Plants under Siege: Investigating the Relevance of 'Ca. P. solani' Cixiid Vectors through a Multi-Test Study.

Crop losses caused by the plant pathogenic bacterium 'Candidatus Phytoplasma solani' (CaPsol) underscore the need to better understand its perplexing epidemiological pathways. Hyalesthes obsoletus (Hemiptera, Cixiidae) is a prominent CaPsol vector with three plant associations in Serbia (ex Urtica dioica/HobsUd; ex Convolvulus arvensis/HobsCa; ex Crepis foetida/HobsCf). Another cixiid planthopper, Reptalus quinquecostatus (Dufour), has been recently confirmed as a noteworthy CaPsol vector. A multi-test study assessed the relevance of H. obsoletus associations and R. quinquecostatus populations from Crataegus monogyna and Prunus spinosa in CaPsol occurrence in sugar beet, maize, and tobacco. Molecular typing of the CaPsol strains transmitted to test plants in experimental trials provided the first evidence of HobsUd transmitting CaPsol tuf-a type to sugar beet, HobsCa infecting maize and tobacco with tuf-b type, and HobsCf transmitting CaPsol tuf-b to maize. Affiliation of R. quinquecostatus with the specific CaPsol genotype, dSTOLg, was reaffirmed in this study. The possible involvement of R. quinquecostatus in maize redness disease and tobacco stolbur was suggested, given that this cixiid was identified as a vector of CaPsol to these crops. The obtained results indicate that the tested vectors pose a threat to cultivated plants in Serbia, underscoring the need to recognize their relevance in CaPsol disease occurrences.

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.

A biotroph sets the stage for a necrotroph to play: 'Candidatus Phytoplasma solani' infection of sugar beet facilitated Macrophomina phaseolina root rot.

'Candidatus Phytoplasma solani' (stolbur phytoplasma) is associated with rubbery taproot disease (RTD) of sugar beet (Beta vulgaris L.), while Macrophomina phaseolina is considered the most important root rot pathogen of this plant in Serbia. The high prevalence of M. phaseolina root rot reported on sugar beet in Serbia, unmatched elsewhere in the world, coupled with the notorious tendency of RTD-affected sugar beet to rot, has prompted research into the relationship between the two diseases. This study investigates the correlation between the occurrence of sugar beet RTD and the presence of root rot fungal pathogens in a semi-field 'Ca. P. solani' transmission experiment with the cixiid vector Reptalus quinquecostatus (Dufour), in addition to naturally infected sugar beet in the open field. Our results showed that: (i) Reptalus quinquecostatus transmitted 'Ca. P. solani' to sugar beet which induced typical RTD root symptoms; (ii) Macrophomina phaseolina root rot was exclusively present in 'Ca. P. solani'-infected sugar beet in both the semi-field experiment and naturally infected sugar beet; and that (iii) even under environmental conditions favorable to the pathogen, M. phaseolina did not infect sugar beet, unless the plants had been previously infected with phytoplasma.

Prevalence of a 'Candidatus Phytoplasma solani'-Related Strain Designated as New 16SrXII-P Subgroup over 'Candidatus Arsenophonus phytopathogenicus' in Sugar Beet in Eastern Germany.

Two phloem-limited pathogens, 'Candidatus Arsenophonus phytopathogenicus' and 'Candidatus Phytoplasma solani', threaten sugar beet production in France, Switzerland, and Germany. Previous studies of these pathogens in Germany had focused on its western and southern regions, leaving a knowledge gap about eastern Germany. Despite their importance, this study is the first to investigate phytoplasmas in sugar beet in Saxony-Anhalt, Germany. A phytoplasma strain related to 'Ca. P. solani' is found predominant in Saxony-Anhalt, unlike in France, where 'Ca. P. solani' has a minor role compared with 'Ca. A. phytopathogenicus'. The phytoplasma strain infecting sugar beet in Saxony-Anhalt was classified into a new subgroup designated as 16SrXII-P. The multilocus sequence analysis (MLSA) of nonribosomal genes of the novel phytoplasma strain showed that it is significantly different from the reference and all previously reported 'Ca. P. solani' strains including the strain from western Germany. Analyses of sugar beet samples from previous years confirmed the presence of the 16SrXII-P strain in sugar beet as early as 2020 and also in Bavaria in southern Germany. Based on 16S rDNA analysis, 'Ca. A. phytopathogenicus' in Saxony-Anhalt is identical to strains in sugar beet in other parts of Germany and France, as well as to a strain in potato from Germany. The presence and prevalence of two phytoplasmas in sugar beet in Germany suggest that more attention should be directed toward understanding phytoplasma infection in sugar beet in this country.

Epidemiological role of novel and already known 'Ca. P. solani' cixiid vectors in rubbery taproot disease of sugar beet in Serbia.

