Nicotiana Rustica Research Articles

Regulation of root-associated microbiomes and root exudates by different tobacco species

BackgroundThe root-associated microbiomes are crucial in promoting plant growth and development through symbiotic interactions with their hosts. Plants may shape their microbiomes by secreting specific root exudates. However, the potential mechanisms how plant species determine root exudates and drive microbiome assembly have been little studied. In this study, three wild tobaccos and one cultivated tobacco were used to investigate the commonalities and differences of both root-associated microbiomes and root exudates.ResultsAmplicon sequencing results suggested that tobacco species significantly affected microbial communities in both the rhizosphere and root endosphere, with the strongest impact on the fungal community in the root endosphere. The microbial networks of wild tobacco species were more stable than that of the cultivated tobacco, and fungal members played a more important role in the networks of wild tobacco species, while bacterial members did so in the cultivated tobacco. The rhizosphere bacteria of wild tobacco species showed a higher functional diversity than that of the cultivated tobacco, while the bacteria in the root endosphere presented a contrary pattern. Metabolomics analysis showed significant differences in the composition and abundance of root exudates among the four tobacco species, and the greatest difference was found between the three wild species and the cultivated one. Correlation analysis showed the strongest correlation between metabolites and rhizosphere bacteria, in which O-benzoic acid (2-methoxybenzoic acid) had the most positive correlations with rhizosphere bacteria, while β-ureidoisobutenoic acid had the most negative correlations with rhizosphere bacteria. The rhizosphere bacteria Streptomyces, Hydrophilus and Roseobacter had the strongest positive correlations with metabolites, and the rhizosphere bacterium Nitrobacter had the most negative correlations with metabolites.ConclusionThis study revealed the differences of microbial communities and root exudates in the rhizosphere and root endosphere of four tobacco species, which can further improve our understanding of plant–microbiome interactions during crop domestication.Graphical

Evaluating the Efficacy of Plant Extracts in Managing the Bruchid Beetle, Callosobruchus maculatus (Coleoptera: Bruchidae).

An estimated 2000 plant species have been employed for pest control worldwide. The use of these botanical derivatives is thought to be one of the most cost-effective and sustainable options for pest management in stored grain. The present study was designed to assess the efficacy of five plant extracts viz; Nicotiana tabacum L., Nicotiana rustica L., Azadirachta indica A. Juss., Thuja orientalis L., and Melia azedarach L. against Callosobruchus maculatus L. Plant species extracts were applied at six different concentrations, i.e., 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% in four replications. The phytochemical analyses of ethanolic extracts of five plant species showed variable amounts of phytochemicals i.e., alkaloids, flavonoids, saponins, diterpenes, phytosterol, and phenols. Total phenolic and flavonoid compounds were also observed. The efficacy of A. indica was highest, characterized by the lowest infestation rate (16.65%), host seed weight loss (7.85%), mean oviposition (84.54), and adult emergence (58.40%). In contrast, T. orientalis was found to be the least effective against C. maculatus, with the highest infestation rate of 25.60%, host seed weight loss of 26.73%, mean oviposition of 117.17, and adult emergence rate of 82.01%. Probit analysis was performed by estimating LC50 and LC90. The toxicity percentages of N. tabacum (LC50 = 0.69%, LC90 = 14.59%), N. rustica (LC50 = 0.98%, LC90 = 22.06%), and A. indica (LC50 = 1.09%, LC90 = 68.52%) were notable in terms of the lower LC50 and LC90 values after the 96-h exposure period against C. maculatus. Repellency was assessed by using the area preference and filter paper method. The repellency of C. maculatus on plant extracts increased with the increasing dose and time, such that it was the highest after 48 h. Likewise, at a 3% concentration, A. indica demonstrated 100.00% (Class-V) repellency followed by N. tabacum (96.00%, Class-V), N. rustica (74%, Class-IV), M. azedarach (70.00%, Class-IV), and T. orientalis (68.00%, Class-IV). Based on the findings of this study, we recommend integrating N. rustica, N. tabacum, A. indica, and M. azedarach for effective management of C. maculatus and highlight the potential of these plant species in the formulation of new biocidal agents.

