First Report of Schlumbergera virus X in Dragon Fruit (Hylocereus spp.) in Spain

Dragon fruit (Hylocereus undatus) is a high-value fruit crop, introduced about a decade ago in the mainland of Spain. In 2021, chlorotic spots were observed on young cladodes in a commercial dragon fruit orchard in the province of Seville (southern Spain). Sap extracts from 4 symptomatic cladodes were used to mechanically inoculate indicator plants: no symptoms were produced in Datura stramonium plants, but Chenopodium amaranticolor reacted with chlorotic local lesions and prickly pear plants (Opuntia ficus-indica) showed irregular yellow ringspot symptoms on young cladodes at 30 days post inoculation. Total RNA was extracted from all 4 symptomatic cladodes as previously described (Pallas et al. 1987). Reverse transcription (RT)-PCR, which was carried out with M-MLV-RT and Go Taq Pol (Promega Biotech Ibérica, SL, Madrid, Spain) and tobamovirus primers (Dovas et al. 2004), failed to produce any amplicons. Electrophoretic analysis of dsRNA, extracted from symptomatic cladodes, yielded a banding pattern similar to the one reported for potexviruses (Valverde et al. 1986). Primers specific for Cactus virus X (Kim et al., 2016) failed to produce amplicons, whereas potexvirus group primers (Potex F5/Potex R2) (van der Vlugt and Berendsen 2002), amplified an expected 584-bp amplicon from RNA extracts of all 4 field-collected samples. The RT-PCR products from the four samples were Sanger-sequenced. All showed identical sequence results (GenBank Accession MZ614940) with a predicted amino acid identity of 99% with the corresponding RNA-dependent RNA polymerase amino acid sequence of Schlumbergera virus X (SchVX) (GenBank Accession No. ACD99908). SchVX-specific primers (431s, 5‘-TTTGAGGAGTTCGTCAGCAAGA-3‘ and 431As, 5‘-TCAAGAGCCCATTGAGAGAGTG-3‘) that were designed based on the new sequence, amplified the expected amplicon of around 430 nucleotides from the total RNA extracts of the four samples. The amplicons were Sanger-sequenced and the expected nucleotide sequence was obtained. This pair of primers were used in RT-PCR tests on subsequent surveys in 2 commercial dragon fruit greenhouses from the province of Seville, and in 1 experimental greenhouse in the province of Almeria. All samples from 25 symptomatic plants of H. undatus, H. hybridum, H. costaricensis, and H. purpusii in Seville and from 1 symptomatic H. undatus plant from Almeria tested positive for SchVX, while 15 asymptomatic plants tested negative. The results obtained in this investigation support that SchVX is present in the cladodes of dragon fruit plants expressing the symptoms. SchVX has been reported previously from H. undatus from Brasil (Duarte et al. 2008) and from prickly pear in Mexico (De La Torre-Almaráz et al. 2016), and to our knowledge, this is the first report of the virus in Spain. These findings suggest that SchVX has been introduced in dragon fruit farms from Spain and propagation of this emerging crop through planting of cuttings should include testing for this virus in order to prevent further spread.

First report of costus stripe mosaic virus infecting Tradescantia spathacea plants in Brazil

