Inoculated Apple Fruit Research Articles

Rapid Detection of Phytophthora cambivora Using Recombinase Polymerase Amplification Combined with CRISPR/Cas12a

Phytophthora cambivora is a major quarantine pathogen that devastates economically important plants across the globe. P. cambivora causes ink disease in chestnut trees and root and stem rot in various fruit trees, resulting in significant yield reductions and plant death. Given the potential dangers of P. cambivora, effective detection methods are needed for both disease management and prevention. In this study, based on the whole-genome screening of specific target genes, a combination of the recombinase polymerase amplification technique (RPA) and CRISPR/Cas12 was established to detect P. cambivora. The RPA-CRISPR/Cas12a assay was able to specifically detect 7 target isolates of P. cambivora but did not detect the following 68 non-target isolates, including 28 isolates of Phytophthora, 3 isolates of Pythium, 3 isolates of Phytopythium, 32 isolates of fungi, and 2 isolates of Bursaphelenchus. The RPA-CRISPR/Cas12a detection method was able to detect 10 pg·μL−1 of P. cambivora genomic DNA at 37 °C within a short time span (60 min). Additionally, this method can identify the presence of P. cambivora in artificially inoculated apple fruits. In summary, compared with conventional detection techniques, the RPA-CRISPR/Cas12a detection method eliminates the need for expensive instruments, long reaction times, and high amounts of raw materials and can detect P. cambivora in imported plants at entry ports, enabling instant prevention and detection.

Reactive Oxygen Species Metabolism Modulation on the Quality of Apple Fruits Inoculated with Penicillium expansum under Different Ambient pHs

Apple blue mold is a significant postharvest disease caused by Penicillium expansum. pH modification in colonized tissues leads to the production of organic substances, the modulation of enzymes, and then increases fungal pathogenicity. This study evaluated Penicillium expansum-inoculated apple fruits’ quality responding to pH treatments ranging from 2.5 to 8.5 and analyzed the reactive oxygen species (ROS) metabolism modulation in inoculated apple fruits at the same pH. The results showed that the fruit quality of the firmness, total soluble solids, and titratable acid displayed a quick loss at pHs 5.0 and 7.0, compared with 2.5 and 8.5. Similarly, higher disease incidence was observed at pHs 5.0 and 7.0. Apple fruits infected with P. expansum at pHs 2.5 and 8.5 had less content of O2•−, H2O2, and malondialdehyde (MDA); lower enzymatic activity of NADPH oxidase (NOX); and greater cell membrane integrity than those at pHs 5.0 and 7.0. The analysis of the antioxidant enzymatic activities showed upregulation of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) at pHs 2.5 and 8.5 compared with those at pHs 5.0 and 7.0. Similar trends were shown in ascorbic acid and glutathione. These results support the hypothesis that inoculated apple fruits at pHs 2.5 and 8.5 improve resistance to P. expansum by modulating ROS metabolism, compared with pHs 5.0 and 7.0.

Fusarium incarnatum Causing Fusarium Wilt on Protea (Protea cynaroides L.) in Turkey

Protea (Protea cynaroides L.) is one of the most important ornamental plants commercially cultivated in Turkey and other parts of the world. In May 2017, protea plants heavily showed wilting symptoms were obtained from the protea growing areas at Hatay province of Turkey. Brown vascular tissue in the stems of the infected plants was observed. To determine the causal agent of the disease, necrotic tissue pieces taken from diseased plants were surface-sterilized with 10% sodium hypochlorite, two times rinsed with sterile distilled water, placed on Petri dishes containing potato dextrose agar (PDA), and incubated at 24 oC for seven days. Microconidia were no septate, ovoid, hyaline, single-celled, and 9 to 12 × 1 to 3 μm. Macroconidia were slightly curved, four to six septate, and 27 to 31 × 3 to 5 μm. Based on the morphology and cultural features, the fungus was identified as F. incarnatum, which belongs to the F. incarnatum-equiseti species complex (FIESC). In addition, a pathogenicity test was made using the apple fruits. The pathogen was re-isolated from inoculated apple fruits and both cultural and morphological characteristics of the pathogen were identical. To confirm the identity of one representative isolate, the internal transcribed spacer (ITS) region including 5.8S rDNA was amplified and sequenced with primers of ITS-1 and ITS-4. Fusarium MLST, Fusarium-ID and GenBank database were used for the identification of sequence. The amplified 465 bp product has 99% nucleotide identity with the sequences of F. incarnatum-equiseti species complex of MLST types and deposited in GenBank under accession number (MH005097). According to the literature, this is the first molecular identification of F. incarnatum on protea plants in Turkey.

