Danijela Ristić

In vitro and in vivo antifungal properties of cysteine proteinase inhibitor from green kiwifruit.

BACKGROUND Higher plants possess several mechanisms of defense against plant pathogens. Proteins actively synthesized in response to those stresses are called defense-related proteins which, among others, include certain protease inhibitors. It is of particular relevance to investigate plant natural defense mechanisms for pathogen control which include cystatins-specific inhibitors of cysteine proteases. RESULTS In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. Immuno-tissue print results indicated that CPI is most abundant in the outer layer of pericarp, near the peel, and the inner most part of the pulp-sites where it could act as a natural barrier against pathogens entering the fruit. The purified protein (15 µmol L(-1)) showed antifungal activity against two phytopathogenic fungi (Alternaria radicina and Botrytis cinerea) by inhibiting fungal spore germination. In vivo, CPI (10 µmol L(-1)) was able to prevent artificial infection of apple and carrot with spore suspension of B. cinerea and A. radicina, respectively. It also exerted activity on both intracellular and fermentation fluid proteinases. CONCLUSION Identification and characterization of plant defense molecules is the first step towards creation of improved methods for pathogen control based on naturally occurring molecules.

Non-persistently aphid-borne viruses infecting pumpkin and squash in Serbia and partial characterization of Zucchini yellow mosaic virus isolates

Cucurbit species grown in the Vojvodina Province, Serbia, were surveyed for the incidence of Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Papaya ringspot virus (PRSV) and Tobacco ringspot virus (TRSV) from 2007 to 2009. Samples from more than 700 pumpkin, squash and bottle gourd plants with virus-like symptoms were analyzed by double-antibody sandwich (DAS)-ELISA. ZYMV, WMV and CMV were detected in 79.2, 32.2, and 12.8% of tested samples, respectively. WMV was prevalent in 2007 and ZYMV in 2008–09. Mixed infections were the most frequent type in 2007–08 in contrast to 2009 when single infection of ZYMV prevailed. ZYMV was the most widespread being found in 33 out of 39 inspected fields. Virus species identification was confirmed in selected samples by conventional reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of their coat protein genes. By comparing the obtained virus isolate sequences with those available in GenBank, the identification of serologically detected viruses was confirmed. Phylogenetic analysis based on complete coat protein (CP) sequences highlighted that Serbian ZYMV isolates were closely related to other Central European ZYMV isolates. Finally, additional testing of ELISA-negative samples by RT-PCR using primers specific to six other mosaic viruses revealed the presence of Tomato spotted wilt virus (TSWV) in winter (Cucurbita maxima) and summer (C. pepo ‘Beogradska’) squash. This is the first report of TSWV natural occurrence on cucurbits in Serbia and on winter squash worldwide.

First Report of Tomato spotted wilt virus on Gerbera hybrida in Serbia

In May 2009, approximately 30% of plants within a greenhouse-grown Gerbera hybrida crop in Vranjska Banja (Pčinj District) in Serbia displayed chlorotic oak-leaf patterns followed by necrosis and distortion of leaves. Symptoms on naturally infected gerbera plants and local necrotic spots on Petunia × hybrida mechanically inoculated with infected gerbera sap using chilled 0.05 M phosphate buffer (pH 7) containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (4) suggested the presence of a Tospovirus. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), and Chrysanthemum stem necrosis virus (CSNV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Sauerlach, Germany). Commercial positive and negative controls and extract from healthy gerbera tissue were included in each ELISA. All 20 tested plants were negative for INSV and CSNV. TSWV was detected serologically in 18 of 20 gerbera samples. The presence of TSWV in ELISA-positive symptomatic gerbera plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) using Serbian tobacco TSWV isolate (GQ279731) and RNA extract from healthy gerbera as positive and negative controls, respectively. Two different sets of TSWV-specific primers, L1 TSWVR/L2 TSWVF (2) and M962/M66 (3), for a 276-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene and a 897-bp fragment of the NSm gene, respectively, were used for both amplification and sequencing. RT-PCR analyses of each tested plant detected the presence of amplification fragments of expected size. The amplified products corresponding to part of the RdRp and NSm genes derived from the isolate 158-Gerb were purified (QIAquick PCR Purification Kit, Qiagen) and sequenced in both directions (GenBank Accession Nos. HQ246452 and HQ246453, respectively). Sequence analysis of the partial RdRp gene, conducted using MEGA4 software, revealed 91.1 to 98% nt identity (95.1 to 98.8% amino acid [aa] identities) with corresponding sequences of TSWV L RNA deposited in GenBank. The highest identity was found with an isolate from globe artichoke (AM940436) in Greece, and isolates from tomato (GQ279732), impatiens (GQ132190), and tobacco isolates (GQ279731, FJ189392, and FJ189393) found within Serbia. Analysis of the NSm sequence of isolate 158-Gerb demonstrated nucleotide identities varying between 90.6 and 99.6% (80.9 and 99.6% aa identities) with those of previously reported TSWV isolates. The highest identity was with tobacco isolate GQ373174 from Serbia. Therefore, while gerbera is one of the principal ornamental hosts of TSWV in the EPPO region (1), to our knowledge, this is the first report infecting gerbera in Serbia, which may have a devastating influence on its production. References: (1) Anonymous. OEPP/EPPO Bull. 29:465, 1999. (2) R. A. Mumford et al. J. Virol. Methods 46:303, 1994. (3) W. P. Qiu et al. Virology 244:186, 1998. (4) P. Roggero et al. Plant Dis. 86:950, 2002.

