Thrip transmission patterns of peanut bud necrosis virus (PBNV) and Tobacco streak virus (TSV) isolates of blackgram and greengram of Andhra Pradesh

Introduction

Blackgram and greengram have been subjected to the attack of several biotic stresses such as fungi, bacteria and viruses, affecting the productivity and among them, viral diseases became great menace and are the great yield reducers. Necrosis caused by viruses is posing a serious threat to Vigna species in Andhra Pradesh. Nearly two decades back i.e., during Kharif of 1990-91, low incidence of leaf curl/necrosis was recorded on these crops of Andhra Pradesh. Over the years, the incidence of the disease gradually increased to about 70-100 per cent. In the recent years, viruses causing necrosis, transmitted by thrips has assumed epidemic proportion and became a serious production constraint in blackgram and greengram especially in upland areas during all the seasons i.e, kharif, rabi and summer in Andhra Pradesh. Of several viral diseases attacking greengram and blackgram, leafcurl disease caused by Peanut bud necrosis virus (PBNV) (= Groundnut bud necrosis virus -GBNV) (Amin et al., 1985) [1] transmitted by Thrips palmi (Karny) in a propagative manner (Sreekanth et al., 2002) [32] was considered to be a major threat, causing 40% yield loss (Nene, 1972) [16] . Recently, Tobacco streak virus (TSV) has also been reported to be a cause of leaf curl symptoms on blackgram (Prasada Rao et al., 2003a, b; Ladhalakshmi et al., 2005) [19,11] and greengram (Bhat et al., 2002; Prasada Rao et al., 2003a, b) [6,19,23] paving confusion in field diagnosis to assess the disease incidence. Although both the viruses cause necrotic symptoms and are transmitted by thrips, the method of transmission and the virus vector relationship vary and hence need different approaches of management practices. It is necessary to understand virus vector relationships and differentiate necrosis-causing viruses and their incidence on blackgram and greengram to follow the appropriate management practices.

Materials and Methods

Collection and identification of thrips

The colonies of thrips species viz., Thrips palmi (Karny), Frankliniella schultzei (Trybom) and Scirtothrips dorsalis (Hood) were initiated with the thrips collected from apparently healthy blackgram and greengram plants in the fields at NBPGR-RS, Rajendranagar, Hyderabad. Large number of terminal leaves and flowers were collected during early morning hours. They were placed in a glass beaker and covered with a funnel attached with a small homeopathic glass vial (Fig. 1). The adult thrips crawled along the walls of the funnel and gathered in the glass vial. The glass vial was changed at 2 h interval (Sakimura et al., 1961) [29] . Thrips were immobilized by placing the vials in a refrigerator at 4 °C for 15 min and were dislodged onto an ice tray (Lewis, 1973) [12] . After immobilization by the cold treatment, thrips were sorted to different species (within 15 min) using a stereobinocular microscope, as per the key characters (Table 1) given by Amin and Palmer (1985) [1] , Palmer et al. (1989) [17] , Reddy et al. (1991) [28] and Vijayalakshmi (1994) [33] . The suspected vectors viz., S. dorsalis (Amin et al., 1978; 1981; Ghanekar et al., 1979) [4,5,8] , F. schultzei (Amin et al., 1981) [5] and Thrips palmi (Palmer et al., 1990; Vijayalakshmi, 1994; Lipa, 1999 and Sreekanth, 2002) [18,33,13,32] were reared on detached cowpea/groundnut leaflets using the method (Fig. 2) developed by Amin et al. (1981) [5] . Small homeopathic glass vials were washed with water and sterilized at 160 °C for 1 h in hot air oven. After sex differentiation (Lewis, 1973) [12] , five females and two males were released gently into each glass vial, held in an inverted position. The thrips moved upward and gathered in the upper portion of the inverted vial. A young cowpea cv. C-152/ groundnut cv. JL-24 leaf let was then introduced and the vial was closed with cork. The vials were kept in an incubator adjusted to 12 h light period at 25 °C and 12 h dark period at 22 °C. After allowing a 24h oviposition access, thrips dislodged were collected into a separate vial. A fresh leaflet was introduced into the vial for further feeding and egg laying by thrips. This process was continued for about 10 days during which 90 per cent of total fecundity was completed. The leaflets with eggs were transferred to a new vial for the incubation of eggs. Thrips from these cultures were frequently released onto susceptible blackgram and greengram plants and the plants were assayed onto cowpea.

