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Groundnut necrosis disease caused by a strain of Groundnut bud necrosis virus
U.P. GUPTA AND LOKESH KUMAR SHUKLA,
Departement of Botany, Harischandra P.G. College, Varanasi 221001, India
ABSTRACT:
Tospovirus infection on Groundnut ( up to 70%) was noticed at Indian Agricultural Research Institute experimental farm, New Delhi in different varietal trials. Symptoms under field conditions included necrosis of plant parts such as leaves, stems. petioles, growing points. buds and pods. Symptomatic groundnut plants showed positive reaction with Groundnut bud necrosis virus (GBNV) and antisera in direct antigen-coated enzyme-linked immunosorbent assay. Groundnut Tospovirus was easily transmitted to Groundnut cv. A.hu , Which could be used as a diagnostic host. The virus was also mechanically transmitted to different plant species belonging to families Fabaceae , Cucurbitaceae. and Solanaceae. The nucleocapsid protein (N) gene of the virus was cloned and sequenced (GenBank Accession number AF 5158)? The sequenced region contained an ORF of 831 nucleotides that could potentially code for N protein of 276 amino acids. Comparative sequence analyses levels respectively, suggesting the Tospovirus isolate from Groundnut to be a strain of (GBNV).
Key words:
Tospovirus, Groundnut , nucleocapsid protein gene, Groundnut bud necrosis virus.
Low productivity of Groundnut (0425 ton per hactore) (Asthana and Chaturvedi, 1999) in the country could e attributed to biotic stresses including viruses. Among viruses (Brunt et al., 1996), Tospoviruses have been recognized as emerging pathogens that can cause significant yields reduction in different crops (Bhat et al., 2001, 2002).
Natural infection of Tospovirus on Groundnut A.H. Wilczek) and other legumes such as Groundnut (Arachis hypogaea (L.) Wilczek) and soybean (Glycine max (L.) Merr.) was recently recorded from Delhi (Bhat et al., 2001). Groundnut Tospovirus was serologically related to Groundnut bud necrosis (GBNV) viruses in direct antigen-coated enzyme linked immunosorbent assay. However, the exact taxonomic status of the virus remained unaddressed. The present study was thus undertaken to evaluate whether Groundnut Tospovirus isolate is a strain of GBNV or a distinct virus species.
MATERIALS AND METHODS
Virus isolate
Groundnut fields at Indian Agricultural Research Institute (IARI) experimental farm, New Delhi were visited at different stages of crop growth for Tospovirus infection and disease incidence was recorded in Initial Varietal (IVT). Advanced Varietal (AVT) and Released Varieties (RVT) trials. Symptomatic Groundnut plants showing leaf chlorosis/necrosis, stem necrosis and bud necrosis were collected and were subjected to bio- and immuno-assays.
The virus was rub-inoculated on to Groundnut at primary leaf stage in a glasshouse using sterilized and chilled pestle and mortar and 0.1 M phosphate buffer (PH 7.2, w/v) containing 0.1% B-mecaptoethanol. For immunoassay. standard direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELiSA) was performed follosing the procedure of Clark and Bar-Joseph (1984). Polyclonal antisera directed against nucleocapsid (N) protein of GBNV.
HOST RANGE
The test plant species belonging to six families were used for host range studies (Table 1). Seedlings of each test plant species were raised in earthen pots (5 per pot) and were rub-inoculated at 2-3 leaf stage. The inoculated seedlings were observed for symptom development up to for 4-6 weeks and also tested for the presence of virus by DAC-ELISA.
RNA isolation
Total RNA from the infected tissues (ca.10mg ) was extracted using RNeasy Kit (Qiagen Inc., Chatsworth. CA, USA) according to the manufacturer’s instructions and was used as a template in the reverse transcription and polymerase chain reaction (RT-PCR) .