First report of Tomato spotted wilt virus on Brugmansia sp. in Serbia.

Brugmansia (Brugmansia spp.), also known as Angels trumpet, is a perennial shrub in the Solanaceae that is a popular landscape plant in the tropics and subtropics, and potted plant in temperate regions. In April 2012, virus-like symptoms including chlorotic leaf patterns and curling followed by necrosis and distortion of leaves were observed on five outdoor-grown brugmansia plants in a private garden in Mackovac, Rasina District, Serbia. Symptomatic leaves were tested for the presence of several common ornamental viruses including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extract from healthy brugmansia leaves were included in each ELISA. TSWV was detected serologically in all five brugmansia samples and all tested samples were negative for INSV, CMV, and TMV. The virus was mechanically transmitted from an ELISA-positive sample (41-12) to five plants of each Petuina × hybrida and Nicotiana glutinosa. Inoculated P. × hybrida plants showed local necrotic lesions and N. glutinosa showed mosaic and systemic necrosis 4 and 12 days post-inoculation, respectively, which were consistent with symptoms caused by TSWV (1). For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the OneStep RT-PCR (Qiagen, Hilden, Germany) using a set of TSWV-specific primers, TSWV CP-f and TSWV CP-r (4), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs from naturally infected brugmansia and symptomatic N. glutinosa plants were extracted using the RNeasy Plant Mini Kit (Qiagen). Total RNAs obtained from the Serbian tobacco isolate of TSWV (GenBank Accession No. GQ373173) and healthy brugmansia plants were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic brugmansia and N. glutinosa but not from healthy tissues. The amplified product derived from the isolate 41-12 was sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen), deposited in GenBank (JX468080), and subjected to sequence analysis by MEGA5 software (3). Sequence comparisons revealed that the Serbian isolate 41-12 shared the highest nucleotide identity of 99.9% (99.5% amino acid identity) with an Italian TSWV isolate P105/2006RB (DQ915946) originating from pepper. To our knowledge, this is the first report of TSWV on brugmansia in Serbia. Due to the increasing popularity and economic importance of brugmansia as an ornamental crop, thorough inspections and subsequent testing for TSWV and other viruses are needed. This high-value ornamental plant may act also as reservoir for the virus that can infect other ornamentals and cultivated crops, considering that TSWV has a very broad host range (2). References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

Plasmopara obducens: A new threat to the production of Impatiens Walleriana in Serbia

During 2010, Impatiens walleriana plants with symptoms of downy mildew were collected in a greenhouse in the vicinity of Mionica, Kolubara District. Disease incidence was extremely high, approaching 100%, and wilting and collapse of affected plants was very rapid, resulting in losses of more than 90%. White downy growth produced on the lower leaf surface consisted of hyaline, thin-walled sporangiophores with monopodial branching and numerous, ovoid and hyaline sporangia. Apical branchlets of sporangiophores were at right angles to the main axis, with no apical thickening. Pathogenicity tests included inoculation of young I. walleriana plants by spraying with a sporangial suspension, and downy mildew symptoms were observed after 13 to 15 days. The absence of well-defined spots on the infected impatiens leaves and straight sporangiophores indicated that the pathogen was P. obducens, which was further supported by molecular identification, the 5’-end of the nuclear DNA coding for the large ribosomal subunit (LSU rDNA) was amplified by PCR, using primers NL1 and NL4. A representative isolate, 28-10, was sequenced and phylogenetic analysis showed its grouping with other P. obducens isolates of different origin. Considering that impatiens downy mildew in Serbia is proved to be caused by P. obducens it is necessary to employ adequate phytosanitary measures to prevent further spread of the pathogen.

