Abstract
Of 70 plant species tested, 50 species were susceptible to Tobacco streak virus (TSV) on sap inoculation. Both localized (necrotic and chlorotic spots) and systemic (necrotic spots, axillary shoot proliferation, stunting, total necrosis and wilt) symptoms are observed by majority of plant species. Eleven new experimental hosts were identified viz., Amaranthus blitum var. oleracea (Chaulai sag), Celosia cristata (Cocks comb), Beta vulgaris var. bengalensis (Palak/Indian spinach), Calendula officinalis (Pot marigold), Chrysanthemum indicum, Cosmos sulphurens (Yellow cosmos), Citrullus lunatus (Watermelon), Lagenaria siceraria (Bottle gourd), Coriandrum sativum (Coriander), Hibiscus subderiffa var. subderiffa (Roselle) and Portulaca oleraceae (Little hogweed). Detected groundnut seed infection with TSV for the first time by Direct antigen coated immunosorbent assay (DAC-ELISA) using whole seed. The seed infection ranged from 18.9 to 28.9% among the seeds collected from naturally infected and sap inoculated groundnut varieties (JL 24, TMV 2, Prasuna, Kadiri 6, Kadiri 9, Anantha and Kadiri 7 Bold) belonging to spanish and virginia types. Further, TSV was detected both in pod shell and seed testa and none of the samples showed the presence of TSV either in cotyledon or embryo. Grow-out and bio-assay tests proved the absence of seed transmission in groundnut and other legume crops. Hence, TSV isolate was not a true seed transmission case under Indian conditions in legumes.
Keywords: Groundnut, Tobacco streak virus, Host range, Seed transmission
Introduction
Tobacco streak virus (TSV) belongs to the genus Ilarvirus of family Bromoviridae and its virions are characterized by quasi-isometric particles measuring about 27–35 nm in diameter [10]. The virus is distributed worldwide including India. Its epidemic on groundnut (Arachis hypogaea) crop was observed during the year 2000–2001 in parts of India, inflicting losses to the tune of 42 million pounds [29]. The virus has also been recorded on sunflower (Helianthus annuus) since 1997 in India, amounting to annual loss of 1.0 lakh pounds [28].
So far, natural occurrence of TSV in India has been reported from sunflower (H. annuus) [3, 27, 28], groundnut (A. hypogaea) [29], cotton (Gossypium hirsutum), sunn-hemp (Crotalaria juncea), mungbean (Vigna radiata) [3], okra (Abelmoschus esculentus), cucumber (Cucumis sativa), gherkin (C. anguria) [21, 22], safflower (Carthamus tinctorious) [4], chilli (Capsicum annuum) [15], urdbean (V. mungo) [23], nizer (Guizotia abyssinica) [1], soybean (Glycine max) [2]. In the similar manner, natural infection of TSV has been reported from other countries: horticultural crops: Black raspberry (Rubus occidentalis) [18], crane berry (Vaccinium macrocarpon) [17], dahlia (Dahlia variabilis) [24]; agricultural crops: Solanaceous crops: pepper (Capsicum spp.) [14], tobacco (Nicotiana tabacum) [11], Potato (Solanum tuberosum) [30], tomato (S. lycopersicum) [8], Legume crops: soybean (G. max) [13], cowpea (V. unguiculata), white clover (Melilotus alba) [19], groundnut (A. hypogaea) [7], Other crops: sunflower (H. annuus) [9, 31], cotton (G. hirsutum) [35].
So far, experimental host range of TSV was reported by authors from different countries [8, 12, 19, 25]. However, host range studies of groundnut TSV isolate was not studied extensively in India as it is a newly emerging virus among oil seed, vegetable and ornamental crops. Hence, present investigation on experimental host range was carried out by taking crop and weed species growing in/proximity of groundnut crop.
TSV transmission has been reported from seeds harvested from both naturally and sap inoculated plants in different countries. Thomas and Graham, 1951 [33] reported the natural seed transmission of TSV in pinto bean cultivars for the first time and subsequently from M. alba [19], Raphanus raphanistrum [8], Phaseolus vulgaris cv Black Turtle Soup bean [20]. The seed transmission has also been recorded from seeds harvested from sap inoculated plants, such as, black raspberry [6], G. max [13], Chenopodium quinoa, Gomphrena globosa, N. clevelandii, V. unguiculata (cv California Black Eye), G. max (cv Bragg) [31], V.unguiculata (cv Adzuki bean), P. vulgaris (cv Black Turtle Soup bean) [20], Frageria vesca [16] and Parthenium hysterophorus [32]. Seed transmission of TSV was not detected from any crop in India even though it was reported from 13 principle crops. So far, detailed study of TSV seed transmission was not carried out in legume crops in India.
Keeping in view of limited experimental host range study of TSV in India and its seed transmission in different crop and weed species in different countries, present investigation was undertaken to ascertain its experimental host range and seed transmission in groundnut and other legume crops using groundnut TSV isolate.
Materials and Methods
Virus Culture
From different parts of Andhra Pradesh, TSV infected groundnut leaf samples showing necrotic spots on young leaves were collected. The virus presence was confirmed by direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) [5] and then maintained on cowpea (cv. Pusa Komal) following three successful single lesion transfer by sap inoculations at National Phytotron Facility (NPF), Indian Agricultural Research Institute (IARI), New Delhi. This is the source of TSV inoculum used for both host range and seed transmission tests.
Host Range
Seventy plant species belonging to 18 families growing in and around groundnut crop were selected for host range studies (Table 1). Three replications were maintained for each plant species. In each replication, 3–5 seedlings were maintained per pot (12.5–22.5 cm) filled with autoclaved sterilized potting mixture containing river sand, clay and coconut peat (1:1:2). The experiment was carried out at NPF, IARI, New Delhi and maintained at temperature of 26–28°C.
