Tomato Yellow Leaf Curl Virus DNA Research Articles

Non-Feeding Transmission Modes of the Tomato Yellow Leaf Curl Virus by the Whitefly Bemisia tabaci Do Not Contribute to Reoccurring Leaf Curl Outbreaks in Tomato.

Tomato yellow leaf curl virus (TYLCV) causes significant yield loss in tomato production in the southeastern United States and elsewhere. TYLCV is transmitted by the whitefly Bemisia tabaci cryptic species in a persistent, circulative, and non-propagative manner. Unexpectedly, transovarial and sexual transmission of TYLCV has been reported for one strain from Israel. In this study, the potential contribution of the B. tabaci B cryptic species transovarial and sexual transmission of TYLCV (Israel strain, Georgia variant, Georgia, USA) to reoccurring outbreaks was investigated by conducting whitefly-TYLCV transmission assays and virus DNA detection using end point PCR, DNA quantitation via real-time PCR, and virion detection by immunocapture PCR. TYLCV DNA was detectable in four, two, and two percent of first-generation fourth-instar nymphs, first-generation adults, and second-generation adults, respectively, following transovarial acquisition. Post-mating between viruliferous counterparts, the virus's DNA was detected in four percent of males and undetectable in females. The accumulation of TYLCV DNA in whiteflies from the transovarial and/or sexual experiments was substantially lower (100 to 1000-fold) compared with whitefly adults allowed a 48-hr acquisition-access period on plants infected with TYLCV. Despite the detection of TYLCV DNA in whiteflies from the transovarial and/or mating experiments, the virions were undetectable by immunocapture PCR-a technique specifically designed to detect virions. Furthermore, tomato test plants exposed to whitefly adults that presumably acquired TYLCV transovarially or through mating remained free of detectable TYLCV DNA. Collectively, the extremely low levels of TYLCV DNA and complete absence of virions detected in whiteflies and the inability of the B. tabaci cryptic species B to transmit TYLCV to test tomato plants following transovarial and mating acquisition indicate that neither transovarial nor sexual transmission of TYLCV are probable or epidemiologically relevant for TYLCV persistence in this pathosystem.

First Report of Tomato Yellow Leaf Curl Virus Infecting Pepper and Tomato in Oklahoma.

