Infected Tobacco Plants Research Articles

Catalases mediate tobacco resistance to virus infection through crosstalk between salicylic acid and auxin signaling pathways.

Catalases (CATs) play important roles in plant growth, development and defense responses. Previous studies have shown that CATs exhibit different or even opposite effects on plant immunity in different plant-pathogen interactions, but little is known about the mechanisms. In this study, Nicotiana tabacum plants with overexpression or knockout of CAT genes, tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) were employed to investigate the role of CAT in compatible plant-virus interactions. The results showed that there were dynamic changes in the effect of CAT on N. tabacum defense responses. Overexpression of catalase 1 (CAT1) and catalase 3 (CAT3) improved N. tabacum resistance in the early stage of virus infection but depressed it during the late stages of pathogenesis, especially in CAT3 overexpressing plants. The lower level of electrolyte leakage, lower contents of malonaldehyde and hydrogen peroxide (H2 O2 ), higher activities of antioxidant enzymes and improved functions of photosystem II corresponded to the milder symptoms and higher resistance of infected tobacco plants. In addition, the infection of TMV and CMV resulted in expression changes of CATs in tobacco plants, and pretreatment with H2 O2 facilitated TMV and CMV infection. Further experiments showed that the content of salicylic acid (SA) and the expression of genes related to SA signaling pathway were positively correlated with plant resistance, whereas auxin and its related signaling pathway were related to the viral susceptibility of plants. Taken together, our results demonstrated that CAT1 and CAT3 mediated tobacco resistance to virus infection through crosstalk between SA and auxin signaling pathways.

Full-length RNA sequencing reveals the mechanisms by which an TSWV-HCRV complex suppresses plant basal resistance.

Viruses deploy numerous strategies to infect plants, typically by forming complexes with another virus, leading to more efficient infection. However, the detailed plant responses to viral infection and the underlying mechanisms of co-infection remain unclear. Previously, we found that tomato spotted wilt orthotospovirus (TSWV) and Hippeastrum chlorotic ringspot orthotospovirus (HCRV) could infect plants in the field by forming a complex. In this study, we found that TSWV infected tobacco (Nicotiana benthamiana) plants in cooperation with HCRV, leading to a more efficient infection rate of both viruses. We then used the in-depth full-length transcriptome to analyze the responses of N. benthamiana to complex infection by TSWV-HCRV (TH). We found that infection with individual TSWV and HCRV triggered plant defense responses, including the jasmonic acid signaling pathway, autophagy, and secondary metabolism. However, TH co-infection could not trigger and even suppress some genes that are involved in these basal resistance responses, suggesting that co-infection is advantageous for the virus and not for the plants. Typically, the TH complex inhibits NbPR1 expression to suppress tobacco resistance. Moreover, the TH complex could alter the expression of microRNAs (miRNAs), especially novel-m0782-3p and miR1992-3p, which directly interact with NbSAM and NbWRKY6 and suppress their expression in tobacco, leading to downregulation of NbPR1 and loss of resistance in tobacco to TSWV and HCRV viruses. Overall, our results elucidated the co-infection mechanisms of TH in tobacco by deploying the miRNA of plants to suppress plant basal resistance and contributed to developing a novel strategy to control crop disease caused by this virus complex.

Small RNA sequencing of Scutellaria baicalensis reveals brassica yellows virus infection and the complementary development of a sensitive and reliable RT-qPCR assay

