First Report of Beet Yellows Virus Causing Virus Yellows in Sugar Beet in Serbia.

Several viruses have been described to infect sugar beet (Beta vulgaris var. saccharifera L.), but virus yellows disease is one of the most important diseases in many sugar beet growing areas. It is caused by four viruses either in single or mixed infection, including the poleroviruses beet western yellows virus (BWYV), beet mild yellowing virus (BMYV), and beet chlorosis virus (BChV), and a closterovirus beet yellows virus (BYV) (Stevens et al. 2005; Hossain et al. 2021). In August 2019, five samples of sugar beet plants showing yellowing on interveinal leaf tissue were collected in a sugar beet crop in the Novi Sad locality (Vojvodina Province, Serbia). Double-antibody sandwich (DAS)-ELISA commercial antisera (DSMZ, Braunschweig, Germany) were used to test the collected samples for the presence of the most common sugar beet viruses: beet necrotic yellow vein virus (BNYVV), BWYV, BMYV, BChV, and BYV. Commercial positive and negative controls were included in each ELISA test. BYV was serologically detected in all sugar beet samples, but no other viruses tested were found. The presence of BYV in sugar beet plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions, and used as template in the RT-PCR. Total RNAs extracted from healthy sugar beet leaves and molecular-grade water were included as negative controls in the RT-PCR analysis. RT-PCR confirmed the presence of BYV in all naturally infected plants using four sets of specific primers (Kundu and Rysánek 2004), whereas no amplification products were obtained in the negative controls. The RT-PCR products derived from isolate 209-19 were purified and directly sequenced in both directions using the same primer pairs as in RT-PCR (accession numbers OQ686792 to OQ686794). Multiple sequence alignment of the L-Pro and N-terminal part of the MET genes showed that the Serbian BYV isolate had the highest nucleotide identity (99.01% and 100%, respectively) with several BYV isolates in GenBank originating from different parts of the world. Sequence analysis of the HSP70 gene showed the highest similarity (99.79%) with the BYV-Cro-L isolate found in Croatia. In a semi-persistent type of transmission test, aphids (Myzus persicae Sulzer) were allowed to feed on BYV-infected leaves of an ELISA-positive sample (209-19) for 48 hours, and then the aphids were transferred to five plants each of Spinacia oleracea cv. Matador and B. vulgaris ssp. vulgaris cv. Eduarda for a three-day inoculation access period. All test plants were successfully infected and exhibited symptoms in the form of interveinal yellowing up to three weeks postinoculation. RT-PCR confirmed the presence of BYV in all inoculated plants. A study by Nikolic (1951) suggested a possible presence of BYV based on symptoms observed on sugar beet plants in fields, but to our knowledge this is the first report of BYV in sugar beet in Serbia. As sugar beet is one of the most important industrial crops in Serbia, the presence of BYV could lead to significant losses, considering that aphid vectors are widespread under Serbian environmental conditions. The discovery of BYV on sugar beet should prompt a more detailed survey and subsequent testing of susceptible hosts to determine the distribution and incidence of BYV in Serbia.

First Report of Yeast-Spot Disease of Soybean Seeds Caused by Eremothecium coryli in Serbia.