PCR amplification
Reverse transcription and amplification (RT-PCR) were performed based on the procedure described by Pappu and his associates (1993), The template was first incubated at 76˚c for 5 min and snap-cooled on wet ice for 2 min. A set of specific primers derived from the first and last 21 bases of the coding region of the N gene of GBNV (Satyanarayana et al. 1996) used to prime the amplification RT-PCR was performed in a single tube in an automated thermal cycle. (Power Block II, Ericomp Inc., San Diego, CA, USA) programmed for one cycle of 42˚c for 45 min for cDNA synthesis and 40 cycles of amplification with 30s of denaturation at 94˚c, 1 min of annealing at 56˚cand 1 min of extension at 72˚c followed by one cycle of final extension for 60 min at 72˚c. Following PRC, amplicons were analyzed by 1% agarose gel electrophoresis in Tris-acetate EDTA (TAE) containing ethidium bromide (Sambroook and Russell. 2001), The gel was observe under ultraviolet trans-illuminator and photographed on the thermal paper
CLONING AND SEQUENCING
The PCR products were ligated into pGEM-T Easy vector (Promega, Madison, WI, USA) and competent Escherichia coli cells (strain DH 5α) were transformed by following standard molecular biology procedures (Sambrook and Russell, 2001). Selected recombinant clones with an insert of N gene(~ 800bp) of Groundnut Tospovirus isolate were sequenced at the automatic DNA sequencing facility at Department of Biochemistry, South Campus, University of Delhi, Delhi, India. Sequence data were initially compiled using SeqAid II ™ version 3.60(Rhoads and Roufa, 1985). Multiple sequence alignments were generate using CLUSTAL W (Thompson et al. , 1994). Sequnce phylograms were constructed using TREEVIEW software (Bootstap analysis with 1000 replicates) (Page, 1996) . N gene sequences of other known Tospoviruses (Table 2 were collected from GenBank (Benson et al. 1999) Both nucleotide and amino acid sequences of N gene of different Tospovirus species were compared and the corresponding phylogenetic trees were generated.
RESULTS AND DISCUSSION
Under field conditions, symptoms on groundnut included necrosis of all plant parts including leaves, stems, petioles, growing points, buds and pods. Early infected plants were severely stunted with reduced internodal length and many axillary shoots. Maximum Tospovirus infection was observed in RVT followed by AVT and IVT. In RVT, disease incidence was maximum in Pusa 2072 ( 71%) , followed by Pusa Bold (63%) , Pusa 105 (46%) , and Pusa Vishal(20%) . Incidence of Tospovirus infection in IVT and AVT ranged from 14-38% and 19-44% respectively.
The Tospovirus from symptomatic groundnut plans was mechanically transmitted onto Groundnut diagnostic assay host for Tospovirus (Bhat et al. 2001) Both localized as well as systemic infections on groundnut were observed localized infection, inoculated leaves showed chlorotic lesions three days after inoculation, which turned necrotic tow days later, This was followed by veinal necrosis of the inoculated leaves, which finally became chlorotic or pale yellow in color before senescence. Newly emerging leaves showed systemic infection symptoms which consisted mild mosaic, concentric chlorotic ringspots and necrotic spots. Necrosis affected plants reacted with the polyclonal antiserum directed against nucleocapsid protein of GBNV (A 405 = 0.75) , suggesting that the groundnut Tospovirus.
Table 1. Host Range of Groundnut Tospovirus isolate.
