Mosambi
4. CROP PROTECTION
4.1.3 Viral Diseases
4.1.3.1 Natural occurrence of viruses in mungbean and urdbean
Natural infection of Mungbean yellow mosaic virus (MYMV), Urdbean leaf crinkle (UBLC) and Groundnut bud necrosis virus (GBNV) on mungbean (18) and urdbean (9) cultivars was detected by symptomatology, transmission, electron microscopy, RT-PCR and ELISA.
All the mungbean cultivars showed mixed infection of MYMV+UBLC. Except cultivars ML 668 and ML 818, all showed mixed infection of MYMV+GBNV, and except ML 668, ML 818 and MH 96-1, all showed mixed infection of UBLC+GBNV. Similarly, urdbean cultivars were infected by single and multiple infection. Except cultivars KU 301 and KU317, all showed mixed infection of MYMV+ GBNV and UBLC+GBNV.
4.1.3.2 Viral genomics
Citrus tristeza virus. The complete coat protein (CP) gene from 25 CTV isolates (19 Darjeeling hills isolates, viz., CTK 2, CTK 4, CTK 5, CTK 6, CTK 7, CTK 9, CTK 8, CTK 10, CTK 13, CTK 15, CTK 16, CTK 17, CTK18, CTK 20, CTK 22, CTK 23, CTK 24, CTK 27 and CTK 30; four Delhi isolates, viz., CTB, CTP, CTD, CTN and two Bangalore isolates, viz., BNG-1 and BNG-2) and a 404 nucleotide (nt) fragment of 5 ORFIa variable region gene (VRG) (positioned from 52 between 1074 nt and 1478 nt) from 19 Darjeeling hills isolates were cloned and sequenced for determining genetic diversity. CTV isolates shared 90- 99% identity in CP gene sequences and were grouped into three different clusters. CTV isolates also shared 83-98%
identity in 52 ORFla VRGs and many Indian CTV isolates were genetically related to exotic isolates, SY568 (California, severe) and VT (Israel, severe).
In continuation of the activity for search of a mild strain of CTV, citrus samples from Pune and other regions of Maharashtra were collected and screened by monoclonal antibodies (MCA13). Three isolates that were negative with monoclonal antibody reaction were tested by RT-PCR using CTV 5 region specific primers. The sequences of these three isolates were related to severe strain of CTV from Bangalore sharing up to 98% identity, suggesting that the MCA13 negative isolates are not mild as observed from the CTV isolates from other countries, and that the Indian CTV isolates were different.
Sequencing of complete genome of Indian citrus ringspot virus. The complete genome of Indian citrus ringspot virus (ICRSV) infecting Kinnow mandarin of Hoshiarpur (Punjab)(ICRSV-H) was sequenced using 7 different sets of primers designed from the ICRSVK1 (Kinnow mandarin, Delhi isolate) sequence available in GenBank. ICRSV-H genome was 7560 nt long and showed 99.6% identity in nucleotide sequence with ICRSVK1.
ICRSV-H also showed six ORFs, identical to that of ICRSVK1. However, both nucleotide and amino acid substitutions were observed in ORF 1 (putative RNA dependent RNA polymerase), ORF 2, ORF 3 and ORF 4 (triple gene block). Only substitution in nucleotide but not in amino acid was observed in ORF 5 (Coat protein gene).
No substitution either in nucleotide or amino acid was found in ORF 6 (nucleic acid binding protein). The sequence analysis further revealed that ICRSVK1 and ICRSV-H were more close to Potexviruses than to other groups of filamentous viruses such as Carlaviruses, Fovreaviruses, Vitiviruses, Trichoviruses and Capilloviruses.
Complete genome sequence of Banana streak virus (BSV). Complete genome of BSV from a banana (Poovan) isolate from Trichy was sequenced using different sets of primers. BSV genome was 6994 nt long and shared 85%
nucleotide sequence with a Chinese isolate of BSV. Further analysis of the sequence is in progress.
Sequencing of 16S, 23S and 16S-23S intergenic spacer region of ribosomal DNA of greening citrus bacterium. 16S (1417 bp), 23S (247 bp) and 16S-23S intergenic spacer region (620 bp) of ribosomal DNA of greening bacterium {Candidatus liberibacter asiaticus (Cla)} in Kinnow mandarin (Ludhiana) were sequenced for the first time in India and showed maximum identity with corresponding region of Cla sequenced from other countries.