Over the last 15 years, the area planted with soybeans (Glycine max) in Serbia has increased drastically, from 131,000 hectares in 2005 to 230,000 in 2019, and the average yield reached 3.2 t/ha in 2020. The Province of Vojvodina is the most important soybean production region with 95% of the total soybean area in Serbia (www.stat.gov.rs). During the 2021 growing season, soybean seeds with various kinds of symptoms including colour changes, light and dark brown spots, blotching, necrosis, and shriveling were collected from soybean field before harvest of soybean cv. Dukat in the Tamis locality (South Banat District, Vojvodina Province: GPS: 44°56'12.936"N 20°43'24.216"E) in Serbia. The incidence of symptomatic seeds was estimated at 6.4%. Symptomatic soybean seeds were surface disinfected with 2% NaOCl for 2 min, rinsed in sterile water, dried on sterile filter paper, placed on potato dextrose agar (PDA) and were incubated at 25°C in the dark for 10 to 14 days. The identification of fungi at the genus level based on morphological characteristics revealed the presence of species of Macrophomina, Botrytis, Cercospora and Alternaria, which were previously reported as pathogens of soybean seed in Serbia (Krsmanovic et al. 2020). Also, seven white to slightly creamy colonies with yeast-like morphology were observed around seeds expressing discoloration and necrotic and sunken spots. Ten days later, microscopic observations of yeast-like colonies revealed the presence of globose budding cells (diameter of 20 to 28 µm) mostly single or rarely in short chains. Also, two to eight needle-shaped ascospores (52 to 80 µm in length) were arranged lengthwise in many cylindrical to naviculate asci (60 to 96 x 8 to 12, avg. 72.4 x 9.2 µm). Ascospores were with a unilateral, slender, flexuous, whip-like appendage. The morphology of the different fungal structures indicated that the pathogen was Eremothecium coryli (Pelgion) Kurtzman and it was further supported by molecular identification. Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). Sequence analysis of ITS region revealed that the Serbian isolate ND2/21 (GenBank Accession No. OL958602) shared the highest nucleotide identity of 100% with E. coryli isolate (Accession No. KY103387). For pathogenicity test, fresh soybean seeds (cv. Sava) were surface-disinfected with 2% NaOCl and rinsed in sterile water before inoculation. The seeds were pierced 3-4 times with a sterile insect pin through a drop of yeast suspension (concentration 106 ascospores/ml) of one selected single-spore isolate (ND2-21). Similarly, control seeds were pierced with sterile insect pins through a drop of sterile distilled water. Five inoculated seeds and control (five replicates per treatment) were arranged uniformly in a Petri dish (9 cm diameter) and incubated at 22 to 25°C in the dark and kept under >95% relative humidity during the first 48 h. Twenty days after inoculation, small brown necrotic lesions were visible on the soybean seeds. Re-isolation from symptomatic seeds on PDA dishes yielded yeast-like colonies with the same morphological characteristics as those used for inoculation, thus confirming Koch's postulates. The control seeds had no symptoms. This fungus is widely known as a pathogen of yeast spot disease on soybean seeds (Heinrichs et al. 1976; Kimura et al. 2008), but to our knowledge, it has never been reported in Serbia. Considering that invasive species Nezara viridula L. and Halyomorpha halys (STÅL, 1855), the vectors of this fungus, were reported in our country (Keresi et al. 2012; Seat 2015) and that their mass appearance has been documented in recent years (Konjevic et al. 2020), the presence of this pathogen has the potential to cause considerable damage and severe yield losses, resulting in significant economic impact on soybean production in Serbia.

Detection of Four New Tomato Viruses in Serbia Using Post Hoc High-Throughput Sequencing Analysis of Samples From a Large-Scale Field Survey.

Tomato production worldwide is affected by numerous plant virus species. The early and accurate detection of viruses is a critical step for disease control. However, the simultaneous detection of the most known tomato viruses can be difficult because of the high number and diversity of tomato-infecting viruses. Here, we have identified four new viruses in Serbia by applying target-independent small RNA high-throughput sequencing (HTS). HTS was applied on pools of samples and separate samples, in total comprising 30 tomato samples that exhibited (severe) virus-like symptoms and were collected in Serbia during three annual surveys (2011 to 2013). These samples had previously tested negative for the presence of 16 tomato viruses using targeted detection methods. Three divergent complete genome sequences of Physostegia chlorotic mottled virus were obtained from different localities, indicating for the first time that this virus is widespread in Serbia and might represent an emergent viral pathogen of tomato. The tomato torrado virus was detected at one locality with devastating yield losses. The southern tomato virus was detected at two localities, and the spinach latent virus was detected at one locality. In addition, we detected the presence of one already-known virus in Serbia, the tomato spotted wilt orthotospovirus. All the HTS results were subsequently confirmed by targeted detection methods. In this study, the successful application of post hoc HTS testing of a limited number of pooled samples resulted in the discovery of new viruses. Thus, our results encourage the use of HTS in research and diagnostic laboratories, including laboratories that have limited resources to resolve disease etiology.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of Gnomoniopsis idaeicola, the Causal Agent of Canker and Wilting of Blackberry in Serbia.