Evaluating the Forensic Inferences of Oral Nicotine Ingestion: Autopsy Case Review, Animal Experimentation, and Legal Implications

Nicotine, a highly lipophilic alkaloid derived from Nicotiana tabacum and Nicotiana rustica, can be lethal even in small doses when ingested in concentrated forms like e-cigarette liquids. This study examined a murder case in South Korea where a 52-year-old man was allegedly killed by oral administration of nicotine. Despite the absence of typical signs, such as chemical burns or vomiting, the case posed questions about the lethal concentration of nicotine and the presence of gastrointestinal symptoms in nicotine poisoning. Through literature review, autopsy analysis, and animal experiments, this study evaluated the forensic evidence presented in court. The results indicated that lethal nicotine levels could be lower than established previously, and death could occur without gastrointestinal injury or vomiting. Our findings supported the court's conviction decision, emphasizing the variability of symptoms in nicotine toxicity and the importance of scientific evidence in legal proceedings. The results of this study highlight the role of forensic medicine in clarifying scientific uncertainties in judicial contexts, particularly in cases involving toxic substances.

Jasmonate/ethylene- and NaWRKY6/3-regulated Alternaria resistance depends on ethylene response factor 1B-like in Nicotiana attenuata.

Biosynthesis of the phytoalexins scopoletin and scopolin in Nicotiana species is regulated by upstream signals including jasmonate (JA), ethylene (ET), and NaWRKY3 in response to the necrotrophic fungus Alternaria alternata, which causes brown spot disease. However, how these signals are coordinated to regulate these phytoalexins remains unknown. By analyzing RNA sequencing data and RNAi, we identified NaERF1B-like (NaERF1B-L) as a key player in Nicotiana attenuata during A. alternata infection by regulating the transcripts of Feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), encoding a key enzyme for scopoletin biosynthesis, and NaVS1-like (NaVS1-L), a putative biosynthetic gene of the phytoalexin solavetivone. We further demonstrated that the synergistic induction of these two genes by JA and ET signaling is mediated by NaERF1B-L. Additionally, we found that the two closely related proteins, NaWRKY6 and NaWRKY3, physically interact to enhance NaERF1B-L expression by directly binding to and activating the NaERF1B-L promoter. Collectively, our current results demonstrate that NaERF1B-L plays a positive role in resistance to A. alternata by modulating phytoalexin biosynthesis through the integration of JA/ET and NaWRKY6/3 signaling. Our findings reveal a fine-tuned transcriptional regulatory hierarchy mediated by NaERF1B-L for brown spot disease resistance in wild tobacco.

The Influence of Health Education on Community Knowledge about the Dangers of Smoking in the Working Area of the Paal Merah I Community Health Center, Jambi City in 2024

Smoking is a bad habits that can have a bad impact on your own health and that of others. Cigarettes are wrapped tobacco products that come from plants such as Nicotiana tabacum and Nicotiana rustica, or they can also be synthetic products containing nicotine and tar, with or without the addition of other ingredients. This research aims to determine the effect of health education on public knowledge about the dangers of smoking in the Paal Merah I Community Health Center Working Area, Jambi City in 2024. This research uses the Wilcoxon test which shows that there is a significant difference between before and after the education was implemented. These findings confirm that health education is effective in increasing public knowledge about the dangers of smoking. Using a semi-experimental method, this research involved a pretest and posttest on 19 respondents selected from three areas that had the highest smoking rates, namely RT.11, RT. 25, and RT.35 Paal Merah Village. The results of the normality test showed that the data was not normally distributed, which means there was a correlation between before the counseling was carried out and after the counseling was carried out. Thus, health education efforts have the potential to change smoking behavior and contribute to efforts to prevent tobacco-related diseases. It is hoped that the intervention steps taken will help reduce the prevalence of smoking at the household level and improve overall public health in the area.

Exploring floral scent in wild tobacco: comparison of volatile compounds across pollinator functional groups and Nicotiana sections

Exploring floral scent in wild tobacco: comparison of volatile compounds across pollinator functional groups and Nicotiana sections

Gambaran Nikotin pada Pra Lansia Perokok Linting (Tembakau) di Desa Pohgading Timur Kecamatan Pringgabaya

Background: Cigarettes are a type of tobacco product produced from the Nicotiana Tabacum, Nicotiana Rustica and other species. Cigarettes are cylinder-shaped from paper measuring between 70 – 120 mm long with a diameter of around 10 mm containing chopped tobacco leaves. There are two types of cigarettes, namely hand-rolled cigarettes and electronic cigarettes which are usually called vape or vapor. Objective: To find out the description of nicotine in hand-rolled (tobacco) smokers in East Pohgading village, Pringgabaya subdistrict. Method: The research is a descriptive analytical method. By taking samples using purposive sampling technique. Results: The results of the research that was carried out obtained samples of 42 elderly people who used hand-rolled cigarettes (tobacco), 41 samples were positive and 1 was negative for nicotine. Conclusion: Results from the urine of pre-elderly respondents who smoked hand-rolled (tobacco) were positive for nicotine as many as 41 people and 1 person was negative.