Table 1.
Reaction of different plant species against Tobacco streak virus (TSV) by sap inoculation
| Test plant | Incubation period (DPI) | No of symptomatic plants/No of plants inoculated | Symptomsa | DAC-ELISAb A405nm (1 h) |
||
|---|---|---|---|---|---|---|
| Local | Systemic | Local (7 DAS) | Systemic (14-20 DAS) | |||
| Family: Fabaceae | ||||||
| Arachis hypogaea cv JL24(Groundnut) | 3 | (15/15) | 2 | 2, 7, 8, 9, 10, 11, 12, | 3.00 (0.09) | 3.0 (0.09) |
| Cajanus cajan cv. Pusa -2002-2 (Pigeon pea) | 5 | (15/15) | 1, 2, 4 | 2, 12 | 0.52 (0.09) | 2.53 (0.04) |
| Cicer arietinum (Chick pea) | 7 | (15/15) | 6 | 13 | 2.57 (0.03) | 0.07 (0.06) |
| Dolichos lablab cv. Pusa Early Prolific(Sem) | 5 | (13/15) | 1, 2 | 3 | 0.24 (0.07) | 0.01 (0.02) |
| Macrotyloma uniflorum (Horse gram) | 3 | (15/15) | 2, 4 | 11 | 3.00 (0.10) | 0.40 (0.08) |
| Glycine max cv. JS-335 (Soybean) | 6 | (15/15) | 1 | 4, 12 | 0.37 (0.04) | 2.60 (0.04) |
| Phaseolus vulgaris cv. Selection-9 (Kidney bean) | 2 | (15/15) | 2 | 5, 12 | 1.63 (0.13) | 0.10 (0.05) |
| Trigonella foenum-graecum cv Lam Selection 1 (Methi) | 5 | (15/15) | 1 | 8, 9, 11, 12 | 3.00 (0.13) | 3.00 (0.09) |
| Vigna mungo cv. T9 (Black gram) | 5 | (15/15) | 2 | 2, 8, 9, 11 | 1.25 (0.09) | 3.0 (0.09) |
| Vigna mungo cv. PDM-139 (Green gram) | 5 | (15/15) | 1, 2 | 2, 8, 9, 11 | 3.0 (0.13) | 3.0 (0.13) |
| Vigna unguiculata cv. Pusa Komal (Cowpea) | 2 | (15/15) | 2, 4 | 2, 10, 12 | 3.00 (0.12) | 3.00 (0.08) |
| Pisum sativam (Pea) | - | 0/15 | 13 | 13 | 0.06 (0.05 | 0.07 (0.04) |
| Cyamopsis tetragonoloba (Cluster bean) | - | 0/15 | 13 | 13 | 0.09 (0.05) | 0.08 (0.06) |
| Lens culinaris cv TL-O4 (Lentil) | - | 0/15 | 13 | 13 | 0.05 (0.05) | 0.06 (0.06) |
| Family: Compositae (Asteraceae) | ||||||
| Calendula officinalis (Pot marigold) | 7 | 15/15 | 2 | 8, 9, 11 | 0.93 (0.03) | 2.50 (0.19) |
| Carthamus tinctorius (Safflower) | 5 | 13/13 | 1, 2 | 8, 9, 11 | 3.0 (0.02) | 3.0 (0.04) |
| Chrysanthemum indicum | 7 | 13/15 | 1 | 8, 9, 11 | 1.50 (0.08) | 0.20 (0.04) |
| Cosmos sulphurens (Yellow cosmos) | 7 | 10/15 | 1, 2 | 8, 9, 11 | 1.54 (0.11) | 3.00 (0.11) |
| Guizotia abyssinica (Niger) | 4 | 15/15 | 1, 2 | 2, 11 | 0.76 (0.11) | 0.72 (0.08) |
| Helianthus annuus (Sunflower) | 3 | 13/15 | 2 | 5, 10, 11 | 3.00 (0.02) | 1.23 (0.01) |
| Lactuca sativa – (Letuce) | 7 | 15/15 | 1 | 3, 8, 11 | 3.00 (0. 02) | 3.0 (0.30) |
| Parthenium hysterophorus (Rag weed) | 7 | 8/8 | 13 | 13 | 1.20 (0.02) | 0.50 (0.03) |
| Tagetes erecta (Marigold) | 3 | 15/15 | 2 | 2, 8, 9, 10, 11, 12 | 1.33 (0.08) | 3.0 (0.14) |
| Tridax procumbence (Daisy) | 7 | 8/10 | 13 | 13 | 1.07 (0.20) | 0.35 (0.00) |
| Family: Solanaceae | ||||||
| Nicotiana benthamiana (Tobacco) | 7 | 11/12 | 2 | 8 | 3.00 (0.03) | 3.00 (0.05) |
| Nicotiana glutinosa (Tobacco) | 7 | 11/12 | 1, 3 | 13 | 3.00 (0.15) | 0.01 (0.0) |
| Nicotiana tabacum (Tobacco) | 7 | 10/12 | 2 | 13 | 3.00 (0.06) | 0.12 (0.03) |
| Petunia axillaries (White petunia) | 5 | 15/15 | 1,3 | 8, 9, 11 | 3.00 (0.08) | 3.00 (0.10) |
| Solanum melongena (Brinjal) | 7 | 13/15 | 2 | 13 | 3.00 (0.07) | 0.40 (0.04) |
| Capsicum annuum (Chilli) | - | 0/15 | 13 | 13 | 0.04 (0.03) | 0.05 (0.04) |
| Solanum lycopersicon (Tomato) | - | 0/15 | 13 | 13 | 0.09 (0.07) | 0.03 (0.04) |
| Family: Cucurbitaceae | ||||||
| Citrullus lunatus (Watermelon) | 7 | 8/12 | 13 | 13 | 0.42 (0.14) | 0.18 (0.15) |
| Cucumis melo (Muskmelon) | 14 | 8/12 | 13 | 13 | 0.38 (0.11) | 0.31 (0.11) |
| Cucumis melo var momordica (Kachri/Snap melon) | 5 | 10/12 | 2 | 5, 8, 12 | 0.80 (0.12) | 0.49 (0.13) |
| Cucumis melo var utilissimus (Kakri/Serpent melon) | 5 | 11/12 | 2 | 12 | 1.44 (0.12) | 0.48 (0.13) |
| Cucumis sativus (Cucumber) | 14 | 10/12 | 13 | 13 | 0.44 (0.12) | 0.27 (0.11) |
| Cucurbita maxima (Summer squash) | 7 | 10/12 | 1, 2 | 13 | 2.17 (0.13) | 0.20 (0.17) |
| Cucurbita moschata (Pumpkin) | 4 | 12/12 | 2 | 5 | 3.00 (0.13) | 0.28 (0.20) |
