Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah,Saudi Arabia

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.     

Global status and future prospects of research in cotton leaf curl disease

Abstract

Begomoviruses are economically important plant viruses with a wide host range infecting numerous vegetables, legumes and fibre crops worldwide. Pakistan cotton suffered with cotton leaf curl disease (CLCuD) epidemic in the 1990s resulting in huge crop losses. Since then CLCuD has become a continuous threat to cotton industry in Pakistan. Several monopartite begomovirus species and satellite molecules are associated with CLCuD. There is threat of disease spreading to other parts of the world which are currently free from CLCuD due to agricultural trade and spread of vector, Bemisia tabaci. Natural resistance in tetraploid cotton, Gossypium hirsutum against CLCuD is very limited and is not durable under field conditions due to emergence of new viral strains. Genetically engineered resistance towards CLCuD has had limited success due to transformation limitations in cotton and the diversity of begomoviruses. Molecular approaches and conventional breeding are underway for developing cotton with CLCuD resistance. There is need to introgress multiple resistance genes in cotton to produce durable resistance which would lost several years. The review is an update on the current status and future prospects of CLCuD.     

Molecular in silico structure and recombination analysis of betasatellite in Calotropis procera associated with begomovirus

The uncharacterised betasatellite of begomovirus associated with Calotropis procera was characterised by using molecular and in silico tools and techniques. Attempts to identify the presence of a DNA-β in the infected C. procera samples, using rolling circular amplification (RCA) followed by restriction digestion, produced a ca. 1.4 kb product, corresponding to that expected for a full-length amplicon from a betasatellite, which was sequenced (accession number HQ631430). During BLASTp, analysis of second reading frame of HQ631430 (HQ631430/2-f) against Protein Databank revealed 35% identity with Tryptophanyl–tRNA synthetase of Giardia lamblia (3FOC). Ramachandran plot of HQ631430/2-f.pdb had only 57.1% residues in the most favoured region while 3FOC.pdb had 94.2% residues in the most favoured region; therefore, only template 3FOC.pdb model could be placed in good quality category. The protein binding function was predicted for HQ631430/2-f as an important functional site of the model with 0.29 confidence level through 3d2GO. The Croton yellow vein mosaic betasatellite (GU111995 CroYVMB) serve as major parent and Croton yellow vein mosaic betasatellite-Panipat 8 (HM143908 PaLCuVM) as minor parent for HQ631430. Perhaps this is the first report of recombination in Croton yellow vein mosaic betasatellite (HQ631430).     

Tomato leaf curl geminivirus associated with cantaloupe yellow leaf disease in Thailand

Geminivirus associated with yellow leaf disease of cantaloupe plants was detected using polymerase chain reaction (PCR) with geminivirus-specific degenerate primers which anneal within the AC1 ORF (replication initiator protein gene) and the AV1 ORF (coat protein gene). A DNA fragment of 1.2 kbp was amplified, cloned and sequenced. The 32-base stem loop region was found in the amplified fragment. This included the conserved nonanucleotide sequence TAATATTAC present in all geminiviruses. The nucleotide sequence of the intergenic region (IR) was compared with 28 whitefly-transmitted geminiviruses. The geminivirus associated with yellow leaf disease of cantaloupe plants showed 96.2% sequence identity with DNA A of tomato leaf curl geminivirus from India (ToLCV-In2). These data suggest that cantaloupe yellow leaf disease was caused by ToLCV.     

Two different begomovirus species are associated with yellow vein mosaic disease of okra in Sri Lanka

Molecular Biology Reports - Yellow vein mosaic disease is the major biotic constraint of okra cultivation in Sri Lanka. Identification and detailed molecular characterization of associated pathogen...     

Pyramiding Ty-1/Ty-3 and Ty-2 in tomato hybrids dramatically inhibits symptom expression and accumulation of tomato yellow leaf curl disease inducing viruses

Tomato yellow leaf curl disease is a major constraint for tomato production worldwide and availability of new resistant materials is of great importance for breeding programmes. A phenotypic survey was undertaken to evaluate the level of resistance to the main tomato yellow leaf curl disease-inducing viruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus, in several commercial tomato cultivars, never characterised before. Seven weeks post inoculation, two cultivars resulted in high resistant phenotypes to both begomoviruses, and four were tolerant to at least one of them. In the two highly resistant hybrids (SJ12, RFT112), symptoms were completely absent and viral DNA was from 10² to 10⁵ fold lower than in susceptible plants. Molecular marker analysis revealed that these genotypes harbour the resistant genes Ty-1/Ty-3 and Ty-2. Given their high resistance, they can be considered good candidates for cultivation and breeding in areas where incidence of TYLCD is very elevated.     