Tomato yellow leaf curl virus (TYLCV), (genus Begomovirus and family Geminiviridae) is one of the devastating viruses infecting tomatoes (Solanum Lycopersicon) and could have a significant impact on tomato production worldwide (Moriones and Navas-Castillo, 2000; Rybicki, 2015; Perez-Padilla et al. 2019). TYLCV was first reported in the United States (US) in 1997 in Florida (Polston et al. 1999) and since then it has been reported in several other states but not in Oklahoma (de Sa et al. 2008). Both pepper (Capsicum spp.) and tomato are grown in >50 counties of Oklahoma for fresh produce in the local market, and for processing industries. During a survey in 2021, pepper (bell pepper cv SV3964) and tomato (cv grand marshall) plants grown in a commercial field in Bixby, Oklahoma, showed typical virus-like symptoms including yellowing, leaf curling, cupping, twisting, and mottling (Fig. 1). Estimated disease incidence was 10% in both crops. In addition, whiteflies (Bemisia tabaci) were also observed on these plants. Twenty symptomatic samples from pepper (n=16) and tomato (n=4) plants, and four asymptomatic from pepper (n=2) and tomato (n=2) plants were collected and brought to the Plant Virology Lab at the University of Tulsa for further analysis Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, LOT: SLCG7913). RNA from two symptomatic samples, one each from pepper and tomato (named BX6 and BX11, respectively) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 22,385,404 (average length 73.6 bp) and 19,012,605 (average length 73.7 bp) trim pair-end for both samples (BX6 and BX11) were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. The three contigs: 393 bp (coverage 363.24X), 670 bp (coverage 489.92X), and 990 bp (coverage 112.96X) for BX6 isolate, and four contigs: 393 bp (coverage 341.64X), 668 bp (coverage 457.23X), 814 bp (coverage 107.86X), and 990 bp (coverage 93.71X), for BX11 isolate, showed from 96 to 99% nucleotide (nt) identities with movement protein, capsid protein, C3, V2, C4, AC1, AC2, AC3 protein genes of the TYLCV isolates from Australia, China, Iran, Mexico, South Korea, and the US. The HTS data did not reveal any other viral sequence in these two samples. To further confirm the presence of TYLCV in these samples, three overlapping TYLCV-specific primer pairs (Supplementary Table 1) were designed based on the alignment of complete genome sequences of TYLCV isolates present in the Genbank. Total DNA was extracted from fresh leaf tissues of BX6 and BX11 samples using the Plant DNA Kit (Omega BIO-TEK,) and used in the PCR assay. The expected PCR products (814 bp, 1,085 bp and 1,068 bp) were generated from both samples providing further evidence of TYLCV infection. To obtain the complete genome sequence, these PCR amplified DNA fragments of both isolates were gel extracted and cloned using the pGEM-T® Easy Vector system. Three independent clones of each fragment were sequenced and analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the GenBank and had the highest identities (99-99.2%) with TYLCV sequences. The overlapping consensus nt sequence of the BX6 isolate was 2,782 nt (Accession no ON321843), whereas that of BX11 was 2,777 nt (Accession no ON785706) showed 99.2% and 99.0% nt identities with the corresponding sequence of TYLCV isolates (KX347141 and KX347142) respectively from Australia. The nt identity between BX6 and BX11 was 99.7%. Further screening by PCR of the remaining 18 symptomatic field samples (pepper, n=15 and tomatoes, n=3) revealed TYLCV infection in two pepper and all three tomato samples consistent with the greater susceptibility of tomato. None of the asymptomatic pepper and tomato samples were positive for TYLCV. DNA from both BX6 and BX11 samples was also used in the rolling circle amplification (RCA) assay (TempliPhi Amplification Kit, Cytiva) and the presence of circular TYLCV DNA was detected. Our results demonstrated the presence of TYLCV infection in these symptomatic pepper and tomato plants. This is the first report of TYLCV infecting pepper or tomato in open fields in Oklahoma. Further surveys are needed to determine the incidence of TYLCV and the presence of its vector in other counties throughout Oklahoma.

Revisiting Seed Transmission of the Type Strain of Tomato yellow leaf curl virus in Tomato Plants

Isolates of the Tomato yellow leaf curl virus (TYLCV) species (genus Begomovirus, family Geminiviridae) infect tomato crops worldwide, causing severe economic damage. Members of the whitefly Bemisia tabaci sibling species group are the vector of begomoviruses, including TYLCV. However, transmission of isolates of the type strain (Israel [IL]) of TYLCV (TYLCV-IL) by tomato seed has recently been reported based on infections occurring in Korea. Because of the consequences of this finding on the epidemiology and control of the disease caused by TYLCV and on the seed market, it was considered essential to revisit and expand those results to other tomato-growing areas. TYLCV DNA content was detected in tomato and Nicotiana benthamiana seed collected from plants naturally or experimentally infected with TYLCV-IL, supporting its seedborne nature. The TYLCV-IL replication detected in tomato and N. benthamiana flower reproductive organs demonstrated close association of this virus with the seed during maturation. However, the significant reduction of TYLCV DNA load after surface disinfections of tomato seed suggests that most of the virus is located externally, as contaminant of the seed coat. Transmission assays, carried out with seven tomato genotypes and more than 3,000 tomato plants, revealed no evidence of seed transmission from "surface-disinfected" or untreated seed for two Mediterranean isolates of TYLCV-IL. Similar results were also obtained for seed collected from TYLCV-IL-infected N. benthamiana plants. The results support the conclusion that TYLCV-IL is seedborne but is not seed transmitted in tomato or N. benthamiana, suggesting that transmission through seed is not a general property of TYLCV.