Rubbery taproot disease (RTD) of sugar beet was recently associated with the plant pathogenic bacterium 'Candidatus Phytoplasma solani' (CaPsol) and reported throughout the Pannonian Plain with variations in severity. Tracing CaPsol epidemiological pathways was performed in the experimental sugar beet field in Rimski Sancevi (Serbia) in 2020-2021, where an RTD outbreak was recently recorded. A molecular epidemiology approach was applied to the study of three RTD occurrence scenarios: epidemic, non-epidemic and 'absence of RTD'. As a result, Hyalesthes obsoletus ex Convolvulus arvensis was detected as a CaPsol vector to sugar beet, while two other cixiids were identified for the first time as vectors of the CaPsol-induced plant disease in crops: Reptalus quinquecostatus and R. cuspidatus. R. quinquecostatus was proposed culpable for the 2020 RTD epidemic outbreak in Rimski Sancevi when dSTOLg CaPsol strain predominated in the RTD-affected sugar beet, whereas R. cuspidatus had a negligible role in RTD occurrence and displayed ambiguous involvement in CaPsol epidemiology on a wider scale. The temporal discrepancy of the offset of CaPsol dissemination and disease occurrence is the main obstacle in predicting CaPsol-induced diseases. Predicting disease occurrence and severity can only be achieved by gaining a better understanding of CaPsol epidemiological pathways and insect vectors involved in disease outbreaks.

Multilocus Genotyping of 'Candidatus Phytoplasma solani' Associated with Rubbery Taproot Disease of Sugar Beet in the Pannonian Plain.

Rubbery taproot disease of sugar beet (RTD), associated with 'Candidatus Phytoplasma solani', appeared in 2020 on an epidemic scale in northern Serbia and southern Slovakia, situated at opposite edges of the Pannonian Plain. In the affected locations where the disease was assessed, symptomatic sugar beets were analysed for phytoplasma infection. Additionally, multilocus sequence analyses of 'Ca. P. solani' strains on epidemiologically informative marker genes (tuf, stamp and vmp1) were performed. Symptomatic sugar beets from other countries of the Pannonian Plain (Croatia, Hungary and Austria), one sample from Germany, and red beets from Serbia were included in the analyses. 'Ca. P. solani' was detected in sugar beet in all assessed countries, as well as in red beet. Molecular analyses revealed the high genetic variability of 'Ca. P. solani' with the presence of all four tuf-types (a, b1, b2 and d), 14 stamp genotypes (seven new) and five vmp1 profiles (one new). The most common multilocus genotype in Serbia, Slovakia, Croatia, and Hungary was dSTOLg (tuf-d/STOL/V2-TA). It was dominant on sites with epidemic RTD outbreaks in the Pannonian Plain and in several sugar beet fields with non-epidemic RTD occurrence suggesting the prevalence of a particular epidemiological pathway during the epidemic's phases.

Rubbery Taproot Disease of Sugar Beet in Serbia Associated with 'Candidatus Phytoplasma solani'.

Rubbery taproot disease (RTD) of sugar beet was observed in Serbia for the first time in the 1960s. The disease was already described in neighboring Bulgaria and Romania at the time but it was associated with abiotic factors. In this study on RTD of sugar beet in its main growing area of Serbia, we provide evidence of the association between 'Candidatus Phytoplasma solani' (stolbur phytoplasma) infection and the occurrence of typical RTD symptomatology. 'Ca. P. solani' was identified by PCR and the sequence analyses of 16S ribosomal RNA, tuf, secY, and stamp genes. In contrast, the causative agent of the syndrome "basses richesses" of sugar beet-namely, 'Ca. Arsenophonus phytopathogenicus'-was not detected. Sequence analysis of the stolbur strain's tuf gene confirmed a previously reported and a new, distinct tuf stolbur genotype (named 'tuf d') that is prevalent in sugar beet. The sequence signatures of the tuf gene as well as the one of stamp both correlate with the epidemiological cycle and reservoir plant host. This study provides knowledge that, for the first time, enables the differentiation of stolbur strains associated with RTD of sugar beet from closely related strains, thereby providing necessary information for further epidemiological work seeking to identify insect vectors and reservoir plant hosts. The results of this study indicate that there are differences in hybrid susceptibility. Clarifying the etiology of RTD as a long-known and economically important disease is certainly the first step toward disease management in Serbia and neighboring countries.

Effect of Sugar Beet Genotype, Planting and Harvesting Dates and Their Interaction on Sugar Yield.