Expression of the Full‐length Coat Protein Gene of Tomato leaf curl Taiwan virus is Not Necessary for Recovery Phenotype in Transgenic Tomato

Transgenic tomato plants expressing full‐length (CPV1) and truncated coat protein (CP) gene (CPV2) of Tomato leaf curl Taiwan virus (ToLCTWV) were generated by Agrobacterium‐mediated transformation. Transgene integration and expression was confirmed by PCR and Southern blotting and Northern analysis, respectively. Resistance was evaluated both in plants of T0 and T1 progenies using viruliferous whiteflies under two different inoculum pressures (10–15 and 40–50 whiteflies/plant). Upon inoculation with ToLCTWV using viruliferous whiteflies, various levels of phenotypic reaction were observed. No complete resistance was observed in any of the plants tested. The reaction of the transgenic tomato lines carrying full‐length and truncated CP gene to ToLCTWV phenotype was (i) susceptible as non‐transgenic control, (ii) delayed symptom expression, (iii) complete susceptible (from delayed symptom expression phenotype) and (iv) recovered phenotype (either plants from symptom expression as non‐transgenic plants or delayed symptom expression phenotype). Dot blot quantification of the ToLCTWV using the replicase gene as a probe revealed that the recovered phenotypes accumulated a low level of ToLCTWV, and virus concentration was gradually reduced from 10 to 14 weeks postinoculation. The possible mechanisms of CP‐mediated resistance are discussed.     

Three variants of cotton leaf curl begomoviruses with their satellite molecules are associated with cotton leaf curl disease aggravation in New Delhi

Cotton leaf curl disease (CLCuD), caused by monopartite begomoviruses and its satellite molecules, is one of the serious constrains in cultivation of cotton in India. In the present study, five CLCuD-begomovirus and its associated satellite molecules were characterized based on rolling circle amplification and sequencing of complete genome. Sequence analysis showed 82–99 % nucleotide identity among them. The phylogenetic analysis and nt identity matrix determined that of the five CLCuD-begomovirus isolates, three IARI-34, IARI-42 and IARI-50 were members of Cotton leaf curl Multan virus (CLCuMuV)-Rajasthan isolates, designated as CLCuMuV-Rajasthan-34 and two, IARI-30 and IARI-45 of Cotton leaf curl Kokhran virus (CLCuKoV)-Burewala isolates, designated as CLCuKoV-Burewala-45. The present CLCuMuV-Rajasthan-34 is recombinant isolate showing recombination events in IR, C1 and C4 regions of its genome with high probality (P = 9.9 × 10^?10–3.2 × 10^?6). Same species of betasatellite (1371 nt) molecules obtained from both the present isolates was related with cotton leaf curl Multan betasatellite by 89–97 % nt identity. Three alphasatellites (1366–1396 nt) related to Cotton leaf curl Burewala alphasatellite and Gossypium darwinii symptomless alphasatellite by 86 % nt identity were also obtained. This is the first report of appearance of CLCuKoV-Burewala isolate and CLCuD associated alphasatellites in New Delhi. The present study demonstrated that CLCuD in New Delhi is caused by three kinds of variants, two are strains of CLCuMuV and one of CLCuKoV, either by single or mixed infection along with beta- and alpha-satellite molecues.     

番茄黄化曲叶病毒对温室白粉虱适合度的影响

【目的】温室白粉虱Trialeurodes vaporariorum(Westwood)是为害北方地区花卉蔬菜的主要粉虱种类,烟粉虱Bemisia tabaci(Gennadius)则在近些年逐渐频繁的花卉贸易活动中扩散至黑龙江省部分地区并取代了白粉虱成为当地温室害虫的优势物种,番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)是烟粉虱传播的一种重要双生病毒,对作物的危害十分严重,然而该病毒对本地白粉虱的影响及对烟粉虱与白粉虱种间竞争关系的影响尚待研究。【方法】本研究观察记录感染番茄黄化曲叶病毒的番茄植株上温室白粉虱和烟粉虱的种群动态及番茄形态和部分生理指标的变化。【结果】结果表明:1)有烟粉虱滋生的番茄植株矮小,根系发达;2)有白粉虱滋生的番茄略微矮粗,影响较小;3)感染病毒的番茄矮粗或矮小,根部无明显变化;4)染毒带虫相对于带虫处理而言,在白粉虱试验中,番茄植株矮小,根系生物量也骤减;与此相反,在烟粉虱试验中,番茄的反应相对缓和;5)此外,不同酶类对植株染毒的响应不同:Ach E酶活高不利于植株,而GST酶活高则有利于植株。【结论】总体而言:烟粉虱单独作用很不利于苗期番茄,白粉虱对苗期番茄没有明显的直接影响;而感染病毒会缓解烟粉虱对番茄的强烈刺激,而加重白粉虱对番茄的作用,即染毒使带白粉虱的苗期番茄生长发育受到明显抑制。与番茄变化情况相对应的是,染毒番茄上烟粉虱产卵较少,但在发育前期(从卵到伪蛹)存活率较高;染毒番茄上白粉虱产卵较多,但在前期存活率较低。本研究可为高纬度地区粉虱综合防控提供参考。     