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

ABSTRACTCRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

Flue-cured tobacco confirmed as a reservoir host plant for Tomato yellow leaf curl virus by agro-inoculation and Bemisia tabaci MED-mediated transmission.

Tomato yellow leaf curl virus (TYLCV) causes great losses in tomato production. In addition to tomato, TYLCV infects many crops or weeds as alternative hosts. These alternative hosts may serve as reservoirs for TYLCV survival and spread. Here, we tested the capability of cultivated, flue-cured tobacco to act as a reservoir host plant for TYLCV. TYLCV DNA was detected in nine flue-cured tobacco cultivars inoculated with an infectious TYLCV clone, although no visible symptoms developed on TYLCV-infected tobacco plants. The percentage of whiteflies with viral DNA increased with an increasing acquisition access period (AAP) and reached 100% after a 12 h AAP on infected tobacco plants. Using infected tobacco plants as virus resources, TYLCV was capable of being transmitted to tobacco and tomato plants by whiteflies, and typical symptoms of TYLCV infection were observed on infected tomato plants but not on infected tobacco plants. Our results suggest that flue-cured tobacco can serve as a reservoir host plant for TYLCV and may play an important role in the spread of TYLCV epidemics in China.

Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperatures.

Cultured tomatoes are often exposed to a combination of extreme heat and infection with Tomato yellow leaf curl virus (TYLCV). This stress combination leads to intense disease symptoms and yield losses. The response of TYLCV-susceptible and resistant tomatoes to heat stress together with viral infection was compared. The plant heat-stress response was undermined in TYLCV infected plants. The decline correlated with the down-regulation of heat shock transcription factors (HSFs) HSFA2 and HSFB1, and consequently, of HSF-regulated genes Hsp17, Apx1, Apx2 and Hsp90. We proposed that the weakened heat stress response was due to the decreased capacity of HSFA2 to translocate into the nuclei of infected cells. All the six TYLCV proteins were able to interact with tomato HSFA2 in vitro, moreover, coat protein developed complexes with HSFA2 in nuclei. Capturing of HSFA2 by viral proteins could suppress the transcriptional activation of heat stress response genes. Application of both heat and TYLCV stresses was accompanied by the development of intracellular large protein aggregates containing TYLCV proteins and DNA. The maintenance of cellular chaperones in the aggregated state, even after recovery from heat stress, prevents the circulation of free soluble chaperones, causing an additional decrease in stress response efficiency.

Degradation mechanisms of the Tomato yellow leaf curl virus coat protein following inoculation of tomato plants by the whitefly Bemisia tabaci

Tomato yellow leaf curl virus (TYLCV) is a begomovirus infecting tomato cultures worldwide. TYLCV is transmitted to plants by the whitefly Bemisia tabaci. Once in the plant, the virus is subjected to attack by the host-plant defences, which may include sequestration in aggregates, proteolysis, ubiquitination, 26S proteasome degradation and autophagy. Elucidating how the virus avoids destruction will make it possible to understand infection and possibly devise countermeasures. The accumulation of viral coat protein (CP) and of viral DNA in plants is a marker of a successful virus transmission by B. tabaci. In response to infection, tomato tissues display multiple ways of degrading TYLCV proteins and DNA. In this study it is shown that CP (in soluble and insoluble states) is the target of protease digestion, 26S proteasome degradation and autophagy. The highest degradation capacity was detected among soluble proteins and proteins in large aggregates/inclusion bodies; cytoplasmic extracts displayed higher activity than nuclear fractions. The very same fractions possessed the highest capacity to degrade viral genomic DNA. Separately, 26S proteasome degradation was associated with large aggregates (more pronounced in the nuclear than in the cytoplasmic fractions), which are indicators of a successful abduction of plants by viruses. Autophagy/lysosome/vacuole degradation was a characteristic of intermediate aggregates, sequestering the CP in the cytoplasm and retarding the development of large aggregates. Chloroplast proteases were active in soluble as well as in insoluble protein extracts. To the best of the authors' knowledge, this study is the first attempt to identify elements of the virus-targeted degradation machinery, which is a part of the plant response to virus invasion.