| Cucurbita pepo (Zucchini) (Yellow squash) | 4 | 11/12 | 1, 2 | 5 | 3.0 (0.16) | 0.14 (0.08) |
| Lagenaria siceraria (Bottle gourd) | 7 | 9/12 | 13 | 13 | 0.64 (0.11) | 0.38 (0.11) |
| Luffa acutangula (Ridge gourd) | 6 | 12/12 | 1, 2 | 5 | 0.89 (0.08) | 0.10 (0.10) |
| Citrullus vulgaris var fistulosus (Tinda/Indian squash) | - | 0/12 | 13 | 13 | 0.06 (0.09) | 0.09 (0.09) |
| Luffa cylindrica | - | 0/12 | 13 | 13 | 0.05 (0.05) | 0.04 (0.0.05) |
| Momordica charantia | - | 0/15 | 13 | 13 | 0.05 (0.06) | 0.06 (0.06) |
| Family: Malvaceae | ||||||
| Abelmoschus esculentus (Lady’s finger) | 4 | 15/15 | 4, 6 | 4, 5, 8, 11 | 3.00 (0.13) | 3.00 (0.15) |
| Gossypium herbaceum (Cotton) | 7 | 15/15 | 2 | 13 | 0.82 (0.13) | 0.30 (0.10) |
| Hibiscus subderiffa var. subderiffa (Roselle) | 5 | 15/15 | 2 | 13 | 1.79 (0.14) | 0.07 (0.20) |
| Family: Amaranthaceae | ||||||
| Amaranthus blitum var. oleraceae –Amaranth (Chaulai sag) | 7 | 10/15 | 2 | 13 | 0.51 (0.11) | 0.05 (0.04) |
| Amaranthus viridis (Pig weed) | 5 | 7/10 | 2 | 13 | 3.00 (0.10) | 0.39 (0.11) |
| Celosia cristata (Cocks comb) | 14 | 10/15 | 13 | 13 | 0.21 (0.10) | 0.14 (0.10) |
| Gomphrena globosa (Globe amaranth) | 3 | 15/15 | 1 | 8, 11 | 3.00 (0.10) | 0.13 (0.12) |
| Family: Chenopodiaceae | ||||||
| Beta vulgaris (Beet root) | 7 | 10/15 | 2 | 13 | 0.18 (0.06) | 0.06 (0.04) |
| Beta vulgaris var.bengalensis (Palak/Indian spinach) | 5 | 12/15 | 2 | 12 | 2.03 (0.12) | 0.13 (0.07) |
| Chenopodium amaranticolor | 4 | 8/8 | 1 | 1, 3, 8, 9, 11 | 3.0 (0.03) | 1.05 (0.21) |
| Family: Portulacaceae | ||||||
| Portulaca oleraceae (Little hogweed) | 7 | 15/15 | 1, 2 | 8, 9, 11 | 1.93 (0.20) | 0.29 (0.20) |
| Family: Aizoaceae | ||||||
| Trianthema portulacastrum (Giant pig weed) | 3 | 8/8 | 1, 2 | 1 | 3.0 (0.19) | 0.73 (0.18) |
| Family: Commelinaceae | ||||||
| Commelina benghalensis (Day flower) | 7 | 6/8 | 13 | 13 | 0.21 (0.03) | 0.21 (0.08) |
| Family: Umbelliferae (Apiaceae) | ||||||
| Coriandrum sativum (Coriander) | 7 | 13/15 | 13 | 13 | 0.98 (0.18) | 1.81 (0.14) |
| Daucus carota (Carrot) | – | 0/15 | 13 | 13 | 0.08 (0.06) | 0.07 (0.07) |
| Family: Crusiferae (Brassicaceae) | ||||||
| Brassica nigra (Black mustard) | – | 0/15 | 13 | 13 | 0.09 (0.05) | 0.07 (0.06) |
| Brassica oleraceae var.botrytis (Cauliflower) | – | 0/15 | 13 | 13 | 0.08 (0.03) | 0.07 (0.05) |
| Brassica oleraceae var.capitata (Cabbage) | – | 0/15 | 13 | 13 | 0.05 (0.05) | 0.06 (0.06) |
| Raphanus sativus (Radish) | – | 0/15 | 13 | 13 | 0.08 (0.05) | 0.08 (0.06) |
| Family: Alliaceae | ||||||
| Allium cepa (Onion) | – | 0/15 | 13 | 13 | 0.08 (0.05) | 0.08 (0.06) |
| Family: Polygonacea | ||||||
| Rumex vesicarius | – | 0/15 | 13 | 13 | 0.06 (0.04) | 0.09 (0.08) |
| Family: Pedaliaceae | ||||||
| Sesamum ndicum (Gingelly) | – | 0/15 | 13 | 13 | 0.06 (0.04) | 0.09 (0.08) |
| Family: Apocyanaceae | ||||||
| Vinca rosea | – | 0/15 | 13 | 13 | 0.08 (0.06) | 0.04 (0.07) |
| Family: Euphorbiaceae | ||||||
| Ricinus communis (Castor) | – | 0/15 | 13 | 13 | 0.06 (0.06) | 0.08 (0.07) |
| Family: Poaceae | ||||||
| Zea mays (Maize) | – | 0/15 | 13 | 13 | 0.05 (0.05) | 0.05 (0.06) |
| Pennisetum typhoides (Bajra) | – | 0/15 | 13 | 13 | 0.06 (0.06) | 0.04 (0.03) |
a1 Chlorotic spot, 2 Necrotic spot, 3 Veinal chlorosis, 4 Veinal necrosis, 5 Mosaic, 6 Leaf necrosis, 7 Bud necrosis, 8 Little leaf, 9 Axillary shoot proliferation, 10 Necrotic streaks on stem, 11 Stunting, 12 Wilting, 13 No symptoms, DPI Days post inoculation, b Values in parentheses were healthy values
Virus sap was prepared using TSV infected cowpea leaves (2–3 days post inoculation) (DPI) and ground (1:1 w/vol) with ice cold 0.01 M potassium phosphate buffer with pH 7.20 and containing 0.2% sodium sulfite and 0.01 M β mercapto ethanol using a chilled pestle and mortar. The virus sap prepared above was used for sap inoculation to the first true leaf of the seedlings by dusting Celite prior to inoculation. Virus sap was applied directly on to the upper surface of the leaves with the help of pestle. After inoculation, the seedlings were washed with tap water.
