First report of Colletotrichum fioriniae causing anthracnose on pear fruit in Serbia.

European pear (Pyrus communis L.) is one of the most important fruits in Serbia, with a total production of 55,938 tonnes in 2021 (Statistical Office of the Republic of Serbia). In November 2021, pear fruits (cv. Santa Maria) with typical anthracnose symptoms were collected in one storage facility in the area of Smederevo, Serbia. The disease incidence was about 2%. A total of nine infected fruits had dark, circular, sunken necrotic lesions with concentric rings of orange conidial masses produced in black acervuli. The tissue pieces from the edges of lesions (3 to 5 mm2) were surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed twice in sterilized distilled water, air-dried, and incubated on potato dextrose agar (PDA) at 25°C. One isolate/diseased fruit was obtained (nine in total). Colony morphology of all isolates on PDA was light gray to pinkish on the upper side, and pink to vinaceous on reverse after 7 days of incubation at 25°C in the dark. Average growth rate was 64 ± 2 mm. The conidia were hyaline, fusiform, aseptate, smooth-walled, with both ends acute, and measured 12.8 to 16.6 µm × 3.5 to 4.5 µm (n =100). Appressoria were single, smooth-walled, dark brown, clavate to irregular outline, 5.5 to 9.6 × 4.8 to 7.2 µm (n =20). These morphological characterstics are consistent with the description of Damm et al. (2012) for Colletotrichum fioriniae. To confirm this identification, genomic DNA of representative isolate SZ-21-36 was extracted and the ITS region of the rDNA, partial beta-tubulin gene (BenA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial actin gene (ACT) were amplified using the primers ITS1/ITS4, Bt2a/Bt2b, GDF1/GDR1, and ACT512F/ACT783R, respectively (Hassan et al. 2019). The nucleotide sequence of each locus has been deposited in GenBank under Accession Nos. ON171625 (ITS), ON186696 (BenA), ON186697 (GAPDH), and ON186698 (ACT). Multilocus phylogenetic analysis based on the above mentioned molecular markers placed Serbian isolate of C. fioriniae with other isolates of this species deposited in GenBank (bootstrap support of 100%). Pathogenicity test was conducted on symptomless, detached pear fruits (cv. Santa Maria). Five surface-sterilized fruits were wound-inoculated with 50 µl of a conidial suspension (1 × 106 conidia/ml). After 10 days of incubation in a plastic chamber (25°C, 90% relative humidity, and 12-h photoperiod), typical anthracnose lesions developed on inoculated fruits. Five control fruits inoculated with sterile distilled water remained asymptomatic. The experiment was repeated once. Fungal colonies reisolated from the lesions showed the same morphological features as the original isolate. Pathogen was not reisolated from the control fruits. C. fioriniae was previously reported on European pear fruits in Croatia (Ivic et al. 2013), France (Da Lio et al. 2017), China (Fu et el. 2019), and in the USA (Pavlovic et al. 2019). To our knowledge, this is the first report of anthracnose of pear fruit caused by C. fioriniae in Serbia. Currently, the economic impact of anthracnose caused by this pathogen is minimal, but its presence decreases commercial value and quality of pear fruits, and can be a risk to the further spread of C. fioriniae to other plant species.

Penicillium and Talaromyces Species as Postharvest Pathogens of Pear Fruit (Pyrus communis) in Serbia.

Pears are one of the oldest and the third most important fruit species grown in temperate regions. They are consumed because of their nutritional and health benefits, in fresh form or as various processed products. This article resolves the etiology of the Penicillium-like mold symptoms on pear fruits in Serbia. Samples of pear fruits with blue mold and other Penicillium-like mold symptoms were collected in Serbia from 2016 to 2019, from four storages. The recovered isolates were identified and characterized according to a polyphasic approach. Morphological and physiological analyses were performed on three media and five temperatures, respectively. Four loci (internal transcribed spacer, beta-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit) were used for sequencing, genetic identification, and phylogenetic analyses. The results of the identification by conventional and molecular methods were in agreement, and they revealed that the obtained isolates belong to five species: Penicillium crustosum, P. expansum, P. italicum, Talaromyces minioluteus, and T. rugulosus. In a pathogenicity test, P. crustosum, P. expansum, T. minioluteus, and T. rugulosus produced decay on artificially inoculated pear fruits, and P. italicum induced tissue response lesions. The results of this study are the first reports of T. minioluteus and T. rugulosus as postharvest pear pathogens. Also, these are the first world records of T. minioluteus, T. rugulosus, and P. italicum on fruits of European pear. Furthermore, this is the first finding of P. crustosum, P. expansum, P. italicum, T. minioluteus, and T. rugulosus on pear fruit in Serbia.

First report of Penicillium olsonii causing postharvest fruit rot on tomato in Serbia.