Blackberry cane diseases with the symptoms of necrosis, canker, and wilting are caused by several fungi worldwide. Surveys conducted from 2013 to 2016 in Serbia revealed the occurrence of Gnomoniopsis idaeicola, the causal agent of cane canker and wilting, which was found to be distributed in almost half of the surveyed orchards, in three blackberry cultivars, and with disease incidence of up to 80%. Wide distribution and high disease incidence suggest that G. idaeicola has been present in Serbia for some time. Out of 427 samples, a total of 65 G. idaeicola isolates were obtained (isolation rate of 34.19%). Reference isolates, originating from different localities, were conventionally and molecularly identified and characterized. G. idaeicola was detected in single and mixed infections with fungi from genera Paraconiothyrium, Colletotrichum, Diaporthe, Botryosphaeria, Botrytis, Septoria, Neofusicoccum, and Discostroma, and no diagnostically specific symptoms could be related directly to the G. idaeicola infection. In orchards solely infected with G. idaeicola, blackberry plant mortality was up to 40%, and yield loses were estimated at 50%. G. idaeicola isolates included in this study demonstrated intraspecies diversity in morphological, biological, pathogenic, and molecular features, which indicates that population in Serbia may be of different origin. This is the first record of a massive outbreak of G. idaeicola infection, illustrating its capability of harmful influence on blackberry production. This study represents the initial step in studying G. idaeicola as a new blackberry pathogen in Serbia, aiming at developing efficient control measures.

Monilinia spp. Causing Brown Rot of Stone Fruit in Serbia.

Brown rot is one of the most important pre- and postharvest fungal diseases of stone fruit worldwide. In Serbia, where production of stone fruit is economically important, Monilinia laxa and M. fructigena are widely distributed. In surveys from 2011 to 2013, 288 isolates of Monilinia spp. were collected from 131 localities in 16 districts and from six hosts in Serbia. Using multiplex polymerase chain reaction, phylogenetic analysis, and morphological characterization, three species of Monilinia were identified as the causal agents of brown rot of stone fruit: M. laxa (89% of isolates), M. fructigena (3%), and M. fructicola (8%). In 2011, M. fructicola was reported for the first time on stone fruit in Serbia, with only one isolate detected. More isolates of M. fructicola were detected in 2012 (2 isolates) and 2013 (20 isolates). The presence of M. fructicola, as well as its increased frequency of detection during the survey, may indicate a change in the population structure of these pathogens, which could have an important impact on brown rot disease management in Serbia.

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.

Peach Rusty Spot Is Caused by the Apple Powdery Mildew Fungus, Podosphaera leucotricha.

Peach rusty spot, an economically important disease of peach (Prunus persica var. persica), appears as necrotic spots on fruit. The etiology of the disease is still not well understood, although it has long been suspected that the causal agent is the apple powdery mildew pathogen, Podosphaera leucotricha. This work confirmed this hypothesis based on cross-inoculation experiments and analysis of rDNA internal transcribed spacer sequences polymerase chain reaction amplified from rusty spot and peach powdery mildew lesions. Cross-inoculations of apple and peach leaves with P. leucotricha and P. pannosa, the causal agent of peach powdery mildew, showed that (i) young peach fruit, up to 5 cm in diameter, developed symptoms typical of rusty spot following inoculation with P. leucotricha; (ii) leaves of 'Jonagold' apple seedlings developed powdery mildew infections when inoculated by touching young rusty spot lesions to their surfaces; (iii) P. leucotricha sporulated on young peach fruit up to 5 cm in diameter; and (iv) peach leaves and young shoots were not susceptible to P. leucotricha, whereas P. pannosa infected all the green parts of peach. A field experiment revealed that there was only a 2- to 3-week period of time during early peach fruit development when the epidermis was susceptible to P. leucotricha. An outcome of this study is that now a clear distinction can be made between the symptoms caused by P. pannosa and P. leucotricha on peach.

Phytophthora ramorum Occurrence in Ornamentals in Serbia.