Tradescantia spathacea (family Commelinaceae) is cultivated worldwide as an ornamental (Golczyk et al., 2013) and as medicinal plant (Tan et al., 2020). In 2019, 90 of ~180 plants of T. spathacea, grown in two beds of 4 m2 and exhibiting leaf mosaic were found in an experimental area at ESALQ/USP (Piracicaba municipality, São Paulo state, Brazil). Potyvirus-like flexuous filamentous particles were observed by transmission electron microscopy in foliar extracts of two symptomatic plants stained with 1% uranyl acetate. Total RNA was extracted using the Purelink viral RNA/DNA kit (Thermo Fisher Scientific) from leaves of two symptomatic plants and separately subjected to a reverse transcription polymerase chain reaction (RT-PCR). The potyviruses degenerate pairs of primers CIFor/CIRev (Ha et al. 2008), which amplifies a fragment corresponding to part of the cylindrical inclusion protein gene, and WCIEN/PV1 (Maciel et al. 2011), which amplifies a fragment containing part of the capsid protein gene and the 3′ untranslated region, were used. The expected amplicons (~700bp) were obtained from both total RNA extracts. Two amplicons from one sample were purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega) and directly sequenced in both directions at Macrogen Inc (Seoul, South Korea). The obtained nucleotide sequences (GenBank MW430005 and MW503934) shared 95.32% and 97.79% nucleotide identity, respectively, with the corresponding sequences of the Brazilian isolate of the potyvirus costus stripe mosaic virus (CoSMV, MK286375) (Alexandre et al. 2020). Extract from an infected plant of T. spathacea was mechanically inoculated in 10 healthy plants of T. spathacea and two plants each of the following species: Capsicum annuum, Chenopodium amaranticolor, Commelina benghalensis, Datura stramonium, Gomphrena globosa, Nicandra physaloides, Nicotiana tabacum cvs. Turkish and Samsun, Solanum lycopersicum, T. palida, and T. zebrina. All T. spathacea plants exhibited mosaic and severe leaf malformation. C. benghalensis plants developed mild mosaic, whereas infected T. zebrina plants were asymptomatic. The plants of other species were not infected. RT-PCR with specific CoSMV primers CoSMVHC-F and CoSMVHC-R (Alexandre et al. 2020) confirmed the infection. Nucleotide sequences of amplicons obtained from experimentally inoculated T. spathacea and T. zebrina (MW430007 and MW430008) shared 94.56% and 94.94% identity with the corresponding sequence of a Brazilian CoSMV isolate (MK286375). None of eight virus-free plants of T. spathacea inoculated with CoSMV using Aphis craccivora exhibited symptoms, nor was CoSMV detected by RT-PCR. Lack of CoSMV transmission by A. solanella, Myzus persicae, and Uroleucon sonchi was previously reported (Alexandre et al. 2020). T. spathacea plants are commonly propagated vegetatively, and by seeds. Virus-free seeds, if available, can provide an efficient and easy way to obtain healthy plants. Only three viruses were reported in plants of the genus Tradescantia: Commelina mosaic virus, tradescantia mild mosaic virus, and a not fully characterized potyvirus (Baker and Zettler, 1988; Ciuffo et al., 2006; Kitajima 2020). CoSMV was recently reported infecting Costus spiralis and C. comosus (Alexandre et al. 2020). As far as we know, this is the first report of CoSMV infecting T. spathacea plants.

First Report on Pea Seed-borne Mosaic Virus in Hungary

One of the most important diseases of pea is caused by Pea seed-borne mosaic virus (PSbMV), which has a relatively wide host range. Since there are few varieties with resistance against the virus, and spraying insecticides is not very effective, the determination of the disease and the pathogen in the seeds is very important. Inoculum prepared from pea seeds showing typical virus symptoms caused very mild symptoms on Chenopodium amaranticolor and C. quinoa, but several chlorotic/necrotic lesions on bean (Phaseolus vulgaris) cv. Scarlet, and systemic symptoms with mosaic and curling of top leaves on bean cv. Maxidor. The detection of the virus was carried out by PCR using universal primers and virus sequence analysis. According to the phylogenetic analysis the PSbMV isolate identified in Hungary belongs to the pathotype P1 and associated with the cluster 2 isolates.

DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

Yellow mosaic stunt disease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. The disease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of the disease may be carried over to imported seeds and necessary to identify as a basic information for developing control strategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents was conducted using electron microscope with quick dipping method and the molecular detection was done using nested PCR with TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculation showed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and vein banding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sized approximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis, the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, a plant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the first report of ReMV in Indonesia infecting Petunia as ornamental plant.

DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

Detection and identification of yellow mosaic stunt disease on Petunia sp. using nested PCR method. Yellow mosaic stuntdisease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. Thedisease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of thedisease may be carried over to imported seeds and necessary to identify as a basic information for developing controlstrategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents wasconducted using electron microscope with quick dipping method and the molecular detection was done using nested PCRwith TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculationshowed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and veinbanding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sizedapproximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis,the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, aplant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the firstreport of ReMV in Indonesia infecting Petunia as ornamental plant.

