Mild Leaf Research Articles

Natural occurrence of mesta yellow vein mosaic virus and DNA-satellites in ornamental sunflower (Helianthus spp.) in Pakistan.

Weeds and ornamental plants serve as a reservoir for geminiviruses and contribute to their dissemination, genome recombination and/or satellite capture. Ornamental sunflower (Helianthus spp.) plants exhibiting mild leaf curl symptoms were subjected to begomovirus and DNA-satellites isolation. The full-length genome of the isolated begomovirus clone (Od1-A) showed 96.8% nucleotide (nt) sequence identity with mesta yellow vein mosaic virus (MeYVMV; accession no. FR772081) whereas, alphasatellite (Od1-a) and betasatellite (Od1-b) clones showed their highest nt sequence identities at 97.4% and 98.2% with ageratum enation alphasatellite (AEA; accession no. FR772085) and papaya leaf curl betasatellite (PaLCuB; accession. no. LN878112), respectively. The evolutionary relationships, average evolutionary divergence and the recombination events were also inferred. The MeYVMV exhibited 9.5% average evolutionary divergence and its CP and Rep had 9.3% and 12.2%, concomitantly; the alphasatellite and the betasatellite had 8.3% and 5.2%, respectively. The nt substitution rates (site-1 year−1) were found to be 6.983 × 10-04 and 5.702 × 10-05 in the CP and Rep of MeYVMV, respectively. The dN/dS ratio and the Tajima D value of MeYVMV CP demonstrated its possible role in host switching. The absolute quantification of the begomovirus demonstrated that mild symptoms might have a correlation with low virus titer. This is the first identification of MeYVMV and associated DNA-satellites from ornamental sunflower in Pakistan. The role of sequence divergence, recombination and importance of MeYVMV along with DNA-satellites in extending its host range is discussed.

First report of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 infecting watermelon (Citrullus lanatus) in the United States.

Watermelon (Citrullus lanatus) and other cucurbits are cultivated globally, and Texas ranks among its top 5 producers in the U.S. In July 2020, plants with virus-like disease symptoms consisting of mild leaf crinkling and yellow mosaic patterns were observed in a 174-ha watermelon field in Burleson Co., TX; disease incidence was visually estimated at 5%. Total nucleic acids were extracted from leaf tissues of 5 randomly sampled plants (Dellaporta 1983) and their equimolar amounts were made into a composite sample that was used for cDNA library construction with TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina). The cDNA library was sequenced on the Illumina NextSeq 500 platform, generating ~37M single-end reads (each 75 nt), which were analyzed as per Al Rwahnih et al. (2018). Of these, 58,200 and 27,500 reads mapped to the genomes of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 (Xin et al. 2017), respectively, along with 4 other virus-specific reads (data not shown). The near complete RNA1-RNA3 segments of WCLaV-1 (354-652X) and WCLaV-2 (144-258X) were generated from the mapped reads and they shared ≥96% nt identities with published RNA segments of both viruses. The results were verified by RT-PCR using newly designed primers WCLaV-1vRP: 5'-GGTGAGTTAGTGTGTCTGAAGG/WCLaV-1cRP: 5'-GAGGTTGCCTGAGGTGATAAG to target 881 bp of the RNA1-encoded RNA-dependent RNA polymerase (RdRP), WCLaV-1vMP: 5'-GAAGGTTTGCTCCCTTGAAATG/WCLaV-1cMP: 5'-GACTGTGGCTGAAGAGTCTATG target 538 bp of the RNA2-encoded movement protein (MP), and WCLaV-1vNP: 5'-CGAATAGACTCTGGAGGGTAGA/WCLaV-1cMP: 5'-GAAAGCAAGAAAGCTGGCTAAA target 786 bp of the RNA3-encoded nucleoprotein (NP). Similarly, the WWCLaV-2-specific primers WCLaV-2vRP: 5'-GTCTCACATTCCTGCACTAACT/WCLaV-2cRP: 5'-ATCGGTCCTGGGTTATTTGTATC target 968 bp of the RdRP, WCLaV-2vMP: 5'-GACTTCAGAACCTCAACATCCA/WCLaV-2cMP: 5'-CAAGGGAGAGTGCTGACAAA target 562 bp of the MP, and WCLaV-2vNP: 5'-ATTCCCAGTGAGAGCAACAA/WCLaV-2cMP: 5'-GAGGTGGAGGTAGGAAAGAAAG target 449 bp of the NP. Fresh cDNA synthesized from the 5 samples with PrimeScript First Strand cDNA synthesis kit (Takara Bio) were tested by PCR with all 6 primer pairs using the PrimeSTAR GXL DNA Polymerase kit (Takara Bio). Three of the 5 samples were positive for both viruses and one sample was positive for each virus. The obtained products from 4 samples were cloned individually into pJET1.2/Blunt vector (Thermo Scientific, USA), followed by bidirectional Sanger-sequencing of the plasmids with the GenElute Five-Minute Plasmid Miniprep kit (Sigma-Aldrich). In pairwise comparisons, the partial RNA1-RNA3 sequences of WCLaV-1 (GenBank accession nos. MW559074-82) shared 100% nt/aa identities with each other and with corresponding sequences of WCLaV-1 isolate KF-1 from China (KY781184-86). The partial RNA1-RNA3 sequences of WCLaV-2 (MW559083-91) shared 97-100% nt/96-100% aa identities with each other and with corresponding sequences of WCLaV-2 isolate KF-15 from China (KY781187-89). This is the first report of WCLaV-1 and WCLaV-2 in Texas and the first record of both viruses in the U.S. and elsewhere outside of China. Both negative-sense, single-stranded RNA viruses represent a novel taxon in the family Phenuiviridae (order Bunyavirales) (Xin et al. 2017). While aspects of the biology of both viruses are yet to be elucidated, our results expand their geographical range. The detection primers developed here will be useful for screening cucurbits germplasm to avert their spread.