In a survey to determine the presence of Phytophthora ramorum in Serbia, ornamentals from garden centers, nurseries, and private and public gardens, as well as imported plant material, were inspected. In total, 577 plant, soil, and potting media samples were tested using various detection methods: lateral flow diagnostic test, enzyme-linked immunosorbent assay, conventional polymerase chain reaction, and isolation, followed by identification based on growth characteristics in culture and morphological features. P. ramorum was not detected in any of the 162 soil or potting media tested by the baiting method. P. ramorum was detected in 12 Rhododendron samples from one private garden in Zemun (City of Belgrade District) exhibiting symptoms of leaf necrosis and blight and petiole necrosis, and in three samples of Pieris spp. from one garden center exhibiting symptoms of leaf necrosis. Eight Phytophthora isolates were obtained from the positive Rhododendron plants and three isolates from Pieris plants, and all were identified as P. ramorum on the basis of their uniform morphological and growth characteristics. P. ramorum conformation was also made by sequencing of the internal transcribed spacer regions for a single isolate taken from one infected rhododendron and one pieris plant. Serbian isolates were determined as A1 mating type, due to formation of a few typical sexual structures when crossed with the A2 mating type of P. cinnamomi and P. cryptogea. Pathogenicity test on nonwounded detached leaves of 19 popular ornamentals, as well as the most frequently imported ones, revealed that 10 host species were susceptible, including Robinia pseudoacacia, which is widely distributed in Serbia. During this study, Cotoneaster horizontalis and C. dammeri were determined to be new experimental hosts of P. ramorum. This article provides evidence of P. ramorum introduction into Serbia. Although P. ramorum has not been detected in Serbian production nurseries, its presence outdoors might cause severe damages on susceptible common urban plants in public green and natural ecosystems.

Incidence and Distribution of Iris yellow spot virus on Onion in Serbia.

In a survey to determine the presence and distribution of Iris yellow spot virus (IYSV) in greenhouse ornamentals and onion field crops in 14 districts of Serbia as well as on imported ornamental plants, 1,574 samples were collected and analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). IYSV was not detected in nearly 1,200 plant samples collected from 39 genera of ornamentals grown in greenhouses in Serbia or imported from other countries during 2005 to 2007. The virus was detected in samples from an onion seed crop in the Sirig locality (South Backa District) that showed symptoms resembling those caused by IYSV and in samples without IYSV-like symptoms from an onion bulb crop in the Obrenovac locality (City of Belgrade District). Mechanical transmission of IYSV isolates was difficult, and only the isolate 605-SRB could infect four plant species, but not in all replications. No virus transmission could be demonstrated in 5,000 tested seeds originating from IYSV-infected onion crops. For further confirmation of IYSV, the nucleotide sequence of its nucleocapsid (NC) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) in symptomatic onion samples as well as in symptomless leaves of Nicotiana benthamiana. Four previously developed primers were tested to determine their suitability for routine detection of Serbian IYSV isolates. Phylogenetic analysis showed clustering of isolates 605-SRB and 622-SRB from the onion seed crop and isolate 283-SRB from the onion bulb crop into two distant clades. The analysis indicated that Serbian isolates of IYSV do not share a recent common ancestor and that they represent two distinct lineages of IYSV in Serbia. Considering that onion is one of the most important and traditionally grown vegetable crops in Serbia, IYSV represents a potentially devastating pathogen in this country.

Elimination of TSWV from impatiens hawkerii Bull. and regeneration of virus-free plant

The possibility for obtaining virus free plants from Impatiens hawkerii Bull. shoots infected with Tomato spotted wilt virus (TSWV) through meristem-tip culture was examined. TSWV presence in I. hawkerii plants was detected by DAS-ELISA and RT-PCR and identification of the virus was confirmed by sequencing one of the chosen isolate (GenBank Accesion CQ132190). Meristem-tip explants (0.3-1.5 mm) from virus-infected shoots are cultured on MS media supplemented with different concentrations of the cytokinins, CPPU or TDZ (0.01-1.0 uM), respectively. Using this system, a large number of in vitro shoots could be produced from a single explant. Also, cytokinins showed a stimulatory effect on the length, fresh and dry weights of the newly formed shoots. Plant pigments content in I. hawkerii shoots increased significantly in the presence of cytokinins. Rooting of shoots was spontaneous on the same media. Rooted plantlets were transferred to soil where 97 percent successfully acclimatized. By DAS-ELISA and RT-PCR, 80 percent of the in vitro plantlets were shown to be a virus-free. Considering these, the present protocol seems to be an efficient method for in vitro generation of virus-free I. hawkerii plantlets by meristem tip cultures.