Ivana Vico

First report of Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus in bottlegourd (Lagenaria siceraria) in Serbia.

During a cucurbit disease survey in August 2004, severe symptoms resembling those caused by viruses were observed on bottlegourd (Lagenaria siceraria (Molina) Standl.) in the Vojvodina region of Serbia. Symptoms included stunting, mosaic, green veinbanding, blistering, yellowing, chlorotic spots, leaf deformation, and fruit distortion. Leaf samples from 25 symptomatic plants were collected from two localities for virus identification using mechanical transmission and serological testing. Crude sap extract from leaf samples was mechanically inoculated onto bottlegourd and pumpkin (Cucurbita pepo) under greenhouse conditions. Field-collected bottlegourd and inoculated plants were tested using double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA). Positive reactions were obtained on collected and inoculated plants with polyclonal antiserum (Loewe Biochemica, Sauerlach, Germany) to Zucchini yellow mosaic virus(ZYMV) in 23 samples, with antiserum to Watermelon mosaic virus (WMV) in eight samples, and with antiserum to Cucumber mosaic virus (CMV) in seven samples. Each of the three viruses was detected in single as well as in mixed infections with the other two viruses. Biological characterization of viruses detected in single infections was done on the following indicator plants: Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Cucumis melo, Citrullus lanatus, Nicotiana glutinosa, and N. tabacum cv. Samsun. The symptoms observed on indicator plants for each isolate corresponded to the results of DAS-ELISA (2,3). All three viruses are known to be important pathogens of cucurbit plants and were previously reported in pumpkin in Serbia (1). To our knowledge, this is the first report of ZYMV, WMV, and CMV in bottlegourd in Serbia. References: (1) N. Dukić et al. J. Agric. Sci. 47:149, 2002. (2) D. E. Lesemann et al. Phytopathol. Z. 108:304, 1983. (3) H. Rahimian and K. Izadpanah. Phytopathol. Z. 92:305, 1978.

Antifungal Activity of Three Essential Oils against Colletotrichum acutatum, the Causal Agent of Strawberry Anthracnose

Abstract The antifungal effects of thyme, cinnamon bark and clove bud essential oils (EOs) were investigated in vitro on Colletotrichum acutatum mycelial growth, conidial germination, appressoria formation, and in vivo on strawberry fruit disease incidence. All tested EOs, incorporated in potato-dextrose agar, inhibited C. acutatum mycelial growth, and had a fungistatic effect at concentration 667 µl/l of medium. Volatiles of cinnamon bark, thyme and clove bud EOs completely prevented conidial germination at the lowest concentrations of 1.53, 15.3 and 76.5 µl/l of air, respectively, and disabled appressoria formation at concentration of 1.53 µl/l of air. On inoculated strawberry fruit, thyme and cinnamon bark EO volatiles reduced anthracnose incidence at concentrations above 15.3 and 76.5 µl/l of air, respectively. GC-FID and GC-MS analysis showed that major components of thyme EO were p-cymene, thymol, α-terpineol, carvacrol; cinnamon bark EO: trans- cinnameldehyde, trans-cinnamyl acetate; clove bud EO: eugenol and β-caryophyllene. Our results suggest that volatiles of thyme and cinnamon bark EOs are effective against C. acutatum both in vitro and in vivo.

First Report of Penicillium polonicum Causing Blue Mold on Stored Onion (Allium cepa) in Serbia