Lamium amplexicaule (Lamiaceae): a weed reservoir for tomato yellow leaf curl virus (TYLCV) in Korea

After the first identification of tomato yellow leaf curl virus (TYLCV) in the southern part of Korea in 2008, TYLCV has rapidly spread to tomato farms in most regions of Korea. From 2008 to 2010, a survey of natural weed hosts that could be reservoirs of TYLCV was performed in major tomato production areas of Korea. About 530 samples were collected and identified as belonging to 25 species from 11 families. PCR and Southern hybridization were used to detect TYLCV in samples, and replicating forms of TYLCV DNA were detected in three species (Achyranthes bidentata, Lamium amplexicaule, and Veronica persica) by Southern hybridization. TYLCV transmission mediated by Bemisia tabaci from TYLCV-infected tomato plants to L. amplexicaule was confirmed, and TYLCV-infected L. amplexicaule showed symptoms such as yellowing, stunting, and leaf curling. TYLCV from infected L. amplexicaule was also transmitted to healthy tomato and L. amplexicaule plants by B. tabaci. The rate of infection of L. amplexicaule by TYLCV was similar to that of tomato. This report is the first to show that L. amplexicaule is a reservoir weed host for TYLCV.

Recruitment of the Host Plant Heat Shock Protein 70 by Tomato Yellow Leaf Curl Virus Coat Protein Is Required for Virus Infection

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.

토마토 작물의 TYLCV 저항성 평가에 이용할 수 있는 감염성 클론 개발

국내에서 채집한 5종의 토마토황화잎말림바이러스(Tomato yellow leaf curl virus, TYLCV) 분리주들에 대하여 PCR법을 이용하여 염기서열을 결정하였다. 전체 유전체의 1.9배 복제수를 가지는 감염성 클론을 제작하기 위하여, PCR로 증폭된 TYLCV DNA를 식물형질전환용 벡터에 각각 도입시켰다. TYLCV 저항성 평가를 위하여 저항성 토마토 시판 품종과 육성 계통을 각각 TYLCV 분리주를 가지고 있는 Agrobacterium tumefaciens로 접종하였다. 감수성 품종인 'Super-sunroad' 품종은 접종 30일 후에 상엽에 황화, 위축 증상을 나타낸 반면, TYLCV에 대한 저항성유전자들로 알려진 Ty-1 또는 Ty-3 유전자를 포함하고 있는 TY12, GC9, GC171, GC173 등의 토마토 육성 계통은 전신 잎에 TYLCV 병징 발현이 없었으므로 이들 계통은 TYLCV 저항성으로 판별되었다. Agroinfiltration을 통한 접종 30일에 감수성 품종과 저항성 품종 간에 TYLCV 병징 차이가 뚜렷하였다. 특이적 프라이머들을 이용한 실시간 PCR법에 의해 TY12, GC9, GC171, GC173 육성 계통에서 TYLCV DNA가 검출되었지만, 저항성 계통에서 TYLCV DNA 축적 수준은 감수성 토마토 품종에서의 TYLCV DNA 축적 수준보다 매우 낮았다. 유사한 병징 세기 및 TYLCV DNA 축적 수준이 담배가루이를 통하여 감수성과 저항성 토마토 품종 및 육성 계통에 TYLCV를 감염시켰을 때에도 관찰되었다. Agroinfiltration 시 아그로박테리움의 농도는 토마토 품종의 TYLCV 저항성 반응에 영향을 미치지 않았다. 따라서 본 연구결과들은 agroinfiltration을 통한 TYLCV 접종이 담배가루이를 통한 TYLCV 감염처럼 효과적이며 TYLCV 저항성 토마토 육성 프로그램에 활용 가능하다는 것으로 보여주었다. Five isolates of Tomato yellow leaf curl virus (TYLCV) collected from various regions of Korea were amplified using PCR and determined the sequences of full-length genome, respectively. The PCR-amplified DNA of each TYLCV isolate was introduced into a binary vector to construct infectious clone containing 1.9 copies of the corresponding viral genome. Various cultivars and breeding lines of tomato were inoculated with Agrobacterium tumefaciens harboring infectious clone of each TYLCV isolate to assess resistance against TYLCV. Susceptible cultivar 'Super-sunread' revealed typical yellowing and narrowing of the upper leaves. In contrast, breeding linesTY12, GC9, GC171, and GC173, which contained the TY-1 and/or TY-3 genes that confer resistance against TYLCV in nature, were completely symptomless, suggesting that the lines were resistant to challenging TYLCV isolates. Symptoms of TYLCV in susceptible tomato cultivars are significantly different from those of TYLCV in the resistant tomato cultivars at 30 days after agroinfiltration. Although genomic DNAs of TYLCV were detected from the breeding lines TY12, GC9, GC171, and GC173 using real-time PCR analysis with specific primers, levels of TYLCV DNA accumulation in the resistant breeding lines were much lower than those of TYLCV DNA accumulation in susceptible tomato cultivars. Similar symptom severity and levels of TYLCV DNA accumulation were observed from TYLCV infections mediated by Bemisia tabaci in the resistant and susceptible tomato cultivars. Concentration of agrobacterium did not affect the response of tomato cultivars against TYLCV inoculation. Taken together, these results suggest that TYLCV inoculation via agroinfiltration is as effective as inoculation through Bemisia tabaci and is useful for breeding programs of TYLCV-resistant tomato.

