Occurrence and Diagnosis of Tomato chlorosis virus in Portugal

Tomato chlorosis virus (ToCV), a new whitefly-transmitted and phloem-limited Crinivirus infecting tomatoes in Europe, is reported for the first time in Portugal. Tomato plants with symptoms of interveinal chlorosis, collected during autumn 1998 and summer and autumn 1999 in Algarve, southern Portugal, were positive in RT-PCR assays using ToCV-specific primers. The amplified 439bp fragment was sequenced and showed 99% homology with the ToCV sequence in the GenBank database. A digoxigenin–DNA probe was produced and tested in dot-blot with total RNAs extracted from tomato samples. Both the RT-PCR and dot-blot hybridisation procedures enabled rapid and reliable detection of ToCV from field samples.     

Typing of Tomato Yellow Leaf Curl Viruses in Europe

Tomato yellow leaf curl disease is spreading in southern Europe, where it has quickly become a serious problem. In recent years, several virus isolates have been characterised. Although with some genetic variability, all isolates found in Europe belong to one of two species Tomato yellow leaf curl-Sardinia (TYLCV-Sar) or Tomato yellow leaf curl-Israel (TYLCV-Is). Several methods were tested to identify and type TYLCV isolates from field samples: (1) RFLP of a DNA fragment amplified from the coat protein gene; (2) PAGE of a fragment amplified from the C2 gene; (3) dot-blot hybridisation. All methods enabled the detection of the TYLCVs and provided good indications for attributing them to one species or the other. However, for typing purposes, the RFLP method was the most reliable, due to the easily recognisable pattern produced by the two virus species present in Europe. Dot-blot hybridisation is less expensive for identifying TYLCVs in large numbers of samples, particularly when a mixture of two probes is used. PAGE of the C2 fragment is the fastest of the methods tested.     

Spread of Tomato yellow leaf curl virus in Sicily: Partial Displacement of Another Geminivirus Originally Present

The geminivirus Tomato yellow leaf curl virus (TYLCV) was reported for the first time in Italy in 2002. We have followed its spread in Sicily, where Tomato yellow leaf curl Sardinia virus (TYLCSV), another tomato-infecting geminivirus, is endemic and has been causing severe crop losses since 1989. The presence of the two viruses was monitored in the main tomato growing area, the Ragusa province, analyzing samples with yellow leaf curling symptoms. At first (spring–summer 2002) both viruses were always found in mixed infections, but in 2003 and 2004 18–35% of plants were found infected by TYLCV alone and 8–28% by TYLCSV alone, with 41–69% carrying both viruses. TYLCV has spread quickly in the area, demonstrating, as in other parts of the world, its high virulence and invasiveness; however it has not, so far, completely displaced TYLCSV. An infectious clone of TYLCV from Sicily (TYLCV-IT) was sequenced. The nucleotide sequence was 97% identical to other TYLCV strains of the ‘severe’ type, found in many countries worldwide.     

Field Evaluation of Tomato Hybrids Engineered with Tomato spotted wilt virus Sequences for Virus Resistance, Agronomic Performance, and Pollen-Mediated Transgene Flow

ABSTRACT Tomato hybrids obtained from homozygous progeny of line 30-4, engineered for Tomato spotted wilt virus (TSWV) resistance, were tested under field conditions in two locations with their corresponding nontransgenic hybrids. No transgenic hybrid became infected, but 33 to 50% of plants of each nontransgenic hybrid became infected with a severe reduction of marketable fruit production. The transgenic hybrids conformed to the standard agronomic characteristics of the corresponding nontransgenic ones. Fruit were collected from the nontransgenic plots included in the experimental field and from border rows, and seed were used to estimate the flow of the transgene via pollen. No transgene flow was detected in the protected crops; however, in the open field experiment, 0.32% of tomato seedlings were found to contain the genetic modification. Immunity to TSWV infection in 30-4 hybrids was confirmed in laboratory conditions using mechanical inoculation and grafting. Thrips inoculation in leaf discs of line 30-4 demonstrated that TSWV replication was inhibited at the primary infection site but not in leaf discs of a commercial hybrid containing the naturally occurring resistance gene Sw-5. Due to the high economic value of tomato crops worldwide and the importance of TSWV, the engineered resistance described here is of practical value for breeding into cultivars of commercial interest, because it could be combined with naturally occurring resistance, thus greatly reducing the ability of the virus to develop resistance-breaking strains.     

Tomato infectious chlorosis virus causes leaf yellowing and reddening of tomato in Italy

Since autumn 2000, severe and widespread chlorosis, sometimes associated with redness, has been observed in greenhouse tomatoes in different regions of Italy. A total of 104 samples were analyzed for tomato infectious chlorosis virus (TICV), by a one-step RT-PCR procedure. In some areas of central Italy and Sardinia, the symptom was consistently correlated with the presence of TICV. The RT-PCR procedure enabled rapid and reliable detection of TICV from field samples. Sequence analysis of the amplified 501-bp fragment, part of the HSP70 coding region, revealed an identity of 99% with the TICV sequence in the GenBank database. A digoxigenin-labeled DNA probe was also produced and successfully tested in dot blot assays. This is the first report of TICV causing epidemics in Europe.     

Genetic transformation of Eustoma grandiflorum Griseb. by microprojectile bombardment

Summary Foreign DNA was introduced into cell suspension cultures and leaf tissue of Eustoma grandiflorum Griseb. (lisianthus) by microprojectile bombardment. For this purpose a low-cost bombardment device that uses a helium flux to accelerate microprojectiles was built. When cell suspensions were used, an average of 4.1 Kan resistant calli were recovered per shot after 4 months' cultivation on selective medium. Most of the Kan resistant plants regenerated from calli were positive to GUS assay. Both the nptII and gus genes were successfully amplified from alkali-treated leaves of putative transgenic plants by PCR analysis. Transgenic plants were not recovered from bombarded leaves. Considering the host range specificity of Agrobacterium, and the response of the species to plant regeneration from suspension culture, microprojectile bombardment is, at present, the most efficient procedure for genetic transformation of lisianthus.Abbreviations BA 6-benzyladenine - Cx cefotaxime - 2,4 D (2,4-dichlorophenoxy) acetic acid - FDA fluorescein diacetate - gus -glucuronidase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2iP (2-isopentenyl) adenine - Kan kanamycin - nptII neomycin phosphotransferase II - PCR polymerase chain reaction