Characterization of two Cucumber mosaic virus isolates infecting Allium cepa in Turkey

Cucumber mosaic virus (CMV) is polyphagous, infecting plants in several families. CMV has occurred as a minor pathogen in Allium crops in several Mediterranean countries, but little was known of the virus naturally infecting Allium spp. This study completed molecular and biological characterization of CMV-14.3Po and CMV-15.5Po, two newly identified CMV isolates infecting onion (Allium cepa L.) in Turkey. Phylogenetic, and nucleotide and amino acid sequence identity analyses of partial RNA2 and RNA3 of the two isolates showed that they were very similar to other CMV isolates from Mediterranean, European, and East Asian countries. Phylogenetic analysis of the partial sequence of RNA3 also showed that the onion isolates belong to subgroup IA. Onion isolates were mechanically transmissible, and caused mild leaf malformation on onion, severe leaf malformation and stunting on garlic (Allium sativus L.), and mosaic and mottle on cucumber (Cucumis sativus L.) and melon (Cucumis melo L.).

Interaction of a tomato leaf curl New Delhi virus with a betasatellite enhances symptom severity in field-infected tomato plants

Two field-collected tomato plants, with severe and mild tomato leaf curl disease symptoms, were shown to be infected with tomato leaf curl New Delhi virus. The plant with severe symptoms was shown to additionally contain tobacco leaf curl betasatellite (TobLCuB). Inoculation of Nicotiana benthamiana plants with the cloned components showed the severe symptoms to be due to the presence of TobLCuB. A shorter latent period was associated with the presence of TobLCuB except in the presence of the DNA-A and DNA-B components of the severe type. The DNA-B component from the mild type also reduced the latent period, more so than the DNA-B from the severe type, except in the interaction with the DNA-A from the mild type. These differences in the effects of the virus components from the two isolates may possibly be due to mutations in the DNA-B from the severe type. The results show that betasatellites can enhance the virulence of bipartite begomoviruses, even for isolates that induce quite mild symptoms.

High Efficacy and Low Risk of Phytotoxicity of Sulfur in the Suppression of Strawberry Powdery Mildew