Inhibition of Rehmannia mosaic virus Infection by Ganoderma sp. Extract

In fruiting bodies and mycelia of several fungi belonging to a Basidiomycetes group, among others, Ganoderma contains active polysaccharides and has potential as an antiviral substances. This study aimed to determine the effect of mycelium and fruiting body extract of Ganoderma sp. against Rehmannia mosaic virus (ReMV) infections on Chenopodium amaranticolor with variations of dilution and application times. The results showed that mycelium and fruiting body extract of Ganoderma sp. could inhibit ReMV infections. Antiviral extract which were applied 4 hours before, and at the same time with virus inoculation resulted in a higher viral inhibition rates than when applied 4 hours after virus inoculation. Fruiting body extract at 10-1 and 10-2 dilutions delayed symptom appearance caused by ReMV infection when applied 4 hours before virus inoculation. Mycelium extract at dilution of 10-2 and 10-3 applied 4 hours before virus inoculation resulted in the highest inhibition rate (100%) which was indicated as the number of local lesions on Chenopodium amaranticolor leaves. Meanwhile the fruiting body extract dilution of 10-1 resulted in highest inhibition (99.55%) when applied 4 hours before virus inoculation.

First Report of Alfalfa mosaic virus in Chayote in Italy

Chayote (Sechium edule (Jacq.) Sw.) is a vigorous perennial and climbing cucurbits, native to Mesoamerica, and cultivated for alimentary purposes in the American continent, Australia, New Zealand, South Europe, Asia and Africa. During spring 2019, some chayote plants showing bright yellow vein banding rings and lines were observed in a private garden in South Italy (Campania region). Symptoms coalesced in some leaves, covering almost the whole foliar area. Double-stranded RNAs were extracted from symptomatic leaves of a single chayote plant and reverse-transcribed, randomly amplified, and submitted to Illumina sequencing (Marais et al., 2018). Reads were assembled using CLC Genomics Workbench 11.1 (http://www.clcbio.com). Contigs were then annotated by Blastn and Blastx comparison with the Genbank database, which allowed the identification of eight contigs of between 380 and 980 nucleotides sharing significant identity with alfalfa mosaic virus (AMV) genomic RNAs. No other viral contigs were identified. Mapping of reads on AMV genomic RNAs identified 4,209 AMV reads (1.26% of total reads) and allowed the scaffolding of the contigs into three scaffolds corresponding to the three AMV genomic RNAs. To complete the sequence of the AMV chayote isolate genome (named See-1), primers were designed from the contig sequences and used to amplify RACE PCR products spanning the 5′ and 3′ terminal regions of the three genomic RNAs using the SMARTer™ RACE cDNA Amplification Kit (Clontech, China). All amplicons were cloned into the pGEM-T vector (Promega, USA) and sequenced (three clones for each amplicon) by Microsynth Seqlab (Microsynth AG, Switzerland). Finally, the complete genomic sequences of the three RNAs were assembled by MacVector 17.5 (MacVector Inc., USA). The RNA1, RNA2 and RNA3 of See-1 are 3,643, 2,593 and 2,037 nt respectively (GenBank accession Nos. MT093209 to MT093211), and share the highest nt sequence identity with the RNA1 and RNA3 of AMV isolate (HZ) from tobacco (99.5% for RNA1, HQ316635; 98.7% for RNA3, HQ316637) and with the RNA2 of isolate AMV-Gym from Gynostemma pentaphyllum (98.1%, MH332898), both from China. AMV isolate See-1 was classified as belonging to subgroup I based on the presence of a BamH I and two AvaII sites in the CP ORF (Parrella et al., 2000). Reverse transcription polymerase chain reaction, using primers targeting the CP gene (Parrella et al., 2000), confirmed AMV infection in three symptomatic cayote plants including that used for Illumina sequencing, with 100% of nt sequence identity of amplicons. Three plants each of Chenopodium amaranticolor, Nicotiana benthamiana and Solanum lycopersicon were mechanically inoculated with sap from isolate See-1 infected plant, leading to the appearance of typical AMV symptoms in all three hosts ten days post-inoculation (Jaspars & Bos, 1980). This note describes the first detection of AMV in cayote in Italy and, to the best of our knowledge, in the world. In some areas of Southern Italy, climatic conditions are favorable enough to allow chayote development in the wild. Further studies would be desirable to determine the distribution and incidence of AMV in chayote and to understand the possibility that this species may play a role in AMV epidemiology, representing a threat to other susceptible crops.