Phenotypic and molecular screening of some tomato germplasm for resistance to tomato yellow leaf curl virus disease in Ghana

Tomato yellow leaf curl virus (TYLCV) is a major tomato virus in Ghana and Africa as a whole. In this study, 30 accessions of Solanum lycopersicum L. with reported TYLCV-resistance from AVRDC were assessed for resistance to TYLCV in Ghana. Plants were grown in a field, a hot spot of the disease and the reactions of plants were evaluated based on the disease symptoms when they were 30, 45 and 60 days after transplanting. Molecular screening was also done to re-confirm the phenotypic evaluation. All the tomato accessions demonstrated various degrees of disease symptoms. Phenotypic evaluation was confirmed by amplification of TYLCV DNA fragment in all tested accessions. Based on the phenotypic and molecular evaluations, no accession provided complete resistance to TYLCV in Ghana. However, accessions with milder symptoms of TYLCV in the field were considered as tolerant. The high level of susceptibility to viral infection noted in the field was not observed in the molecular screening. The viral DNA was detected using six different primers and the primers indicated polymorphism. TYLCV was detected in 23 accessions using primer pair GhF and GhR. The results suggested that accessions that indicated symptoms of the disease on the field but had no TYLCV DNA amplification could be due to other viruses or virus strain. Accessions with reported resistance in other countries but collapsed in Ghana could be attributed to genotype – environment interactions and or the emergence of new mutants of the TYLCV in Ghana. Key words: Molecular screening, tomato, tomato yellow leaf curl virus, virus resistance, DNA, primer.

Rapid spread of tomato yellow leaf curl virus in China is aided differentially by two invasive whiteflies.

Background Tomato yellow leaf curl virus (TYLCV) was introduced into China in 2006, approximately 10 years after the introduction of an invasive whitefly, Bemisia tabaci (Genn.) B biotype. Even so the distribution and prevalence of TYLCV remained limited, and the economic damage was minimal. Following the introduction of Q biotype into China in 2003, the prevalence and spread of TYLCV started to accelerate. This has lead to the hypothesis that the two biotypes might not be equally competent vectors of TYLCV.Methodology/Principal FindingsThe infection frequency of TYLCV in the field-collected B. tabaci populations was investigated, the acquisition and transmission capability of TYLCV by B and Q biotypes were compared under the laboratory conditions. Analysis of B. tabaci populations from 55 field sites revealed the existence of 12 B and 43 Q biotypes across 18 provinces in China. The acquisition and transmission experiments showed that both B and Q biotypes can acquire and transmit the virus, however, Q biotype demonstrated superior acquisition and transmission capability than its B counterparts. Specifically, Q biotype acquired significantly more viral DNA than the B biotype, and reached the maximum viral load in a substantially shorter period of time. Although TYLCV was shown to be transmitted horizontally by both biotypes, Q biotype exhibited significantly higher viral transmission frequency than B biotype. Vertical transmission result, on the other hand, indicated that TYLCV DNA can be detected in eggs and nymphs, but not in pupae and adults of the first generation progeny.Conclusions/SignificanceThese combined results suggested that the epidemiology of TYLCV was aided differentially by the two invasive whiteflies (B and Q biotypes) through horizontal but not vertical transmission of the virus. This is consistent with the concomitant eruption of TYLCV in tomato fields following the recent rapid invasion of Q biotype whitefly in China.