Sulfur is a multisite inhibitor of strawberry powdery mildew (Podosphaera aphanis) recommended for fungicide resistance management, yet growers may refrain from its use due to purported phytotoxicity under conditions often qualitatively defined as “hot”, or “during periods of high temperature”. Our objectives were to (i) determine phytotoxicity of sulfur on three strawberry cultivars treated in controlled environments at temperatures of 30, 35, and 40°C for periods of 1, 2, 4, and 8 h after the sulfur application; and (ii) evaluate phytotoxicity and efficacy of sulfur on strawberry grown in open fields and high tunnels in Florida. Under controlled conditions, cultivars Florida127 and Florida Beauty exhibited minor phytotoxicity when exposed to sulfur at 30°C for 2 to 8 h. Cultivar Florida Radiance, however, was more sensitive, with greater phytotoxicity as exposure time increased. We found that weekly applications of sulfur on cultivar Florida127 significantly reduced P. aphanis foliar and fruit disease as well as or better than the standard fungicide treatment, and resulted in 80 to 300% higher yields (compared with nontreated controls) in open field and high tunnel trials, respectively. Ambient temperature did not exceed 35°C in open field trials, where no phytotoxicity was observed. In the high tunnel, where only mild leaf phytotoxicity was observed, temperatures exceeded 40°C for a total of 20 h across 9 days. Our results suggest that sulfur has benefits for disease suppression and fungicide resistance management that greatly surpass the minimal phytotoxicity observed only at temperatures above 35°C for a period of at least 2 h.

First report of lettuce necrotic leaf curl virus infecting cultivated lettuce in France.

Lettuce necrotic leaf curl virus (LNLCV, genus Torradovirus, family Secoviridae) has a bipartite single-stranded RNA genome and has so far only been reported in the Netherlands in open field lettuce (Verbeek et al. 2014). It was the first Torradovirus described from non-tomato host and, contrary to whitefly-transmitted tomato torradoviruses, aphids are its natural vectors (Verbeek et al. 2017). In October 2019, a symptomatic lettuce (JG3, cv. "Tregoney") was collected in an open field in southwestern France. Symptoms included stunted and deformed leaves with light necrosis and yellow spotting along minor veins of older leaves. Double-stranded RNAs were purified from JG3 leaves as described (Marais et al. 2018) and a cDNA library prepared and analyzed by Illumina NovaSeq sequencing. Analysis of sequence data identified two nearly fully assembled RNAs integrating respectively 28.9% and 60.9% of the sequencing reads and sharing respectively 85.5% and 83.3% nucleotide (nt) identity with the RNAs 1 and 2 of the LNLCV reference isolate, (NC_035214 and NC_035219, respectively). To confirm the presence of LNLCV in the original JG3 plant, it was used to mechanically inoculate indicator Nicotiana benthamiana, Chenopodium quinoa and C. amaranticolor plants. Only N. benthamiana developed symptoms, in the form of smaller and yellowed leaves. All inoculated plants were tested one month post-inoculation for the presence of LNLCV. Total RNAs were extracted according to Foissac et al. (2005) and used for RT-PCR tests with primers designed from the alignment between NC_035214 and our RNA1 sequence (LNLCV-S 5'-ATATTTTCCAAGTTGGAGGCTC-3' and LNLCV-R 5'-AGTRACAAAGGGACTAACTG-3'). LNLCV was detected in 3 out of 4 inoculated N. benthamiana plants. The full length RNA1 sequence (7577 nt) and the near complete RNA2 (5286 nt, lacking 3 nt at the 5' end as compared to NC_035219) could be assembled from the JG3 sequencing data and have been deposited in GenBank (MW172270 and MW172271, respectively). The lettuce JG3 isolate RNA1 shows 86.5% nt identity with the reference isolate while the taxonomically informative protease-polymerase regions share 96.8% aa identity. JG3 RNA2 shares 84.8% nt identity with NC_035219 while the movement protein and capsid subunits share respectively 92.5% and 98.3% aa identity. The smaller upstream ORF that slightly overlaps with the large MP-CP1/2/3 ORF is also conserved and shows 94.8% aa identity with the reference isolate. To our knowledge, this represents the first report of a natural infection of LNLCV in cultivated lettuce in France and anywhere outside the Netherlands. Since no other viruses were detected in the sequence dataset, LNLCV is most likely responsible for the mild necrosis and leaf deformation symptoms observed on the JG3 plant that appear to be similar to those initially described for LNLCV (Verbeek et al. 2014). While the pathogenicity of LNLCV in lettuce appears to be firmly established, further studies are needed to establish its distribution and prevalence, to understand why this pathogenic and aphid-transmitted virus is not more widely reported and whether it has the potential to increase in impact as a potential emerging agent on field lettuce crops.

First report of Leaf Wilt caused by Fusarium proliferatum and F. brachygibbosum on Date Palm (Phoenix dactylifera) in Tunisia.

