsoid, hyaline, 15.2 × 10.1 μm, and produced in branched monilioid chains (2). Preliminary morphological identification of fungi resembling M. fructicola was confirmed by PCR using genomic DNA extracted from the mycelia of pure cultures. The DNA was amplified with a common reverse primer and three species-specific forward primers (3) obtained from a sequence characterized amplified region and a product of 535 bp, diagnostic for the species M. fructicola, was obtained. BLAST analysis of the amplified sequence (GenBank Accession No. FI569728) showed 96% similarity to the sequence of a M. fructicola isolated from Canada (GenBank Accession No. AF506700), 15% similarity to M. laxa ATCC11790 (GenBank Accession No. AF506702), and 35% similarity to a M. fructigena sequence isolated in Italy (GenBank Accession No. AF506701). Moreover, two sequences obtained through the amplification of ribosomal region ITS1-5.8S-ITS2, showing 100% similarity to the same ribosomal sequence of M. fructicola, were deposited in GenBank (Accession Nos. FJ411109 and FJ411110). The pathogen was detected on some mummified fruit from the same orchards in November of 2008. Pathogenicity was tested by spraying 103 conidia/ml on 10 surface-sterilized artificially wounded nectarines per strain of M. fructicola. After 5 days of incubation at 20 ± 2°C, typical, brown, rot symptoms developed on inoculated fruit. M. fructicola was reisolated from the inoculated fruit on PDA. Symptoms did not appear on control fruit. To our knowledge, this is the first report of M. fructicola in Italy. Its occurrence in Europe has been reported sporadically in Austria and France, and in 2006, it was detected in Hungary and Switzerland on peaches and nectarines imported from Italy and Spain (1,4).

A Rapid Method to Quantify Fungicide Sensitivity in the Brown Rot Pathogen Monilinia fructicola.

Management of brown rot of stone fruit relies upon the application of effective fungicides that may be compromised by the development of fungicide resistance. We evaluated fungicide resistance in the brown rot pathogen, Monilinia fructicola, using Alamar blue (AB) dye, or resazurin, a chromogenic substrate that can be used as an indicator of respiration, in a 96-well microtiter format. We compared the AB method to traditional mycelial growth assays for resistance screening using 10 isolates of M. fructicola that represented a range of sensitivities to fenbuconazole. Using traditional mycelial growth assays, isolate sensitivity ranged from 17.7 to 115.3% growth on medium amended with fenbuconazole at 0.03 μg/ml relative to that on nonamended medium. Concordant results between both assays were obtained (R2 = 0.9943, P < 0.0001), but the AB method provided results within 24 h, as opposed to the 3- to 5-day period required for mycelial growth assays. We found that sensitive isolates reduced AB less than resistant isolates in the presence of fungicide. Spore density influenced the reduction of AB by M. fructicola; spectrophotometric discrimination of fungicide sensitivity was best achieved at a density of 105 spores/ml.

Population dynamics ofEpicoccum nigrum, a biocontrol agent against brown rot in stone fruit

To study the population dynamics of Epicoccum nigrum on peaches and nectarines and to enhance its colonization on fruit surfaces to improve its biocontrol efficacy against brown rot. Twelve surveys were performed to study E. nigrum populations and their effect on the number of the pathogenic Monilinia spp. conidia in peach orchards in Spain and Italy between 2002 and 2005. Fresh conidia and five different formulations of E. nigrum conidia were applied three to six times to peach and nectarine trees from full flowering to harvest. The size of the E. nigrum populations was determined from the number of colony-forming units and conidial numbers per flower or fruit. Treatment with all conidial formulations increased the size of the indigenous conidial population on peach surfaces. Formulations of E. nigrum having high viability are most effective against conidia of the pathogen when applied at pit hardening and during the month immediately before fruit harvest. Application of an E. nigrum conidial formulation decreased the number of conidia of Monilinia spp. on fruit surfaces during the growing season to the same extent as fungicides.

Effects of potassium sorbate on postharvest brown rot of stone fruit.

The effect of potassium sorbate (K-sorb), a low-toxicity chemical, to control Monilinia spp. was investigated. Preliminary in vitro studies found the MIC of K-sorb for conidial germination and mycelial growth was, respectively, 260 and 1,250 mg/ liter. Immersion of naturally infected peach and nectarine fruit in a solution (15 g/liter) of K-sorb for 120 s reduced brown rot by over 80% in four of five trials. Although treated fruits showed a significant reduction in firmness with respect to the control, they did not reach the overripe stage and retained acceptable quality parameters. In an attempt to elucidate the mechanism of action for K-sorb, the inhibition of enzymatic activity by K-sorb was also tested. In a radial diffusion assay, the addition of K-sorb to agarose reduced polygalacturonase (PG) activity across the concentrations considered. The greatest reduction (54.3%, with respect to the control) was obtained at a sorbate concentration of 15 g/liter. PG kinetic activity of Monilinia laxa observed by a spectrophotometric assay peaked after 40 min in all samples tested. PG activity was significantly higher in the control than in the samples with increased K-sorb concentrations. In conclusion, based on these findings, K-sorb can be recommended as a low-toxicity antifungal compound against Monilinia spp. in peaches and nectarines with its mode of action probably depending in part on the inhibition of PG activity in M. laxa.

Quantification of allele E198A in beta‐tubulin conferring benzimidazole resistance in Monilinia fructicola using real‐time PCR

Thiophanate-methyl, a member of the benzimidazole class of fungicides, is used in California to control brown rot of stone fruit caused by Monilinia fructicola (G. Wint.) Honey. The goal of this study was to develop a real-time polymerase chain reaction (PCR) assay as an efficient method to quantify the E198A allele of beta-tubulin that confers benzimidazole resistance. Using the real-time PCR assay, the frequency of allele E198A (FEA) in a population was determined from the quantities of DNA amplified with the E198A allele-specific primer pair HRF/HRR and the M. fructicola-specific primer pair MfF6/MfR6. The average proportions of highly resistant isolates determined with the conventional fungicide sensitivity method were within the range of average FEA values determined with the real-time PCR assay. We also determined the FEAs of M. fructicola populations sampled from 21 stone fruit orchards in California. Only one orchard showed a high FEA over 0.20, seven orchards had values between 0.01 and 0.1, and 13 orchards had values less than 0.01. The real-time PCR assay developed in this study provides a potentially useful tool to efficiently quantify benzimidazole resistance for large M. fructicola populations.