Transmission of Tomato Yellow Leaf Curl Virus by two invasive biotypes and a Chinese indigenous biotype of the whitefly Bemisia tabaci

The global invasion of certain Bemisia tabaci biotypes provides opportunities to compare the competency of virus transmission between invasive and indigenous biotypes. Here we report on the acquisition, retention and transmission of Tomato Yellow Leaf Curl Virus (TYLCV) by the invasive B, Q and indigenous ZHJ2 biotypes of B. tabaci from Zhejiang, China. For all whitefly biotypes, TYLCV DNA was detected within a 30-min acquisition access period (AAP) to infected leaves. The percentage of adults with viral DNA increased with the length of AAP and reached 100% after 10–12 h. Following acquisition, viruliferous B, Q and ZHJ2 adults retained TYLCV DNA for the rest of their lives. Transmission was achieved with one B/Q adult per plant at the frequency of 50–55%, which rose to 100% with 10 insects per plant. In contrast, transmission of the virus was not observed with one ZHJ2 adult per plant in the experiments, and the transmission frequency rose to only 30–45% when whitefly adults increased to 5–10 per plant. These new data will help in the determination of the pest status of the whitefly biotypes as virus vectors in the regions of invasion, and so help in the development of management strategies.

Low frequency of horizontal and vertical transmission of two begomoviruses through whiteflies exhibits little relevance to the vector infectivity

Transmissions of plant viruses between individuals of their vector insects through mating are rare events. Recently, three begomoviruses were found to be transmitted between males and females of the whitefly Bemisia tabaci through mating, and two viruses were shown to be transmitted transovarially to progeny. However, results between reports were not consistent. Here we examined the horizontal and vertical transmission of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl China virus (TYLCCNV) by the B and Q biotypes of B. tabaci, using virus isolates and whitefly colonies established recently in China. Both TYLCV DNA and TYLCCNV DNA were shown to be transmitted horizontally and vertically by each of the two biotypes of the whitefly, but frequency of transmission was usually low. In transovarial transmission, virus DNA was detected in eggs and nymphs but not in the adults of the first generation progeny, except in the combination of TYLCV and Q biotype whitefly where 2–3% of the offspring adults contained the virus DNA. We also showed that the first generation adults, which developed from eggs of viruliferous whiteflies, were not infective to plants. These results demonstrated that for the viruses and whiteflies tested here low frequency of horizontal and vertical transmission can be expected but these two modes of transmission are unlikely to have much epidemiological relevance in the field.

Broomrape Can Acquire Viruses from Its Hosts

Broomrapes (Phelipanche, formerly Orobanche) are parasitic plants that physically connect with the vascular systems of their hosts through haustorial structures. In this study, we found that Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Potato virus Y (PVY), and Tomato yellow leaf curl virus (TYLCV) translocate from infected host plants to Phelipanche aegyptiaca. In order to examine whether these viruses, and specifically CMV, replicate in the parasite, we tested several replication parameters. We detected accumulation of both plus and minus strands of CMV genomic RNA and CMV-derived siRNAs in the shoots of Phelipanche grown on CMV-infected tobacco and tomato plants. We purified CMV particles from Phelipanche grown on CMV-infected plants. These particles were present in amounts comparable to those found in the hosts' leaves. These data indicate that CMV replicates in Phelipanche tissues. In addition, viable ToMV and PVY were observed, and the plus and minus strand RNAs of ToMV were detected in Phelipanche shoots grown on infected hosts. However, we found only low levels of ToMV coat protein and did not detect any PVY coat protein. We also detected genomic TYLCV DNA in shoots of Phelipanche grown on TYLCV-infected tomato. Thus, for the first time, we demonstrate that broomrape is a host for at least one plant virus CMV, and possibly various other viruses.