Since 2017, a new leaf wilt syndrome was observed in plantations of date palm in Tunisia. Its incidence increases sharply from year to year, especially in 'Deglet Nour' trees, aged between 5 and 15 years. In severe cases, the large number of dried leaves per tree can lead to complete cessation of date production. Symptoms appear on one or more leaves in the center of the crown. Whitening and drying start at the top of the leaflets and proceed to their base, while the midrib remains green. Then the whole leaf dies. Small white-creamy leaflet fragments and roots were collected from five different regions in the Djerid Oases. They were disinfected with diluted bleach (0,8 % NaOCl) and ethanol (80%) (each 2 min), rinsed with sterile distilled water, dried and finally plated in Petri dishes containing Potato Dextrose Agar (PDA) amended with 50mg/l neomycin. After incubation for 7 days at 25ºC±2, emerging fungal colonies were single-spored by serial dilution. They were transferred to PDA, Carnation Leaf Agar (CLA) and Spezieller Nahrstoffarmer Agar (SNA) for morphological identification. Based on the colony color on PDA, conidial morphology and phialide structures on CLA and/or SNA, of the 85 Fusarium isolates, around 90% were identified as F. proliferatum and around 10% as F. brachygibbosum (Leslie and Summerell, 2006). Fusarium proliferatum colonies rapidly developed white aerial mycelium that became purple in old cultures. Microconidia were abundant in the aerial mycelium and formed chains of variable length, on monophialides and polyphialids, a characteristic that distinguishes F. proliferatum from F. verticilloides. Less often, they were observed in false heads. Chlamydospores were absent. On CLA, microconidia were mostly 2 × 15 µm in size, a large number of sickle shaped macroconidia (2 × 25 µm) had one septum, some were larger (2 × 50 µm) with 3 septa and tips at both ends. Molecular identification was carried out based on elongation factor (EF-1α) gene sequencing. The region between the EF1 and EF2 primers (O'Donnell et al., 1998) was amplified and the sequences were compared to Fusarium reference sequences (GenBank). The sequences of the isolates Fus 1953 (539 bp), Fus 1962 (618 bp), and Fus 1965 (605 bp) shared respectively 100%, 99.51% and 99.51% homology with that of F. proliferatum JF740713.1 and were deposited in GenBank with the following accession numbers: MT630418, MT630419, and MT630420, respectively. The sequences of isolates 7F, 28F, Fus 1955 and Fus 1956 shared 100 % homology with that of F. brachygibbosum (GQ505418.1) while those of Fus 1955 and Fus 1956 showed 99.02 and 98.91 % identity, respectively, with F. brachygibbosum JX118981.1. The sequences of 7F (535 bp), 28F (535 bp), Fus 1955 (608 bp), and Fus 1956 (647 bp) were deposited in GenBank with the following accession numbers: MT630409, MT630410, MT630411, and MT630412, respectively. Two ml suspension of 106 conidia / ml of each isolate was sprayed separately or in combinations on in vitro cloned 'Deglet Nour' plants, placed in a greenhouse at 28°±2 °C and 70% R.H.. Isolates of F. proliferatum led to dryness and wilting leaflets after 3 weeks. Fusarium brachygibbosum only induced mild leaf yellowing, while in combination they were more virulent. Fungal isolates of both species were re-isolated and their identity confirmed to be the same of those isolated from leaflets infected in the open field, confirming Koch's postulates. Control plants lacked symptoms. Fusarium proliferatum is known as date palm pathogen in many countries (Saleh et al. 2017), however, to our knowledge, this is the first report of F. proliferatum and also F. brachygibbosum causing Leaf Wilt symptoms on P. dactylifera in Tunisia.

Pseudomonas syringae pv. actinidiae: Ecology, Infection Dynamics and Disease Epidemiology.

Since 2008, the kiwifruit industry has been devastated by a pandemic outbreak of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker. This disease has become the most significant limiting factor in kiwifruit production. Psa colonizes different organs of the host plant, causing a specific symptomatology on each of them. In addition, the systemic invasion of the plant may quickly lead to plant death. Despite the massive risk that this disease poses to the kiwifruit industry, studies focusing on Psa ecology have been sporadic, and a comprehensive description of the disease epidemiology is still missing. Optimal environmental conditions for infection, dispersal and survival in the environment, or the mechanisms of penetration and colonization of host tissues have not been fully elucidated yet. The present work aims to provide a synthesis of the current knowledge, and a deeper understanding of the epidemiology of kiwifruit bacterial canker based on new experimental data. The pathogen may survive in the environment or overwinter in dormant tissues and be dispersed by wind or rain. Psa was observed in association with several plant structures (stomata, trichomes, lenticels) and wounds, which could represent entry points for apoplast infection. Environmental conditions also affect the bacterial colonization, with lower optimum values of temperature and humidity for epiphytic than for endophytic growth, and disease incidence requiring a combination of mild temperature and leaf wetness. By providing information on Psa ecology, these data sets may contribute to plan efficient control strategies for kiwifruit bacterial canker.Electronic supplementary materialThe online version of this article (10.1007/s00248-019-01459-8) contains supplementary material, which is available to authorized users.