Sequencing of small (s) RNA of Groundnut bud necrosis virus (GBNV). Genome organization and complete nucleotide (nt) sequence of the small (s) (AY871098) RNA segment of GBNV (mungbean isolate) was determined. sRNA was 3057 nt long and had two ORFs in an ambisence arrangement separated by an intergenic region (IGR) of 773 nt. The large ORF (1320 nt) was on viral sense strand (v) and encodes non-structural protein (NSs) of 440 aa long. The small ORF (831 nt) on viral complementary sense (Vc) encodes 277 aa nucleocapsid protein (N). The size of the 5 and 3 NCRs were 66 nt and 67 nt, respectively.
4.1.3.3 Molecular diagnosis
Simultaneous detection of citrus pathogens. Duplex PCR for simultaneous detection of citrus pathogens in naturally infected Sathgudi sweet orange, viz., Indian citrus ring spot virus (ICRSV) and Citrus tristeza virus (CTV), Citrus mosaic virus (CMBV) and greening bacterium (Cla) and CMBV and ICRSV was standardized.
Natural infection of CMBV and ICRSV in Sathgudi sweet orange orchard in Nagari (A.P.) was confirmed by electron microscopy.
Nucleic acid extraction protocol. A cost effective RNA extraction protocol was earlier standardized for efficient detection of ICRSV and CTV from bark and leaf tissue. This extraction protocol also worked for template preparation for the detection of a DNA virus (CMBV). A multipurpose kit for template preparation of both RNA and DNA viruses in citrus is under evaluation.
4.1.3.4 Transgenic resistance
Agrobacterium-mediated transformation of tomato.
To develop transgenic tomato against bud blight caused by Groundnut bud necrosis virus (GBNV), putative tomato transformants of CO 3, Pusa Early Dwarf (PED) and Pusa Ruby (PR) generated through Agrobacterium- mediated transformation using nucleo-capsid protein (N) gene sense and antisense constructs were screened for the presence of N gene. Thirteen of 45, 5 of 23 and 17 of 43 putative transformants from CO 3, PED and PR, respectively, were PCR positive. Of the 35 PCR positive transformants, nine transformants were Southern positive.
Lines CO3S1, CO3S12, PRS1, PRS3, PRA2 and PRA4 possessed single copy of the transgene, while lines CO1S2, PRA1 and PRA5 possessed two copies of the transgene. Five of nine lines were positive for N gene expression in DAC-ELISA.
Agrobacterium-mediated transformation of groundnut. Transgenic groundnut (Arachis hypogaea) lines with sense and antisense coat protein (CP) gene of Tobacco streak virus (TSV) were generated by Agrobacterium-mediated transformation of de- embryonated cotyledons of cultivar JL 24. Approximately 180-200 days were required between explant transformation and hardening of transformants in the glasshouse. Genomic Southern analysis revealed two transgenic lines (S15, AS2) with single copy and one transgenic line (S3) with two copies of the CP gene. CP expression was observed in transgenic lines with CP gene (S3, S15) by ELISA. Evaluation of the transgenic lines for resistance to TSV is in progress.
Development of Papaya ringspot virus (PRSV) gene constructs. Coat protein (CP) gene of PRSV isolates originating from two different locations (Bilaspur in Chattisgarh; and Coimbatore in Tamil Nadu) was used as transgene to confer resistance. CP genes cloned in pDrive vector (3.85 kb) were mobilized into binary vectors (pBI121 and pBinAR) both in sense and antisense orientations.
Constructs were then mobilized into Agrobacterium tumefaciens, and the conjugants analysed by colony PCR were supplied to the Central Institute of Subtropical Horticulture (CISH), Lucknow for papaya transformation. To confer broad spectrum resistance against PRSV, truncated CP gene constructs were also prepared.
Simultaneous detection of CMBV and ICRSV. Lane M: marker;
lanes 1-3: CMBV+ICRSV; lane 4: CMBV; and lane 5: only ICRSV
Molecular analysis of putative transgenic of tomato varieties with N gene of GBNV
Variety Putative Genomic Number ELISA
transformants Southern of insert
CO 3 S1T0 + 1 +
S2T0 + 2 +
S12T0 + 1 +
PR S1T0 + 1 +
S3T0 + 1 +
A1T0 + 2 NT
A2T0 + 1 NT
A4T0 + 1 NT
A5T0 + 2 NT
*Absorbance at 405 nm more than two times of the transformed control was considered positive; + : positive; -: negative; NT: not tested
M 1 2 3 4 5
Development of CTV gene construct. CTV isolates from Darjeeling hills (CTK5, CTK9, CTK 10 and CTK13), Bangalore (BNG1 and BNG2) and Delhi (CTB and CTP) were selected for developing CP gene constructs in sense and antisence direction. Sense and antisense CTV CP constructs (four each) were developed in pBinAR binary vector.