|
Plant species
|
No. Plants infected/ inoculated
|
Observation
|
|
Visual*
|
Serological (A405 nm)**
|
|
Local
|
Systemic
|
|
Fabaceae
Arachis hypogaea (Groundnut)
Cajanus cajan (Redgram)
Cicer arietinum(Chickpea)
Glycine max (Soyabean)
Phaseolus vulgaris (French bean)
Pisum sativum (Pea)
Vigna radiate (Mungbean)
V.Unguiculata (Cowpea)
Cucurbitaceae
Cirulus vulgaris (Tinda)
Cucumis melo (Musk Melon)
C. sativus (Cucumber)
Lagenaria Siceraria (Bottle gourd)
Solanaceae
Nicotiana benthamiana
Physalis floridana (Physalis)
Solanum tuberosum (Potato)
|
3/9
6/20
14/15
3/10
4/10
5/17
14/15
19/19
6/10
0/10
5/11
2/10
7/8
7/7
0/7
|
1,2,3
1,
1,2
1
1,2
1,2
1,2
1,2,3
1,2,3
-
1,2,3
1,
1,2,3
1,2,3
-
|
4,7,8,10
-
7,8,10
-
4
7,8,10
5,6,7,8,9,10
4,5,6,7,8,9,10
-
-
-
-
4,5,6,7,10
4,5,6,7,10
-
|
0.65(0.26)
0.51(0.12)
0.37(0.18)
0.53(0.14)
0.57(0.20)
2.29(0.29)
1.09(0.21)
0.53(0.11)
0.26(0.13)
0.20(0.11)
0.22(0.11)
0.38(0.19)
0.71(0.03)
0.41(0.00)
0.18(0.17)
|
The virus was easily sap-transmissible to the members of Fabaceae and Solancaceae and both localized and systemic infection was observed. Symptoms exhibited by different hosts included chlorotic/necrotic lesions and ringspots, followed by leaf yellowing, veinal necrosis, leaf deformation Other symptoms were strunting, bud necrosis, stem necrosis And wilting Only localized infection ( Chlorosis, necrosis, ringspot)was observed in plant species belonging to Cucurbitaceae, Malvacaea and Compositae (Table 1) . ELISA reactions were consistent with visual observation. Intensity of ELISA reactions varied with plant species. Members of Fabaceae and Solanaceae showe higher absorbance values (0.37-2.29). Whereas those of Cucurbitaceae, compositae and Malvaceae showed lower absorbance values (0.22 0.38) These hot species could serve as potential reservoirs for groundnut Tospovirus. Host range studies suggests that the Groundnut Tospovirus was closely related to GBNV .
The nucleotide and the translated amino acid sequences of the N gene of the Groundnut Tospovirus revealed that the sequenced region contained a single open reading frame (ORF) of 831 nucleotides that could potentially code for a protein of 276 amino acids. The nucleotide sequence data were submitted to the GenBank under accession number AF515819.
The N gene sequence of groundnut Tospovirus isolate was compared with corresponding genes from other recognized Tospovirus species at nucleotide and amino acid levels. Cluster dendrograms revealed that Groundnut Tospovirus isolate was most closely related to GBNV. forming one cluster Comparative sequence analyses also revealed that Groundnut Tospovirus isolate shared maximum sequence identity with GBNV at nucleotide (97%) as well as amino acid (99%) levels In contrast. 79-81% nucleotide sequence identity was observed with N genes. Nucleotide identities with eleven other Tospovirus species were in the range of 41- 64%. Similarly, comparison of amino acid sequences of n genes revealed that the N gene of groundnut Tospovirus shared 82-84% sequence in contrast to 16-58% identity with other Tospovruses .
Isolates in the Tospovirus genus with greater than 90% N gene sequence identity are classified as strains of the same virus (Moyar, 1999) Since the biological characteristics and N gene sequences of groundnut Tospovirus were similar to GBNV ,it is proposed that the groundnut Tospovirus should be regarded as a strain of GBNV belonging to WSMV serogroup , henceforth designated as GBNV-Mb. This is the first report of the presence of GBNV on groundnut under natural condition. Considering that GBNV has long been endemic to India on groundnut and has a wide host range (Ghanekar et al. 1979), it is possible that GBNV has spread from groundnut . Recently, soybean was also identified as natural host of GBNV (Bhat et al. 2002) and GBNV-Sb shared 97% amino acids sequence identity with GBNV- Mb Thus, natural infection of GBNV on other crops should also be monitored.
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