PBNV -Thrip transmission

PBNV infected portions of young leaflets of blackgram and greengram were separated and kept floating on water in separate Petri dishes and fifteen freshly emerged first instar larvae (Fig. 3), from the lab cultures of T. palmi, F. schultzei and S. dorsalis, were released separately onto these diseased leaflet portions with the help of a fine camel hairbrush and allowed for two days to acquire the virus. Then ten larvae from the infected source were transferred to individual glass vials each containing healthy blackgram/greengram leaflets. The vials were kept in an incubator (22-26 °C) for adult emergence. After adult emergence, ten thrips of each species were released per plant. A vial containing ten adult thrips of same species, which were given PBNV acquisition at larval stage, was fixed in such a way that the open end of the vial was touching the stem of the seedling. Since the thrips have the tendency of moving upwards, they move to the stem and then leaves. After releasing the thrips, they were restrained on the test plants by placing a cage over the plants (Fig. 4). The thrips were given inoculation access period (IAP) on the test plants for two days, then the insect cages were removed and sprayed with dimethoate @ 1.5ml/l to kill the insects and repeated at weekly intervals to keep the plants free from insect pests, particularly thrips. The seedlings were kept in greenhouse for a maximum period of 20 days to observe for symptoms and to record the frequency of transmission.

TSV -Thrip transmission

All the thrip species reared in the laboratory were used for the transmission of TSV viz., F. schultzei, S. dorsalis and T. palmi.

Collection of infective pollen from mechanically inoculated plants

Young parthenium plants collected from the field and tested negative to TSV by DAC-ELISA were transplanted in glasshouse. The transplanted healthy plants were inoculated by TSV-BG and TSV-GG isolates and subjected to DAC-ELISA seven days post inoculation. Pollen was collected from tested TSV positive plants and inoculated on cowpea cv. C-152 for the confirmation of presence of TSV in pollen and used as source of infective pollen for the transmission studies.

Pollen associated thrip transmission

Each ten plants (2 plants/pot) of susceptible checks (Blackgram cv. LBG-20 and greengram cv.K-851), groundnut cv. JL-24, sunflower cv. PAC-36 and cowpea cv. C-152 grown in glasshouse were used to perform thrip transmission. Infective pollen from parthenium plants was collected in a Petri plate by gently tapping the flowers. The infective pollen collected was dusted on the leaves of test plants by using a camel brush. Ten thrips of each species were released per plant. A vial containing ten adult thrips of same species was fixed in such a way that the open end of the vial was touching the stem of the seedling. Since the thrips have the tendency of moving upwards, they move to the stem and then leaves. After releasing the thrips, they were restrained on the test plants by placing a cage over the plants (Fig. 8). The thrips were allowed for feeding on the test plants for 24 hours, then the insect cages were removed and sprayed with dimethoate @ 1.5ml/l to kill the insects and repeated at weekly intervals to keep the plants free from insect pests, particularly thrips. Transmission experiments with TSV infective pollen were also carried out with mixed populations of thrip species. Control seedlings were exposed to pollen only or to thrips only, fed on infected leaves. The plants were observed for symptom development and indexed for TSV four to five days post-inoculation by both DAC-ELISA and transmission to cowpea cv.C-152.

