Tomato Brown Rugose Fruit Virus Pandemic.

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus. It was first reported in 2015 in Jordan in greenhouse tomatoes and now threatens tomato and pepper crops around the world. ToBRFV is a stable and highly infectious virus that is easily transmitted by mechanical means and via seeds, which enables it to spread locally and over long distances. The ability of ToBRFV to infect tomato plants harboring the commonly deployed Tm resistance genes, as well as pepper plants harboring the L resistance alleles under certain conditions, limits the ability to prevent damage from the virus. The fruit production and quality of ToBRFV-infected tomato and pepper plants can be drastically affected, thus significantly impacting their market value. Herein, we review the current information and discuss the latest areas of research on this virus, which include its discovery and distribution, epidemiology, detection, and prevention and control measures, that could help mitigate the ToBRFV disease pandemic.

First Report of Fusarium verticillioides Causing Fusarium Ear Rot of Corn in Jordan.

Sweet corn (Zea mays L.) is one of the most popular crops grown in Jordan. Fusarium verticillioides (Sacc.) is a major pathogen of corn and a producer of mycotoxin fumonisins (Blacutt et al. 2018). During September and October 2019, ear rot symptoms were observed on Ì´30% of the sweet corn variety Mis Dolce grown in the Jordan Valley. The disease caused substantial losses, including damage to greater than 50% of the kernels within 15-20 days after harvesting. A total of 350-corn kernels were randomly taken from 70 plants distributed in five fields with a total area of 2 ha. About 35% of the samples showed typical symptoms of the disease. Discolored corn kernels were surface sterilized with 5% NaOCl solution for 1 min, then rinsed three times with sterilized distilled water (SDW), plated on potato dextrose agar (PDA) at 25°C, and incubated in the dark for 7 days. Twelve putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar (SNA) with a ca. 1 × 2-cm strip of sterile filter paper on the agar surface (Nirenberg 1976). Cultures were incubated for 10 to 14 days at 20°C in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined under the microscope at 400x. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Macroconidia were abundant, falcate to straight, three- to five-septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 µm. Microconidia produced on polyphialides and aggregating in heads, were unicellular, ovoidal, or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 µm (Fig. 1A, B, C, D, E, and F). Based on morphological characteristics, isolates were tentatively identified as F. verticillioides (Al-Hatmi et al. 2016; Guarro 2013). Two representative isolates were DNA extracted and the translation elongation factor 1-α gene (TEF1) was amplified (O'Donnell et al. 1998), and sequenced from both directions at Macrogen Inc, South Korea. The consensus sequences of the two isolates Fvcorn2021JO-03 (OK040159) and Fvcorn2021JO-04 (OK040160) were used as BLASTn query on the NCBI website and were 100% and 99% similar with F. verticilloides JF740717 and JF740737 accessions, respectively. Similarly, the two isolates were 100% and 99.85% similar with F. verticilloides reference sequences MH582332 and MH582327 on the Fusarium MLST database, respectively. The pathogenicity of the two isolates was tested on 15 cobs by injecting 2 ml of a 2.5 × 105 conidia/ml suspension into the silk channel and into kernel wounds of the primary ear (three replicates) for each treatment (Reid and Hamilton 1996). Inoculated kernels were incubated at 25°C for 2 weeks in plastic boxes. The healthy kernels were injected with 2 ml of SDW as a negative control. Grains started to rot after 2 weeks, in the form of a thick, cottony, crimson-looking growth between the ear and its covers, with only some grains or a group of a adjacent grains rotting, and then white lines appear on the outer skin of the grain, yielding symptoms similar to those in the field (Fig. 2A, B, C, and D). The fungus was re-isolated from the inoculated kernels and was morphologically identified as F. verticilloides thus fulfilling Koch's postulates. The fumonisins-producing potential of the isolated F. verticillioides was confirmed using the AgroQuant Total Fumonisins Assay (Romer Labs, Singapore). To our knowledge, this is the first report of F. verticillioides causing Fusarium ear rot on corn in Jordan. Further investigation is needed to gain a better understanding of the spatio-temporal dynamics of this novel pathogen.

New Weed Hosts for Tomato Brown Rugose Fruit Virus in Wild Mediterranean Vegetation.