Utilization of waste glycerol for the production of biocontrol agents nigericin and niphimycin by Streptomyces hygroscopicus: bioprocess development

ABSTRACT Search for more environment-friendly methods for controlling plant diseases that would contribute to the goal of sustainability in agriculture is in focus. In the present study, the potential of Streptomyces hygroscopicus isolated from soil sample in the production of biocontrol agents, nigericin and niphimycin, effective against Alternaria alternata storage apple pathogen was examined. Also, modelling and optimization of medium composition for biocontrol agent biosynthesis was performed. The results showed that the optimum amount of C3H8O3, (NH4)2SO4 and K2HPO4 in the medium for Streptomyces hygroscopicus biosynthesis is 20, 0.25 and 1.46 g/L, respectively. Scale-up and validation of the obtained results performed in the 3 L laboratory-scale bioreactor showed that on the optimized medium at an aeration rate of 0.7 vvm and an agitation speed of 200 rpm, produced nigericin and niphimycin, showed high activity. Under the same conditions, cultivation of S. hygroscopicus was performed in a 7 L laboratory bioreactor in a medium with waste glycerol instead of pure glycerol. Results showed that the methanol extract of S. hygroscopicus cultivation liquid, containing nigericin and niphimycin, was high effective against two Alternaria isolates. This was confirmed in vitro by obtaining large inhibition zone diameters on A. alternata KA10 (47 mm) and T1Jg3 (44.33 mm) isolates. After successful in vitro analysis, in planta testing was performed. It was found that necrosis diameters that were measured on artificially inoculated apple fruits with A. alternata compared to necrosis diameter measured on untreated, control fruits, were 4.47 and 3.56 times smaller.

Unraveling Interactions of the Necrotrophic Fungal Species Botrytis cinerea With 1-Methylcyclopropene or Ozone-Treated Apple Fruit Using Proteomic Analysis.

Gray mold caused by the necrotrophic fungus Botrytis cinerea is one of the major postharvest diseases of apple fruit. The exogenous application of 1-methylcyclopropene (1-MCP) and gaseous ozone (O 3) is commonly used to ensure postharvest fruit quality. However, the effect of these treatments on the susceptibility of apple fruit to postharvest pathogens remains largely unknown. Herein, the effect of O 3 and 1-MCP treatments on the development of gray mold on apple fruit (cv. “Granny Smith”) was investigated. Artificially inoculated apple fruits, treated or not with 1-MCP, were subjected for 2 months to cold storage [0°C, relative humidity (RH) 95%] either in an O3-enriched atmosphere or in a conventional cold chamber. Minor differences between 1-MCP-treated and control fruits were found in terms of disease expression; however, exposure to ozone resulted in a decrease of disease severity by more than 50% compared with 1-MCP-treated and untreated fruits. Proteomic analysis was conducted to determine proteome changes in the mesocarp tissue of control and 1-MCP- or O3-treated fruits in the absence or in the presence of inoculation with B. cinerea. In the non-inoculated fruits, 26 proteins were affected by 1-MCP, while 51 proteins were altered by ozone. Dynamic changes in fruit proteome were also observed in response to B. cinerea. In O3-treated fruits, a significant number of disease/defense-related proteins were increased in comparison with control fruit. Among these proteins, higher accumulation levels were observed for allergen, major allergen, ACC oxidase, putative NBS-LRR disease resistance protein, major latex protein (MLP)-like protein, or 2-Cys peroxiredoxin. In contrast, most of these proteins were down-accumulated in 1-MCP-treated fruits that were challenged with B. cinerea. These results suggest that ozone exposure may contribute to the reduction of gray mold in apple fruits, while 1-MCP was not effective in affecting this disease. This is the first study deciphering differential regulations of apple fruit proteome upon B. cinerea infection and postharvest storage treatments, underlying aspects of host response related to the gray mold disease.