Results

PBNV -Thrip transmission

Transmission tests with laboratory reared cultures of T. palmi, F. schultzei and S. dorsalis indicated that only T. palmi could transmit PBNV-BG and PBNV-GG to an extent 66.6% and 70% in blackgram cv. LBG-20 (Fig. 5), 50% and 66.6% in greengram cv. K-851 (Fig. 6) and 60% and 87.5% in cowpea cv. C-152 (Fig. 7), respectively. F. schultzei and S. dorsalis did not transmit the virus to the test plants (Table 2).

TSV -Thrip transmission

Pollen from both DAC-ELISA tested TSV positive parthenium plants, collected from field (natural infection) and glasshouse (mechanically inoculated with TSV-BG and TSV-GG) produced symptoms typical of TSV on assay host, cowpea cv.C-152 indicating the presence of the virus in the pollen. The results of the thrip assisted pollen transmission experiments with TSV infective pollen (Table 3) showed that TSV-BG, TSV-GG were transmitted by all the four thrip species tested. All 'thrips only' which were fed on TSV infected leaves and 'pollen only' control seedlings remained healthy. Varied percent transmission was observed in blackgram cv. LBG-20 (Fig. 8), greengram cv. K-851 (Fig. 9), cowpea cv.C-152 (Fig. 10), groundnut cv. JL-24 (Fig. 11), and sunflower cv.PAC-36 (Fig. 12) with TSV-BG and TSV-GG. All the four thrip species have showed transmissions to all test plants. All test plants that expressed symptoms were positive in DAC-ELISA tests confirming the transmission of TSV.

Discussion

PBNV -Thrip transmission

Different authors reported different thrips to be vectors of PBNV. Amin et al. (1978, 1981) [4][5] and Ghanekar et al. (1979) [8] reported S. dorsalis as the vector of the disease on groundnut, while Prasada Rao et al. (1980) [21] reported in tomato. Amin et al. (1981) [5] indicated that F. schultzei was responsible for the transmission. F. schultzei was reported as vector of TSWV (considered to be PBNV) in pea (Prasada Rao et al., 1985) [22] . Involvement of T. palmi as a vector of peanut bud necrosis disease (PBND) was also suspected by Palmer et al. (1990) [18] and later confirmed by Vijayalakshmi (1994) [33] . Findings of the present investigation provided unequivocal evidence that T. palmi, as the vector of PBNV on blackgram and greengram. It was also shown that F. schultzei and S. dorsalis failed to transmit the virus. This finding was in accordance with the reports PBNV transmission by T. palmi in groundnut (Vijayalakshmi, 1994; Lipa, 1999) [33,13] , in greengram Sreekanth (2002a) [32] and in blackgram (Rajkumar, 2004) [25] . Obviously, some degree of specificity existed with regard to transmission of viruses belonging to tospovirus group. Experimental evidence to this extent stems from the observations that TSWV was transmitted by F. schultzei (Sakimura, 1961; Amin, 1985a; Mulder et al., 1991) [29,1,15] and Peanut yellow spot virus (PYSY) by S. dorsalis but not by T. palmi (Cho et al., 1991; Mau et al., 1991) [7,14] . Under natural conditions, the stationary tendency of larval thrips, non-feeding pre-pupal and pupal stages were not likely to play a role in the spread of PBNV. Thus, the adult thrips only play major role in the spread of the virus (Sreekanth, 2002a) [32] .