Tomato brown rugose fruit virus (ToBRFV; genus, Tobamovirus, family, Virgaviridae) was first reported in 2015 infecting tomatoes grown under protected cropping in the Jordan Valley. Since then, ToBRFV has been detected in tomatoes grown in both protected and open fields across Jordan. The increased incidence of ToBRFV prompted this investigation of the potential role of natural weed hosts in the dissemination of ToBRFV. A survey was conducted in the Jordan Valley and highlands to determine possible reservoir hosts of ToBRFV in fields and greenhouse complexes in which tomatoes were grown. Detection of ToBRFV infection was made by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and further confirmation by reverse-transcription polymerase chain reaction (RT-PCR), followed by DNA cloning and sequencing, and bioassays. Thirty weed species belonging to twenty-six genera from sixteen families were tested. Twelve species belonging to eight families were infected of which ten species are newly reported hosts for ToBRFV. Seed transmission of ToBRFV in Solanum nigrum was confirmed in a grow-out experiment. To our knowledge, this is the first report of the natural occurrence of ToBRFV on weed hosts. Identification of natural reservoirs of ToBRFV can help to develop management practices focused on weed plant species to prevent ToBRFV transmission. The extent to which ToBRFV survives in diverse alternate weed host species outside tomato growing seasons in different world regions requires further research in order to establish the risk associated with the possible contribution of weeds as a reservoir for primary infections in tomato crops.

Isolation and molecular characterization of a tomato brown rugose fruit virus mutant breaking the tobamovirus resistance found in wild Solanum species.

A new tobamovirus named tomato brown rugose fruit virus (ToBRFV) overcomes the effect of the Tm-1, Tm-2, and Tm-22 resistance genes introgressed from wild Solanum species into cultivated tomato (Solanum lycopersicum). Here, we report the isolation and molecular characterization of a spontaneous mutant of ToBRFV that breaks resistance in an unknown genetic background, demonstrated recently in Solanum habrochaites and Solanum peruvianum. The wild isolate ToBRFV-Tom2-Jo and the mutant ToBRFV-Tom2M-Jo were fully sequenced and compared to each other and to other ToBRFV sequences available in the NCBI GenBank database. Sequence analysis revealed five nucleotide substitutions in the ToBRFV-Tom2M-Jo genome compared to ToBRFV-Tom2-Jo. Two substitutions were located in the movement protein (MP) gene and resulted in amino acid changes in the 30-kDa MP (Phe22 → Asn and Tyr82 → Lys). These substitutions were not present in any of the previously described ToBRFV isolates. No amino acid changes were found in the 126-kDa and 183-kDa replicase proteins or the 17.5-kDa coat protein. Our data strongly suggest that breaking the newly discovered resistance in wild tomatoes is associated with one or two mutations on the MP gene of ToBRFV.

Screening of Solanum (sections Lycopersicon and Juglandifolia) germplasm for reactions to the tomato brown rugose fruit virus (ToBRFV).

The reaction of 636 Solanum (sections Lycopersicon and Juglandifolia) accessions were evaluated under greenhouse conditions after mechanical inoculation with a Jordanian isolate of the new tobamovirus tomato brown rugose fruit virus (ToBRFV). Local and systemic infections were assayed by symptoms evaluation and virus detection via biotests and RT-PCR. All cultivated tomatoes (Solanum lycopersicum) and the great majority of wild tomato accessions proved susceptible to ToBRFV. They showed a wide range of symptoms (mosaic, leaf deformations, mottling, shoestring, and stunting). Twenty-six accessions representing S. lycopersicum var. cerasiforme, S. pimpinellifolium, S. habrochaites, and S. chilense were tolerant. High levels of resistance have been demonstrated in three accessions of S. ochrantum, a close relative to wild tomatoes (member of the sect. Juglandifolia) not only to ToBRFV but also to the tobamoviruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). After mechanical inoculation, the three tobamoviruses could be detected only in inoculated leaves in the accessions LA2160, LA2162, and LA 2166, which remained symptomless. However, two other S. ochrantum accessions PI 473,498 and PI 230,519 reacted unusually. They were demonstrated highly resistant to TMV and ToMV, but proved transiently susceptible to ToBRFV showing mild systemic mosaic followed by total recovery from symptoms and the virus. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s41348-021-00535-x.