Inoculated seedlings were observed daily for symptom development and continued up to 4–6 weeks post inoculation. Biological parameters, such as, incubation period, per cent infection and symptoms were recorded for all plant species. Virus titer was also monitored by DAC-ELISA both in localized and systemic infection from 7 to 20 DPI.
Production of TSV Infected Groundnut Pods
Healthy seeds from seven popular groundnut varieties (JL 24, TMV 2, Prasuna, Kadiri 6, Kadiri 9, Anantha and Kadiri 7 Bold) were sown (4–5 seeds/pot) in plastic pots (22.5 cm). Seedlings were sap inoculated with TSV at 25, 30, 35 and 40 days after sowing (DAS) by maintaining 20 pots per age group per variety separately. Similarly, healthy controls (20 pots) were maintained for each age group of variety. Inoculation was made to the first true leaf of the seedlings. Carborundum (320 grit) was used as abrasive due to rough texture of groundnut leaf. The pods were harvested at maturity from both sap inoculated and healthy plants and stored up to 2 months at 4°C after proper drying for 10–15 days under shade to bring the moisture to 9.0%. Above seed material was used for detection of seed infection and seed transmission tests.
In the similar manner, the legume crops, namely, V. mungo (cv T9), V. radiata (cv PDM 139), G. max (cv JS 335), D. lablab (cv Pusa Early Prolific), P. vulgaris (cv Selection 9) and Trigonella foenum-graecum (cv Lam Selection1) were sown and TSV sap inoculated at 10–15 DAS. The collected seed material was used for detection of both seed infection and seed transmission of TSV.
Collection of Naturally TSV Infected Groundnut Pods
Three popular groundnut varieties (JL24, TMV2, Kadiri 6) were tagged prior to flowering under field conditions after confirmation by DAC-ELISA for TSV. Infected pods were collected from the above varieties at maturity stage and stored at 4°C up to 2 months after drying. The seed material thus collected was used to check the seed infection and seed transmission.
Detection of Seed Infection
Stored groundnut seed material collected from sap inoculated and naturally field infected plants of different varieties were used for testing the seed infection. Whole seed was used to detect the seed infection using DAC-ELISA test by maintaining negative controls using healthy seed collected from un inoculated plants. Similar procedure was followed for other legume crops tested.
Detection of TSV in Different Parts of Pod
Different parts of groundnut pod namely, pod shell, seed coat (testa), cotyledon and embryo were tested using seed material collected from sap inoculated plants under glasshouse conditions. Seeds were dissected after soaking in the sterile deionized water for overnight. Different parts of seed were separated with sterilized scalpel and used to detect the presence of virus by DAC-ELISA after washing under tap water for 3–5 min.
TSV Seed Transmission Tests
The seed material collected from sap inoculated seedlings (JL 24,TMV 2, Prasuna, Kadiri 6, Anantha) belonging to different age groups in each variety was bulked and used for grow-out test. Similarly, seed material collected from naturally infected groundnut varieties (JL 24, TMV 2, Kadiri 6) were also used. Ten to twenty pots were maintained for each variety along with their healthy controls. Seed material was sown in plastic pots (5–7 seeds/pot). The percent germination was recorded in each variety and seedlings grown were tested by DAC-ELISA.
The leaf samples collected randomly at 20, 40 and 70 DAS from grow-out test seedlings were bulked separately and used for bio-assay test by sap inoculating on cowpea (cv Pusa Komal) indicator host. Leaf samples collected randomly from sap inoculated cowpea plants were checked by DAC-ELISA. Followed similar method for other legume crops.