Biolistic inoculation of plants with Tomato yellow leaf curl virus DNA

Tomato yellow leaf curl virus (TYLCV) full-length DNA was amplified by PCR and cloned into a bacterial plasmid. The cloned TYLCV DNA was excised from the plasmid, ligated and the resulting monomeric circular double-stranded TYLCV DNA was used to inoculate tomato ( Solanum lycopersicom) and datura ( Datura stramonium) plants by particle bombardment. The bombarded plants produced typical disease symptoms, similar to those produced following whitefly-mediated inoculation, albeit 5–7 days later than whitefly-inoculated plants. The success rate of inoculating tomato plants by particle bombardment averaged 37%, whereas with datura plants, it averaged 85%. With whitefly-mediated inoculation of TYLCV, the success rate of inoculation was also higher in datura plants than in tomato plants. Bombardment of datura plants with a linear form of TYLCV DNA also resulted in viral infection, with an inoculation success rate similar to that with the closed-circular TYLCV DNA. Bombarding datura plants with the bacterial plasmid containing the cloned TYLCV DNA did not result in viral infection, but bombardment with a bacterial plasmid containing a cloned dimer of TYLCV DNA yielded an infection rate of 50–100%. This is the first report of TYLCV inoculation of plants using particle bombardment of a cloned monomeric linear or closed-circular form of TYLCV double-stranded DNA.

Tomato yellow leaf curl virus in Tomato in Texas, Originating from Transplant Facilities

Tomato yellow leaf curl virus (TYLCV), a monopartite virus in the genus Begomovirus (family, Geminiviridae) from the Middle East, is one of the most damaging whitefly-transmitted viruses of tomato (Lycopersicon esculentum) worldwide. TYLCV was first identified in the United States in 1997 in Florida (4), and most recently, in the Pacific Coast states of Mexico where fresh market tomatoes are grown for the U.S. market (1). During September 2006, tomatoes grown from transplants in Waller County, TX exhibited shortened internodes, stunting and puckering of leaflets, green vein banding, and diffuse chlorosis. The disease incidence in two fields (4 ha total) was 95% and yield was substantially reduced. Many of the transplants were symptomatic at planting. The transplants originated from two facilities in Hidalgo County, TX. Both facilities had experienced heavy infestations of the whitefly, Bemisia tabaci (Genn.), during transplant production. At the same time, transplants produced in Uvalde and Bexar counties, TX, where whitefly infestations were also prevalent, had similar virus symptoms. Total DNA was extracted from the leaves of symptomatic tomato plants from 10 samples from these four counties and amplified by PCR (2). DNA samples from Waller, Hidalgo, and Uvalde counties were cloned, and a partial fragment of the viral coat protein gene (core Cp) was sequenced. BLAST analysis of the core Cp sequences of each sample confirmed the presence of TYLCV. No other begomovirus was detected, and all attempts to amplify a bipartite begomovirus by PCR using degenerate DNA-B specific primers (3) were unsuccessful. The full-length TYLCV DNA was amplified from three samples using the rolling circle amplification method as described (1), cloned, and the sequences were determined. The three sequences shared 99.6 to 100% nt identity and so only one sequence was deposited in the NCBI GenBank database (Accession No. EF110890) (1). Analysis of the complete genome nucleotide sequence corroborated TYLCV identity predicted by core Cp analysis that was 98.1% identical with TYLCV from Egypt (GenBank Accession No. AY594174) and Spain (GenBank Accession No. AJ489258), 97.6% with TYLCV from Mexico (GenBank Accession No. DQ631892), and 96.5% with TYLCV-Is (GenBank Accession No. X15656). Additionally, a Southern blot with TYLCV as the probe detected replicating (double-stranded) TYLCV DNA in all samples consisting of three plants from Uvalde County and 21 plants from Bexar County. To our knowledge, this is the first report of TYLCV in Texas that occurred in two transplant production areas approximately 400 km apart. Transplants produced in Uvalde and Bexar counties were planted there, while Hidalgo County transplants were shipped outside of the usual range of the whitefly. Hidalgo County has a subtropical climate, which can allow overwintering of TYLCV and the whitefly vector, allowing the establishment and spread of this virus in the future.

