Characterization of an isolate of Lettuce big-vein associated virus (LBVaV) detected in naturally infected tomato (Solanum lycopersicum L.) in Slovakia

A tomato plant (Solanum lycopersicum Linnaeus, labelled KVE) displaying virus-like symptoms, tested negative for common tomato viruses, was subjected to high-throughput sequencing (HTS) on the Illumina MiSeq platform using ribosomal RNA-depleted total RNA as a template. The analysis has revealed the contigs mapping to Lettuce big-vein associated virus (LBVaV). The near complete LBVaV-KVE sequence of RNA1 and RNA2 revealed 95.0 and 94.9% identity with the reference sequence, the same length of translated products and a typical varicosavirus genome organisation. After initial long-term maintenance of LBVaV-KVE in the original plant, the virus could be detected by RT-PCR or nanoLC-ESI-Q-TOF in new plants generated from lateral shoot cuttings or inoculated by stem chips, although not uniformly. So far, LBVaV was reported to infect lettuce and related species. Our study expands the natural host range of the LBVaV to tomato.

Controlling Alternaria cerealis MT808477 Tomato Phytopathogen by Trichoderma harzianum and Tracking the Plant Physiological Changes

Plant responses during the pathogen infection and the pathogen control reflect its strategies to protect its cells. This work represents the Alternaria cerealis MT808477 as a phytopathogen causing leaf spot disease in tomatoes. A. cerealis was identified morphologically and genetically by 18SrRNA, and its pathogenicity was confirmed by light and scanning electron microscopy. Trichoderma harzianum has the ability to control A. cerealis MT808477 by stimulating various cell responses during the controlling process. The cell behavior during the biological control process was observed by analyses of total phenol, flavonoids, terpenoids, antioxidant, malondialdehyde and antioxidant enzymes (catalase and peroxidase). The extracts of infected tomato leaves were tested against plant and human pathogenic microorganisms. Results showed that the biological control process activates the defense cell strategies by increasing the plant tolerance, and activation of plant defense systems. The total phenol, flavonoids, terpenoids, antioxidant and malondialdehyde were increased after 48 h. Catalase and peroxidase were increased in infected tomato plants and decreased during the biological control process, reflecting the decrease of cell stress. Leaves extract inhibited the growth of nine plant and human pathogenic microorganisms. Biological control represents a safe and effective solution to phytopathogens that decreases plant cell stress by stimulating various defensive agents.

Species-Specific Molecular Detection of the Root Knot Nematode Meloidogyne luci

Meloidogyne luci has been identified in various countries around the world parasitizing economically important crops and, due to its potential to cause serious damage to agriculture, was included in the European and Mediterranean Plant Protection Organization Alert List in 2017. This species shares morphological and molecular similarities with M. ethiopica and M. inornata, and a M. ethiopica group was established. Although specific primers for DNA amplification of species belonging to the M. ethiopica group have been developed previously, primers were not species-specific and molecular markers for the specific detection of M. luci are still needed. The objective of this study was to develop a SCAR marker, for detection of M. luci and discrimination from other Meloidogyne spp., based on the intraspecific variability found in RAPD markers. RAPD screening of M. luci and M. ethiopica genome was used for the identification of a specific amplification product on M. luci, which was cloned, sequenced and converted into a SCAR marker. The specificity of the designed primers (Mlf/r) was tested and produced a fragment (771 bp) for all nine M. luci isolates with no amplification for the other nine Meloidogyne spp., including M. ethiopica and M. inornata. Additionally, the proper amplification of the M. luci SCAR-marker was also successful with DNA from galls of M. luci infected tomato roots. The results obtained in this study reveal that the specific molecular detection of M. luci was achieved and the developed methodology can be used for routine diagnosis purposes, which are essential to monitor M. luci distribution and spread, in order to implement future effective and integrated nematode pest management programs.