Characterization of a New Isolate of Chilli ringspot virus in Yunnan, China

Virus diseases are the major limiting factors of chilli production in China. A virus isolate of chilli ringspot virus-Yunnan (ChiRSV-YN) from chilli (Capsicum annuum L.) showing mild mosaic, leaf crinkling, ringspot, and vein banding symptoms was recently identified in Yunnan, China. The electron microscopy confirmed the virus as a typical potyvirus having flexuous filamentous particles ca. 780 nm in length. Several chilli and tobacco varieties were tested through mechanical inoculation and found to be susceptible to the virus isolate ChiRSV-YN. The viral genome was sequenced through RNA sequencing. The complete genomic RNA of the isolate (GenBank Acc. No. KX258620) consisted of 9,652 nucleotides (nt) excluding the poly (A) tail at the 3′ end. The isolate contains a large ORF which encodes a polyprotein of 3,086 amino acids (aa) with Mr. of 349.21 kDa. It had a typical genomic organization of potyviruses. The complete nucleotide and polyprotein sequences of ChiRSV-YN had 87.6 and 93.2% identities with the ChiRSV-HN/14, respectively. The result showed ChiRSV-YN is a new isolate of ChiRSV and it is the first full-length genome characterization of ChiRSV in Yunnan, China. Understanding of the characteristics of this virus isolate might provide the basis for disease diagnosis and control in the future.

Diversity and occurrence of chickpea chlorotic dwarf virus on legumes from Iran

Chickpea chlorotic dwarf virus (CpCDV; genus Mastrevirus, family Geminiviridae) is one of the most important legume‐infecting viruses with a wide host range and geographic distribution in Africa and Asia. In Iran, CpCDV is common in chickpea (Cicer arietinum), but there is limited information about diversity and infections in plants of other legume species. In the current study, a total of 1671 leaf samples from different pulse crops with symptoms were collected in nine provinces of Iran, and the CpCDV infection status was tested by PCR and/or rolling circle amplification (RCA), resulting in the detection of CpCDV in samples of chickpea, lentil (Lens culinaris) and faba bean (Vicia faba) from different regions. Sequence analysis of complete genomes of 18 isolates recovered by digestion of RCA products revealed infection with isolates of the strains CpCDV‐A and CpCDV‐F in chickpea, lentil and faba bean. Phylogenetic analysis showed that the Iranian isolates of CpCDV were closely related to previously sequenced isolates of CpCDV‐A and CpCDV‐F. To the authors' knowledge, this is the first report of CpCDV‐F in Iran. Using agroinoculation with infectious clones for one isolate each of CpCDV‐A and CpCDV‐F, infectivity was confirmed in both faba bean and chickpea, with plants developing leaf curling and/or yellowing. Both infectious clones also successfully infected Nicotiana benthamiana resulting in mild yellowing and intensive leaf curling for CpCDV‐A, and dark‐green mosaic, dwarfing and mild leaf curling for CpCDV‐F.

VdOGDH is involved in energy metabolism and required for virulence of Verticillium dahliae.