DAC-ELISA
DAC-ELISA, as described by Clark and Bar-Joseph (1984) was followed [5]. The samples (leaf/seed/seed parts) were ground in 0.1 M coating buffer (1:1 w/v; pH 9.2) containing 2% polyvinyl pyrollidone (PVP, MW 40,000). For each sample, two replications were maintained. Samples were loaded in ELISA plate (Costar, Sigma, USA) and kept at 37°C for 1 h incubation. Followed by three washings using Phosphate buffer saline (PBS) having 0.05% Tween -20. For blocking, 1% bovine serum albumin (BSA) was used. Later kept at 37°C for 1 h and washed three times as above. Crude antiserum developed against TSV at Unit of Virology, IARI, New Delhi was used after cross adsorption with healthy extract (1:20). Above crude antiserum was loaded after dilution (1:4000) in PBS containing tween-20 (0.05%), polyvinyl pyrollidone (2%) and egg ova albumin (0.2%) and kept at 37°C for 2 h followed by washings for three times. Goat anti-rabbit immunoglobulin conjugated to alkaline phosphatase (Sigma chemicals, St Louis, USA) was diluted in antibody diluent (PBS-Tween, polyvinyl pyrollidone ova albumin, PBS-TPO) used (1:30,000). Later kept at 37°C for 2 h and washed the plate three times. The substrate buffer (10% Diethanolamine) containing p-nitro phenyl phosphate (0.5 mg/ml) (Sigma, USA) was used. Absorbance was recorded at 405 nm after 1 h of incubation with a substrate at 37°C using a Teacan Sunrise (version 1.2) ELISA reader. The mean absorbance value of the two wells was compared with the absorbance values of healthy control. Absorbance values more than 2 times of healthy control are considered as positive for TSV.
Results
Host Range of TSV
Seventy plant species belonging to 18 families were tested by TSV sap inoculation and out of them 50 species belonging to Fabaceae (11), Asteraceae (10), Solanaceae (5), Cucurbitaceae (10), Malvaceae (3), Amaranthaceae (4), Chenopodiaceae (3), Umbelliferae (1), Aizoaceae (1), Commelinaceae (1), and Portulacaceae (1) were susceptible to TSV. The remaining 20 plant species, namely, Allium cepa (Alliaceae), Brassica nigra, B. oleraceae var. botrytis, B. oleraceae var. capitata, Raphanus sativus (Crusiferae), C. annuum, S. lycopersicon (Solanaceae), Citrullus vulgaris var fistulosus, Luffa cylindrica, Momordica charantia (Cucurbitaceae), Cyamopsis tetragonoloba, Lens culinaris (cv TL-O4), Pisum sativam (Fabaceae), Rumex vesicarius (Polygonaceae), Sesamum indicum (Pedaliaceae), Daucus carota (Umbelliferae), Vinca rosea (Apocyanaceae), Ricinus communis (Euphorbiaceae) Pennisetum typhoides, and Zea mays (Poaceae) were insusceptible to TSV as they neither exhibited clear symptoms nor supported virus multiplication.
Necrotic and chlorotic spots as localized symptoms and necrotic spots, axillary shoot proliferation, stunting, total necrosis and wilt of plant as systemic symptoms were observed in majority of plant species, such as, A. esculentus, A. hypogaea, Calendula officinalis, C. tinctorius,C. amaranticolor, Cosmos sulphurens, Macrotyloma uniflorum, G. max, G. globosa, G. abyssinica, H. annuus, Petunia axillaries, P. vulgaris, Portulaca oleraceae, Tagetes erecta, T. foenum-graecum, V. mungo and V. unguiculata. Among different family members tested, majority of cucurbitaceous members did not show systemic infection. Some of the susceptible plant species belonging to different families namely, Celosia cristata, C. benghalensis, C. lunatus, C. melo, C. sativus, Coriandrum sativum, L. siceraria, P. hysterophorus and Tridax procumbence reacted symptomlessly and are likely to serve as symptomless carriers of TSV (Table 1).
The new experimental hosts identified were Amaranthus blitum var. oleracea (Chaulai sag), C. cristata (Cocks comb) (Amaranthaceae), Beta vulgaris var. bengalensis (Palak/Indian spinach) (Chenopodiaceae), C. officinalis (Pot marigold), Chrysanthemum indicum, C. sulphurens (Yellow cosmos) (Asteraceae), C. lunatus (Watermelon), L. siceraria (Bottle gourd) (Cucurbitaceae), C. sativum (Coriander) (Umbelliferae), Hibiscus subderiffa var. subderiffa (Roselle) (Malvaceae) and Portulaca oleraceae (Little hogweed) (Portulacaceae) (Table 1; Fig. 1).
Fig. 1.
New experimental hosts of Tobacco streak virus:a–c: Amarantus blitum var. oleracea (Chaulai sag) (a healthy, b necrotic spots, c Symptom less); d–fBeta vulgaris var bengalensis (Palak) (d healthy, e necrotic spots, f wilting); g–iCalendula officinalis (g healthy, h Necrotic spots, i auxillary shoots proliferation); j–lChrysanthemun indicum (j healthy, k chlorotic spots, l auxillary shoots proliferation) m–oCosmos sulphurenes (m healthy, n chlorotic and necrotic spots, o auxillary shoots proliferation); p–r: Hibiscus subderiffa var. subderiffa (p healthy, q necrotic spots, r Symptom less); s–uPortulaca oleraceae (s healthy, t chlorotic spots, b auxillary shoots proliferation)
Production of TSV Infected Pods and Symptoms
Produced both localized and systemic symptoms upon sap inoculation with TSV to the different aged group of groundnut seedlings (25, 30, 35, 40 DAS) belonging to different varieties. Localized symptoms were observed at 1–2 DPI. Initial symptoms included pale necrotic spots which turned to necrotic spots within a day followed by yellowing and wilting of inoculated leaf. Systemic symptoms included necrotic spots on young leaves, necrosis of top growing bud, necrotic streaks on stem, axillary shoot proliferation, peg necrosis, reduction of pod and kernel size, necrotic streaks on pod and discoloration of seed. Younger age group of seedlings (25 DAS) in all the varieties of groundnut tested were severely infected and majority of them produced small size pods with shriveled kernels. Localized and systemic symptoms observed were similar among different varieties of groundnut tested.