Evaluation of resistance in transgenic potato lines. Seven lines of transgenic potato cv.
Kufri Giriraj were developed using coat protein (CP) gene from Potato virus Y (PVY). Based on the genomic Southern, two transgenic lines, 2.2 and 3.4 were selected for the evaluation of resistance to PVY in environmentally controlled plant growth chamber (Conviron) in the National Phytotron Facility at IARI. The transgenic line- 2.2 had poor growth as leaves were narrow and stems were thin and lanky, whereas the transgenic line-3.4 had luxuriant growth with bold leaves and stems. The inoculated non-transgenic plants developed mild greenish mosaic symptoms on the
upper leaves, and petiole necrosis on the lower leaves, whereas no symptoms were observed in the inoculated transgenic plants. Electron microscopic analysis of the inoculated plants showed the presence of PVY in the nontransgenic plants, whereas no PVY virus particles were observed in the transgenic plants. As transgenic plants contained CP gene, virus infection following challenge inoculation was judged by PCR using non-CP primer from NIb region which showed specific amplification of 660 bp fragment in the inoculated non-transgenic plants but not in any inoculated transgenic plants tested. The absence of symptoms and virus by EM and RT-PCR in PVY challenged transgenic potato plants indicates resistance to PVY.
The transgenic line-3.4, which showed excellent growth and resistance to PVY in plant growth chamber, was micro- propagated 10 times successively over a period of nine months and then examined for the presence of transgene in five randomly selected plants. PCR showed specific amplification of CP gene in all these selected plants indicating the stability of transgene.
Characterization of RNAi suppressor of ToLCNDV. The AC4 gene of Tomato leaf curl New Delhi virus (ToLCNDV) was sequenced. The sequence analysis revealed 96% homology with severe strain of ToLCV, and 75-85% homology with other mono and bipartite viruses causing leaf curl disease in India.
The presence of certain conserved amino acids and motifs similar to other viral suppressors and the PAZ domain of dicer
Viral gene constructs developed for papaya transformation
Viral Gene Orientation Vector Mobilized to Remarks
gene source E. coli A. tumefaciens
Full CP PRSV Sense pBI121 + + Shared with
(861 bp) (Bilaspur Antisense pBinAR + + CISH, Lucknow
isolate) for papaya
transformation
Full CP PRSV Sense pBinAR + +
(858 bp) (Coimbatore Antisense pBI121 + +
isolate)
Truncated CP PRSV Truncated pBinAR + + Ready to share with
(~410bp) CISH, Lucknow
Challenge inoculated transgenic potato plants of cv. Kufri Giriraj (line 3.4) with PVY in the growth chambers
Non-inoculated Inoculated Non-inoculated Non-transgenic control Transgenic (3.4 line)
Phenotypic observation of AC4 in N. tabacum, S. lycopersicon, and N. benthamiana:
A- transformed, and B- degenerated
AC4 Transformation
A B A B A B
N. tabacum S. lycopersicon N. benthamiana
protein directly reflects its role in binding with small RNAs and hence playing a crucial role in development and defence.
For establishing its direct involvement in plant development, the ToLCNDV-AC4 was used to transform N. tabacum and the transformants obtained showed severe developmental abnormalities suggesting its direct role in plant development.
Resistance evaluation of existing promising leaf curl resistant transgenic lines under contained trials. Molecular analysis of transgenic lines PEDAR-12 and PEDAR-26 from T-1 to T-4 generations suggested non-Mendalian inheritance.
Non-Mendelian inheritance of the transgene may be due to the positional effect or recombinational rearrangements.
Further studies with larger sample size are in progress to establish gene inheritance in transgenic lines.
Development of transgenic resistance to Mungbean yellow mosaic virus in soybean. Replication initiation protein construct of Mungbean yellow mosaic virus (MYMV) in antisense orientation (MYMV0Rep-AS) under 35S promoter was used to transform soybean cultivar JS 335 through Agrobacterium. Of the 535 putative transformants from embryonic tip (397) and cotyledonary explants (138), thirty- two transformants developed roots and molecular analysis of these transformants is in
progress. In order to circumvent the problem of rooting, biolostic delivery of the MYMV-Rep-AS was attempted, and a total of 62 plantlets were obtained.