Verticillium dahliae, a soil-borne fungus, can invade plant vascular tissue and cause Verticillium wilt. The enzyme α-oxoglutarate dehydrogenase (OGDH), catalyzing the oxidation of α-oxoglutarate in the tricarboxylic acid cycle (TCA), is vital for energy metabolism in the fungi. Here, we identified the OGDH gene in V. dahliae (VdOGDH, VDAG_10018) and investigated its function in virulence by generating gene deletion mutants (ΔVdOGDH) and complementary mutants (ΔVdOGDH-C). When the ΔVdOGDH mutants were supplemented with different carbon sources, vegetative growth on Czapek Dox medium was significantly impaired, suggesting that VdOGDH is crucial for vegetative growth and carbon utilization. Conidia of the ΔVdOGDH mutants were atypically rounded or spherical, and hyphae were irregularly branched and lacked typical whorled branches. Mutants ΔVdOGDH-1 and ΔVdOGDH-2 were highly sensitive to H2O2 in the medium plates and had higher intracellular ROS levels. ΔVdOGDH mutants also had elevated expression of oxidative response-related genes, indicating that VdOGDH is involved in response to oxidative stress. In addition, the disruption of VdOGDH caused a significant increase in the expression of energy metabolism-related genes VdICL, VdICDH, VdMDH, and VdPDH and melanin-related genes Vayg1, VdSCD, VdLAC, VT4HR, and VaflM in the ΔVdOGDH mutants; thus, VdOGDH is also important for energy metabolism and melanin accumulation. Cotton plants inoculated with ΔVdOGDH mutants exhibited mild leaf chlorosis and the disease index was lower compared with wild type and ΔVdOGDH-C strains. These results together show that VdOGDH involved in energy metabolism of V. dahliae, is also essential for full virulence by regulating multiple fungal developmental factors.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan.

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer's field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch's postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.

Identification of Tomato yellow leaf curl virus, Pepper huasteco yellow vein virus and Pepper golden mosaic virus associated with pepper diseases in northern Mexico

New diseases in pepper plantations were discovered in La Comarca Lagunera (CL) region in September 2014, the severity of which increased by October 2016. Pepper plants exhibited mild and severe yellow leaf mosaic, deformation, stunting and chlorotic leaves. In addition, whiteflies were observed on symptomatic plants, suggesting a possible begomovirus aetiology. In this study, naturally infected pepper plants were collected during three consecutive years to identify the potential begomovirus present in pepper in CL. PCR detection using degenerate and specific primers indicated that 47 out of 49 pepper plants were infected by begomoviruses mainly in mixed infection. The complete begomovirus genomes were isolated from a representative symptomatic pepper plant and two clones for each begomovirus were fully sequenced for the corresponding year of collection (2014 to 2016). Phylogenetic analysis of complete genomes of CL begomovirus pepper isolates indicated a close homology with Tomato yellow leaf curl virus (designated TYLCV-CL) displaying 99.9–100% identity with TYLCV Sinaloa isolate, and the bipartite Pepper huasteco yellow vein virus (designated PHYVV-CL) displaying 94.5% and 84.2% identity with first PHYVV isolate from Tamaulipas for DNA A and DNA B, respectively, and 97–98% identity with PHYVV Sinaloa isolate for DNA B. In 2016, Pepper golden mosaic virus (designated PepGMV-CL) was also found that consisted of DNA-A and DNA-B genome displaying 97% and 93.5% identity with PepGMV isolate Tamaulipas. To our knowledge, this is the first report of a pepper disease associated with TYLCV in double or triple infection either with PHYVV and/or PepGMV in Mexico.

Molecular characterization of Mungbean yellow mosaic India virus infecting tomato (Solanum lycopersicum L.)

The severe mosaic and mild leaf curl symptoms were observed on tomato crops in the agricultural farm at Chitrakoot, Madhya Pradesh, India. Based on typical viral symptoms and high populations of whiteflies in the field, the association of begomovirus was suspected. The identification of a bipartite begomovirus associated with the disease was performed by sequence analyses of their cloned full-length genome. The complete genome sequences were submitted in GenBank under accession numbers MF683072 (DNA-A) and MF683073 (DNA-B). The sequence analyses of both genome of bipartite begomovirus revealed highest nucleotide sequence identity and close phylogenetic relationships with Mungbean yellow mosaic India virus (MYMIV) reported from India, therefore, following the ICTV guidelines, the begomovirus under study was identified as an isolate of MYMIV. Best of our knowledge, this is the first report of MYMIV infecting tomato.