Detection of Seed Infection
Of 505 seeds collected from different groundnut varieties sap inoculated with TSV under glasshouse conditions, 130 seeds showed TSV seed infection in DAC-ELISA test. Maximum seed infection observed in cultivar TMV 2 (28.9%) followed by Prasuna, JL 24 (26.5%), Kadiri 9 (25.0%), Anantha (23.4%), Kadiri 6 (22.2%) and Kadiri 7 Bold (18.9%). Virus titer ranged from 0.20 to 3.00 (A405nm, 1 h) in all the varieties tested. Of 407 seeds collected from naturally infected groundnut cultivars, 84 seeds were positive for TSV seed infection. Maximum seed infection observed with the variety JL 24 (21.9%), followed by TMV 2 (20.9%) and Kadiri 6 (19.1%). The virus titer ranged from 0.23 to 1.80 (A405nm, 1 h) in all the varieties tested. The absorbance values (A405nm, 1 h) of 400 healthy seeds collected from glasshouse and field samples belonging to different varieties ranged from 0.04 to 0.09 in DAC-ELISA (Table 2).
Table 2.
Detection of seed infection of Tobacco streak virus in seeds collected from sap inoculated and naturally infected groundnut varieties by DAC-ELISA
| Method of seed collection | Variety | Seed infection | A405nm (1 h)b (Range) | |
|---|---|---|---|---|
| Noa. | Percent | |||
| I. Sap inoculated | JL-24 | 40/151 | 26.5 | 0.20–3.00 (0.04–0.09) |
| TMV2 | 42/145 | 28.9 | 0.22–2.00 (0.03–0.05) | |
| Prasuna | 9/34 | 26.5 | 0.23–1.50 (0.04–0.09) | |
| Kadiri 6 | 14/63 | 22.2 | 0.20–1.70 (0.02–0.06) | |
| Kadiri 9 | 7/28 | 25.0 | 0.31–0.50 (0.06–0.08) | |
| Anantha | 11/47 | 23.4 | 0.48–3.00 (0.03–0.09) | |
| Kadiri 7 Bold | 7/37 | 18.9 | 0.55–1.37 (0.03–0.05) | |
| Total | 130/505 | 25.7 | ||
| II. Naturally infected | JL-24 | 27/123 | 21.9 | 0.23–1.80 (0.04–0.06) |
| TMV2 | 31/148 | 20.9 | 0.71–1.51 (0.02–0.06) | |
| Kadiri 6 | 26/136 | 19.1 | 0.23–1.20 (0.04–0.06) | |
| Total | 84/407 | 20.6 | ||
a Number of seeds positive in DAC-ELISA/total number of seeds tested
b DAC-ELISA absorbance values and figures in parentheses were healthy values
Detection of TSV in Different Parts of Pod
To know the virus distribution in different parts of seeds, 460 pods were collected from seven TSV sap inoculated groundnut varieties. TSV was detected both in pod shell and seed coat (testa). In DAC-ELISA, absorbance values (A405nm, 1 h) ranged from 0.38 to 3.00 and 0.19 to 1.81 in pod shell and seed coat, respectively. No virus was detected in either cotyledons or embryos obtained from infected seeds from all the varieties tested. The absorbance values (A405nm, 1 h) of different healthy parts of pod (225) collected from different groundnut varieties ranged from 0.04 to 0.09 in DAC-ELISA (Table 3).
Table 3.
Detection of Tobacco streak virus by DAC- ELISA in different parts of groundnut pod collected from TSV sap inoculated groundnut varieties
| Variety | No. of samples tested | DAC-ELISA (A405nm, 1 h)-#Range | |||
|---|---|---|---|---|---|
| Pod shell | Seed coat (Testa) | Cotyledon | Embryo | ||
| JL 24 | 86 | 0.76–3.00 (0.02–0.09) | 0.19–1.72 (0.04–0.05) | 0.04–0.05 (0.03–0.05) | 0.04–0.05 (0.03–0.06) |
| Prasuna | 45 | 0.89–3.00 (0.03–0.08) | 0.20–1.40 (0.02–0.06) | 0.03–0 .04 (0.03–0.05) | 0.04–0.06 (0.03–0.06) |
| TMV2 | 73 | 0.38–2.50 (0.04–0.09) | 0.23–1.20 (0.03–0.06) | 0.08–0.09 (0.04–0.06) | 0.08–0.10 (0.05–0.08) |
| Kadiri 6 | 80 | 1.80–2.26 (0.03–0.07) | 1.20–1.81 (0.02–0.06) | 0.03–0.05 (0.04–0.05) | 0.02–0.05 (0.02–0.05) |
| Anantha | 48 | 0.85–2.10 (0.02–0.05) | 0.69–0.91 (0.02–0.07) | 0.05–0.09 (0.03–0.08) | 0.05–0.09 (0.04–0.09) |
| Kadiri 9 | 87 | 0.65–1.30 (0.03–0.08) | 0.71–0.91 (0.03–0.08) | 0.04–0.08 (0.04–0.08) | 0.05–0.09 (0.04–0.07) |
| Kadiri 7 Bold | 41 | 0.81–1.21 (0.02–0.05) | 0.69–1.12 (0.03–0.07) | 0.04–0.05 (0.04–0.08) | 0.05–0.06 (0.04–0.05) |
| Total | 460 | ||||
# Figures in parentheses were healthy values
TSV Seed Transmission
Grow-out test was conducted using pods harvested from TSV sap inoculated (275 seeds) and naturally TSV infected (132 seeds) plants. The seed germination ranged from 88.0 to 91.2% against 100% germination using 407 healthy seeds collected from glasshouse and field from different varieties. None of the seedlings raised from infected seeds showed symptoms of TSV and were ELISA negative. Bio-assay test using cowpea (cv Pusa Komal) indicator host did not show any TSV symptoms by sap inoculation and were ELISA negative. The absorbance values (A405nm, 1 h) of seedlings in grow-out and bio-assay tests were similar to healthy controls in DAC-ELISA (Table 4).