TSV -Thrip transmission

In the present study, all the four thrips species viz., Frankliniella schultzei, Scirtothrips dorsalis, Megalurothrips usitatus and Thrips palmi transmitted TSV-BG and TSV-GG in presence of infected pollen. In laboratory tests, transmission was achieved with pollen from parthenium and sunflower deposited on leaves of blackgram cv. LBG-20, greengram cv. K-851, groundnut cv. JL-24 and cowpea cv.C-152 and sunflower cv.PAC-36 and colonized by any of the four vector thrip species. The results of the present study confirm the findings of the previous reports from India and elsewhere in the world, on the crucial role played by pollen in thrip transmission. Costa and Lima (1976) reported the transmission of TSV by thrips (Frankliniella sp.) from A. polystachya to tobacco and soybean in Brazil while, Kaiser et al. (1982) [10] reported the transmission of TSV by a mixture of thrips (T. tabaci and F. occidentals) from naturally infected M. alba to C. quinoa and M. alba in Washington, USA. Rana et al. (1987) [26] reported that C. quiona plants inoculated with the pollen collected from TSV infected Clematis vitalba (TSV-Cle) became infected and Sdoodee and Teakle (1988) [30] also reported that transmission of TSV by thrips might be through their wounding of plants during feeding and the presence of infected plant pollen in the vicinity rather than a specific virusvector interaction. In India, Prasada Rao et al. (2003b) [23] reported that more than one thrips species can act as a vector of TSV. Adults of all the four thrip species (Frankliniella schultzei, Scirtothrips dorsalis, Megalurothrips usitatus and Thrips palmi) were shown to transmit TSV in groundnut, sunflower and cowpea in the presence of pollen from TSV infected parthenium, sunflower or marigold. The thrips fed on infected leaves alone did not transmit the virus. As infective pollen is required for TSV transmission under field conditions it occur in two ways viz., pollen carrying thrips from flowers of TSV infected weeds or crop plants when colonize groundnut plants transmit the virus, and windblown pollen from TSV infected weeds or crop plants settle on groundnut plants and when these plants are colonized by thrips transmit the virus. Thrips, during their feeding, cause injury to both leaf tissue and deposited pollen and thus facilitate virus infection of the plant. The mechanism of TSV transmission by thrips is mechanical rather than a specific virus-vector interaction as observed in peanut bud necrosis tospovirus (Prasada Rao et al., 2003b, 2003c, 2005; Shukla et al., 2005) [23,24,20,31] . In the present study, thrips free of pollen did not transmit the virus confirming the findings by Sdoode and Teakle (1988) [30] , Greber et al. (1991) [9] , Reddy et al. (2002) [27] and Prasada Rao et al. (2003b, 2003c) [23,24] .

Table 1:

Identification of characters of different thrip species given by Amin and Palmer (1985) [1] , Palmer et al. (1989) [17] , Reddy et al. (1991a) [28] and Vijayalakshmi (1994) [33] . 0/9 0.00 0/10 0.00 a-1 st /2 nd instar larvae were given 2 days acquisition access period (AAP) and allowed for adult emergence in the vials incerted with healthy groundnut/cowpea leaf. b-Ten adults were released per seedling allowing for 2 days Inoculation access period (IAP).

