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Our previous studies reported that phytoplasma was the causative agent of the pine disease in the Curonian Spit, Lithuania. In this study, insects from diseased pine trees and their adjacent areas were collected from 2016 to 2019 to further identify potential insect vectors that spread phytoplasmas. A total of 1018 phloem-feeding insects (order Hemiptera) were identified, 98.62% of which were aphids (Aphididae), and no known phytoplasma vectors were found. The results from semi-nested polymerase chain reaction (PCR) using phytoplasma-specific universal primers revealed that phytoplasmas were detected in Scots pine aphids (Cinara pini (Linnaeus, 1758)), waxy grey pine needle aphids (Cinara pineti (Fabricius, 1781)), and species-unknown aphids. Further sequence analysis and virtual RFLP (restriction fragment length polymorphism) analysis of aphid-harbored phytoplasma strains indicated that they were closely related to ‘Candidatus Phytoplasma pini’ (16SrXXI-A), but mainly 16SrXXI-A variants, which were also main strains identified in diseased pine trees. In addition, three new phytoplasma subgroups were delineated in the present study. Subgroups 16SrXXI-C and 16SrXXI-D were unveiled from previously identified (but classification was overlooked) Lithuanian pine phytoplasma strains. Subgroup 16SrXXI-E was discovered from the newly identified aphid-harbored phytoplasmas. Further transmission trial studies on these aphids will provide insights into the epidemiology and pathosystem of pine phytoplasma diseases, as well as disease management.

n this study we investigated the secA gene as possible marker to supplement the classification scheme based on 16S rRNA sequences. We cloned and sequenced secA gene and its flanking region of 'Ca. P. asteris' CPh strain phytoplasma, as well as 1997 bp length secA gene fragment of Canada peach X-disease CX and 1327 bp fragment of 'Ca. P. ziziphi' JWB. This helped us to select a primer pair for a single step PCR that can amplify similar to 1200 kb length fragment of secA gene from different phytoplasma groups. We propose that this fragment could be used in RFLP analysis to quickly identify and distinguish 16SrI-A, I-B, I-C, I-D, III-A, III-B, III-E, III-F, III-H, V-B, XII-B and XXI-A subgroup phytoplasmas using just two restriction endonucleases.

Protozoans of genus Sarcocystis are widespread parasites infecting mammals, birds, and reptiles. Morphology of their sarcocysts is an important criterion for species identification. However, as more and more morphologically similar Sarcocystis species are being found and described, additional methods for their routine diagnostics are needed. We investigated restriction fragment length polymorphism (RFLP) as potential alternative to sequencing data analysis for the identification of Sarcocystis species using birds as an intermediate host. The internal transcribed spacer 1 (ITS1) region sequences of seventeen Sarcocystis species (S. albifronsi, S. anasi, S. calchasi, S. columbae, S. cornixi, S. corvusi, S. cristata, S. halieti, S. falcatula, S. fulicae, S. kutkienae, S. lari, S. lindsayi, S. rileyi, S. turdusi, S.wenzeli, and S. wobeseri) of interest were analysed and five best-fitting endonucleases generating most informative restriction fragments were selected for routine testing. In general, RFLP analyses are always inconclusive as they target very short DNA sequences. However, it can be an irreplaceable technique when fast and cheap identification and discrimination of known species are required, which was our main goal and preliminary results indicate that RFLP could be successfully used when identifying closely related avian Sarcocystis species with just two nucleases.

Fusarium head blight (FHB) is an important crop disease worldwide and is mainly caused by members of the Fusarium graminearum species complex. F. graminearum sensu stricto is the most common cosmopolitan and predominant FHB causal agent in Europe. Thus far, the majority of studies have focused on the primary hosts (wheat and barley) of this pathogen, while the relationships between other sources of infection remain unclear. We monitored and sampled test fields over the course of 3 years and acquired 804 F. graminearum isolates from different sources: primary hosts and other plant species included in the crop rotations, weeds from the test fields, decaying plant residue, soil samples, and crop seed. Of these isolates, 73.3% had the 15-acetyldeoxynivalenol genotype and 26.7% had the 3-acetyldeoxynivalenol genotype. F. graminearum isolation rates from weeds (>50%) were much higher than from soil (< 10%) or decaying plant matter (4%). Variable number of tandem repeat markers were used for population analysis. Noticeable genetic differentiation was detected between isolates from living plants and soil biome. In contrast, absence of any noticeable division between primary and alternative plant host communities indicates the importance of weeds and other segetal plants for FHB control and prevention.