Characterization of Waste Nicotiana rustica L. (Tobacco) Fiber Having a Potential in Textile and Composite Applications.

Nicotiana rustica L. (NRL) is a type of tobacco plant, and its stalk waste is a potential lignocellulosic source for obtaining cellulose fibers freely available in nature. However, they are left in fields after harvesting, and this study provides a green and sustainable method to reuse tobacco waste. Fiber was obtained by retting the plant stalks in water and decomposing them naturally in three weeks. NRL fiber was characterized by comparing it with known bast fibers, and tests were applied to examine its physical, chemical, mechanical, morphological, and thermal properties. With its high cellulose content (56.6 wt%), NRL fiber had a high tensile strength (113.4 MPa) and a good crystallinity index (70%) that helped it to bond with other fibers in the composite matrix. Furthermore, the fiber is an environmentally friendly alternative to synthetic fibers with a diameter of 36.88 μm and low density (1.5 g/cm3). The NRL fiber was found to have a semi-crystalline structure and large crystalline size, which makes it hydrophobic. The thermal gravimetric analysis showed that it can be durable (353.9 °C) in higher temperatures than the polymerization temperature. As a result, it can be concluded that NRL fiber has the potential to be used as a reinforcement in polymer composites, technical textiles, and agricultural applications.

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5–7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

NaWRKY70 is a key regulator of Nicotiana attenuata resistance to Alternaria alternata through regulation of phytohormones and phytoalexins biosynthesis.

Jasmonate (JA) and abscisic acid (ABA) are two major phytohormones involved in pathogen resistance. However, how their biosynthesis is regulated is not well understood. We silenced NaWRKY70 in wild tobacco Nicotiana attenuata and determined its role in regulating genes involved in the production of JA, ABA and the phytoalexin capsidiol in response to the fungal pathogen Alternaria alternata using techniques including electrophoretic mobility shift, chromatin immunoprecipitation, transient overexpression and virus-induced gene silencing. Silencing NaWRKY70 dramatically reduced both basal and A. alternata-induced jasmonoyl-isoleucine (JA-Ile) and ABA. Further evidence showed that NaWRKY70 directly binds to the W-boxes of the promoters of NaAOS and NaJAR4 (JA biosynthesis), NaNCED1 and NaXD1-like (ABA biosynthesis), and NaMPK4 (ABA signaling) to activate their expression, while binding but repressing the expression of NaCYP707A4-like3 (ABA degradation). Additionally, NaWRKY70 regulates capsidiol production through its key enzyme genes NaEASs and NaEAHs, and interacts with its regulator NaERF2-like to enhance their expression, whereas ABA negatively regulates capsidiol biosynthesis. Our results highlight the key role of NaWRKY70 in controlling both JA-Ile and ABA production, as well as capsidiol production, thus providing new insight into the defense mechanism of plant resistance to A. alternata.

Abstract B015: Tobacco-derived miRNAs and their crosstalk with human transcripts towards carcinogenesis: A cross kingdom approach

Abstract There are more than 70 different species of Nicotiana reported around the world; however, Nicotiana tabacum (common tobacco) and Nicotiana rustica (aztec tobacco) are widely used for snuffing, chewing, smoking, etc., worldwide. Early reports have described that tobacco causes cancer and is associated with many neurological and cardiac disorders. The current study was an effort to understand the paradoxical regulatory mechanism of tobacco-derived miRNAs on human transcripts at 3’ untranslated regions (UTRs) through a computational and experimental genomics approach. This is the first attempt at cross-kingdom analysis where tobacco-specific miRNAs were predicted from different available sources like express sequence tags (EST), genome-wide approaches, and deep sequencing. The miRNA-derived plant targets were identified using stringent bioinformatics methods. A total of 164 mature reported miRNAs, 38 miRNAs from EST datasets, and 21 miRNAs from the genome were studied in detail for cross-kingdom regulation. The potential predicted and reported tobacco-derived miRNAs were analysed against human transcripts. Interestingly, we found tobacco-derived miRNAs such as miR156, miR403, miR7997, miR8011, and miR8036 target tumour suppressor genes and have a putative role in pathways in cancer. Collectively, we have identified a comprehensive set of miRNAs from small RNA sequencing of N. rustica and revealed 842 new miRNAs that have not been reported yet and have putative targets in cancer pathways like AMPK and WNT signalling pathways. Conclusively, this study is the first report of intra- and interspecies relationships using N. tabacum miRNAs with Human and has created a clear road map to focus on specific cancer targets that are modulated by tobacco-derived miRNAs for future studies. Citation Format: Saumya Patel, Manasi Bhavsar, Rakesh Rawal. Tobacco-derived miRNAs and their crosstalk with human transcripts towards carcinogenesis: A cross kingdom approach [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B015.