Characteristics

References

1. Amin PW. Studies on arthropod vectors of groundnut viruses, their etiology and control. Progress Report for 1978-1983, ICRISAT; c1985. p. 166.
2. Amin PW, Palmer JM. Identification of groundnut Thysanoptera. Trop Pest Manag. 1985;31:286-91.
3. Amin PW, Ghanekar AM, Rajeswari R, Reddy DVR. Tomato spotted wilt virus as the causal pathogen of leaf curl of mungbean, Vigna radiata (L.) Wilczek and urdbean (Vigna mungo (L.) Hepper) in AP, India. Indian J Plant Protect. 1985;13:9-13.
4. Amin PW, Reddy DVR, Ghanekar AM. Transmission of bud necrosis virus (BNV) of groundnut by chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) (Abst.). Indian Phytopathol. 1978;31:118.
5. Amin PW, Reddy DVR, Ghanekar AM, Reddy MS. Transmission of Tomato spotted wilt virus, the causal agent of bud necrosis of peanut by Scirtothrips dorsalis and Frankliniella schultzei. Plant Dis. 1981;65:663-5.
6. Bhat AI, Jain RK, Kumar A, Ramiah M, Varma A. Serological and coat protein sequence studies suggest that necrosis disease on sunflower in India is caused by Tobacco streak virus. Arch Virol. 2002;147:651-8.
7. Cho JJ, Mau RFL, Ullman DE, Custer BM. Detection of Tomato spotted wilt virus (TSWV) within thrips. In: ARSUS Department of the USDA workshop "Virus-thrips-plant interactions of Tomato spotted wilt virus"; c1991 Apr 1819;
8. Ghanekar AM, Reddy DVR, Lizuka N, Amin PW, Gibbons RW. Bud necrosis of groundnut (Arachis hypogea (L.)) is caused by Tomato spotted wilt virus. Ann Appl Biol. 1979;93:173-9.
9. Greber RS, Klose MI, Teakle DS, Milne JR. High incidence of Tobacco streak virus in tobacco and its transmission by Microcephalothrips abdominalis and pollen from Ageratum houstonianum. Plant Dis. 1991;75:450-2.
10. Kaiser WJ, Wyatt SD, Pesho GR. Natural hosts and vectors of Tobacco streak virus in eastern Washington. Phytopathology. 1982;72:1508-12.
11. Ladhalakshmi D, Ramaiah M, Ganapathy T, Krishnareddy M, Khabbaj SE, Merin Babu, Kamalakannan A. First report of natural occurrence of Tobacco streak virus on blackgram (Vigna mungo). Plant Pathol. 2005;55:1395.
12. Lipa JJ. Analysis of risk caused by Thrip palmi to glass house plants in England, conclusions for Poland. Ochrona Roslin. 1999;43:25-6.
13. Mulder PG, Cole CL, Karner MA, Bolte R. Seasonal prevalence of the Thysanoptera in an Oklahoma peanut ecosystem and potential for Tomato spotted wilt virus. Southwestern Entomol. 1991;16:108-16.
14. Palmer JM, Reddy DVR, Wightman JA, Rao GVR. New information on the thrip vectors of Tomato spotted wilt virus in groundnut crops in India. Int Arachis Newslett. 1990;7:24-5.
15. Prasada Rao RDVJ, Jyothirmai Madhavi K, Varaprasad KS, Khetarpal R. Present status of stem necrosis disease of groundnut caused by Tobacco streak virus. Indian J Virol. 2005;16:66.
16. Prasada Rao RDVJ, Lijuka N, Raghunathan V, Joshi NC. Occurrence of Tomato spotted wilt virus on tomato in Andhra Pradesh. Indian Phytopathol. 1980;33:463-9.
17. Prasada Rao RDVJ, Rajeswari R, Rao MVS, Raghunathan V, Joshi NC. Spotted wilt of pea in India. Indian Phytopathol. 1985;38(1):90-3.
18. Rana GL, Krajacic M, Stefanac Z, Plese N, Rubino L, Milicic D. Properties of a new strain of Tobacco streak virus from Clematis vitalba (Ranunculaceae). Ann Appl Biol. 1987;111:153-60.
19. Reddy AS, Prasada Rao RDVJ, Thirumala Devi K, Reddy SV, Mayo MA, Roberts I, Satyanarayana T, et al. Occurrence of Tobacco streak virus on peanut (Arachis hypogea) in India. Plant Dis. 2002;86:173-8.
20. Sakimura K. Techniques for handling thrips in transmission experiments with the Tomato spotted wilt virus. Plant Dis Reporter. 1961;45:766-71.
21. Sdoodee R, Teakle DS. Transmission of Tobacco streak virus by Thrips tabaci, a new method of plant virus transmission. Plant Pathol. 1988;36:377-80.
22. Shukla S, Kalyani G, Kulkarni N, Waliyar F, Nigam SN. Mechanism of transmission of Tobacco streak virus by Scirtothrips dorsalis, Frankliniella schultzei, Megalurothrips usitatus in groundnut, Arachis hypogea L. J Oilseed Res. 2005;22:215-7.
23. Sreekanth M, Sreeramulu M, Prasada Rao RDVJ, Sarath Babu B, Ramesh Babu T. Effect of sowing date on Thrip Palmi Karny population and Peanut Bud Necrosis virus incidence in greengram (Vigna radiata (L.) Wilczek). Indian J Plant Protect. 2002;30:16-21.