Intron–hairpin RNA Derived from Replication Associated Protein C1 Gene Confers Immunity to Tomato Yellow Leaf Curl Virus Infection in Transgenic Tomato Plants

The whitefly-transmitted Tomato Yellow Leaf Curl Virus (TYLCV) is the major pathogen of tomato crop in Cuba and one of the most outstanding viral diseases of plants worldwide. In this work, we have developed transgenic tomato plants, transformed with an intron-hairpin genetic construction to induce post- transcriptional gene silencing against the early TYLCV replication associated protein gene (C1). The intron-hairpin RNA produced involves 726 nts of the 3' end of the TYLCV C1 gene as the arms of the hairpin, and the castor bean catalase intron. Transgenic tomato plants belonging to line 126, which harbor a single transgene copy, showed immunity to TYLCV, even in extreme conditions of infection (4-leaf-stage plants and 300 to many hundreds viruliferous whiteflies per plant during 60 days). Dot blot hybridization of these plants showed no TYLCV DNA presence 60 days after inoculation. Small interfering RNA molecules were detected in both inoculated and non-inoculated plants from line 126. These transgenic tomato plants of the otherwise very TYLCV-susceptible Campbell-28 tomato cultivar, are the first report of resistance to a plant DNA virus obtained by the use of the intron-hairpin RNA approach.

Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction

The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato ( Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies ( Bemisia tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of amplification of TYLCV genomic DNA without gel electrophoresis.

Effect of Host Plant Resistance to Tomato yellow leaf curl virus (TYLCV) on Virus Acquisition and Transmission by Its Whitefly Vector

ABSTRACT The effect that Tomato yellow leaf curl virus (TYLCV)-infected resistant tomato plants may have on virus epidemiology was studied. Four tomato genotypes that exhibit different levels of viral resistance, ranging from fully susceptible to highly resistant, served as TYLCV-infected source plants. Viral acquisition and transmission rates by white-flies following feeding on the different source plants were evaluated. TYLCV transmission rate by whiteflies that had fed on infected source plants 21 days postinoculation (DPI), shortly after the appearance of TYLCV symptoms, was negatively correlated with the level of resistance displayed by the source plant. Therefore, the higher the resistance, the lower the transmission rate. In addition, TYLCV DNA accumulation was shown to be lower in the resistant source plants compared with the susceptible plants. Whitefly survival rate, following feeding on source plants 21 DPI, was similar for all the cultivars tested. Significant differences in whitefly survival were found, however, following feeding on the infected source plants at 35 DPI; here, whitefly survival rate increased with higher levels of resistance displayed by the source plant. At 35 DPI, the susceptible plants had developed severe TYLCV disease symptoms, and transmission rates from these plants were the lowest, presumably due to the poor condition of these plants. Transmission rates from source plants displaying a medium level of resistance level were highest, with rates declining following feeding on source plants displaying higher levels of TYLCV resistance. TYLCV DNA accumulation in whiteflies following feeding on infected source plants at both 21 and 35 DPI was directly correlated with viral DNA accumulation in source plants. Results show that, in essence, the higher the resistance expressed, the less suitable the plant was as a viral source. Consequently, following acquisition from a highly resistant plant, TYLCV transmission by whiteflies will be less efficient.