Molecular evidence for association of Cotton leaf curl Alabad virus with yellow vein mosaic disease of okra in North India

Two virus isolates (OY77 and OY81B) from okra plants showing yellow vein mosaic, downward curling and vein twisting symptoms were collected from different farmer's fields in Karnal, Haryana state, India. The genomes of the two isolates were amplified, cloned, sequenced and analysed. The analysis indicated that the isolates are similar with 89.2% nucleotide sequence identity. Based on the current threshold cut-off value for taxonomy distinguishing the genus begomoviruses species from strains, the two isolates are designated as strains of Cotton leaf curl Alabad virus (CLCuAV) which shared nucleotide sequence identity of >90% with CLCuAV infecting cotton in Pakistan. Phylogenetic and recombination analyses of the major genome component of OY77 and OY81B is derived from different begomviruses (CLCuAV, BYVMV, CLCuMuV) as the foremost parents for evolution of these new recombinant strains.     

The merging of two dynasties--identification of an African cotton leaf curl disease-associated begomovirus with cotton in Pakistan

Cotton leaf curl disease (CLCuD) is a severe disease of cotton that occurs in Africa and Pakistan/northwestern India. The disease is caused by begomoviruses in association with specific betasatellites that differ between Africa and Asia. During survey of symptomatic cotton in Sindh (southern Pakistan) Cotton leaf curl Gezira virus (CLCuGV), the begomovirus associated with CLCuD in Africa, was identified. However, the cognate African betasatellite (Cotton leaf curl Gezira betasatellite) was not found. Instead, two Asian betasatellites, the CLCuD-associated Cotton leaf curl Multan betasatellite (CLCuMB) and Chilli leaf curl betasatellite (ChLCB) were identified. Inoculation of the experimental plant species Nicotiana benthamiana showed that CLCuGV was competent to maintain both CLCuMB and ChLCB. Interestingly, the enations typical of CLCuD were only induced by CLCuGV in the presence of CLCuMB. Also in infections involving both CLCuMB and ChLCB the enations typical of CLCuMB were less evident. This is the first time an African begomovirus has been identified on the Indian sub-continent, highlight the growing threat of begomoviruses and particularly the threat of CLCuD causing viruses to cotton cultivation in the rest of the world.     

Occurrence of Cotton leaf curl Multan virus and associated betasatellites with leaf curl disease of Bhut-Jolokia chillies (Capsicum chinense Jacq.) in India

Molecular Biology Reports - Geminiviridae comprises the largest family of plant viruses which causes severe crop losses in India. The highest pungency chilli Bhut-Jolokia or ghost pepper (Capsicum...     

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

Tomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.     

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India

Background

Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.

Results

We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.

Conclusion

This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.     

Role of leaf extracts of some medicinal plants in the management of fungal diseases of okra (Abelmoschus esculentus (L.) Moench)

In order to manage the fungal pathogens in okra, seeds of variety Arka Anamika were subjected to soaking treatment with the aqueous leaf extracts of Coleus aromaticus, Adathoda vesica, Vitex negundo, Solanum nigrum, Leucas aspera, Ocimum sanctum and Catharanthus roseus. Among the extracts used, C. aromaticus, V. negundo extracts were found superior in reducing the incidence of mycoflora. These leaf extract treatments also resulted in increased seed germination and vigour of the seedlings. Both in green-house and field conditions also these extracts were proved their efficient in the enhancement of biomass, number of leaves, number of seeds per fruit, seed density and ascorbic acid content in the raw fruits.     

Association of tomato leaf curl Palampur virus with yellow mosaic disease of Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) in India

Natural occurrence of yellow mosaic disease was observed on Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) with disease incidences of ~36 and ~27%, respectively. Association of tomato leaf curl Palampur virus (ToLCPV) with the disease was investigated by Polymersae chain reaction (PCR) using begomovirus-specific primers. Full-length genome was amplified by rolling circle amplification (RCA) method from representative samples of C. melo and C. callosus. RCA products obtained were cloned and sequenced. Analyses of sequence data revealed the presence of full-length begomoviral genome of 2756 nucleotides with the gene arrangement of a typical begomovirus: HQ848383 (C. melo) and GU253914 (C. callosus). Both the isolates shared 99% sequence identity together and high 97–99% identities and the closest phylogenetic relationships with ToLCPV strains reported worldwide, hence identified as two new members of ToLCPV. Natural occurrence of ToLCPV on C. melo and C. callosus is the first report.