Molecular detection of phytoplasmas infecting date palm in Saudi Arabia and Jordan

Date palm is one of the most important cash crops in Saudi Arabia and Jordan. Symptoms of mild leaf stunting, yellow streaking and a reduction in fruit and stalk size had been observed in date palm orchards in both countries. In Jordan a total of 262 healthy and symptomatic samples were collected from different date palm cultivars, weeds growing under date palm trees and other different plant species possible phytoplasma hosts growing in the orchards in addition to leafhoppers, while 567 leaf samples were collected from Saudi Arabia. Nested PCR was performed using phytoplasma P1/P7 and R16F2n/R2 primer pairs. BLAST analysis of the obtained sequences of samples from Jordan revealed over 98% sequence identity with different reference phytoplasma strains deposited in the GenBank and 98–100% sequence identity with ‘Candidatus Phytoplasma ulmi’-related strains. A 16SrII phytoplasma, ‘Candidatus Phytoplasma aurantifolia’-related strain was identified in the samples from Saudi Arabia.

First Report of Citrus viroid V Naturally Infecting Grapefruit and Calamondin Trees in California.

The Citrus Clonal Protection Program–National Clean Plant Network, University of California, Riverside (UCR), received citrus budwood samples in June 2016 from the California Department of Food and Agriculture (CDFA) for virus and viroid testing under the mandatory California (CA) §3701 Citrus Nursery Stock Pest Cleanliness Program. Samples originated from nonsymptomatic budwood sources, two redblush grapefruit (RG) (Citrus paradisi Macfadyen) and four variegated calamondin (VC) (C. madurensis Lour.), and were found in a nursery in Tulare County, CA. TRIzol-extracted RNA was tested with a SYBR green I reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the universal detection of citrus viroids (Osman et al. 2017). Amplicons with melting temperatures similar to the Citrus viroid V (CVd-V) control (Vidalakis and Wang 2013) were produced in 50% of the RG and 25% of the VC samples. Citron (C. medica L.) Arizona 861-S-1 indicators were graft-inoculated with RG and VC blind buds (two citrons each) and maintained under warm greenhouse conditions (27 to 41°C). By 8 months, all citrons had developed mild stunting and leaf bending coupled with midvein necrosis, indicative of viroid infection. Sequential polyacrylamide gel electrophoresis of the symptomatic citrons identified circular and linear viroid-like RNAs with electrophoretic mobility similar to CVd-V. RT-PCR with primers specific for the full CVd-V genome (Wang et al. 2013) produced amplicons of the expected size of 294 bp from both the RG and VC samples and from the four indicator citrons. Cloning and sequencing of the RT-PCR CVd-V amplicons produced complete viroid genome sequences (GenBank MF477857 to MF477876). The RG isolates (MF477857 to MF477866) had 99% similarity with the KM isolate (JQ348924, Pakistan), with one nucleotide change (A₆₅ > U). The VC isolates (MF477867 to MF477876) had 99% similarity with the MO isolate (JQ348925, Pakistan), with two nucleotide changes (G₆₉ > A and A₂₅₁ > U). To further test the infectivity of the identified viroid, RNA extracts from the RG and VC graft-inoculated citrons were slash-inoculated onto four new citrons budded on rough lemon (C. jambhiri Lush.) rootstock. All citrons developed symptoms, and CVd-V was identified with RT-PCR, cloning, and sequencing. The CVd-V identification results were independently verified by CDFA’s Plant Pest Diagnostic Center, and the original source trees were destroyed. CVd-V was first reported in Spain (Serra et al. 2008a) and has been detected in various samples including a source from UCR, but the source of the UCR field material was not recorded (Serra et al. 2008b). This note reports the natural occurrence of CVd-V in CA and corroborates the CVd-VCᴬ variant report from the UCR source (Serra et al. 2008b). This note also demonstrates the importance of regulatory testing of propagative materials and elimination of infected citrus germplasm source trees to prevent the spread of viroids into commercial citrus.

Accumulation and transmission of alphasatellite, betasatellite and tomato yellow leaf curl virus in susceptible and Ty-1-resistant tomato plants