Climate changes are affecting the plant production, including sugar beet growing especially in the southern and central parts of the Europe. Modifying the sowing and harvesting dates are one of the most often used adaptations in sugar beet cultivation. The aim of this study was to assess the interactions between planting date and sugar beet genotypes for different harvest dates with recommendation for duration of vegetation period for specific hybrids in order to achieve the best performance and to evaluate influence of climatic factors on sugar yield. Three-way analysis of variance and AMMI (Additive main effect and multiple interactions) analysis were performed to investigate interaction between main factors. Analysis of variance revealed that genotypes (G), planting date (PD), harvest date (HD) and interaction G × PD significantly affected sugar yield in 2016. In 2017 genotypes, planting date, harvest date and G x PD interaction significantly affected sugar yield on probability level of 1%, while PD × HD interaction had significant effect on probability level of 5%. Results of AMMI analysis enabled discrimination of genotypes with the highest level of stability in certain planting dates. Hybrids with combined yield and sugar content (NZ type) should have the advantage in earlier planting dates compared to of sugar beet hybrids with higher sugar content (Z type). However, in shortened vegetation period Z type hybrids are more stable and with better sugar yield results. Results of our study suggest that delaying the harvest date decreases differences between sugar yields obtained from hybrids sown in different planting dates. Major factors in the study affecting sugar yield were growing degree days, insolation and number of days from planting to harvest.

Sampling Error in Relation to Cyst Nematode Population Density Estimation in Small Field Plots.

Cyst nematodes are serious plant-parasitic pests which could cause severe yield losses and extensive damage. Since there is still very little information about error of population density estimation in small field plots, this study contributes to the broad issue of population density assessment. It was shown that there was no significant difference between cyst counts of five or seven bulk samples taken per each 1-m2 plot, if average cyst count per examined plot exceeds 75 cysts per 100 g of soil. Goodness of fit of data to probability distribution tested with χ2 test confirmed a negative binomial distribution of cyst counts for 21 out of 23 plots. The recommended measure of sampling precision of 17% expressed through coefficient of variation (cv) was achieved if the plots of 1 m2 contaminated with more than 90 cysts per 100 g of soil were sampled with 10-core bulk samples taken in five repetitions. If plots were contaminated with less than 75 cysts per 100 g of soil, 10-core bulk samples taken in seven repetitions gave cv higher than 23%. This study indicates that more attention should be paid on estimation of sampling error in experimental field plots to ensure more reliable estimation of population density of cyst nematodes.

Estimation of genetic diversity and relationship in sugar beet pollinators based on SSR markers

Background: Genetic diversity studies are important for the selection of parents with a greater combination capacity that, when crossed, increase the chances of obtaining superior genotypes. Thus, 26 polymorphic simple sequence repeat (SSR) primers were used to assess the genetic diversity of 140 individual samples from 12 diploid sugar beet pollinators (pollen parents) and two cytoplasmic male sterile (cms) lines (seed parents). Eight pollinators originated from three research centers in the United States Department of Agriculture, while four pollinators and cms lines were from the Institute of Field and Vegetable Crops, Novi Sad, Serbia. Results: In total, 129 alleles were obtained, with a mean of 3.2 alleles per SSR marker. The observed heterozygosity ranged from 0.00 to 0.87 (mean = 0.30). Expected heterozygosity and Shannon's information index were the lowest for marker BQ590934 and the highest for markers SB15s and FDSB502s; the same markers were the most informative, with PIC values of 0.70 and 0.69, respectively. Three private alleles were found in pollinator EL0204; two in pollinator C51; and one in pollinators NS1, FC221, and C93035. Molecular variance showed that 77.34% of the total genetic variation was attributed to intrapopulation variability. Cluster and correspondence analysis grouped sugar beet pollinators according to the breeding centers, with few exceptions, which indicate that certain amount of germplasm was shared, although centers had their own breeding programs. Conclusions: The results indicate that this approach can improve the selection of pollinators as suitable parental components and could further be applied in sugar beet breeding programs.

Drought Impact Is Alleviated in Sugar Beets (Beta vulgaris L.) by Foliar Application of Fullerenol Nanoparticles.

Over the past few years, significant efforts have been made to decrease the effects of drought stress on plant productivity and quality. We propose that fullerenol nanoparticles (FNPs, molecular formula C60(OH)24) may help alleviate drought stress by serving as an additional intercellular water supply. Specifically, FNPs are able to penetrate plant leaf and root tissues, where they bind water in various cell compartments. This hydroscopic activity suggests that FNPs could be beneficial in plants. The aim of the present study was to analyse the influence of FNPs on sugar beet plants exposed to drought stress. Our results indicate that intracellular water metabolism can be modified by foliar application of FNPs in drought exposed plants. Drought stress induced a significant increase in the compatible osmolyte proline in both the leaves and roots of control plants, but not in FNP treated plants. These results indicate that FNPs could act as intracellular binders of water, creating an additional water reserve, and enabling adaptation to drought stress. Moreover, analysis of plant antioxidant enzyme activities (CAT, APx and GPx), MDA and GSH content indicate that fullerenol foliar application could have some beneficial effect on alleviating oxidative effects of drought stress, depending on the concentration of nanoparticles applied. Although further studies are necessary to elucidate the biochemical impact of FNPs on plants; the present results could directly impact agricultural practice, where available water supplies are often a limiting factor in plant bioproductivity.