Development of Polyclonal Antibodies-Based Serological Method for the Detection of Calanthe Mild Mosaic Virus and Application in Virus Certification Programme.

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807 bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His6BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His6BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids.

Association between job control and psychological health in middle-level managers.

The present study analyses the effect of job control at work on psychological stress for Indian Middle-Level Managers (MLMs) of a public telecom organisation. Two hundred ten MLMs from different parts of India have participated in the survey. Three dimensions of job control visualize control over work (CoW), control over working time (CoT1) and control over working days (CoT2), were considered. The validity and reliability were confirmed using Factor and reliability analysis. A Binary Logistics Regression (BLR) was performed to find the effect of job control on behavioural, somatic and cognitive stress controlling for age, gender, and experience. The Odds Ratio and Adjusted Odds ratio were calculated. 56% of the participants reported suffering from psychological stress. Results showed that CoT1 had a significant association with somatic stress while CoT1 and CoT2 with cognitive stress. Low CoW and low CoT2 were associated with high psychological stress among middle-level managers while low CoT1 to low psychological stress. The findings indicate that job control have both positive and negative relationships with psychological well-being depending on its dimension. Increasing job control cannot entirely ensure the psychological well-being of employees. Therefore, organisations need to assess different dimensions of job control carefully before providing work flexibility to employees.

Genetic diversity and population structure of 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing in India based on the prophage types.

Huanglongbing (HLB), also known as 'citrus greening', is an extremely destructive disease of citrus worldwide. HLB is associated with three species of the fastidious proteobacterium, Candidatus Liberibacter asiaticus (CaLas), Ca. L. africanus and Ca. L. americanus with CaLas being the most widely distributed around the world and the only species detected and described so far in India, one of the major global citrus fruit producers. Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. Three types of prophages, Type 1, Type 2 and Type 3 have been identified and described in CaLas so far. In the present study, 441 CaLas isolates sampled across 18 Indian states were used for prophage typing. Based on detection of three prophage types by PCR, all the eight probable combinations of CaLas prophages were identified, including single Type 1 (26.5%), single Type 2 (18.8%), single Type 3 (1.4%), Type 1 + Type 2 (20.4%), Type 1 + Type 3 (12.5%), Type 2 + Type 3 (4.8%), Type 1 + Type 2 + Type 3 (11.3%) and None type (4.3%). Prophage types were confirmed by PCR amplicon sequencing and subsequent phylogenetic analysis. By discovery of all 3 prophages and based on genetic identity and genetic distance, CaLas populations from eighteen citrus growing states were separated into two major Prophage Typing Groups (PTGs): PTG1 and PTG2. The PTG1 comprised of CaLas from North-West India and PTG2 from rest of the country (North-East, Central and South India), and both major groups were further divided into two (PTG1-A, PTG1-B) and three (PTG2-A, PTG2-B and PTG2-C) subgroups respectively. The findings of CaLas population patterns provide evidence for independent origins of HLB-associated CaLas. CRISPR (clustered regularly interspaced short palindromic repeats) array was also detected in CaLas isolates. This is the first report evaluating the genetic variation of a large population of CaLas bacterium in India using the PCR markers from the prophage regions which would certainly assist the ongoing HLB management efforts in India.

Analyses of 3' half genome of citrus tristeza virus reveal existence of distinct virus genotypes in citrus growing regions of India.

Citrus tristeza virus (CTV, genus Closterovirus) is one of the most serious pathogens responsible for huge loss of citrus trees worldwide. Four Indian CTV isolates, Kat1 (C. reticulata/Central India), D1 (C. sinensis/North India), B5 (Citrus limettoides/South India) and G28 (C. lemon/Northeast India) collected from different regions of India were characterized based on sequencing of 3' half genome (~ 8.4 kb) comprising 10 open reading frames (ORFs2-11) and 3' UTR and the sequences were submitted to NCBI database as Acc. No KJ914662, HQ912022, HQ912023 and KJ914661, respectively. The present and previously reported Indian isolates Kpg3 and B165 were analyzed and compared with other Asian and international CTV isolates. The Indian CTV isolates had 92-99% nt identities among them. The phylogenetic analysis generated overall ten genogroups/lineages. Of them, all the Asian isolates fell into seven genogroups, whereas the Indian isolates into four. Indian isolates Kat1, D1 and Kpg3 grouped together, termed "Kpg3Gr", along with Florida severe isolate T3. The Indian isolates B5, and G28 were found to be two distinct and separate lineages, indicating that these isolates are two new CTV entities. Based on phylogenetic analysis, Kpg3Gr was identified as "Indian VT" subtype which is distinct from the Asian and the Western VT subtype within diversified VT genotype. The recombination detecting-program, RDP4 detected Indian isolates Kat1, B5, B165 and G28 as recombinants, where G28 as strong recombinant. The present study determined the occurrence of at least four CTV genotypes, B5 (distinct), B165 (T68 type) G28 (distinct) and Kpg3Gr in citrus growing regions of India.