To identify the potential causal agent that has caused Scutellaria baicalensis to exhibit yellowing and other morbid symptoms, we sampled from this medicinal plant in Gansu Province, Northwest China, namely, that plant's main growing area. All 18 contings obtained from small RNA sequencing (sRNA-Seq) were homologous to brassica yellows virus (BrYV), with 93%–100% nucleotide similarity. A full-length genome (5693 bp) was obtained using segmentation amplification and Rapid Amplification of cDNA Ends (RACE), which was subsequently identified as BrYV-HQ. Genomic similarity between BrYV-HQ and other isolates ranged from 80.64% to 90.58%. Observed variation was mainly in the coding region of the ORF 3–5 fusion proteins. Evolutionary tree analysis indicated that the kinship of BrYV-HQ may be linked to BrYV-NtabQJ (MK057527.1), known to infect tobacco plants produced in China. Recombination analysis indicated that BrYV-HQ may have derived through recombination between BrYV-NtabQJ and a BrYV isolate (KY310572.1) that infects Brassica napus in China. Based on the coat protein (CP) gene design of the RT-qPCR primer set obtained, the linearity of the standard curve equation y = −2.987*Log(x) + 41.75, which we established for quantitative detection, was good (ranging from 4.43 × 1010 copies/μl∼4.43 × 103 copies/μl) for the quantitative detection of BrYV-HQ. This study marks the first time that BrYV has been confirmed to infect S. baicalensis and upon which a quantitative detection method was established, providing a molecular basis for the research and management of S. baicalensis viral disease in China.

Induction of resistance to Myzus persicae-nicotianae in Cucumber mosaic virus infected tobacco plants using silencing of CMV-2b gene

Aphids such as tobacco aphid Myzus persicae-nicotianae, are among the most important plant viral vectors and plant viruses encode genes to interact with their vectors. Cucumber mosaic virus (CMV) encodes 2b protein as a suppressor of plant immune and it plays a vital role in CMV accumulation and susceptibility to aphid vectors. In this study, the resistance of tobacco plants (Nicotiana tabacum) to M. p. nicotianae was evaluated by silencing of 2b in CMV-infected plants. However, the pFGC-C.h silencing gene construct was transiently expressed using Agrobacterium tumefacience, LBA 4404 in tobacco leaves, and four days later, the plants were mechanically inoculated by CMV (Kurdistan isolate), and then, 15 days post-inoculation 1 nonviruliferous aphid was placed on each leaf for evaluation of resistance to M. p. nicotianae. To evaluate the tobacco plants resistance and susceptibility to M. p. nicotianae, the number of aphids existent per tobacco leaf, life table and, demographic parameters were recorded and used as a comparison indicator. The obtained results were analyzed using the age-stage, two-sex life table. The highest number of aphids was recorded on the control CMV-infected plants, while the lowest number on CMV infected leaves expressing CMV-2b silencing construct (pFGC-C.h). The obtained data revealed the lowest rate for all of intrinsic rate of natural increase (rm) (0.246/day), the rate of reproduction (r0) (17.04 females/generation), and finite rate of increase (λ) (1.279/day), on the pFGC-C.h treatment. The maximum generation time (T) (11.834 days) was observed on (V) treatment. However, the collected data revealed induction of resistance to tobacco aphids by silencing of CMV-2b in CMV infected plants.

Metabolic Profiling of Resistant and Susceptible Tobaccos Response Incited by Ralstonia pseudosolanacearum Causing Bacterial Wilt

The causal agent of bacterial wilt, Ralstonia pseudosolanacearum, can cause significant economic losses during tobacco production. Metabolic analyses are a useful tool for the comprehensive identification of plant defense response metabolites. In this study, a gas chromatography-mass spectrometry (GC-MS) approach was used to identify metabolites differences in tobacco xylem sap in response to R. pseudosolanacearum CQPS-1 in two tobacco cultivars: Yunyan87 (susceptible to R. pseudosolanacearum) and K326 (quantitatively resistant). Metabolite profiling 7 days post inoculation with R. pseudosolanacearum identified 88 known compounds, 42 of them enriched and 6 depleted in the susceptible cultivar Yunyan87, while almost no changes occurred in quantitatively resistant cultivar K326. Putrescine was the most enriched compound (12-fold) in infected susceptible tobacco xylem, followed by methyl-alpha-d-glucopyranoside (9-fold) and arabinitol (6-fold). Other sugars, amino acids, and organic acids were also enriched upon infection. Collectively, these metabolites can promote R. pseudosolanacearum growth, as shown by the increased growth of bacterial cultures supplemented with xylem sap from infected tobacco plants. Comparison with previous metabolic data showed that beta-alanine, phenylalanine, and leucine were enriched during bacterial wilt in both tobacco and tomato xylem.