Penicillium expansum – Induced release of branched-chain volatile compounds in apple fruit by increasing amino acids accumulation

Abstract Amino acids (AAs) play a critical role in resistance response and biosynthesis of volatile compounds (VOCs) in fruit. In this study, we inoculated apple fruits (cv. Delicious) with Penicillium expansum to investigate the effect of different AA accumulation on the biosynthesis of branched-chain VOCs at different stages of colonization. Generally, infection significantly increased the total AAs in fruit by 38.65 %. Of the 17 AAs detected in fruit, the contents of Asp, Val, Leu, Ala, Ser, Cys, Thr, His, Agr, Glu, Met, Gly, and Pro increased in the early and middle stages of colonization ( 6 DAI). Moreover, infection promoted higher activities of PDC and PDH involved in branched-chain VOCs biosynthesis, leading to increased contents of branched-chain VOCs. These results suggest that P. expansum increases AA’s accumulation and release of branched-chain VOCs in apple fruit.

Precipitation Impacts Dissemination of Three Sooty Blotch and Flyspeck Taxa on Apple Fruit.

The spatial dissemination of three prevalent taxa of sooty blotch and flyspeck (SBFS) fungi under several levels of precipitation was compared during 2015 and 2016 in an Iowa apple orchard. Overhead irrigation was used to supplement ambient precipitation in order to insure SBFS spore dissemination and colony development. There were five irrigation levels, involving 1-min-long periods of irrigation that were imposed either once or twice per hour at intervals of 3, 6, or 12 h, as well as a nonirrigated control. Preselected apple fruit were inoculated with one of the three SBFS taxa to serve as sources of inoculum. Dissemination from these inoculated apple fruit was assessed at harvest by counting SBFS colonies on water-sprayed and nontreated fruit. As a further control, additional fruit were enclosed in fruit bags throughout the fruit development period. In both 2015 and 2016, the number of colonies of the SBFS fungus Peltaster gemmifer per apple increased sharply as the duration of irrigation increased, whereas the number of colonies of Microcyclosporella mali increased to a lesser extent and Stomiopeltis sp. RS1 showed no increase. In 2015, the linear relationship between the duration of irrigation-imposed precipitation levels and the number of colonies on the water-sprayed apple fruit was similar for P. gemmifer (slope = 0.09), Stomiopeltis sp. RS1 (slope = 0.07), and Microcyclosporella mali (slope = 0.13); whereas, in 2016, the slope was higher for P. gemmifer (0.28) than for Stomiopeltis sp. RS1 (-0.09) or M. mali (0.06). The results indicated that dissemination of P. gemmifer increased sharply in response to increased irrigation-imposed precipitation, and that dissemination patterns differed considerably among the three SBFS taxa. The apparent advantage of P. gemmifer in precipitation-triggered dissemination may stem from its ability to produce spores rapidly by budding. To our knowledge, this is the first article to assess splash dispersal by SBFS fungi in the field and the first to document taxon-specific patterns of dissemination in this pathogen complex.

Biocontrol of Monilinia fructigena, causal agent of brown rot of apple fruit, by using endophytic yeasts