Penicillium polonicum K. Zaleski is an economically important airborne fungus with a broad host range including cereals, peanuts, onions, dried meats, citrus fruits, and yam tubers (2,4). Secondary metabolites produced by this species include harmful mycotoxins penicillic acid, verucosidin, and nephrotoxic glycopeptides, which may play a role in Balkan Endemic Nephropathy (2,5). In January 2013, decayed onion bulbs (Allium cepa L. cv. Meranto) with blue mold symptoms were found causing significant economic losses at a storage facility in Stara Pazova, Serbia, and were collected. The decayed area of the bulbs was pale yellow to light brown, and tissue was soft and watery. Bluish green sporulation was abundant on the surface and inside the bulb, between decayed scales. Two isolates (designated L1a and L4p) were obtained and further characterized using morphological and molecular methods. Colonies on potato dextrose agar (PDA), Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) media at 25°C after 7 days were blue green, velutinous, with clear exudate present on CYA. Colony reverse color on CYA and YES for both isolates were cream to yellow brown. The mean colony diameter on PDA for L1a was 29.89 ± 0.96 mm, and for L4p was 26 ± 0.37 mm; on CYA 32.56 ± 0.53 mm for L1a and 30.11 ± 2.42 mm for L4p; and on YES 33.86 ± 1.59 mm for L1a and 31.17 ± 1.83 mm for L4p. No growth was observed on CYA when isolates were incubated at 37°C. Conidiophores of both isolates were terverticillate, stipes were septate with smooth to finely roughened walls, and phialides were ampulliform. Conidia were globose to subglobose, smooth-walled, and borne in columns. Conidial dimensions for L4p were 2.72 to 3.82 (3.26) × 2.36 to 3.42 (2.95) μm, and for L1a were 2.87 to 4.39 (3.58) × 2.53 to 3.79 (3.16) μm (n = 50). Both isolates tested positive for the production of cyclopiazonic acid and other alkaloids, as indicated by a violet reaction for the Ehrlich test. Morphological characters of L1a and L4p were in accordance with those described for P. polonicum K. Zaleski (2). Genomic DNA was isolated using CTAB extraction method (1) and molecular identification was completed using gene specific primers for the β-tubulin locus (Bt-LEV-Up4/Bt-LEV-Lo1) via conventional PCR (3). The nucleotide sequences of amplified products (~800 bp) have been assigned to GenBank (KJ570971 and 72). MegaBLAST of obtained sequences showed a 99% similarity with several sequences of P. polonicum deposited in GenBank, which confirmed the morphological identification. Pathogenicity was tested by wound inoculation of 10 surface sanitized onion bulbs cv. Meranto with 50 μl of a 105/ml conidial suspension from isolates grown on PDA. Ten control onion bulbs were wound-inoculated with Tween-treated sterile distilled water. After 30 days incubation in plastic containers, under high humidity at 22°C, typical symptoms of blue mold developed on inoculated bulbs, while non-inoculated controls remained symptomless. Isolates recovered from inoculated bulbs showed the same morphological characteristics as the original isolates, thus completing Kochs postulates. To our knowledge, this is the first report of P. polonicum on stored onion in Serbia. Results from this study indicate that a holistic approach to control this fungus should be implemented that may include one or all of the following: increased sanitation methods to eliminate inoculum, breeding for resistant onion cultivars, and integration of additional control methods to maintain onion quality during storage. References: (1) J. P. Day and R. C. Shattock. Eur. J. Plant Pathol 103:379, 1997. (2) J. C. Frisvad and R. A. Samson. Stud. Mycol. 49:1, 2004. (3) S. N. de Jong et al. Mycol. Res. 105:658, 2001. (4) W. K. Kim et al. Mycobiology 36:217, 2008. (5) P. G. Mantle. Facta Univ. Ser. Med. Biol. 9:64, 2002.

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 β-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) μm. 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 β-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. [Projekat Ministarstva nauke Republike Srbije, br. III46008]