Combined Transcriptomic and Proteomic Analysis of Myzus persicae, the Green Peach Aphid, Infected with Cucumber Mosaic Virus.

Simple SummaryIn this study, an integrated analysis of the mRNA and protein was performed to identify important putative regulators involved in the transmission of CMV (cucumber mosaic virus) by aphids. At the level of transcription, a total of 20,550 genes (≥2-fold expression difference) were identified as being differentially expressed genes (DEGs) 24 h after healthy aphid transfer to infected tobacco plants using the RNA-seq approach. At the protein level, 744 proteins were classified as being differentially abundant between virus-treated and control Myzus persicae using iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The combined mRNA and protein analysis enabled the identification of some viral putative regulators, such as cuticle proteins, ribosomal proteins, and cytochrome P450 enzymes. The results show that most of the key putative regulators were highly accumulated at the protein level. Based on those findings, we can speculate that the process by which aphids spread CMV is mainly related to post-translational regulation rather than transcription.Aphids transmit CMV (cucumber mosaic virus) in a non-persistent manner. However, little is known about the mechanism of CMV transmission. In this study, an integrated analysis of the mRNA and protein was performed to identify important putative regulators involved in the transmission of CMV by aphids. At the level of transcription, a total of 20,550 genes (≥2-fold expression difference) were identified as being differentially expressed genes (DEGs) 24 h after healthy aphid transfer to infected tobacco plants using the RNA-seq approach. At the protein level, 744 proteins were classified as being differentially abundant between virus-treated and control M. persicae using iTRAQ (isobaric tags for relative and absolute quantitation) analysis. The combined mRNA and protein analysis enabled the identification of some viral putative regulators, such as cuticle proteins, ribosomal proteins, and cytochrome P450 enzymes. The results show that most of the key putative regulators were highly accumulated at the protein level. Based on those findings, we can speculate that the process by which aphids spread CMV is mainly related to post-translational regulation rather than transcription.

Small RNA profiling analysis of two recombinant strains of potato virus Y in infected tobacco plants

Plant viral infections lead to accumulation of virus-derived small interfering RNAs (vsiRNAs) as a result of host defense mechanisms. High-throughput sequencing technology enables vsiRNA profiling analyses from virus infected plants, which provide important insights into virus-host interactions. Potato virus Y (PVY) is a detrimental plant pathogen that can infect a variety of solanaceous crops, e.g., potato, tobacco, tomato, and pepper. We analyzed and characterized vsiRNAs derived from Nicotiana tabacum cv. Samsun infected with two recombinant PVY strains, N-Wi and NTN. We observed that the average percentage of vsiRNAs derived from plants infected with N-Wi was higher than from plants infected with NTN, indicating that N-Wi invokes a stronger host response than NTN in tobacco. The size distribution pattern and polarity of vsiRNAs were similar between both virus strains with the 21 and 22 nucleotide (nt) vsiRNA classes as most predominant and the sense/antisense vsiRNAs ratio nearly equal in the 20−24 nt class. However, the percentage of sense vsiRNAs was significantly higher in the 25−26 nt long vsiRNAs. Distinct vsiRNA hotspots, identifying highly abundant reads of different unique vsiRNA sequences, were observed in both viral genomes. Previous studies found an A or U bias at the 5′ terminal nucleotide position of 21 nt vsiRNAs; in contrast, our analysis revealed a C and U nucleotide bias. This study provides insights that will help further elucidate differential processing of vsiRNAs in plant antiviral defense.

Identifying Early Warning Signals for the Sudden Transition from Mild to Severe Tobacco Etch Disease by Dynamical Network Biomarkers.

