Anupam Varma

Global Emergence and Spread of Whitefly (Bemisia tabaci) Transmitted Geminiviruses

Whitefly-transmitted geminiviruses (WTGs) have emerged as the most destructive pathogens, particularly in the tropics and subtropics. The epidemics caused by the emerging WTGs have spread even in the regions that were free from these viruses earlier. The most seriously affected crops include cassava, cotton, grain legumes, and cucurbitaceous, malvaceous and solanaceous vegetables. Cassava mosaic disease pandemic in East Africa in the early 1990s was caused by the emergence of a highly virulent recombinant WTG, having sequences from African and East African cassava mosaic viruses. Since then, the recombinant WTGs, aided by sharp increase in whitefly population, have spread over large cassava growing areas of Africa, leading to acute food shortages in the affected region. Cotton leaf curl disease has been endemic in the Sudan for a long time. In the 1990s it caused severe epidemics in the north-western region of the Indian subcontinent resulting in enormous economic losses. The expanding host range of WTGs infecting legumes, along with their spread to new geographical regions, limits production of this important group of crops. The most dramatic emergence of WTGs affecting tomato has been in Asia and the Americas during the last two decades. The major contributory factors for the emergence and spread of new WTGs are (a) evolution of variants of WTGs through mutations, recombination and pseudo-recombination, (b) acquisition of satellite-like DNA molecules, (c) appearance of aggressive biotypes and increase in populations of Bemisia tabaci, (d) changes in the cropping systems, (e) introduction of new crops, (f) introduction of host susceptibility genes, and (g) the movement of infected planting materials. In addition, favourable climatic changes and human activity have also played an important role in the emergence of serious WTG associated diseases across the globe. This chapter highlights the emergence and spread of WTGs in selected crops threatened by this large group of plant viruses.

Variability in the coat protein gene of Papaya ringspot virus isolates from multiple locations in India.

Summary.The coat protein (CP) sequences of eleven Papaya ringspot virus (PRSV) isolates originating from different locations in India were determined, analysed and compared with the sequences of other isolates of PRSV. The virus isolates from India exhibited considerable heterogeneity in the CP sequences. The CP-coding region varied in size from 840–858 nucleotides, encoding protein of 280–286 amino acids. Comparative sequence analysis revealed that the PRSV isolates originating from India were divergent up to 11%. Though the PRSV isolates were differentiated in to two clusters, yet the sequence variation could not be correlated with the geographical origin of the isolates. Implication of the sequence variation in the coat protein derived transgenic resistance in papaya is discussed.

Purification and properties of growth stage-dependent antiviral proteins from the leaves of Celosia cristata.

Two antiviral glycoproteins, active against mechanical transmission of two tobamoviruses, tobacco mosaic virus and sunnhemp rosette virus, and citrus ring spot virus (ungrouped), were purified from the dried leaves of Celosia cristata. These proteins, called CCP-25 and CCP-27, have M(r) 25 and 27 kDa, respectively. Their concentration was found to vary between the pre-flowering and post-flowering stages of C. cristata90% lesion formation at a concentration of 20-30 µg ml(-1). They were resistant to proteases in the native state, but were readily digested when denatured. Both of them imparted actinomycin D sensitive resistance by inhibiting local lesions on Nicotiana tabacum cv. Samsun NN by tobacco mosaic virus. Their application, individually, also resulted in high resistance in systemic hosts to sunnhemp rosette virus, and citrus ring spot virus, respectively.

Molecular diversity of the DNA-β satellites associated with tomato leaf curl disease in India

DNA-β satellites, referred to here as betasatellites, were found associated with tomato leaf curl disease (ToLCD) in India. The size of eight betasatellites isolated from different geographical locations in India varied from 1353 to 1424 nt; these molecules had an ORF βC1, an adenine-rich region, and a satellite conserved region. Their nucleotide sequence identity varied from 45 to 93%. In phylogenetic analysis, these betasatellites grouped according to their geographic locations rather than the host species. Two new betasatellites, tomato leaf curl Bangalore betasatellite and tomato leaf curl Maharashtra betasatellite, were identified.

Identification of sources of resistance in Lycopersicon species to Tomato leaf curl geminivirus (ToLCV) by agroinoculation

A total of 90 genotypes of Lycopersicon species were tested forresistance to the Tomato leaf curlgeminivirus (ToLCV) by agroinoculation andthe vector whitefly (Bemisia tabaciGenn.) inoculation techniques underinsect-proof glasshouse conditions. Therate of infection in the inoculated plantswas determined by detection of the viralDNA in individual plants by the nucleicacid spot hybridization (NASH). Of the 38cultivars and 11 breeding lines of L.esculentum Mill. tested, none was highlyresistant or resistant while three andseven were moderately resistant whenexposed to the cloned virus DNAs byagroinoculation and whitefly inoculationrespectively. On the other hand, among the38 commercial cultivars screened, 16(42.1%) were highly susceptible in vectorinoculations and 31 (81.6%) inagroinoculation. Among the exoticcollection (EC) accessions six were highlyresistant, eleven resistant to whiteflyinoculation and none was highly susceptiblein either of the two tests, indicating thepresence of resistance among the ECaccessions. A higher degree of resistancewas observed in other species of Lycopersicon. While only one accession ofL. cheesmanii Riley was tested, itcould not be infected by either of the twomethods. L. pimpinellifolium (Jusl.)Mill. genotype EC 251580 was similarlyresistant. In L. peruvianum (L.)Mill., five EC accessions could not beinfected by whitefly inoculation, withthree of these being resistant and twomoderately resistant in agroinoculation.This study demonstrates the importance ofthe agroinoculation technique in the virusresistance screening programs andidentifies several good sources ofresistance to the Tomato leaf curlvirus in Lycopersicon species.