In this study; an isolate of Monilinia fructigena the causal agent of apple fruit brown rot was isolated from rotten apple fruits cv. Golden delicious. In the in vivo pathogenicity assay, it presented significant disease incidence on the inoculated apple fruit, thus confirming its virulence. Five endophytic yeasts were isolated from healthy apple fruits; identified according to their microscopic characteristics as; Schwanniomyces vanrijiae, Galactomyces geotrichum, Pichia kudriavzevii, Debaryomyces hansenii, and Rhodotorula glutinis. The first three isolates showed appreciable inhibitory potential against M. fructigena by in vitro test; moreover, they caused a significant inhibition of germination of pathogen conidia by 67.6–89.2%. Identification of these three potent yeasts in addition to M. fructigena isolate was confirmed by PCR analysis through amplification of ITS region. In in vivo assays, fruits were inoculated with each of the 5 BCAs and M. fructigena simultaneously; 24 h before, and 24 h after pathogen inoculation, showing a noticeable reduction of disease incidence by 84.02–89.5%, 80.1–86.9%, 56.3–86.9% respectively, compared with the controls. By in vitro plate assays, BCAs were able to produce fungal cell wall hydrolyzing enzymes such as: chitinases, pectinases, β-1,3-glucanase, and protease, in addition to the production of a killer toxin. At 10 mg/L of Fe+3 in PDA, the three BCAs recorded significant in vitro inhibition of M. fructigena growth. The three potent BCAs do not express human pathogenicity traits. The aims of the current study were to isolate and characterize yeasts able to inhibit the growth of M. fructigena during apple storage.

Effect of hot water treatments on apple fruit rot caused by Fusarium spp.

Hot water treatments of apple fruits prior to long-term storage have a high potential as a physical measure against apple fruit storage rot caused by plant pathogenic fungi. Fusarium species are increasingly found as causal agents of postharvest apple fruit rot. The effect of high temperatures on casual agents of Fusarium fruit rot, F. avenaceum and F. graminearum, was tested in this article. In in vitro assay, spore suspensions of F. avenaceum and F. graminearum were exposed to high temperatures (ranging from 45 to 90 °C) at different duration (ranging from 30 s to 20 min) and the treatments that significantly inhibited mycelial growth of the isolates (53 and 57 °C for 3 and 5 min) were tested in vivo by hot water treatment of inoculated apple fruits of Fuji cultivar. The obtained results showed that the most promising hot water treatment should be between 53 and 57 °C and between 3 and 5 min of the exposure period.

Effects of developed thyme and oregano essential oil formulations on Monilinia laxa and Monilinia fructicola

Essential oils have been well-known for their antimicrobial properties for a very long time. Some of them have been effectively used in human medicine for decades. Our earlier investigation revealed a great potential of thyme and oregano essential oils as crop protectants against some postharvest fruit pathogens. The effects of formulated thyme and oregano essential oils on Monilinia laxa and Monilinia fructicola were studied in vitro and in vivo. In vitro antagonistic assays were performed on solidified PDA medium using a slightly modified agar overlay technique, while in vivo experiments were conducted on inoculated apple fruits. In vitro essays showed that the developed formulations (emulsifiable concentrates - EC) significantly inhibited the mycelial growth of Monilinia spp. Experiments in vivo, performed on inoculated apple fruits, revealed that the developed formulations provided a significant level of Monilinia spp. suppression. To our knowledge, another EC formulation of oregano essential oil intended for use in Monilinia spp. control has never been developed before. The presented results are initial findings and evaluation of the activity of the developed products should therefore proceed under field conditions to determine their efficacy and activity spectrum, and to estimate economic aspects of their use.

Use of local bioresources for post-harvest management of white rot of apple caused by Botryosphaeria dothidea

Post-harvest fungal pathogens are reported to cause 10 to 25 percent losses in apple. Among various pathogens, white rot caused by Botryosphaeria dothidea is one of the important post-harvest diseases. Incidence of white rot was recorded between3.0-14.1 percent in important marketing yards of Himachal Pradesh. Golden Delicious variety of apple has been found to be most susceptible to white rot pathogen. Two botanical formulations by mixing equal quantity of leaves of Karu (Roylea elegans), Artemisia (Artemisia roxburghiana), Neem (Azadirachta indica), Bana (Vitex negundo), Tulsi (Ocimum sanctum) and seeds of Darek (Melia azedarach) made in water and cowurine were evaluated against the white rot pathogen. Fruit dip for 30 minutes in cow urine based formulation did not allowed the disease development to form lesions in artificially inoculated apple fruits. Also, fruits dipped in cow urine based formulation followed by their storage in card board boxes at room temperature (25± 30C) for 30 days storage had minimum incidence (2.8%) of white rot disease. Therefore, dip treatment of apple fruits with different bio -resources including water and cow urine based botanical formulation separately proved effective in the management of white rot (Botryosphaeria dothidea) of apple.