Genetic Cause of Hybrid Lethality Observed in Reciprocal Interspecific Crosses between Nicotiana simulans and N. tabacum.

Hybrid lethality, a type of postzygotic reproductive isolation, is an obstacle to wide hybridization breeding. Here, we report the hybrid lethality that was observed in crosses between the cultivated tobacco, Nicotiana tabacum (section Nicotiana), and the wild tobacco species, Nicotiana simulans (section Suaveolentes). Reciprocal hybrid seedlings were inviable at 28 °C, and the lethality was characterized by browning of the hypocotyl and roots, suggesting that hybrid lethality is due to the interaction of nuclear genomes derived from each parental species, and not to a cytoplasmic effect. Hybrid lethality was temperature-sensitive and suppressed at 36 °C. However, when hybrid seedlings cultured at 36 °C were transferred to 28 °C, all of them showed hybrid lethality. After crossing between an N. tabacum monosomic line missing one copy of the Q chromosome and N. simulans, hybrid seedlings with or without the Q chromosome were inviable and viable, respectively. These results indicated that gene(s) on the Q chromosome are responsible for hybrid lethality and also suggested that N. simulans has the same allele at the Hybrid Lethality A1 (HLA1) locus responsible for hybrid lethality as other species in the section Suaveolentes. Haplotype analysis around the HLA1 locus suggested that there are at least six and two haplotypes containing Hla1-1 and hla1-2 alleles, respectively, in the section Suaveolentes.

Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N.alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

Plant Chemical Defenses against Insect Herbivores—Using the Wild Tobacco as a Model

Plant Chemical Defenses against Insect Herbivores—Using the Wild Tobacco as a Model

A Qualitative Study on Attitude Towards Smoking and Quitting Among Smokers in Tribal Gypsies Dwelling from Chennai

Background: Tobacco happens to be as old as human civilization since the cultivation of tobacco plants, nicotiana rustica, and nicotiana tabacum, dates back to 8000 years before the American Indians spread that. Smoking tobacco discloses the consumer to more than 7000 chemicals, of which almost 250 and nearly 69 chemicals were proven to be harmful and carcinogenic correspondingly. A particular tribal community is called Narikuravars, which is partially a nomadic group that was present across the borders and has been interpreted with low educational levels, low socio-economic status, and a dearth of pertinent access to health care. Aim: To assess the attitudes towards smoking and quitting among smokers in tribal gypsies population dwelling from Chennai. Materials and methods: An in-depth interview for 15 current smokers and a focus group discussion of 15 current smokers were conducted with a guide and moderator, which was audio recorded. About 9, 13, and 68 subjects from primary, middle, and high school literates participated in the study. Their tobacco consumption and dependence using the Fagerstrom Test for Nicotine Dependence scale (FTNDS) was assessed. Results: High nicotine dependency was observed among 80% of middle school and 62% of primary school literates. Similarly, a Low dependency rate was observed among primary and high school literate with a rate of 40% and 20%, respectively. All the participants were aware of the health effects of tobacco, which affects the lungs and causes cancer. Some of them have had their relatives and known people who had suffered from cancer due to smoking. However, they had no knowledge or idea about COTPA. Conclusion: An interdisciplinary approach is required to prevent tobacco usage along with the government's support by introducing new schemes and programs and creating awareness about the ill effects of tobacco usage. Hence, tribal gypsy people should be educated about the consequences of smoking, and multiple cessation programs are to be conducted for the betterment of their lifestyle.