Complex systems exhibit critical thresholds at which they transition among alternative phases. Complex systems theory has been applied to analyze disease progression, distinguishing three stages along progression: (i) a normal noninfected state; (ii) a predisease state, in which the host is infected and responds and therapeutic interventions could still be effective; and (iii) an irreversible state, where the system is seriously threatened. The dynamical network biomarker (DNB) theory sought for early warnings of the transition from health to disease. Such DNBs might range from individual genes to complex structures in transcriptional regulatory or protein–protein interaction networks. Here, we revisit transcriptomic data obtained during infection of tobacco plants with tobacco etch potyvirus to identify DNBs signaling the transition from mild/reversible to severe/irreversible disease. We identified genes showing a sudden transition in expression along disease categories. Some of these genes cluster in modules that show the properties of DNBs. These modules contain both genes known to be involved in response to pathogens (e.g., ADH2, CYP19, ERF1, KAB1, LAP1, MBF1C, MYB58, PR1, or TPS5) and other genes not previously related to biotic stress responses (e.g., ABCI6, BBX21, NAP1, OSM34, or ZPN1).

TuMV as an efficient transient vector for expressing heterologous proteins in Nicotiana tabacum and N. benthamiana

Nowadays the production of recombinant proteins such as drugs and commercial protein compounds in plants is called molecular farming. It has some benefits such as fast and large quantity production of recombinant proteins with low cost. In this research, the green fluorescent protein (GFP) was transiently expressed in two tobacco species via turnip mosaic virus (TuMV) derived vector, a virus which can infect a wide range of plant species. Florescence microscopy results indicated that TuMV could infect tobacco plants and accumulate GFP protein in plant leaves. In addition, RT-PCR, Dot-Blot and ELISA assays demonstrated the recombinant gene transcription, translation and stability. This is the first report of using TuMV-based viral vectors for producing recombinant proteins in tobacco. Optimized TuMV-based viral vectors could be used for producing recombinant proteins in tobacco.

Biosynthesis of novel phytochemicals in tobacco plant infected with tobacco mosaic tobamovirus (TMV)

The main purpose of this work was to study the effect of TMV infection on physiology of active gradient photochemicals and protein expression in infected tobacco plants. Impact of Tobacco mosaic tobamovirus (TMV) on active gradient photochemicals quantitative and qualitative was evaluated in Nicotiana tobaccum cv. white burly. First, the TMV samples were isolated from single local lesions of infected leaves of N. glutinosa. Then, the N. tobaccum cv. white burly plants were inoculated with TMV. The infected plants showed severe systemic mosaic symptoms and reduction of leave size. We used Datura metel as a diagnostic tool-plant (indicator) for mosaic virus because of its vast exhibitory ability to show the symptoms incited by viruses. It was confirmed that these symptoms were due to the effect of TMV virus, comparing with Datura plant (as control). Analysis of TMV infected leaves by GC-mass detected biosynthesis of novel photochemicals (2-cyclopenten-1-one, Furfural, Indene, Pyrrole, Benzonitrile, Guaiacol and Oxime, methoxy-phenyl) that could not be detected in healthy plants. Furthermore, a 56.17% decreased in nicotine content was observed in infected plants compared with healthy ones. Also, increase of soluble protein contents was observed in infected leaves in response to TMV infection, compared with healthy ones. Alterations in protein patterns were observed in N. tabaccum leaves in response to TMV infection using SDS PAGE. Several secondary bioactive compounds were also found to hold important functions in infected plants. For example, flavonoids could protect against free radicals generated during photosynthesis. Terpenoids may attract pollinators or seed dispersers, or inhibit competing plants. Alkaloids usually ward-off herbivore animals or insect attacks (phytoalexins).