Members of the family Corvidae are ecologically flexible omnivorous birds, particularly adaptive to urban habitats, and living in proximity to humans, these birds may serve as definitive hosts (DH) for Sarcocystis spp., but research about this is lacking. In the present study, intestinal samples from 91 corvids collected in Lithuania were molecularly tested by species-specific PCR targeting the ITS1 and cox1 genes and subsequently sequenced for the presence of Sarcocystis spp. Under a light microscope, oocysts of Sarcocystis spp. were observed in 43 samples (47.3%), while molecular methods, detected Sarcocystis spp. in 77 birds (84.6%). Eleven Sarcocystis spp. (S. columbae, S. cornixi, potentially pathogenic S. halieti, S. kutkienae, S. lari, S. turdusi, S. wobeseri, S. arctica, S. lutrae, S. ovalis, and S. oviformis) were identified in the intestinal samples from six corvid species from Lithuania. Infections with multiple Sarcocystis spp. were detected in 79.2% of the infected corvid birds. Three of the identified Sarcocystis spp. use corvids as intermediate hosts (IH), therefore, corvids may serve as IH and DH of the same Sarcocystis species. Based on molecular results and on corvid diet, omnivorous corvids may play an important role in transmitting Sarcocystis spp.

Canids and scavenger birds were shown to act as definitive hosts of numerous Sarcocystis species using members of the Cervidae family as an intermediate host, whereas definitive hosts spreading closely related S. elongata, S. entzerothi, S. japonica, S. matsuoae, S. rangiferi, S. truncata, S. silva and S. tarandi remain unknown. In the current study, the intestine samples of 40 American minks (Neovison vison) were molecularly tested for the presence of the above-mentioned Sarcocystis spp. Species-specific PCR of cytochrome c oxidase subunit I (cox1) fragments and subsequent sequencing revealed the presence of sporocysts/oocysts of five species, S. elongata (n=2), S. entzerothi (n=10), S. japonica (n=4), S. silva (n=13) and S. truncata (n=21) in the analysed samples. Sarcocystis infection was confirmed in 32/40 (80%) examined samples. In addition, half of the infected animals (50%) were infected with multiple Sarcocystis species suggesting that American minks had access to meat of different deer species, such as roe deer, red deer and sika deer. This causes concern about compliance of hunters and game processing companies with game waste management rules. Further research on the involvement of mustelids in the transmission of various Sarcocystis spp. from different geographical locations is needed.

Mountain pine (Pinus mugo Turra) is a coniferous native to the highlands of central Europe. Our previous study revealed that mountain pine proliferation decline (MPPD) disease in the Curonian Spit of Lithuania is caused by a ‘Candidatus Phytoplasma pini’-related strain (16SrXXI-A). However, the insect vector of MPPD has not been identified. In this study, we conducted a survey to determine potential insect vectors of MPPD phytoplasma for three consecutive years (2016–2019). More than 1000 insect samples were collected from four locations in the Curonian Spit. These insects were identified as belonging to six families and ten genera. The presence of phytoplasma in insect samples was examined by nested polymerase chain reaction (PCR) using phytoplasma-specific primers (P1A/16S-SR and R16F2n/R16R2n). Phytoplasmas were detected in Cinara (Cinara) pini (Scots pine aphid), Cinara (Cinara) piniphila and Cinara (Schizolachnus) pineti (waxy grey pine needle aphid) insect samples. Subsequent restriction fragment length polymorphism (RFLP) analysis showed that the PCR-RFLP profile of these positive insect samples was consistent with that of the MPPD of diseased pine trees. These results suggest that C. (C.) pini, C. (C.) piniphila and C. (S.) pineti may be potential insect vectors of MPPD phytoplasma. The findings from this survey will provide useful information for the management of MPPD disease.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) of small grain cereals, but the importance of weeds in the FHB disease cycle and the establishment of F. graminearum in agroecosystems are still not fully understood. The objective of this study was to determine the potential role of weeds present within cereal crop rotations as alternative hosts. F. graminearum was isolated from different organs of asymptomatic weeds sampled from six fields with cereal-crop rotations in Lithuania for two consecutive years (2015 and 2016). The fungi were identified using morphological and molecular methods. Out of 57 weed species that were investigated, 41 (71.9%) harboured F. graminearum isolates. Twenty five weed species were identified as new, previously undocumented, hosts. The majority (73.3%) of the isolates of F. graminearum from this study belonged to the 15ADON genotype while a smaller proportion (23.4%) belonged to the 3ADON genotype. All F. graminearum isolates that were assessed induced FHB symptoms on artificially inoculated spring wheat tested in the field.