Tomato (Solanum lycopersicum, L.) is one of the most important vegetable crop in Serbia, with a total production of 111,639 t in 2019 (Statistical Office of the Republic of Serbia). In July 2020, six tomatoes (cv. Balkan) with symptoms of fruit rot were collected from market in Belgrade, Serbia. The incidence of disease was about 2%, and the symptomatic samples were stored for 10 days after harvest. The initial symptoms on fruits were small circular, slightly sunken and water-soaked spots with white mycelia, that progressively expanded into larger grey lesions following the occurrence of sporulation. Isolations were conducted from one spot/fruit. Small pieces (2 to 3 mm2) from the margins of lesions were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and cultivated on potato dextrose agar (PDA) at 25°C. The isolation frequency of Penicillium-like colonies was 100%. In total, six monosporic isolates were obtained and two isolates (SZ-20-6 and SZ-20-7) were selected as representative for morphological and molecular identification, and pathogenicity test. Morphological characteristics of both isolates were observed after growth on malt extract agar (MEA) for 7 days at 25ºC. On MEA, mycelia were white and colonies turned greyish-green with abundant sporulation. On the reverse sides colonies were pale yellow. The mean colony diameter on MEA for isolate SZ-20-6 was 25 ± 1.2 mm and 26 ± 1.0 mm for isolate SZ-20-7. The colony texture was velvety, without exudates and pigmentation. The conidiophores of both isolates were terverticillate, unbranched; phialides were flask shaped with a short neck, and conidia were smooth, greenish and subglobose to ellipsoidal. The conidial diameter for isolate SZ-20-6 was 3 to 4 × 2.5 to 3 µm, and for isolate SZ-20-7 was 3.5 to 4 × 2.5 to 3.5 µm (n =50). Based on these characteristics, isolates were identified as Penicillium olsonii (Pitt 1979). To confirm the morphological identification, genomic DNA was extracted from isolates (SZ-20-6 and SZ-20-7), and the rDNA ITS region and partial ß-tubulin gene (BenA) were amplified using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. All sequences showed 99 to 100% similarity to P. olsonii and were deposited in GenBank (ITS, MW130235 and MW130236; BenA, MW145147 and MW145148). In multilocus phylogenetic analysis (ITS+BenA), isolates from this study clustered together with other P. olsonii sequences with 100% bootstrap support. To complete Koch's postulates, asymptomatic fruits of tomato cv. Balkan (five fruits per isolate) were superficially sterilized with 70% ethanol, wounded with a sterile needle and inoculated with 10 µl of a spore suspension (1 × 106 spores/ml). Five control fruits were inoculated with 10 µl of sterile distilled water. The experiment was repeated twice. After 7 days of incubation in a moisture chamber at 25°C, typical grey lesions developed on inoculated fruits. The control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as the original isolates. P. olsonii was previously reported on tomato fruit only in Canada (Chatterton et al. 2012) and Pakistan (Anjum et al. 2018). To our knowledge, this is the first report of P. olsonii causing postharvest fruit rot on tomato in Serbia, and in Europe, as well. Therefore, it is essential to monitor spreading of P. olsonii on tomato and other crops in storages, and develop efficient disease management strategies. References: Anjum, N. et al. 2018. Plant Dis. 102:451. Chatterton, S., et al. 2012. Can. J. Plant Pathol. 34:524. Glass, N. L. and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. Pitt, J. I. 1979. The Genus Penicillium and its Teleomorophic States Eupenicillium and Talaromyces. Academic Press, London, U.K. Statistical Office of the Republic of Serbia. https://www.stat.gov.rs/en-US/ White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Funding: This research was financed by the Ministry of Education, Science and Technical Development of the Republic of Serbia, grant 451-03-68/2020-14/200010.

Talaromyces minioluteus: New Postharvest Fungal Pathogen in Serbia.

Talaromyces minioluteus is one of the important species of genus Talaromyces, which has cosmopolitan distribution and is encountered on a wide range of different habitats. This species has not been considered as an important plant pathogen, even though it has been isolated from various plant hosts. Fruits and vegetables with Penicillium-like mold symptoms were collected from 2015 to 2017 from markets in Serbia. Isolates originating from quince, tomato, and orange fruits, onion bulbs, and potato tubers were identified and characterized on a morphological, physiological, and molecular level. Morphological and physiological examination included observing micromorphology, testing growth on six different media and at five different temperatures, and production of three enzymes. Molecular identification and characterization were performed using four molecular markers: internal transcribed spacer, ß-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit. The results of morphological and molecular analyses were in agreement, and they proved that the obtained isolates are T. minioluteus. In the pathogenicity assay, T. minioluteus was confirmed as a pathogen of all species tested with the exception of potato tubers. This is the first report of T. minioluteus as a postharvest plant pathogen on quince, tomato, and orange fruit and onion bulbs. Also, this is the first record of T. minioluteus in Serbia.