Table 4.
Detection of Tobacco streak virus by grow-out and bio-assay tests with seedlings raised from virus infected seeds collected from sap inoculated and naturally infected groundnut varieties
| Method of seed collection | Variety | Grow-out tests | Bio-assay tests | |||
|---|---|---|---|---|---|---|
| No.a | Germination Percent | A405nm (1 h)b (Range) | No.c | A405nm (1 h)b (Range) | ||
| I. Seeds collected from sap inoculated plants | JL-24 | 71/80 | 88.8 | 0.03–0.08 (0.02–0.05) | 0/20 | 0.05–0.09 (0.04–0.05) |
| TMV2 | 73/80 | 91.2 | 0.05–0.08 (0.04–0.09) | 0/20 | 0.03–0 .05 (0.04–0.06) | |
| Prasuna | 32/37 | 86.5 | 0.06–0.11 (0.05–0.08) | 0/10 | 0.05–0.16 (0.03–0.09) | |
| Kadiri 6 | 40/45 | 88.9 | 0.05–0.06 (0.03–0.05) | 0/20 | 0.04–0.08 (0.03–0.06) | |
| Anantha | 29/33 | 87.9 | 0.05–0.09 (0.03–0.06) | 0/13 | 0.05–0.11 (0.03–0.07) | |
| II. Naturally infected seeds | JL-24 | 40/45 | 88.9 | 0.02–0.07 (0.02–0.04) | 0/20 | 0.04–0.08 (0.03–0.05) |
| TMV2 | 41/45 | 91.1 | 0.03–0 .07 (0.02–0.07) | 0/23 | 0.05–0 .08 (0.03–0.07) | |
| Kadiri 6 | 37/42 | 88.0 | 0.05–0.09 (0.03–0.05) | 0/13 | 0.05–0.12 (0.02–0.08) | |
a Number of seeds germinated/total number of infected seeds sown
b DAC-ELISA absorbance values and figures in parentheses were healthy values
c Number of samples positive by indexing on cowpea/total number of samples
Detection of Seed Infection and Seed Transmission in Legumes
Of 59 seeds of V. mungo (cv T9) tested, 15 seeds (25.0%) were positive for seed infection by DAC-ELISA and absorbance values ranged from 0.21 to 0.28 (A405nm, 1 h). In the similar way, of 47 seeds tested, 11 seeds (23%) were infected in P. vulgaris (cv Selection 9). From 45 seeds tested, 9 seeds were positive (20.0%) in the crop T. foenum- graecum (cv. Lam Selection 1). No seed infection was detected in other legume crops, namely, V. radiata (cv PDM 139), G. max (cv JS335), D. lablab (cv. Pusa Early prolific). Absorbance values (A405nm, 1 h) ranged from 0.02 to 0.08 using 60 healthy seeds belonging to different varieties. In grow-out tests, none of the seedlings raised from infected seed of different legumes showed symptoms of TSV and they were ELISA negative as absorbance values were similar to healthy seedlings. Bio-assay test also showed none of the TSV symptoms by sap inoculation on cowpea (cv Pusa Komal) and reacted negatively in DAC- ELISA (Table 5).
Table 5.