Complete nucleotide sequence of a begomovirus associated with an alphasatellite and a betasatellite naturally infecting okra in Jordan.

The complete nucleotide sequences of a monopartite begomovirus and an associated alphasatellite and betasatellite isolated from naturally infected okra (Abelmoschus esculentus) plants originating from Jordan were determined. The sequences of the begomovirus, alphasatellite, and betasatellites were determined to be 2,764, 1,307, and 1,354 nucleotides in length, respectively. Sequence Demarcation Tool (SDT) and phylogenetic analysis revealed that the begomovirus isolate shared the highest (99.5-99.8%) nt sequence identity with isolates of cotton leaf curl Gezira virus (CLCuGeV), a begomovirus found to exclusively infect cotton in Africa, and recently, in Asia and the Middle East. The DNA sequences of the alphasatellite and betasatellite exhibited the highest nt sequence identity (98.7-98.9% and 92.2-95.3%, respectively) to cotton leaf curl Gezira alphasatellite and cotton leaf curl Gezira betasatellite, respectively. This is the first identification of an African begomovirus, associated with DNA satellites, infecting okra in Jordan.

Carrot mottle virus ORF4 movement protein targets plasmodesmata by interacting with the host cell SUMOylation system.

Plant virus movement proteins (MPs) facilitate virus spread in their plant hosts, and some of them are known to target plasmodesmata (PD). However, how the MPs target PD is still largely unknown. Carrot mottle virus (CMoV) encodes the ORF3 and ORF4 proteins, which are involved in CMoV movement. In this study, we used CMoV as a model to study the PD targeting of a plant virus MP. We showed that the CMoV ORF4 protein, but not the ORF3 protein, modified PD and led to the virus movement. We found that the CMoV ORF4 protein interacts with the host cell small ubiquitin-like modifier (SUMO) 1, 2 and the SUMO-conjugating enzyme SCE1, resulting in the ORF4 protein SUMOylation. Downregulation of mRNAs for NbSCE1 and NbSUMO impaired CMoV infection. The SUMO-interacting motifs (SIMs) LVIVF, VIWV, and a lysine residue at position 78 (K78) are required for the ORF4 protein SUMOylation. The mutation of these motifs prevented the protein to efficiently target PD, and further slowed or completely abolished CMoV systemic movement. Finally, we found that some of these motifs are highly conserved among umbraviruses. Our data suggest that the CMoV ORF4 protein targets PD by interacting with the host cell SUMOylation system.

Phylogenetic classification of a group of self-replicating RNAs that are common in co-infections with poleroviruses.

Tombusvirus-like associated RNAs (tlaRNAs) are positive-sense single-stranded RNAs found in plants co-infected with viruses of the genus Polerovirus. TlaRNAs depend upon capsid proteins supplied in trans by the co-infecting polerovirus vector for transmission and intra-host systemic movement. Here, the full-length genomes of five tlaRNAs were determined using a combination of RT-PCR and next-generation sequencing, and evidence is provided for an additional tlaRNA associated with potato leafroll virus. Phylogenetic analyses based on conserved domains of the RdRp placed tlaRNAs as a monophyletic clade clustering with members of the family Tombusviridae and comprising three different subclades. Full-length clones of tlaRNAs from two of three subclades were confirmed to replicate autonomously, and each produces a subgenomic RNA during infection.

Draft Genome Sequence of the Plant Growth-Promoting Pseudomonas punonensis Strain D1-6 Isolated from the Desert Plant Erodium hirtum in Jordan.

Pseudomonas punonensis strain D1-6 was isolated from roots of the desert plant Erodium hirtum, near the Dead Sea in Jordan. The genome of strain D1-6 reveals several key plant growth-promoting and herbicide-resistance genes, indicating a possible specialized role for this endophyte.

Complete Genome Sequence of Diaphorina citri-associated C virus, a Novel Putative RNA Virus of the Asian Citrus Psyllid, Diaphorina citri.

We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus.

Diverse Array of New Viral Sequences Identified in Worldwide Populations of the Asian Citrus Psyllid (Diaphorina citri) Using Viral Metagenomics.