4.1.3.5 Citrus
Survey, collection and maintenance of virus and virus-like pathogens infecting citrus. Citrus tristeza virus (CTV) isolates were collected from West Sikkim and Lower Chhibo Busti (Kalimpong), indexed and maintained on kagzi lime (Citrus aurantifolia). One isolate of greening pathogen was collected from Sikkim orange (Citrus reticulata) in Zoom (West Sikkim), and two isolates were
collected from Kalimpong, and one each was collected from Darjeeling orange (C. reticulata) and pummelo (Citrus grandis). The isolates were indexed on Darjeeling orange and mausambi (Citrus sinensis).
Surveys of four declining orange orchards in the Zoom region of West Sikkim revealed that the incidence of greening disease is 32.25%. Symptoms of incidence include yellow mottling, pale green discolouration of young leaves, general yellowing and sectorial chlorosis in canopy. Affected trees were sparsely foliated and produced lop-sided fruits with greenish-orange mottle. Greening-affected pummelo trees also exhibited similar foliar symptoms. The fruits from affected trees were lop-sided which, even when ripe, had large greenish patches on the rind.
Biological characterization of Citrus tristeza virus isolates from Darjeeling hills. Of the 20 CTV isolates from Sikkim and Darjeeling hills being maintained at IARI Regional Station, Kalimpong, the results of indexing of seven isolates on five different citrus species, viz., kagzi lime (C.
aurantifolia), Darjeeling orange (C. reticulata), mausambi (C.
sinensis), rough lemon (Citrus jambhiri Lush), Rangpur lime (Citrus limonia Osb.) are tabulated below:
Biological characterization of Citrus tristeza virus isolates from Darjeeling hills
Isolate Symptoms on
Kagzi lime Darjeeling orange Mausambi Rough lemon Rangpur lime Ichchhey Busti – 2 Vein clearing; Mild stem pitting No symptom Mild stem Mild stem
(Source: Kagzi lime) die-back pitting pitting
Kalimpong – 2 Vein clearing; Yellowing of leaves; Stem pitting Vein corking; Yellowing of (Source: Kagzi lime) vein flecking; vein corking; stem pitting midribs;vein
vein corking; Stem pitting corking
stem pitting;
die-back
UBKV Vein corking; No symptom Vein corking Interveinal Interveinal
(Source: Mandarin) die-back chlorosis; vein chlorosis;vein
corking; mild corking; mild stem pitting stem pitting
Upper Dalep Vein clearing; ———- No symptom ——— Inward curling
(Source: Kagzi lime) mild stem of young leaves
pitting;
die-back
CDRS Vein clearing; vein corking; Vein corking; Vein corking; Vein corking (Source: Darjeeling Vein corking die-back die-back
orange) die-back
Chhibo Busti Vein clearing; Vein corking; Vein corking; ——— ———
Source: (Darjeeling vein corking; chlorosis; die-back
orange) die-back die-back
Mirik Vein clearing; Vein corking; ——— Vein corking Vein corking;
(Source: Darjeeling vein corking; die-back die-back
orange) die-back
Monitoring of aphid vector population in kagzi lime plantation by yellow coloured traps placed at different heights of 1', 2', 3', 4', 5' and 6' recorded the maximum of 345 aphids/
trap in the fourth week of January. The trap placed at 1' height recorded the highest aphid catches compared to those placed at other heights. ELISA test identified eight isolates, i.e., M1, M2, Tm, Aphid transmitted, Katol, Shrirampur, Donde and IARI, Pune positive for CTV antisera.
4.1.3.6 Papaya
Visual observations on aphid colonization on papaya revealed the maximum number of 19.6 aphids (nymphs &
adults) per plant on inoculated papaya plants transplanted in October, and these were identified as Myzus persicae, Ceratovacuna lanigera, Macrosyphum species and Toxoptera aurantii. Weed plants grown in and around papaya plantations such as Cucumis melo, Alternanthera sessilis, Datura metal and Xanthium indicum proved to be alternative hosts for Papaya ring spot virus (PRSV). In a screening trial, all the eight cultivars tested showed PRSV incidence (13.2-97.6%) by flowering stage. The variety Madhubala showed the least infection (13.2%) followed by the varieties CO 2 (39.8%) and Pusa Nanha (44.8%) at hundred per cent flowering stage.
Studies on the effect of roguing of PRSV infected plants on the productivity of papaya revealed that plant height at flowering was significantly higher in treatment combinations where PRSV infected plants were uprooted. However, the difference in the collar diameter was statistically non- significant.
It was demonstrated that five rows of banana planted as border crop on the periphery of papaya plantation was successful in reducing the number of aphid-vectors when compared with their number outside the border crop. The number of aphid-vectors found inside the border crop of banana was about 68% less than that found outside the border crop.