Trehalose induces tomato defenses and increases drought tolerance and bacterial wilt disease resistance

Ralstonia solanacearum causes plant bacterial wilt disease, leading to severe crop losses. Xylem sap from R. solanacearum-infected tomato is enriched in host produced trehalose. Water stressed plants accumulate the disaccharide trehalose, which increases drought tolerance via abscisic acid (ABA) signaling networks. Because infected plants have reduced water flow, we hypothesized that bacterial wilt physiologically mimics drought stress, which trehalose could mitigate. Transcriptomic responses of susceptible vs. resistant tomato plants to R. solanacearum infection revealed differential expression of drought-associated genes, including those involved in ABA and trehalose metabolism. ABA was enriched in xylem sap from R. solanacearum-infected plants. Treating roots with ABA lowered stomatal conductance and reduced R. solanacearum stem colonization. Treating roots with trehalose increased ABA in xylem sap and reduced plant water use by reducing stomatal conductance and temporarily improving water use efficiency. Further, trehalose-treated plants were more resistant to bacterial wilt disease. Trehalose treatment also upregulated expression of salicylic acid (SA)-dependent defense genes, increased xylem sap levels of SA and other antimicrobial compounds, and increased wilt resistance of SA-insensitive NahG tomato plants. Additionally, trehalose treatment increased xylem concentrations of jasmonic acid and related oxylipins. Together, these data show that exogenous trehalose reduced both water stress and bacterial wilt disease and triggered systemic resistance. This suite of responses revealed unexpected linkages between plant responses to biotic and abiotic stress and suggests that that R. solanacearum-infected tomato plants produce more trehalose to improve water use efficiency and increase wilt disease resistance. In turn, R. solanacearum degrades trehalose as a counter-defense.

Synergistic Effects of a Tomato chlorosis virus and Tomato yellow leaf curl virus Mixed Infection on Host Tomato Plants and the Whitefly Vector

In China, Tomato chlorosis virus (ToCV) and Tomato yellow leaf curl virus (TYLCV) are widely present in tomato plants. The epidemiology of these viruses is intimately associated with their vector, the whitefly (Bemisia tabaci MED). However, how a ToCV+TYLCV mixed infection affects viral acquisition by their vector remains unknown. In this study, we examined the growth parameters of tomato seedlings, including disease symptoms and the heights and weights of non-infected, singly infected and mixed infected tomato plants. Additionally, the spatio-temporal dynamics of the viruses in tomato plants, and the viral acquisition and transmission by B. tabaci MED, were determined. The results demonstrated that: (i) ToCV+TYLCV mixed infections induced tomato disease synergism, resulting in a high disease severity index and decreased stem heights and weights; (ii) as the disease progressed, TYLCV accumulated more in upper leaves of TYLCV-infected tomato plants than in lower leaves, whereas ToCV accumulated less in upper leaves of ToCV-infected tomato plants than in lower leaves; (iii) viral accumulation in ToCV+TYLCV mixed infected plants was greater than in singly infected plants; and (iv) B. tabaci MED appeared to have a greater TYLCV, but a lower ToCV, acquisition rate from mixed infected plants compared with singly infected plants. However, mixed infections did not affect transmission by whiteflies. Thus, ToCV+TYLCV mixed infections may induce synergistic disease effects in tomato plants.

Molecular diagnosis of new isolate of tomato yellow leaf curl virus (TYLCV) in Iraq

Tomato yield and quality in Iraq have been threatened by a variable range of infections caused by tomato yellow leaf curl virus. In previous studies, the TYLCV isolates were partially characterized using molecular tests for small fragments not the entire length of the virus. Sample of TYLCV-infected tomato has applied in this study to diagnose complete sequence of TYLCV isolate. Three sets of primers that belong to three well-identified strains in Iraq were used in a PCR technique and interestingly the results were negative. A new Iraqi isolate has been characterized as a first novel Iraqi isolate detected ever using next generation (NGS) and bioinformatics techniques. The NGS platform has produced about 78,232,062 paired reads of the TYLCV-infected tomato var. Oula F1. The complete raw reads of the infected variety have been analyzed using RepeatExplorer pipeline and Map to reference. The full sequence of TYLCV was reconstructed and extracted to consist of 2770 nt and then deposited in Genbank under accession number MT583814. Copy numbers and genome proportion of this sequence have been calculated that were 3523 and 0,086% respectively. Phylogenetic analyses of full nucleotide sequences confirmed close relationship to Iranian isolate (TYLCV-Kahnooj) than other published viruses. Additionally, a and β DNA satellites have not discovered in the TYLCV-infected sample.