Phenotypic, molecular and pathogenic characterization of Phlyctema vagabunda, causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.

Effect of storage conditions on virulence of Fusarium avenaceum and Alternaria alternata on apple fruits

AbstractTo overcome difficulty in phytopathogenic fungi control during storage of apple fruits, the effect of different storage conditions on the occurrence and development of Fusarium avenaceum and Alternaria alternata infections on apple cultivar “Cripps Pink” was investigated during and after storage. Inhibitory effects of wild oregano essential oil on apple fruit rots caused by F. avenaceum and A. alternata were also tested as possible rot control measure. Artificially inoculated apple fruits were kept in cold storage with normal (NA) and controlled (CA) atmosphere for 95 days and at room temperature only. The obtained results indicated that different storage conditions significantly affect necrosis development on apple fruits caused by F. avenaceum and A. alternata after storage, as well as during shelf life.

Comparative study of Monilinia fructigena and Monilia polystroma on morphological features, RFLP analysis, pathogenicity and histopathology

In this study, we compared cultural, morphological, pathogenic and biological features of Serbian isolates of Monilia polystroma and Monilinia fructigena from apple fruit. Isolates of M. polystroma formed characteristic stromatal plates throughout the cultures unlike M. fructigena, while mycelial growth rates were not distinguishable features between species. Conidia of M. fructigena were larger than M. polystroma, and both species produced slightly larger conidia on apple fruit than on V8 medium. The existence and morphology of the microconidial state of M. polystroma was described for the first time using light and SEM microscopy. Microconidia were globose (2.19–3.44 μm), similar to M. fructigena. Digestion of polymerase chain reaction (PCR) product of ribosomal ITS1-5.8S-ITS2 region with restriction enzyme HhaI provided a reliable method for specific detection of M. polystroma. In a pathogenicity test, both species produced larger lesions on cv. Golden Delicious, than on cvs. Idared and Granny Smith. On cv. Golden Delicious, the mean lesion size was not significatly different between species, while on cvs. Idared and Granny Smith M. fructigena tended to produce slightly larger lesions compared to M. polystroma. On artificially inoculated apple fruit after 1 and 4 months of incubation, M. polystroma, unlike M. fructigena, was capable of forming thicker and more compact stromata with complex morphology, which consisted of densely interwoven medullary hyphae covered with a discontinuous layer of melanized hyphal rind cells. The dynamics of stromata formation beneath the host’s cuticle also showed differences between two species, where M. polystroma formed this layer after 1 month, while M. fructigena after 4 months of incubation.

Identification, characterization and mycotoxigenic ability of Alternaria spp. causing core rot of apple fruit in Greece

Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece. The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressiveness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequencies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial inoculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation at two different temperatures (2 and 25°C) showed that fruit of Fuji variety were the most susceptible and fruit of Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species. Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially inoculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD). The results showed that most of the isolates of both species were able to produce all the 3 metabolites both in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of apple by-products.

Optimization of cultivation medium for enhanced production of antifungal metabolites by Streptomyces hygroscopicus

Antifungal potential of Streptomyces hygroscopicus metabolites was tested against the isolates of Colletotrichum spp., obtained from rotten apple fruits. Experimental factorial design and response surface methodology were employed to optimize medium composition and enhance antifungal components productivity by S. hygroscopicus. Results of optimization suggest that the optimal medium composition is (g/L): glycerol (13.92), yeast extract (5.00), phosphates (0.63), CaCO3, (3.0), NaCl, (3.0), MgSO4, (0.5). Further research was performed to validate the obtained results and confirm their applicability in planta on artificially inoculated apple fruits. The obtained efficacy after 14 days of incubation was 99.3% against Colletotrichum acutatum and 99.8% against Colletotrichum gloeosporioides. Application of supernatant of S. hygroscopicus cultivation medium, produced under defined conditions, in biological control of apple rot could offer safe and healthy apple fruits on the market, while simultaneously helping to solve the problem of waste glycerol disposal in the biodiesel industry.