Incidence and distribution of leek yellow stripe virus in allium crops in Serbia

Ivan Vučurović1*, Dušan Nikolić2, Nikola Radović2, Ana Vučurović2, Danijela Ristić1, Branka Krstić2 and Ivana Stanković2 1Institute for Plant Protection and Environment, Department of Plant Pathology, Teodora Drajzera 9, 11040 Belgrade, Serbia 2University of Belgrade, Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia *Corresponding author: vucurovic.ivan@gmail.com Received: 24 October 2017 Accepted: 14 November 2017

Molecular identification of Fusarium graminearum, sorghum pathogen in Serbia

A total of 39 samples of sorghum (Sorghum bicolor) with symptoms of stem and root rot were collected and analyzed during 2009-2011 in Backi Petrovac and Cantavir, Serbia. Monosporic cultures were isolated from stem tissue, their pathogenicity was confirmed by the development of symptoms on artificially inoculated sorghum plants, and they were identified on the basis of macroscopic and microscopic morphological features as Fusarium graminearum. Molecular identification was performed utilizing polimerase chain reaction (PCR) with primer pair ef1/ ef2 and by amplification of protein coding TEF 1-alpha gen. Sequence of TEF gene from the selected isolate 535- 10 (JF747146) showed 98-99% nucleotide identity with sequences of 63 Gibberella zeae isolates deposited in NCBI GenBank. Amplification of the barcoding region of F. graminearum genome of sorghum isolate, contributes to the fast and accurate identification and characterization of Fusarium species in Serbia.

Presence and molecular characterization of alfalfa mosaic virus on tobacco in Serbia

Three-year investigation of the presence and distribution of tobacco viruses in Serbia revealed that Alfalfa mosaic virus (AMV) appeared every year with different frequency in tobacco crops. During 2008, the presence of AMV was detected in most of the tested samples (58.82%) and it was the second most common compared to all other viruses which presence was confirmed in Serbia. In 2006 and 2007, AMV was detected in a significantly lower percentage (2.80% and 13.64%, respectively). This study showed that Alfalfa mosaic virus was more commonly found in multiple infections with two, three or even four detected viruses. Single infections were detected only in 2006, in one tobacco field in the locality of Futog. During this investigation, a rapid and simple protocol was optimized and developed for molecular detection of AMV in tobacco leaves, using primers CPAMV1/CPAMV2 and commercially available kits for total RNA extraction as well as for RT-PCR (reverse transcription - polymerase chain reaction). Using RT-PCR and these primers that flank the AMV coat protein gene, a DNA fragment of 751 bp was amplified, sequenced, and compared with the sequences available in GenBank database. The sequence of isolate 196-08 (GenBank Acc. No. FJ527749) proved to be identical at the nucleotide level of 99 to 93% with those from other parts of the world. Phylogenetic analysis of 27 isolates based on 528 bp sequences of the coat protein gene did not show correlation of the isolates with their geographic origin or plant host and showed that these isolates fall into four molecular groups of strains. Serbian AMV isolate from tobacco belongs to group IV, the group that includes most of the isolates selected for phylogenetic analysis.

Characterization of cucumber mosaic virus originating from cucurbits in Serbia

Cucumber mosaic virus (CMV) is considered one of the most economically important plant viruses and has a worldwide distribution and a very wide host range including plants from family Cucurbitaceae. In Serbia, on cucurbits CMV was detected in single and mixed infections with Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Viruses, including CMV, are constantly present in cucurbit crops, but their frequency changes by year and locality. Surveys and sample collections were conducted in cucurbit crops in the period from 2008 to 2009 at 15 localities in Vojvodina province, and sample testing was carried out using the DAS-ELISA method and commercially available antisera for six economically most important cucurbit viruses. In 2008, a total of 51 samples were collected from 13 cucurbit crops of oilseed pumpkin Olinka variety, squash, and bottle gourd and CMV was detected in a total of 55% of tested samples with symptoms of viral infection. The most common infectious type was mixed infection with ZYMV and WMV (35.3%), and then mixed infection with ZYMV (17.7%) and WMV (2%). A total of 599 symptomatic samples of oilseed pumpkin Olinka variety, zucchini squash varieties Beogradska and Tosca, squash, and winter squash were collected in 15 cucurbits crops in 2009. CMV was present in 4.4% of total collected samples, in single infections in 1.3%, and in mixed with WMV or ZYMV in 1.3%, and 1.8%. Five CMV isolates were obtained by mechanical inoculations of N. glutinosa and one of them was selected for further biological characterization. Test plants which were described to be hosts of CMV expressed symptoms characteristic for those caused by CMV after inoculations by isolate 115-08. CMV specific primers Au1u/Au2d were used to amplify an 850 bp fragment using RT-PCR method. Amplified fragment encodes the entire viral coat protein (CP) gene and partial 5’ and 3’ UTRs of two selected CMV isolates. Amplified fragments were sequenced and deposited in the NCBI, where they were assigned accession numbers, HM065510 (115-08) and HM065509 (151-08). The sequences of CMV isolates from Serbia shared the highest nucleotide and amino acid identity with isolates from subgroup IA, from 99.5 to 97.4% and 99.1 to 97.4%, and the lowest identities were with the subgroup II isolates from 66.9 to 64, 5%, from 75.8 to 74.1%.