Degradome Analysis of Tomato and Nicotiana benthamiana Plants Infected with Potato Spindle Tuber Viroid

Viroids are infectious non-coding RNAs that infect plants. During infection, viroid RNAs are targeted by Dicer-like proteins, generating viroid-derived small RNAs (vd-sRNAs) that can guide the sequence specific cleavage of cognate host mRNAs via an RNA silencing mechanism. To assess the involvement of these pathways in pathogenesis associated with nuclear-replicating viroids, high-throughput sequencing of sRNAs and degradome analysis were carried out on tomato and Nicotiana benthamiana plants infected by potato spindle tuber viroid (PSTVd). Both hosts develop similar stunting and leaf curling symptoms when infected by PSTVd, thus allowing comparative analyses. About one hundred tomato mRNAs potentially targeted for degradation by vd-sRNAs were initially identified. However, data from biological replicates and comparisons between mock and infected samples reduced the number of bona fide targets—i.e., those identified with high confidence in two infected biological replicates but not in the mock controls—to only eight mRNAs that encode proteins involved in development, transcription or defense. Somewhat surprisingly, results of RT-qPCR assays revealed that the accumulation of only four of these mRNAs was inhibited in the PSTVd-infected tomato. When these analyses were extended to mock inoculated and PSTVd-infected N. benthamiana plants, a completely different set of potential mRNA targets was identified. The failure to identify homologous mRNA(s) targeted by PSTVd-sRNA suggests that different pathways could be involved in the elicitation of similar symptoms in these two species. Moreover, no significant modifications in the accumulation of miRNAs and in the cleavage of their targeted mRNAs were detected in the infected tomato plants with respect to the mock controls. Taken together, these data suggest that stunting and leaf curling symptoms induced by PSTVd are elicited by a complex plant response involving multiple mechanisms, with RNA silencing being only one of the possible components.

Effects of Combined Application of Potassium Silicate and Salicylic Acid on the Defense Response of Hydroponically Grown Tomato Plants to Ralstonia solanacearum Infection

Bacterial wilt, caused by soilborne pathogenic bacterium Ralstonia solanacearum, is a serious and widespread disease that affects global tomato production. Both silicon (Si) and salicylic acid (SA) play important roles in enhancing tomato resistance against bacterial wilt, however, their combined effects on the defense responses of infected tomato plants remain unknown. Hence, the combined effects of Si and SA on physiological and biochemical parameters of R. solanacearum-infected tomato plants were investigated. The combination treatment of Si and SA significantly decreased disease incidences, lipoxygenase (LOX) activity and ethylene (ET) production. The combined treatments were more prominent in improving the morphological traits of root systems, such as root length, root surface area, average root diameter and root volume. The activities of polyphenol oxidase (PPO) and peroxidase (POD) and the concentrations of total soluble phenolics (TSPs) and lignin-thioglycolic acid (LTGA) derivatives were significantly increased in the plants with combined treatments. Si in combination with SA could significantly enhance neutral invertase (NI) and acid invertases (AI) activities in the leaves of tomato plants at 3 days post-infection (dpi) compared with application of Si alone. Three defense-related genes, PAL, POD and pathogenesis-related protein 1 (PR1), were significantly induced in Si+SA treatment at 7 dpi when compared with individual application of Si or SA. The expression level of salicylic acid-binding protein 2 (SABP2) was significantly higher for combination treatment when compared with treatment of Si or SA alone. The possible mechanisms involved in the synergistic effects of Si and SA on the control of tomato bacterial wilt were proposed. This study indicates that under hypertonic conditions, the combined application of 2.0 mM potassium silicate (K2SiO3) and 0.5 mM SA had a synergistic effect on the control of tomato bacterial wilt.