4.1.3.7 Management of virus diseases in capsicum In capsicum, three varieties, Shanta, Tara and Indra, were screened for the incidence of virus diseases under field conditions. All the three varieties were susceptible to Pepper veinal mottle virus and Cucumber mosaic virus. The variety Indra showed the highest incidence of virus diseases (35- 40%). Virus diseases in the field grown capsicum varieties Shanta, Tara and Indra could be effectively managed by
regular alternate sprays of neem oil (1%) and diamethoate (0.1%) along with 2 applications of Thimet at 40 days’
interval.
4.1.3.8 Peri-urban vegetables
Surveys. Surveys around Pune revealed the occurrence of PRSV-W in various cucurbits, viz., bottlegourd (15-20%), spongegourd (15-18%), ridgegourd (5-8 %) and pumpkin (10- 15%).
In poly-house grown capsicum, the incidence of Pepper veinal mottle virus (30-35%) and Cucumber mosaic virus (42- 47%) in the varieties Bomby and Lario was recorded. In field grown capsicum, the occurrence of Pepper veinal mottle virus (18-20%) and Cucumber mosaic virus (25-30%) was recorded in the variety Indra.
Varietal screening of cucurbits. Four varieties each of bottlegourd, ridgegourd, spongegourd, cucumber and pumpkin were screened for virus diseases under field conditions. A Poty virus was recorded in the bottle gourd varieties Arjun and Samrat. In spongegourd, all four varieties were susceptible to PRSV-W infection. The infection ranged from 8% to 10%.
The occurrence of WBNV in watermelon in February to March sown crop was higher (50-70%) because of the building up of thrips populations during this period as compared to that in the crop sown from the 2nd fortnight of December to January (3-5%).
The occurrence of viral infections was recorded in the cucurbitaceous vegetables, namely, ridge gourd (Luffa acutangula), sponge gourd (Luffa cylindrica), bitter gourd (Momordica charantia) and pumpkin (Cucurbita moschata), grown in and around Kalimpong during kharif. The affected plants exhibited vein clearing, vein banding, light and dark green mosaic and various deformations in leaves. In ELISA, the sap of infected plant materials reacted positively with polyclonal antibodies to Potyvirus group.
4.1.3.9 Large cardamom
Characterization of the virus causing foorkey disease.
Four infected clumps of the large cardamom cultivar Ramsey were used for PCR-amplification of foorkey virus genome.
Tissue samples, namely, fully expanded leaves, youngest unfurled leaves (core leaves), meristem tips, old roots and newly emerging roots were extracted. Genome fragments of the foorkey virus could be amplified using three different sets of primers designed from the Banana bunchy top virus
(BBTV) DNA1, DNA2, DNA3 components and a set of primers from BBTV common stem loop region. The amplified products of putative replicase and coat protein genes were cloned. The putative rep-gene was sequenced.
It had 83% sequence homology with BBTV master rep-gene.
Amplification of viral genome could be achieved only in the tissues from the youngest unfurled leaves and meristem tips.
4.1.3.10 Orchids
Characterization of the virus infecting Cymbidium and Coelogyne. Two important viruses, namely, Cymbidium mosaic virus (CymMV) and Odontoglossum ring spot virus (ORSV) are ubiquitous with orchid cultivation in Sikkim and Darjeeling hills. CymMV and ORSV may infect orchid genera either singly or in combinations. Immunological studies detected mixed infection of CymMV and ORSV in Cymbidium and other orchid genera. Electron microscopic (EM) studies of the infected samples also revealed the presence of both flexuous filamentous particles of CymMV and rigid rods of ORSV. Besides these two viruses, a number of viruses having Rhabdovirus-, Potyvirus- and Badnavirus-like particles were found to be associated with different orchid species. EM studies of the leaf tissue of Cymbidium spp.
infected with mosaic streak disease and the leaf tissue of Coelogyne spp. infected with ring spot disease showed the presence of Rhabdovirus-like particles either alone or in combination with ORSV.
4.1.3.11 Tapping panel dryness of rubber
R-PAGE analysis of grafted trees revealed that TPD is graft transmissible. One hundred per cent (100%) transmission was observed when both stock and scion were from the affected source, up to 70% when the stock source was positive, up to 42% when the stock source was negative and the scion source was positive. In such case where both the stock and scion were from apparently healthy source, small percentage of plants showed the presence of TPD. Characterization of LMW-RNA -like bands associated with TPD is in progress.