Begomoviruses (family Geminiviridae) are frequently associated with alphasatellites and betasatellites in the Old World. Tomato yellow leaf curl virus, one of the most damaging begomovirus species worldwide, was recently found associated with betasatellites in the eastern coast of the Mediterranean Sea, and in the Middle East region. Tomato yellow leaf curl virus (TYLCV)/betasatellite associations were shown to increase TYLCV virulence in experimental conditions. The sustainability of TYLCV/satellite associations in tomato was assessed here by estimating accumulation levels of satellites in comparison to TYLCV, vector transmission efficiency, and by testing how far the popular Ty-1 resistance gene used in most TYLCV-resistant tomato cultivars in the Mediterranean Basin is effective against betasatellites. Three satellites previously isolated from okra in Burkina Faso—of the species Cotton leaf curl Gezira betasatellite, Cotton leaf curl Gezira alphasatellite and Okra leaf curl Burkina Faso alphasatellite—were shown to accumulate at levels similar to, or higher than, the helper virus TYLCV-Mld in tomato plants from 32 to 150 days post inoculation (dpi). Cotton leaf curl Gezira betasatellite (CLCuGB) reduced TYLCV-Mld accumulation whereas alphasatellites did not. Transmission tests were performed with B. tabaci from plants infected with TYLCV-Mld/CLCuGB- or TYLCV-Mld/Okra leaf curl Burkina Faso alphasatellite. At 32 dpi, both satellites were transmitted to more than 50% of TYLCV-infected test plants. Betasatellite transmission, tested further with 150 dpi source plants was successful in more than 30% of TYLCV-infected test plants. Ty-1 resistant tomato plants co-infected with TYLCV (–Mld or –IL) and CLCuGB exhibited mild leaf curling and mosaic symptoms at the early stage of infection associated with a positive effect on TYLCV-IL accumulation, while resistant plants infected with TYLCV only, were asymptomatic. Together with previous experimental studies, these results further emphasize the potential risk of betasatellites to tomato cultivation, including with Ty-1 resistant cultivars.

Evaluation of rice cultivars for resistance to rice yellow mottle virus

Rice yellow mottle virus (RYMV), which is only found in Africa, threatens rice farming on the continent. A local Oryza sativa cultivar collected from Burkina Faso (named BM24), was evaluated with that of well known highly resistant and tolerant cultivars. Firstly, three RYMV isolates were used to characterise the differential interaction within the cultivars. Secondly, disease kinetics of symptom expression and virus titer on leaves at 21 days after inoculation were assessed using the BF1 isolate. Thirdly, the allelic profile of O. sativa varieties using SSR marker RM101 located on chromosome 12 was also assessed. IR64 showed susceptibility to all isolates; while Tog5681 was resistant to all isolates. Ng122 overcame the resistance of Gigante, with mild leaf symptoms at 42 dpi. Azucena and BM24 had, therefore, different resistance level regarding the three isolates (Ng117b, Ng122 and Ng144). When infected with the isolate, BF1, BM24 and Azucena exhibited same resistance patterns in early growth stages with delayed of symptoms appearance, but BM24 outperformed Azucena at later stages. The virus content in the two accessions, at 14 days post inoculation, was statistically different with BM24, showing less virus compared to Azucena. However, the two accessions depicted an identical allelic profile at RM101 locus. Key words: Azucena, Burkina Faso, local variety, Oryza sativa

Minimal genomic variability in Merremia mosaic virus isolates endemic in Merremia spp and cultivated tomato in Puerto Rico.

Merremia mosaic virus (MerMV), a bipartite begomovirus, was identified for the first time as a pathogen of commercial tomato plantings. Infection of tomato by MerMV caused mild leaf curling and yellow foliar mosaic symptoms. Herein, the MerMV was identified in symptomatic Merremia quinquefolia and M. aegyptia (Convolvulaceae) plants exhibiting bright yellow or yellow-green foliar mosaic symptoms, respectively. The full-length begomoviral components were amplified from total DNA isolated from two wild species of Merremia and commercial tomato plants during 1991-1998. The DNA was subjected to rolling circle amplification, restriction digestion, and DNA sequencing. The resultant 19 and 26 apparently full-length DNA-A and DNA-B components were~2557 and~2492 bases, respectively. The 140-base common region was 97.9% identical between DNA-A and -B components, a predictive evidence for cognate DNA-A and -B components. Although the DNA-A components were highly conserved at 96-100%, the DNA-B components diverged at~89 to 100%, respectively. The overall clonal genomic features strongly suggested that MerMV lineage has been under host-selection for some time, and only recently, has undergone a host-shift, putatively, from wild convolvulaceous species to tomato (Solanaceae). Phylogenetically, MerMV grouped with other bipartite begomoviruses indigenous to the Caribbean region, with MerMV DNA-A components forming three clusters, and the DNA-B components grouped in one clade. Both clades contained only one closet relative, an isolate of MerMV from Venezuela, MerMV-VE. Biolistic inoculation of M. quinquefolia and tomato seedlings with the DNA-A and -B components of PR68 and PR80 resulted in development of symptoms like those observed in naturally-infected species, respectively.