UNLABELLED: The Asian citrus psyllid, Diaphorina citri, is the natural vector of the causal agent of Huanglongbing (HLB), or citrus greening disease. Together; HLB and D. citri represent a major threat to world citrus production. As there is no cure for HLB, insect vector management is considered one strategy to help control the disease, and D. citri viruses might be useful. In this study, we used a metagenomic approach to analyze viral sequences associated with the global population of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we showed that the virus-like sequences of D. citri are diverse. We identified novel viral sequences belonging to the picornavirus superfamily, the Reoviridae, Parvoviridae, and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. This is the first comprehensive survey to assess the viral community from worldwide populations of an agricultural insect pest. Our results provide valuable information on new putative viruses, some of which may have the potential to be used as biocontrol agents. IMPORTANCE: Insects have the most species of all animals, and are hosts to, and vectors of, a great variety of known and unknown viruses. Some of these most likely have the potential to be important fundamental and/or practical resources. In this study, we used high-throughput next-generation sequencing (NGS) technology and bioinformatics analysis to identify putative viruses associated with Diaphorina citri, the Asian citrus psyllid. D. citri is the vector of the bacterium causing Huanglongbing (HLB), currently the most serious threat to citrus worldwide. Here, we report several novel viral sequences associated with D. citri.

Occurrence of organochlorine pesticide residues in eggs, chicken and meat in Jordan.

Organochlorine pesticide (OCP) residues in 519 samples; comprising eggs, chicken and meat (lamb and beef), collected from Jordan were determined. All samples were analyzed for their residual contents of aldrin, dichlorodiphenyltrichloroethane and metabolites (DDTs), dieldrin, endosulfan isomers, endrin, hexachlorocyclohexane isomers (HCHs), heptachlor, heptachlor epoxide and hexachlorobenzene (HCB). The samples were Soxhlet extracted for 8h in 250mL petroleum ether. The cleanup of the samples was performed by Florisil column chromatography and analysis was done on a gas chromatography equipped with an electron capture detector (GC-ECD). The results indicated that 28% (38/134), 20% (23/115) and 49% (131/270) of the examined eggs, chicken and meat samples, respectively, were contaminated with OCP residues. HCHs and DDTs are the most prominently noticed compounds, as they were detected at a high incidence. On the other hand, heptachlor, heptachlor epoxide, HCB, aldrin and endrin compounds were only present in less than 7% of the analyzed samples. These residues are present despite complete ban on the use of OCPs for agricultural purposes in Jordan. No residues of op'-DDD, op'-DDT, dieldrin, alpha-endosulfan and beta-endosulfan were detected.

Organochlorine pesticide residues in dairy products in Jordan.

The use of aldrin, dieldrin, endrin, heptachlor and hexachlorobenzene (HCB) has been banned in Jordan officially in 1981, and of dichlorodiphenyltrichloroethane (DDT) in 1995. However, residues of such compounds can still be found in the environment and in foodstuffs. Dairy products are an important exposure route for organochlorine pesticides (OCPs) to humans. For this reason, the presence of OCP residues in 233 dairy product samples; comprising milk, butter, cheese, labaneh and yoghurt collected from Jordan was determined. All samples were analyzed for their residual contents of aldrin, DDT and metabolites (DDTs), dieldrin, endosulfan isomers, endrin, hexachlorocyclohexane isomers (HCHs), heptachlor and HCB. Levels of these compounds were determined by gas chromatography with electron capture detector (GC-ECD). The results indicated that 9% (21/233), 8.5% (20/233), 6% (14/233) and 2.1% (5/233) of the examined samples were contaminated with beta-HCH, pp'-DDE, alpha-HCH and gamma-HCH, respectively. Heptachlor and alpha-endosulfan were only present in less than 2% of the analyzed samples. None of the samples revealed the presence of aldrin, op'-DDD, pp'-DDD, op'-DDE, op'-DDT, pp'-DDT, dieldrin, beta-endosulfan, endrin and HCB at their detection limits. The order for the contamination in the analyzed dairy products was labaneh>cheese>yoghurt>butter>milk. This study has provided the preliminary information on the concentration of OCPs in dairy products for the first time in Jordan. The results will help in a scientific assessment of the implications of pesticide residues with regards to human risks in Jordan.