Weeds and ruderal plants as potential sources of inoculum for vegetable diseases in northern Sinaloa

Weeds and ruderal plants of the families Cucurbitaceae and Solanaceae are addressed as potential sources of inoculum for the development of viral diseases such as Tomato apex necrosis virus (ToANV), zucchini (Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Papaya ring spot virus (PRSV-W) and Cucumber mosaic virus (CMV). Reference is made to weeds and ruderal plants as potential sources of inoculum, including wild sunflower for powdery mildew (Golovinomyces spadiceus), wild tobacco for foliar blight (Alternaria spp.), black nightshade for leaf spot (Curvularia moehlemvekiae), Johnson grass for foliar blight (Alternaria sp.), and wild castor bean for foliar blight (Alternaria ricini) and wild melon for downy mildew (Pseudoperonospora cubensis). Future lines of multidisciplinary research focusing on the determination of pathogenicity in cultivated plants of viruses and fungi associated with wild plants and vice versa are proposed; the spatial-temporal distribution of wild plants that may serve as sources of inoculum, as well as the of potential insect vectors of viral diseases, should also be studied. The implementation of modern molecular techniques, such as High Throughput Sequencing, for the detection of phytopathogens is important. All this will contribute to the implementation of environmentally friendly strategies for disease control in agricultural crops in Sinaloa, for the benefit of the vegetable growers.

Insecticidal Activity of Nicotiana benthamiana Trichome Exudates on the Sweetpotato Whitefly Bemisia tabaci MED (Gennadius)

Trichome is a hair-like structure involved in mechanical and chemical defenses of plants against herbivorous insects. Nicotiana benthamiana, a wild tobacco plant, has well-developed glandular trichomes that secrete sugar esters with potent repellent and insecticidal properties. However, there is a lack of information about the effectiveness of trichome extract in the control of plant-sapping insects. The objective of this study was to investigate the effects of N. benthamiana trichome exudates on Bemisia tabaci MED (Gennadius) (Hemiptera: Aleyrodidae), a highly destructive insect pest that poses significant threats to both vegetable and ornamental plants globally. First, we determined the host preference and mortality of B. tabaci adults using the choice test and feeding assay towards tomato and N. benthamiana plants. B. tabaci preferred tomato over N. benthamiana plants. Second, we extracted N. benthamiana trichome exudates by washing the leaves with either water or ethanol and evaluated their oral toxicities against B. tabaci adults using a parafilm feeding chamber containing 20% sucrose solution. Oral ingestion of both extracts significantly increased mortality in a concentration-dependent manner. Oral ingestion of ethanol-washed 10% trichome extract caused >60% mortality in B. tabaci adults after 36 h. Third, trichome exudates were concentrated by drying to obtain a powder form, which was more potent in killing whiteflies than the liquid form. Oral ingestion of 1% trichome powder was completely lethal to B. tabaci within 36 h. N. benthamiana trichome exudates are highly toxic to B. tabaci through oral ingestion, suggesting that N. benthamiana can be used as a potential natural pesticide for whitefly management.

Molecular and biological investigating of tea plant necrotic ring blotch virus as a worldwide threat

Tea plant necrotic ring blotch virus (TPNRBV) has emerged as a significant threat to tea plantations, primarily in China. Since 2020, similar symptoms have been observed in tea plants in northern Iran, raising concerns about the spread of this viral infection. In this study, we conducted an extensive investigation involving approximately 70 samples collected from both symptomatic and asymptomatic tea plants. Using reverse transcription-polymerase chain reaction with specially designed primers, we successfully amplified DNA fragments from 26 samples, confirming the presence of TPNRBV. Subsequent sequencing of these fragments revealed various segments of the TPNRBV genome. Our phylogenetic analysis revealed that the Iranian TPNRBV isolates formed a distinct sub-cluster alongside Chinese isolates, distinguishing them from Japanese isolates. These finding sheds light on the genetic diversity and relationships of TPNRBV across different regions. Additionally, we explored the potential modes of TPNRBV transmission. Mechanical transmission experiments confirmed the ability of the virus to infect Nicotiana rustica and Chenopodium quinoa seedlings, highlighting the risk of mechanical spread within tea plantations. Moreover, we investigated seed transmission and found evidence of TPNRBV in various parts of tea seeds, suggesting the possibility of seed-borne transmission. Overall, this comprehensive study enhances our understanding of the biological and molecular characteristics of TPNRBV, an emerging threat to global tea production. Our findings provide valuable insights into the virus’s transmission dynamics and genetic diversity, which are essential for developing effective management strategies to mitigate its impact on tea cultivation worldwide.