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

Botryosphaeria dothidea (Moug.: Fr.) Ces. & De Not has a worldwide distribution infecting species from over 80 genera of plants (1). Apart from being an important pathogen of apple trees in many countries, B. dothidea can cause pre- and postharvest decay on apple fruit (2). It has been known to cause canker and dieback of forest trees in Serbia (3), but has not been recorded either on apple trees or apple fruit. In December 2010, apple fruit cv. Idared (Malus × domestica Borkh.) with symptoms of white rot were collected from one storage in the area of Svilajnac in Serbia. The incidence of the disease was low but the symptoms were severe. Affected fruit were brown, soft, and almost completely decayed, while the internal decayed tissue appeared watery and brown. A fungus was isolated from symptomatic tissue of one fruit after surface sterilization with 70% ethanol (without rinsing) and aseptic removal of the skin. Small fragments of decayed tissue were placed on potato dextrose agar (PDA) and incubated in a chamber at 22°C under alternating light and dark conditions (12/12 h). Fungal colonies were initially whitish, but started turning dark gray to black after 5 to 6 days. Pycnidia were produced after 20 to 25 days of incubation at 22°C and contained one-celled, elliptical, hyaline conidia. Conidia were 17.19 to 23.74 μm (mean 18.93) × 3.72 to 4.93 μm (mean 4.45) (n = 50). These morphological characteristics are in accordance with those described for the fungus B. dothidea (4). Genomic DNA was isolated from the fungus and internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The nucleotide sequence has been assigned to GenBank Accession No. KC994640. BLAST analysis of the 528-bp segment showed a 100% similarity with several sequences of B. dothidea deposited in NCBI GenBank, which confirmed morphological identification. Pathogenicity was tested by wound inoculation of five surface-sterilized, mature apple fruit cv. Idared with mycelium plugs (5 mm in diameter) of the isolate grown on PDA. Five control fruit were inoculated with sterile PDA plugs. After 5 days of incubation in plastic containers, under high humidity (RH 90 to 95%) at 22°C, typical symptoms of white rot developed on inoculated fruit, while wounded, uninoculated, control fruit remained symptomless. The isolate recovered from symptomatic fruit showed the same morphological features as original isolate. To the best of our knowledge, this is the first report of B. dothidea on apple fruit in Serbia. Apple is widely grown in Serbia and it is important to further investigate the presence of this pathogen in apple storage, as well as in orchards since B. dothidea may cause rapid disease outbreaks that result in severe losses. References: (1) G. H. Hapting Agriculture Handbook 386, USDA, Forest Service, 1971. (2) A. L. Jones and H. S. Aldwinckle Compendium of Apple and Pear Diseases. APS Press, St. Paul, MN, 1990. (3) D. Karadžic et al. Glasnik Šumarskog Fakulteta 83:87, 2000. (4) B. Slippers et al. Mycologia 96:83, 2004.

Carbon, nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ∼7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.

Distribution & characterization of Monilinia spp. causing apple fruit decay in Serbia

Brown rot, caused by Monilinia spp., is an economically important pre- and postharvest disease of pome and stone fruits worldwide. In Serbia, apple is the most widely grown pome fruit, and the distribution of economically important Monilinia spp. responsible for apple brown rot is unknown. Hence, we conducted a three year survey, from 2010 to 2012, where 349 isolates were obtained from six orchards and four storage facilities from five different apple cultivars with brown rot symptoms. Morphological characterization of the isolates, multiplex PCR, and phylogenetic analysis revealed four species: M. fructigena, M. laxa, M. fructicola, and Monilia polystroma. All species were found in the orchard and in storage, with M. fructigena predominating, followed by M. polystroma. Representative isolates were analyzed in vitro and in vivo where differences in growth rate, sporulation, and virulence on apple fruit were observed. Findings from this investigation demonstrate diversity in the species responsible for pre- and postharvest apple brown rot, which has significant implications for pathogen detection and for developing disease-specific management strategies.

Penicillium polonicum: Causal agent of onion bulb decay in storage

Penicillium polonicum je ekonomski važna gljiva sa širokim krugom domaćina, uključujući žitarice, kikiriki, lukove, sušeno meso, citruse, jabuke i krtole jama. U januaru 2013. godine, u skladištu na teritoriji Stare Pazove prikupljeni su uzorci lukovica crnog luka cv. Meranto sa karakterističnim simptomima plave truleži. Iz obolelih lukovica dobijeni su izolati gljive čija je patogenost potvrđena inokulacijom zdravih lukovica. Na diferencijalnim podlogama izolati su formirali dobro razvijene, guste kolonije, plavozelene boje sa belim obodom, karakterističnog mirisa. Izolati nisu rasli na 37oC, dok je porast zabeležen na 5oC. Utvrđeno je da su izolati stvarali ciklopiazonsku kiselinu i druge alkaloide. Konidiofore izolata su bile terverticilate, stipe septirane, tankih i glatkih zidova, a fijalide cilindrične. Konidije su bile loptaste do elipsoidne, glatke i formirale su se u dugim lancima. Sekvence dobijenih amplikona veličine oko 800 bp dela gena za β-tubulin su u MegaBLAST analizi pokazali najvišu sličnost od 99% sa sekvencama istog regiona vrste P. polonicum. Na osnovu morfoloških i molekularnih osobina kao prouzrokovač propadanja lukovica crnog luka u skladištu identifikovana je gljiva P. polonicum.