Dispersion of Myzus persicae and transmission of Potato virus Y under elevated CO2 atmosphere

AbstractMost phytoviruses use insect vectors to spread and infect the surrounding crop plants. Because atmospheric gas concentrations alter the physiology and metabolism of plants, we hypothesize that the concentration of carbon dioxide (CO2) affects the spread of viruses, due to modifications in the feeding behavior of the vector. Tobacco plants, Nicotiana tabacum L. (Solanaceae), and green peach aphids, Myzus persicae (Sulzer) (Hemiptera: Aphididae), were cultivated under ambient (450 p.p.m., termed aCO2) and elevated (800 p.p.m., eCO2) concentrations of CO2. For each atmospheric condition, we first evaluated the ability of the Potato virus Y to spread in a small experimental design, from a central infected tobacco plant to two surrounding circles of healthy plants in presence of aphid vectors for 7 days. The number of aphids recovered on each plant and the infection status of the plants (i.e., healthy vs. infected) were assessed at the end of the experiment. We also evaluated the ability of aphids to transmit the virus under the two experimental atmospheres, by immediately transferring a single insect from an infected plant to a healthy one. The presence of virus in healthy plants was then determined. We found that aphid dispersal, as well as the associated spread of viruses, did not differ between the two atmospheres. On the other hand, we found that aphids grown under eCO2 were more efficient in transmitting viruses to healthy plants compared to aphids reared under aCO2 conditions. The results of this experiment indicate that: (1) the ability of an aphid vector to spread a phytovirus is not affected by the level of CO2 at short time and spatial scales, but (2) the concentration of CO2 may affect plant defenses or the feeding behavior of herbivorous insects, resulting in more efficient viral transmission from the vector to the host plant.

Flue-cured tobacco confirmed as a reservoir host plant for Tomato yellow leaf curl virus by agro-inoculation and Bemisia tabaci MED-mediated transmission.

Tomato yellow leaf curl virus (TYLCV) causes great losses in tomato production. In addition to tomato, TYLCV infects many crops or weeds as alternative hosts. These alternative hosts may serve as reservoirs for TYLCV survival and spread. Here, we tested the capability of cultivated, flue-cured tobacco to act as a reservoir host plant for TYLCV. TYLCV DNA was detected in nine flue-cured tobacco cultivars inoculated with an infectious TYLCV clone, although no visible symptoms developed on TYLCV-infected tobacco plants. The percentage of whiteflies with viral DNA increased with an increasing acquisition access period (AAP) and reached 100% after a 12 h AAP on infected tobacco plants. Using infected tobacco plants as virus resources, TYLCV was capable of being transmitted to tobacco and tomato plants by whiteflies, and typical symptoms of TYLCV infection were observed on infected tomato plants but not on infected tobacco plants. Our results suggest that flue-cured tobacco can serve as a reservoir host plant for TYLCV and may play an important role in the spread of TYLCV epidemics in China.

Population Structure and Genetic Diversity of Phytophthora nicotianae from Tobacco in Georgia.

Black shank, caused by Phytophthora nicotianae, occurs worldwide and is responsible for significant yield loss in tobacco production in Georgia. Management of the disease has primarily relied on utilization of tobacco cultivars with resistance to race 0 of the pathogen and application of the fungicide mefenoxam. Races of P. nicotianae currently prevalent in tobacco production in Georgia, their sensitivity to mefenoxam, and genetic diversity of the pathogen are largely unknown. To determine population structure and genetic diversity of the pathogen, simple sequence repeat (SSR) markers were used. Three races of P. nicotianae (races 0, 1, and 3) were isolated from infected tobacco plants, with race 3 identified in Georgia for the first time. The majority of isolates were identified as A2 mating type and all isolates were sensitive or intermediately sensitive to mefenoxam at 1 or 10 μg/ml, with effective concentration of mefenoxam for 50% mycelial growth reduction values ranging from <0.01 to 0.12 μg/ml. Bayesian and unweighted pair group method with arithmetic means analyses of 59 isolates using SSR markers grouped the isolates in two major groups. Group I contained 20 isolates, of which 19 isolates were collected from Berrien County. Group II contained 39 isolates collected from Bacon, Cook, Tift, and Toombs Counties as well as one sample from Berrien County. Genetic diversity of the isolates was associated with geographical location of collection, and isolates in group I were primarily (75%) race 1, whereas isolates in group II were primarily (69%) race 0. The presence of a single pathogen mating type at most of the locations implies low probability of sexual recombination that may have contributed to the low genetic diversity at a particular geographical location. Sensitivity of the isolates to mefenoxam indicates that the fungicide remains to be a potent tool for growers to combat the disease. Information generated in the study advances our knowledge about diversity and population structure of P. nicotianae, which facilitates development and implementation of effective disease management programs.