Detection of seed infection of Tobacco streak virus in seeds of legume crop produced by sap inoculation with TSV and confirmation of seed transmission by grow-out and bio-assay tests
| Crop | Seed infection | Grow-out test | Bio-assay test | ||||
|---|---|---|---|---|---|---|---|
| No.a | Percent | A405nmb (Range)-1 h | No.c | A405nm (1 h)b | No.d | A405nmb (1 h) | |
| Vigna mungo cv T9 (Black gram) | 15/59 | 25.0 | 0.21–0.28 (0.02–0.04) | 0/35 | 0.19–0.20 (0.02–0.10) | 0/20 | 0.06–0.09 (0.02–0.05) |
| Vigna radiata cv PDM 139 (Green gram) | 0/62 | 0.00 | 0.04–0.08 (0.03–0.05) | 0/42 | 0.03–0.08 (0.03–0.05) | 0/24 | 0.04–0.06 (0.03–0.04) |
| Glycine max cv JS335 (Soybean) | 0/51 | 0.00 | 0.02–0 .08 (0.02–0.06) | 0/37 | 0.02–0 .08 (0.02–0.05) | 0/15 | 0.06–0.11 (0.04–0.09) |
| Dolichos lablab cv Pusa early prolific (Sem) | 0/42 | 0.00 | 0.03–0.11 (0.02–0.08) | 0/38 | 0.06–0.12 (0.04–0.08) | 0/12 | 0.05–0.08 (0.02–0.08) |
| Phaseolus vulgaris cv Selection 9 (French bean) | 11/47 | 23.0 | 0.22–0.33 (0.03–0.06) | 0/39 | 0.05–0.08 (0.03–0.06) | 0/15 | 0.06–0.10 (0.04–0.06) |
| Trigonella foenum- graecum cv Lam selection 1 (Methi) | 9/45 | 20.0 | 0.13–0.18 (0.03–0.06) | 0/31 | 0.05–0.11 (0.03–0.07) | 0/18 | 0.04–0.10 (0.03–0.07) |
a Number of seeds positive in DAC-ELISA/total seeds
b Range of absorbance values (A405nm) in DAC-ELISA and figures in parentheses were healthy control values
c Number of seedlings positive in DAC-ELISA/total number of seedlings
d Number of samples positive by indexing on cowpea/total number of samples
Discussion
Present study on the experimental host range of groundnut TSV isolate was led to the identification of 11 new experimental hosts in addition to presently known host range of TSV from different countries [8, 12, 19, 25]. Among different plant species tested, the hosts, such as, cowpea (V. unguiculata), groundnut (A. hypogaea), horse gram (M. uniflorum), methi (T. foenum- graecum), green gram (V. radiata) (Fabaceae), sunflower (H. annuus), safflower (C. tinctorius), marigold (T. erecta), lettuce (Lactuca sativa), cosmos (C. sulphurens), tobacco (N. benthamiana), white petunia (P. axillaries) and okra (A. esculentus) were supported high virus titer both in localized and systemic infection and could be used as excellent propagating hosts for virus maintenance. Similarly, V. unguiculata, M. uniflorum, C. amaranticolor, N. tabacum, p. axillaries and G. globosa were identified as excellent local lesion assay hosts. Of 70 plant species tested, 20 plant species were insusceptible to TSV. However, some of the insusceptible hosts identified in the present study such as, B. campestris, P. sativam, L. culinaris, C. tetragonoloba, S. lycopersicum and V. rosea were reported as well known experimental hosts for TSV [8, 12, 19]. This discrepancy may be due to the difference in the TSV isolates used. This study clearly indicates that isolate specific experimental host range of TSV can’t be ruled out.
In the present study, seed infection was recorded both in sap inoculated and naturally infected seeds from different varieties of groundnut tested (Table 2). This is the first report of seed infection of TSV in groundnut crop under natural and glasshouse conditions. Further, to ascertain exact location of TSV, the virus was checked in different parts, such as, pod shell, seed testa, cotyledon and embryo using seeds collected under glasshouse experiment from different varieties of groundnut. The virus was detected in pod shell and seed testa only but not in either cotyledon or embryo (Table 3). In the similar manner, none of the cotyledons and embryos showed presence of TSV in naturally infected groundnut seeds (data not showed). Hence, present study clearly suggests that TSV restricts to seed testa in groundnut kernel even though TSV was sap inoculated at flowering period. Further tests are needed to ascertain barriers which prevent entry of virus into the cotyledons and embryo of kernel.
To ascertain seed transmission of TSV in groundnut, grow-out test was conducted using seeds collected from ELISA positive sap-inoculated and naturally infected plants separately. Germination varied from 88.0 to 91.2% and none of the seedlings showed symptoms of TSV and were ELISA negative. The loss of germination per cent might be due to non viability of severely infected seeds (Table 4). In grow-out tests, some of groundnut seedlings showed mild chlorotic symptoms immediately after germination. However, plants recovered well in a short span of time and produced normal flowers and pods. Bio-assay tests using cowpea (cv Pusa Komal) as indicator host were also negative. The present study suggests that TSV fails to enter into seedlings raised from infected seeds, in spite of presence of virus in the seed coat. Hence, it is concluded that seed transmission is negative in groundnut host. In the similar manner, we are reporting TSV seed infection for the first time in the seeds collected from V. mungo (cv T9) and T. foenum-graecum (cv Lam Selection1) under glasshouse experiments. However, seed transmission tests were negative in all the legume crops tested based on the limited studies conducted by sap inoculation (Table 5).
Seed transmission was very well proved in different TSV isolates worldwide since seed transmission nature of TSV has been documented both in naturally infected and artificially sap inoculated plants belonging to different families. The percent seed transmission rate was less in naturally infected plants when compared to artificially sap inoculated plants. Ghanekar and Schwenk, [13] revealed that seed transmission was obtained with soybean TSV isolate (Glycine max) but not with tobacco TSV isolate. In the similar manner, Walter et al. [34] observed highly seed transmitting (Mel 40) and non or less seed transmitting (Mel F) strains of TSV infecting M. alba (Biennial wild legume). Hence, existence of seed and non-seed transmissible strains of TSV might be the reason for presence or absence of seed transmission.
So far, TSV seed transmission was not proved from any crop or weed hosts in India. Hence, it is concluded that non-seed transmissible strain of TSV might be existing in India. Absence of seed transmission of TSV was also observed by Prasada Rao et al. [26] in crop and weed hosts belonging to Fabaceae and Asteraceae family members in India. Moreover, complete genome sequence is lacking for Indian TSV isolate and comparison has to be made with seed transmissible strain of TSV from different countries as Walter et al. [34] observed extra minor RNAs in non- seed transmissible strain (Mel F) of TSV. So, differentiation has to be made between seed and non seed transmissible strains of TSV with respect to their host range, physiochemical properties and genetic diversity.
Acknowledgments
Greatly indebted to the Indian Agricultural Research Institute, New Delhi and Acharya N.G. Ranga Agricultural University, Andhra Pradesh for financial support and to Dr. R.D.V.J Prasada Rao, Principal Scientist and Dr. M. Johnson, Senior Scientist for their generous help. I thank to Dr. K. Sankar Reddy, Associate Director of Research, and Dr. D. Lokanadha Reddy, Principal Scientist (Groundnut) and Head for critical reading of the manuscript and encouragement.
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