Serological and coat protein sequence studies suggest that necrosis disease on sunflower in India is caused by a strain of Tobacco streak ilarvirus.

Summary. Serological and coat protein sequence studies were conducted to identify an ilarvirus associated with necrosis disease on sunflower in India. In electroblot immunoassay, sunflower ilarvirus reacted strongly only with antiserum to Tobacco streak virus (TSV). The coat protein gene of sunflower ilarvirus was cloned and sequenced. The sequence analyses also showed that the CP gene was most closely related to TSV, the member of subgroup I of Ilarvirus. The sunflower ilarvirus CP shared 90% amino acid sequence identity with TSV. On the basis of serological relatedness and sequence identity, it is proposed that the sunflower ilarvirus from India should be considered a strain of TSV belonging to subgroup I and designated as TSV-SF. This is the first report of the molecular characterization of TSV on sunflower from the Indian subcontinent.

Role of betasatellite in the pathogenesis of a bipartite begomovirus affecting tomato in India

Betasatellites are commonly associated with tomato leaf curl disease caused by begomoviruses in India. This study demonstrates the role of a betasatellite in the pathogenesis of tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus affecting tomato in India. For infection, accumulation, systemic movement and disease induction by ToLCNDV, co-infection by the associated betasatellite was not essential, as the DNA A alone of ToLCNDV could infect tomato and Nicotiana benthamiana and induce mild symptoms, but DNA B or Cotton leaf curl Multan betasatellite (CLCuMB) was required for development of typical leaf curl symptoms. The symptoms were most severe in plants infected with all three components, indicating a role of the betasatellite in the pathogenesis of ToLCNDV. The plants infected with ToLCNDV DNA A alone had limited accumulation of viral DNA, which increased by many times in plants co-infected with DNA B or/and betasatellite. However, the plants infected with all three components accumulated 20 times less betasatellite DNA than the plants infected with DNA A and betasatellite. The increase in the amount of viral DNAs was also reflected in the commensurate increase in symptom severity and transmissibility by whitefly, Bemisia tabaci.

Detection of rice tungro bacilliform virus in field and glasshouse samples from India using the polymerase chain reaction

Rice tungro bacilliform virus (RTBV) together with rice tungro spherical virus (RTSV) is the causal agent for the rice tungro disease. A rapid technique was developed to detect RTBV DNA in the crude extract of freshly collected leaf samples by polymerase chain reaction (PCR). This technique can detect the viral DNA in 1000-fold diluted leaf extract. Detection has been possible in samples stored upto 5 days after the collection. This technique may have wide application for the field diagnosis of RTBV infection.

Allergens/Antigens, Toxins and Polyketides of Important Aspergillus Species

The medical, agricultural and biotechnological importance of the primitive eukaryotic microorganisms, the Fungi was recognized way back in 1920. Among various groups of fungi, the Aspergillus species are studied in great detail using advances in genomics and proteomics to unravel biological and molecular mechanisms in these fungi. Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Aspergillus nidulans and Aspergillus terreus are some of the important species relevant to human, agricultural and biotechnological applications. The potential of Aspergillus species to produce highly diversified complex biomolecules such as multifunctional proteins (allergens, antigens, enzymes) and polyketides is fascinating and demands greater insight into the understanding of these fungal species for application to human health. Recently a regulator gene for secondary metabolites, LaeA has been identified. Gene mining based on LaeA has facilitated new metabolites with antimicrobial activity such as emericellamides and antitumor activity such as terrequinone A from A. nidulans. Immunoproteomic approach was reported for identification of few novel allergens for A. fumigatus. In this context, the review is focused on recent developments in allergens, antigens, structural and functional diversity of the polyketide synthases that produce polyketides of pharmaceutical and biological importance. Possible antifungal drug targets for development of effective antifungal drugs and new strategies for development of molecular diagnostics are considered.

Nine novel DNA components associated with the foorkey disease of large cardamom: evidence of a distinct babuvirus species in Nanoviridae.

Foorkey disease is a serious constraint to the production of large cardamom (Amomum subulatum, family Zingiberaceae). The disease is characterized by profuse proliferation of excessive stunted shoots, which makes the clump totally unproductive. The disease has been known in India since 1936 but the complete genome of the virus had not yet been characterized. In a preliminary study, an associated virus tentatively named as Cardamom bushy dwarf virus (CBDV) was identified based on the partial sequence of a single DNA component (DNA-R). In the present study, a high incidence (37.2-39.3%) of foorkey was recorded in certain plantations in the Darjeeling hills located at lower altitudes (300-1380 m) and CBDV was detected in several field samples by PCR. Nine novel DNA components were isolated and characterized from foorkey affected plants. CBDV contained six major DNA components (DNA-R, -S, -M, -C, -N and -U3) similar to the integral genome components known for the members of the genus Babuvirus in the family Nanoviridae. Additional components, satellite Rep (DNA-sRep1) and unknown components (DNA-Uf1 and -Uf2) were also identified. The size of the genome components ranged from 1028 to 1127. The sequence identity and phylogeny based on the individual components as well as overall genome (59.8-62% identity) distinguished CBDV from the two existing babuvirus species, Banana bunchy top virus and Abaca bunchy top virus. CBDV is the first distinct babuvirus species that affects plant species outside family Musaceae. This study shows further diversity in the genus Babuvirus.