A Virus-Induced Assay for Functional Dissection and Analysis of Monocot and Dicot Flowering Time Genes.

Virus-induced flowering (VIF) uses virus vectors to express Flowering Locus T (FT) to induce flowering in plants. This approach has recently attracted wide interest for its practical applications in accelerating breeding in crops and woody fruit trees. However, the insight into VIF and its potential as a powerful tool for dissecting florigenic proteins remained to be elucidated. Here, we describe the mechanism and further applications of Potato virus X (PVX)-based VIF in the short-day Nicotiana tabacum cultivar Maryland Mammoth. Ectopic delivery of Arabidopsis (Arabidopsis thaliana) AtFT by PVX/AtFT did not induce the expression of the endogenous FT ortholog NtFT4; however, it was sufficient to trigger flowering in Maryland Mammoth plants grown under noninductive long-day conditions. Infected tobacco plants developed no systemic symptoms, and the PVX-based VIF did not cause transgenerational flowering. We showed that the PVX-based VIF is a much more rapid method to examine the impacts of single amino acid mutations on AtFT for floral induction than making individual transgenic Arabidopsis lines for each mutation. We also used the PVX-based VIF to demonstrate that adding a His- or FLAG-tag to the N or C terminus of AtFT could affect its florigenic activity and that this system can be applied to assay the function of FT genes from heterologous species, including tomato (Solanum lycopersicum) SFT and rice (Oryza sativa) Hd3a Thus, the PVX-based VIF represents a simple and efficient system to identify individual amino acids that are essential for FT-mediated floral induction and to test the ability of mono- and dicotyledonous FT genes and FT fusion proteins to induce flowering.

Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

Testing of antiviral characteristics of flammutoxin in transgenic tobacco

Flammutoxin (FTX), a protein from fruiting bodies of edible mushroom Flammulina velutipes, is a hemolytic pore-forming protein and a transepithelial electrical resistance-decreasing protein. FTX also acts as an antiviral protein to inhibit Tobacco mosaic virus (TMV) infection of tobacco plants. To clarify its inhibition mechanism for TMV, the following experiments were performed. The gene FTX was introduced into the vector pBinFTX and then transformed into tobacco plants using Agrobacterium tumefaciens. The transgenic tobacco plant expressing FTX showed antiviral activities, and the development of TMV in tobacco was retarded after inoculated with TMV. Not only was the appearance of mosaic symptoms postponed, but also systemic symptoms were lessened in transgenic tobacco plants than in wild-type controls. The results indicate that FTX has antiviral activities.

Effects of Paracoccus marginatus (Mealy bug) and Myzus persicae (Mite) attack on forming of Exacum ritigalensis (Binara / Ginihiriya) Flower

Seeds of Exacum ritigalensis were taken in Pannala, Sri Lanka and potted in green house at the Department of Agriculture Biology, Faculty of Agriculture, University of Ruhuna, Sri Lanka. Four pots replicates were conducted on E. ritigalensis plants that were infested with Paracoccus marginatus and four pots replicates were conducted on E. ritigalensis plants that were infested with Myzus persicae. Two pots were used as controls. Potting medium was taken 1:1:1; sand, compost and topsoil and one E. ritigalensis plants were cared in each pot. At the flowering stage, two adults of Paracoccus marginatus which were taken from infected papaya plant (Carica papaya) and two aphids which were taken from infected tobacco plant (Nicotiana tabacum L.) introduced to the every branch of E. ritigalensis plants separately. Data were collected from maturity stage of the flower (after blooming 3days). Data regarding E. ritigalensis flowers diameter reduction were subjected to analysis mean average value and compared with the control. There were no significant difference of flowers which plant attacked by aphids and it was shown the reduction of number of flowers per bunch. Mealy bugs and aphids severely damage to E. ritigalensis flower production. Researchers and farmers should have to use control measures to prevent mealy bug and ants entering to the plantation.

