Virus - vector relationships in the transmission of tospoviruses

Abstract

Tomato spotted wilt virus (TSWV), member of the genus Tospovirus within the family Bunyaviridae, ranks among the top ten of economically most important plant viruses. Tospoviruses cause significant yield losses in agricultural crops such as tomato, lettuce, pepper, tobacco, potato and groundnut, but also in ornamentals like chrysanthemum, alstroemeria, gloxinia and impatiens. Currently, more than 650 different plant species belonging to more than 70 distinct botanical families are known to be susceptible to tospoviruses. Following the introduction of the vector Frankliniella occidentalis from the United states in Europe, TSWV and impatiens necrotic spot virus (INSV), a tospovirus mainly occurring in ornamentals, are prevailing in the Netherlands, especially in greenhouse cultivations.To gain more insight in the epidemiology of tospoviruses, information is needed on the kinetics of their transmission. The experiments described in this thesis were done to analyze some of the parameters involved.An efficient local lesion assay was developed to facilitate transmission studies using leaf disks of Petunia x hybrida "Blue Magic". In this assay, transmission could be scored reliably within 2 to 3 days on the basis of local lesion formation. This assay also enabled easy handling of large numbers of thrips. Besides petunia, leaf disks of other plant hosts were found to be suitable as substrate in transmission studies as well.Using this assay it was established that F. occidentalis could acquire and transmit the virus in periods of 5 min. Longer periods were required for more efficient transmission. To quantify virus transmission the median acquisition (AAP 50 ) and inoculation access periods (IAP 50 ), i.e. the periods needed for 50% of the thrips to respectively acquire or inoculate the virus were determined. The value for the AAP 50 was 67 min whereas an IAP 50 of 59 min was found. The leaf disk assay was also employed to determine the efficiency of transmission and the latent period (LP), the period between acquisition and inoculation of the virus, for both TSWV and INSV. The majority of thrips already transmitted virus at the end of the second larval stage at transmission rates for TSWV or INSV of 52.8 % or 80.0%, respectively.The fate of the virus in the thrips after ingestion was studied in different developmental stages of F. occidentalis. The accumulation of two viral proteins, the nucleocapsid (N) and a non-structural (NS S ) protein, as monitored by ELISA en Western blot analyses, increased within two days above the levels initially ingested. Immunocytology of infected adults confirmed that viral products were present in high amounts, especially, in the salivary glands. Electron microscopic studies revealed the presence of many virus particles in the salivary ducts. These results unequivocally demonstrated that TSWV replicated in its insect vector and that the salivary glands were a major site of multiplication. This multiplication was not accompanied by pathological effects on the vector. No effect of virus infection was found on development time, reproduction rate and survival . This study al so revealed that no transovarial transmission of virus to the progeny took place.The vector competence was determined for four thrips and four tospovirus species. The results showed that specificity of transmission, as occurring for other insect-transmitted plant viruses, also exists for tospovirus transmission by thrips. F. occidentalis appeared to be the most efficient vector for all 4 viruses tested, i.e. TSWV, INSV, tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). Frankliniella schultzei transmitted three (TSWV, TCSV and GRSV) of the four tospoviruses, whereas Frankliniella intonsa, not yet reported as tospovirus vector, transmitted TSWV and TCSV. Of Thripstabaci, previously known as an important vector for TSWV, only one out of four populations tested was able to transmit TSWV at, moreover, a low efficiency.Finally, the transmission of mutant tospoviruses, which were generated after successive mechanical transfers of virus, was studied. Defective interfering mutants were found to be transmittable by thrips, albeit at a frequency 10 to 20 times lower than the wild type virus. In contrast, envelope deficient mutants were not transmitted by thrips, indicating the importance of the envelope glycoproteins in virus-vector relationships