Fusarium head blight (FHB) is an important crop disease worldwide and is mainly caused by members of the

Fusarium head blight (FHB) is a devastating disease of cereals worldwide. The primary hosts of this pathogen are wheat and barley, but it also can survive and spread in segetal plants. Data on Fusarium graminearum subpopulations in weeds is lacking. We collected isolates of F. graminearum sensu stricto from segetal plants growing in six separate crop fields and used variable number of tandem repeats (VNTR) markers to gain insights into the diversity of its subpopulations. Ten primer pairs of VNTR markers were tested on 57 F. graminearum isolates obtained from 415 sampled plants representing 52 species. High genetic diversity was found, even in this relatively small number of isolates.

Although mainly known as pathogens that affect angiosperms, phytoplasmas have recently been detected in diseased coniferous plants. In 2008–2014, we observed, in the Curonian Spit of Western Lithuania and in forests of Southern Lithuania (Varena district), diseased trees of Scots pine (Pinus sylvestris) and mountain pine (Pinus mugo) with unusual symptoms similar to those caused by phytoplasmas. Diseased trees exhibited excessive branching, dwarfed reddish or yellow needles, dried shoots and ball-like structures. restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of 16S rRNA gene fragments revealed that individual trees were infected by Candidatus (Ca.) Phytoplasma pini-related strains (members of phytoplasma subgroup 16SrXXI-A) or by Ca. Phytoplasma asteris-related strains (subgroup 16SrI-A). Of the nearly 300 trees that were sampled, 80% were infected by phytoplasma. Ninety-eight percent of the positive samples were identified as Ca. Phytoplasma pini-related strains. Strains belonging to subgroup 16SrI-A were identified from only few trees. Use of an additional molecular marker, secA, supported the findings. This study provides evidence of large-scale infection of Pinus by Ca. Phytoplasma pini in Lithuania, and it reveals that this phytoplasma is more widespread geographically than previously appreciated. This is also the first report of phytoplasma subgroup 16SrI-A in pine trees.

Wheat streak mosaic virus (WSMV) causing viral diseases in graminaceous plants worldwide has been isolated in Lithuania from natural infected winter wheat (Triticum aestivum L.) plants. The diseased wheat plants with pronounced symptoms (chlorotic mottling, streaks of leaves and stunting) were found in various localities of Vilnius and Kaunas regions. Virus isolates were investigated by serology, electron microscopy (EM) and reverse transcription-polymerase chain reaction (RT-PCR) methods. The identification of the virus was based on the results of symptomology on host-plants, positive DAS-ELISA (double-antibody sandwich enzyme-linked immunosorbent assay) test, particle morphology (presence of flexuous, filamentous virus particles ~15 nm in diameter and 700 nm in length) and a specific amplification fragment size (750 bp) of virus RNA (ribonucleic acid) in RT-PCR. Isolate (WSMV-1313) of WSMV from wheat plants was characterized by a coat protein gene sequence analysis. Comparison with other WSMV isolates from the GenBank database showed that the Lithuanian WSMV isolate shares the highest sequence similarity to WSMV isolates from Russia (98.3%) and Italy (98.1%). This is the first report of wheat as a natural host for WSMV in Lithuania.

Two isolates of Pepino mosaic virus (PepMV) from tomato plants grown in different commercial greenhouses in Lithuania were characterized by coat protein (CP) gene sequence analysis. Comparison with other PepMV isolates from the GenBank database showed that both Lithuanian PepMV isolates share 78.3% nucleotide identity and belong to two distinct EU and CH2 genotypes of PepMV. This is the first report on characterization of two PepMV genotypes detected in Lithuania.

Here we report on a plant disease caused by insect-transmitted unculturable plant pathogenic bacteria detected in a private garden in Vilnius region. Samples of symptomatic peas (Pisum sativum L.), nasturtium (Tropaeolum majus L.), strawberry (Fragaria × ananassa Duchesne) and zucchini (Cucurbita pepo var. giromontina) plant tissues were collected. Based on the molecular technique, the Internet tools and phylogenetic analysis, these pathogens were identified as phytoplasmas and classified in phytoplasma RFLP (restriction fragment length polymorphism) group 16SrI, subgroup A. Because this pathogen may be spread by insect-vector that comes from the wild nature, the phytoplasmas could cause a problem in agriculture of Lithuania., Straipsnyje yra pateikta informacija apie augalų ligas, kurias sukelia vabzdžių platinamos, nekultivuojamos, augalams patogeninės bakterijos, aptiktos privačiame sode Vilniaus rajone. Tyrimams buvo surinkti simptomatinių žirnių, nasturtų, braškių ir cukinijų augalų audinių pavyzdžiai. Molekuliniais metodais, internetiniais įrankiais ir filogenetine analize šie patogenai buvo identifikuoti kaip fitoplazmos ir klasifikuoti į 16SrI-A fitoplazmų pogrupį. Identifikuotos fitoplazmos yra platinamos vabzdžių, kurie plinta iš laukinės gamtos, todėl gali sukelti problemų Lietuvos žemės ūkiui.