Paenibacillus lentimorbus Inoculation Enhances Tobacco Growth and Extenuates the Virulence of Cucumber mosaic virus.

Previous studies with Paenibacillus lentimorbus B-30488” (hereafter referred as B-30488), a plant growth promoting rhizobacteria (PGPR) isolated from cow’s milk, revealed its capabilities to improve plant quality under normal and stress conditions. Present study investigates its potential as a biocontrol agent against an economically important virus, Cucumber mosaic virus (CMV), in Nicotiana tabacum cv. White Burley plants and delineates the physical, biophysical, biochemical and molecular perturbations due to the trilateral interactions of PGPR-host-CMV. Soil inoculation of B-30488 enhanced the plant vigor while significantly decreased the virulence and virus RNA accumulation by ~12 fold (91%) in systemic leaves of CMV infected tobacco plants as compared to the control ones. Histology of these leaves revealed the improved tissue’s health and least aging signs in B-30488 inoculated tobacco plants, with or without CMV infection, and showed lesser intercellular spaces between collenchyma cells, reduced amount of xyloglucans and pectins in connecting primary cells, and higher polyphenol accumulation in hypodermis layer extending to collenchyma cells. B-30488 inoculation has favorably maneuvered the essential biophysical (ion leakage and photosynthetic efficiency) and biochemical (sugar, proline, chlorophyll, malondialdehyde, acid phosphatase and alkaline phosphatase) attributes of tobacco plants to positively regulate and release the virus stress. Moreover, activities of defense related enzymes (ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase and catalase) induced due to CMV-infection were ameliorated with inoculation of B-30488, suggesting systemic induced resistance mediated protection against CMV in tobacco. The quantitative RT-PCR analyses of the genes related to normal plant development, stress and pathogenesis also corroborate well with the biochemical data and revealed the regulation (either up or down) of these genes in favor of plant to combat the CMV mediated stress. These improvements led tobacco plant to produce more flowers and seeds with no negative impact on plant health. The present study may advocate the applicability of B-30488 for crop yield improvement in virus infested areas.

Apple Proliferation Phytoplasma Influences the Pattern of Plant Volatiles Emitted Depending on Pathogen Virulence

Apple proliferation (AP) and pear decline (PD) are the most severe diseases in pome fruit growing areas. AP-infected trees show typical symptoms such as witches’ broom, enlarged stipules, tasteless and dwarf fruits. PD-infected pears show a progressive weakening, reduced terminal growth, smaller fruits and die within weeks (quick decline) or years (slow decline). The diseases are caused by the cell-wall lacking bacteria Candidatus Phytoplasma mali (AP phytoplasma) and Ca. P. pyri (PD phytoplasma), respectively. In previous studies it has been shown that AP-infected apple trees emitted higher amounts of the sesquiterpene β-caryophyllene, an attractant of the insect vector Cacopsylla picta (Hemiptera: Psyllidae), thereby facilitating the dispersal of AP phytoplasma. In the present study, volatile organic compounds (VOCs) occurring in the headspace of plants infected with Ca. P. mali strains causing different severity of symptoms in apple plants were collected, analyzed and identified. Headspace samples from healthy and AP-infected model plant tobacco (Nicotiana occidentalis) and apple (Malus domestica) as well as from healthy and PD-infected pear (Pyrus communis) were investigated via thermodesorption and GC-MS analysis. Significantly higher concentrations of ethyl benzoate were produced in all phytoplasma-infected plants compared to healthy ones and an as yet unidentified sesquiterpene differed between the odor bouquets of healthy and by Ca. P. mali infected tobacco plants. Additionally, statistically significant higher amounts of both compounds were measured in the headspace of plants infected by the virulent AP strain. In apple, significantly higher concentrations of ethyl benzoate and methyl salicylate were observed for trees infected with strains of Ca. P. mali. Ethyl benzoate was also detected in the headspace of pear trees infected with Ca. P. pyri.

A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum.

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.