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.

First Report of Brown Rot Caused by Monilia polystroma on Apple in Serbia.

Monilia polystroma van Leeuwen is a new Japanese species, similar to M. fructigena but distinguishable based on morphological and molecular characteristics (3). After its first discovery on apple in Japan, occurance of M. polystroma in Europe has been reported in Hungary, the Czech Republic, and Switzerland (2,3,4). In October 2011, during a survey for apple fungal pathogens in the Bela Crkva district, 15 apple fruit (Malus domestica Borkh.) cv. Golden Delicious were collected. Two isolates of Monilinia polystroma were obtained from apple fruit showing brown rot, covered with small yellowish sporodohia. The pathogen was identified as M. polystroma based on morphological and molecular features (1,3). Upon isolation, colonies cultivated on PDA were white to grayish and the mycelium grew 8.85 mm per day at 22 ± 1°C in 12-h light/12-h dark regime. After 6 to 8 days of incubation, black stromatal plates were observed on the reverse sides of the inoculated petri dishes. Conidia were one-celled, limoniform, hyaline, 14.7 to 21.88 μm (16.2 mean) × 7.85 to 12.92 μm (10.8 mean), and were produced in branched monilioid chains on inoculated apple fruit. Morphological identification was confirmed by PCR (1) using genomic DNA extracted from the mycelium of pure cultures, and amplified products of 425 bp in length, specific for M. polystroma were amplified as expected with primers MO368-5 and MO368-8R. For one isolate, the ribosomal ITS1-5.8S-ITS2 region was obtained, using primers ITS1 and ITS4, and deposited in GenBank (Accession No JX315717). The sequence was 498 bp in length and showed 100% identity with sequences deposited for M. polystroma in NCBI GenBank (JN128835, AM937114, GU067539). Pathogenicity was confirmed by wound-inoculating five surface-sterilized, mature apple fruit with mycelium plugs (5 mm in diameter) of both isolates grown on PDA. Control fruit were inoculated with sterile PDA plugs. After 3 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22 ± 1°C, typical symptoms of brown rot developed on inoculated fruit, while control fruit remained symptomless. Isolates recovered from symptomatic fruit showed the same morphological and molecular characteristics as original isolates. To the best of our knowledge, this is the first report of M. polystroma in Serbia. Further studies are necessary to estimate the economic importance and geographic distribution of this organism in Serbia.

Molecular detection of Monilinia fructigena as causal agent of brown rot on quince

Species of the genus Monilinia are important causal agents of fruit rot on pome and stone fruits in Serbia. The pathogen is very harmful, especially on small properties and cottage plantations where intensive control measures are not applied. Quince is important host for the pathogens of this genus. During spring 2010, intensive occurrence of mummified fruits overwintering on branches of the quince was observed. The pathogen was isolated using standard phytopathological methods. Pathogenicity of eight obtained isolates was tested by artificial inoculation of injured apple fruits. Identification was performed according to pathogenic, morphological and ecological properties, and was confirmed by Multiplex polimeraze chain reaction, PCR. All the isolates studied caused brown rot on inoculated apple fruits. The isolates form light yellow colonies with lobate margins, with single-celled, transparent, elliptical or oval conidia in chains, regardless temperature or light presence. Sclerotia are observed in 14 days old cultures. The highest growth rate of most of the isolates is at 27?C and in dark. Based on studied pathogenic, morphological and ecological characteristics, it was found that the Monilinia fructigena is causal agent of brown rot of quince. Using specific primers (MO368-5, MO368-8R, MO368-10R, Laxa-R2) for detection of Monilinia species in Multiplex PCR reaction, the expected fragment 402 bp in size was amplified, which confirmed that the studied isolates belonged to the species M. fructigena.

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.