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2.

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3' non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.

Partial Sequence and Survey Analysis Identify a Multipartite, Negative-Sense RNA Virus Associated with Fig Mosaic.

RNA and nucleotide sequence-based analyses were used to identify viruses in fig mosaic (FM)-affected fig (Ficus carica) trees. Nucleotide sequence analyses of 267 cloned cDNAs identified sequences corresponding to four viruses representing four distinct taxa from fig trees in California. Virus sequences corresponding to members of the family Closteroviridae were most common (55 sequences). We also found two sequences for an Umbravirus, one sequence corresponding to a Luteovirus-associated RNA, and two sequences that showed homology to European mountain ash ringspot-associated virus (EMARAV). Reverse transcription-polymerase chain reaction (RT-PCR) and northern hybridization analyses were used to confirm the presence of specific virus RNAs in fig trees. A survey of 184 fig trees from a germplasm collection, a commercial orchard, backyards, and feral fig trees showed that one virus was most common (detected in 96% of tested samples), while none of the other virus sequences were detected in more than 36% of the fig trees. Based on its association with FM-affected trees, nucleotide sequence-based phylogenetic association, and previous reported properties, we suggest the name of this virus as Fig mosaic-associated virus (FMaV).

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Complete nucleotide sequences and genome characterization of a novel double-stranded RNA virus infecting Rosa multiflora.

The three double-stranded (ds) RNAs were detected in Rosa multiflora plants showing rose spring dwarf (RSD) symptoms. Northern blot analysis revealed three dsRNAs in preparations of both dsRNA and total RNA from R. multiflora plants. The complete sequences of the dsRNAs (referred to as dsRNA 1, dsRNA 2 and dsRNA 3) were determined based on a combination of shotgun cloning of dsRNA cDNAs and reverse transcription-polymerase chain reaction (RT-PCR). The largest dsRNA (dsRNA 1) was 1,762 bp long with a single open reading frame (ORF) that encoded a putative polypeptide containing 479 amino acid residues with a molecular mass of 55.9 kDa. This polypeptide contains amino acid sequence motifs conserved in the RNA-dependent RNA polymerases (RdRp) of members of the family Partitiviridae. Both dsRNA 2 (1,475 bp) and dsRNA 3 (1,384 bp) contained single ORFs, encoding putative proteins of unknown function. The 5' untranslated regions (UTR) of all three segments shared regions of high sequence homology. Phylogenetic analysis using the RdRp sequences of the various partitiviruses revealed that the new sequences would constitute the genome of a virus in family Partitiviridae. This virus would cluster with Fragaria chiloensis cryptic virus and Raphanus sativus cryptic virus 2. We suggest that the three dsRNA segments constitute the genome of a novel cryptic virus infecting roses; we propose the name Rosa multiflora cryptic virus (RMCV). Detection primers were developed and used for RT-PCR detection of RMCV in rose plants.

Identification and Partial Characterization of a New Luteovirus Associated with Rose Spring Dwarf Disease.

A number of viruses in the genera Ilarvirus and Nepovirus have been shown to be associated with specific diseases in rose, but many graft-transmissible rose diseases still have unknown etiologies. One of these diseases originally was detected by grafting from nonsymptomatic roses to Rosa multiflora indicator plants. Double-stranded RNAs (dsRNAs) were recovered and used as templates for cDNA synthesis and generating a cDNA library. Analysis of deduced amino acid sequences clearly positioned this virus as a member of the family Luteoviridae. The name rose spring dwarf associated virus (RSDaV) is tentatively proposed for the novel virus because the symptoms of this virus on R. multiflora are consistent with previous descriptions of rose spring dwarf disease (RSD). Phylogenetic analysis revealed a close relationship of RSDaV with members of the genus Luteovirus. Aphid transmission studies identified the rose-grass aphid (Metapolophium dirhodum) and yellow rose aphid (Rhodobium porosum) as vectors for this new virus. Host range data showed that RSDaV has a host range including both monocots and dicots. A specific reverse-transcription polymerase chain reaction assay was developed and revealed the presence of the RSDaV in several rose cultivars. RSDaV-inoculated rose plants developed RSD symptoms, confirming its role in the etiology of the disease.