Discovery and genome characterization of a new Nepovirus infecting grapevine

In 2012, dormant canes of a proprietary wine grape (Vitis vinifera L.) accession were included in the collection of the University of California-Davis Foundation Plant Services. No virus-like symptoms were elicited when bud chips from propagated own-rooted canes of the accession were graft-inoculated onto a panel of biological index grape varieties. However, chlorotic ring symptoms were observed on sap inoculated Chenopodium amaranticolor Coste & A. Rein and C. quinoa Willd. plants, indicating the presence of a mechanically transmissible virus. Transmission electron microscopy of virus preps from symptomatic C. quinoa revealed spherical, non-enveloped virions of ~27 nm in diameter. And nepovirus-like haplotypes of sequence contigs were detected in both the source grape accession and recipient C. quinoa plants using high throughput sequencing analysis. A novel bipartite nepovirus-like genome was assembled from these contigs and the termini of each RNA segment were verified by RACE assays. The RNA1 (7,186-nt) of the virus encode a large polyprotein P1 of 231.1 kDa while the RNA2 (4,460-nt) also encode a large polyprotein P2 of 148.9 kDa. Each of the polyadenylated RNA segment is flanked by 5′- (RNA1=156-nt; RNA2=170-nt) and 3′- (RNA1=834-nt; RNA2=261-nt) untranslated region sequences that shared >90% identities between their corresponding sequences. Maximum-likelihood phylogenetic analyses of the conserved Pro-Pol amino acid sequences of Secoviridae species revealed the clustering of the new virus within the nepovirus clade. Considering its biological and molecular characteristics, and based on current criteria, we propose that the novel virus, named as grapevine nepovirus A (GNVA), be assigned as a member of the genus Nepovirus.

The Phytochemical, Antifungal, and First Report of the Antiviral Properties of Egyptian Haplophyllum tuberculatum Extract

In this study, ethanol whole plant extract (WPE) of Haplophyllum tuberculatum was characterized and tested for its antifungal and antiviral activities against Fusarium culmorum, Rhizoctonia solani and tobacco mosaic virus (TMV). High Performance Liquid Chromatography (HPLC) analysis showed that the main phytochemical constituents of H. tuberculatum WPE were resveratrol (5178.58 mg/kg), kaempferol (1735.23 mg/kg), myricetin (561.18 mg/kg), rutin (487.04 mg/kg), quercetin (401.04 mg/kg), and rosmarinic acid (387.33 mg/kg). By increasing H. tuberculatum WPE at concentrations of 1%, 2%, and 3%, all of the fungal isolates were suppressed compared to the two positive and negative controls. Under greenhouse conditions, WPE-treated Chenopodium amaranticolor plants strongly inhibited TMV infection and significantly reduced TMV accumulation levels when compared to non-treated plants. Moreover, the induction of systemic resistance with significant increases in the transcriptional levels of the pathogenesis-related protein-1 (PR-1), chalcone synthase (CHS), and hydroxycinnamoyl-CoA quinate transferase (HQT) genes for treated plants were noticed at 3 and 5 days post-inoculation (dpi) for both assays. To the best of our knowledge, this is the first reported observation of the antiviral activity of H. tuberculatum extract against plant viral infections. Finally, the results obtained suggest that H. tuberculatum WPE can be considered a promising source of both antifungal and antiviral substances for practical use and for developing plant-derived compounds for the effective management of plant diseases.

Cucumber mosaic virus pada Tanaman Lada di Yogyakarta dan Bangka Belitung

Cucumber mosaic virus on Black Pepper in Yogyakarta and Bangka BelitungPepper (Piper nigrum) is spice crop which has been cultivated a long time ago in Indonesia. Stunting is one of disease on pepper caused by cucumber mosaic virus (CMV). The research aimed to diagnose the biological, morphological and nucleaic acid characters of CMV on pepper in Yogyakarta and Bangka Belitung. CMV infection on pepper in both area (Putat dan Kleben village, Yogyakarta and Air Buluh village, Bangka Belitung) showed typical symptoms such as mosaic, narrow leaves and stunting. The disease incidence and disease severity of stunting disease are varies. The virus able to transmitted by cutting, grafting and mechanically on Nicotiana tabacum and Chenopodium amaranticolor. However, it was unable to transmitted mechanically on pepper and by Aphis gossypii. The virus particles were isometric with diameter size 28-30 nm. RT-PCR using coat protein partial gene primer successfully amplified a DNA with size ± 500 bp from all three samples. The homology of nucleotide between three isolates was 98-97%, while the highest homology of those three strains CMV from Yogyakarta and Bangka Belitung was 98% against strains from China in Brassica chinensis. Three strains CMV from pepper were in the same group, and separated from CMV pepper lines from Indonesia and other CMV isolates.