Viruses Demonstrate Selective Survival During Simulated Anaerobic Digestion of Plant Biomass

Objective. This research aimed at laboratory simulation of anaerobic digestion of plant biomass contaminated by a mixture of viruses with a wide host range to evaluate if viruses can retain their infectivity and pose a biohazard during the use of plant-based reusable resources such as digestate. Methods. Anaerobic digestion was simulated in air-tight desiccators using anaerobic bags and indicator strips to ensure proper conditions, and using fresh biomass of naturallyvirus-infected plants. In one of the variants, a commercial mixture of anaerobic bacteria and enzymes was also added to see if it would be more efficient in terms of plant digestion and virus inactivation. For serological detection of the tomato mosaic virus, cucumber mosaic virus, and potato virus Y, DAS-ELISA was used with commercial diagnostic kits and positive controls. Absorbance values were measured at 405 nm. Total RNA was extracted using a RNeasy Plant Mini kit following the manufacturer’s instructions. Molecular detection of viruses was carried out by two-step RT-PCR using specific oligonucleotide primers available from the literature. To check if the viruses can retain their infectivity after the anaerobic digestion of plant biomass waste, a bioassay was used where Nicotiana rustica plants producing necrotic lesions upon infection with many viruses were mechanically inoculated at the stage of 2 true leaves. For statistical processing of data, the mean and standard error were calculated using Microsoft Excel software. Results. After prolonged anaerobic digestion, tomato mosaic virus was still detected in plant residues, but it is important to note that it was also found in high concentration in the liquid phase. Hence, during the anaerobic digestion, a significant proportion of the virus was successfully eluted from plant residues into the digestate. Interestingly and contrary to tomato mosaic virus, cucumber mosaic virus has not practically eluted to the liquid phase. On the other hand, potato virus Y was supposedly completely decomposed during the anaerobic digestion of plant biomass waste, as it was not detected either by ELISA or by PCR. It is likely that not all of the investigated viruses represent a potential biohazard in the context of the use of renewable resources. RT-PCR outcomes showed the presence of tomato mosaic virus and cucumber mosaic virus both in the solid and the liquid phases, augmenting the ELISA data. Lastly, bioassay results have confirmed that at least tomato mosaic tobamovirus may escape the aggressive process of waste decomposition and remain able to infect host plants raising issues about the safety of reusable plant resources. Conclusions. The survival of plant viruses during plant biomass waste processing was studied in the context of biological safety. The preservation of serological markers and genetic signatures of model viruses in the process of anaerobic digestion has been confirmed. For the first time, it was proven that tomato mosaic virus may preserve its infectivity during long-term incubation of plant biomass waste in anaerobic conditions, which requires further attention when using renewable resources. On the example of PVY, it has been shown that potyviruses do not survive during anaerobic digestion and therefore may not pose an epidemic risk in using processed renewable resources. It has been demonstrated that artificial introduction of anaerobic microorganisms does not have a decisive effect on the stability of the studied model viruses.

Evaluating potential of leaf reflectance spectra to monitor plant genetic variation

Remote sensing of vegetation by spectroscopy is increasingly used to characterize trait distributions in plant communities. How leaves interact with electromagnetic radiation is determined by their structure and contents of pigments, water, and abundant dry matter constituents like lignins, phenolics, and proteins. High-resolution (“hyperspectral”) spectroscopy can characterize trait variation at finer scales, and may help to reveal underlying genetic variation—information important for assessing the potential of populations to adapt to global change. Here, we use a set of 360 inbred genotypes of the wild coyote tobacco Nicotiana attenuata: wild accessions, recombinant inbred lines (RILs), and transgenic lines (TLs) with targeted changes to gene expression, to dissect genetic versus non-genetic influences on variation in leaf spectra across three experiments. We calculated leaf reflectance from hand-held field spectroradiometer measurements covering visible to short-wave infrared wavelengths of electromagnetic radiation (400–2500 nm) using a standard radiation source and backgrounds, resulting in a small and quantifiable measurement uncertainty. Plants were grown in more controlled (glasshouse) or more natural (field) environments, and leaves were measured both on- and off-plant with the measurement set-up thus also in more to less controlled environmental conditions. Entire spectra varied across genotypes and environments. We found that the greatest variance in leaf reflectance was explained by between-experiment and non-genetic between-sample differences, with subtler and more specific variation distinguishing groups of genotypes. The visible spectral region was most variable, distinguishing experimental settings as well as groups of genotypes within experiments, whereas parts of the short-wave infrared may vary more specifically with genotype. Overall, more genetically variable plant populations also showed more varied leaf spectra. We highlight key considerations for the application of field spectroscopy to assess genetic variation in plant populations.