Feather degrading, phytostimulating, and biocontrol potential of native actinobacteria from North Eastern Indian Himalayan Region.

Twelve actinobacterial strains were isolated from tomato rhizospheric soil from Manipur, a state in North East Indian Himalayan Region and screened for keratinolytic and plant growth promoting traits. Nine promising isolates were identified as Streptomyces species using partial 16S rRNA gene sequencing. Among the seven isolates showing chicken feather degradation activity, three keratinolytic strains RCM-SSR-2, -6, and -12 were found to be the most efficient feather degrading strains achieving 90% feather weight loss within 48 h of incubation. They also showed maximum keratinase and soluble peptide production. Strain RCM-SSR-2, -5, -6, -8, and -11 showed positive results for all plant growth promoting traits tested. Maximum indole-3-acetic acid production was exhibited by RCM-SSR-6. Strain RCM-SSR-1, -2, -5, -6, -9, and -11 showed antagonistic activity against three important plant pathogens. Feather hydrolysate of RCM-SSR-6 was also evaluated for in vitro seed germination test using garden pea seeds. Higher concentration of feather protein hydrolysate (3 mg ml-1 ) inhibited shoot and root length of the germinating embryo. However, lower concentration (0.01 mg ml-1 ) of feather protein hydrolysate promoted seed germination. Among the 12 strains, four isolates namely RCM-SSR-1, -2, -5, and -6 were found to be promising as multi-traits plant growth promoting rhizobacteria for development of organic fertilizer, phytostimulator, and biocontrol agents.

Sequence analysis of shorter than genome length episomal Banana streak OL virus like sequences isolated from banana in India.

Electron microscopy and sequencing of reverse transcriptase and ribonuclease H (RT/RNase H) region of Badnavirus genome from two banana cultivars: Poovan (triploid: AAB) and Safed velchi (diploid: AB), exhibiting leaf streak symptoms, confirmed the association of Banana streak OL virus (BSOLV). As per ICTV species demarcation threshold of 80 % identity in RT/RNase H region, both the isolates were identified as BSOLV. Rolling circle and end-to-end amplification showed the association of two short episomal BSOLV variants: BSOLV-IN1 and BSOLV-IN2 from Poovan and Safed velchi banana, respectively. The genome sizes of both isolates were 6,950 nucleotides long, but shorter than the typical BSOLV genome of 7,389 bp. Open reading frames (ORFs) 1 and 2 of shorter BSOLV isolates shared almost complete nucleotide identity (>99 %) to that of BSOLV. However, the ORF 3 (5,130 bp) and intergenic region (IGR), 886 bp, showed deletions compared with ORF 3 (5,499 bp) and IGR (956 bp) of BSOLV. In phylogenetic analysis for ORF 3 polyprotein, both the isolates clustered with BSOLV, Banana streak CA virus (BSCAV), and Sugarcane bacilliform GA virus (SCBGAV). Identical ORF 1, ORF 2, and the presence of all the conserved domains in short ORF 3 and promoter elements in IGR indicated that these isolates represent replicationally competent shorter variants of BSOLV. These two shorter-than-BSOLV genome sequences and two other identical banana streak virus sequences in GenBank (BSV-TRY; DQ859899 and BSV-GD; DQ451009) might have evolved due to error-prone reverse transcription and splicing or excision from the integrated sequences by homologous recombination in natural banana hybrids under field conditions.

Complete genome sequence of mandarin decline Citrus tristeza virus of the Northeastern Himalayan hill region of India: comparative analyses determine recombinant.

The complete genome sequence of a mandarin (Citrus reticulata) decline CTV isolate, Kpg3, of the Darjeeling hills of the Northeastern Himalayan region of India is reported for the first time. The complete Kpg3 genome has 19253 nt, and its nucleotide sequence identity ranged from 79% with the Florida CTV isolate T36 to 94% with the Israel isolate VT, whereas its identity to B165, the other Indian isolate, was 89%. Phylogenetic analysis indicated that the Kpg3 genome is closely related to isolate VT and distantly to T36 and B165. Recombination analysis indicated that Kpg3 is recombinant and originated through multiple recombination events in which parts of the genome were exchanged between divergent CTV sequences.