The nematicidal potential of novel fungus, Trichoderma asperellum FbMi6 against Meloidogyne incognita.

One of the most damaging pests in vegetable crops is the root-knot nematode (Meloidogyne incognita) worldwide. The continuous use of nematicide is costly and has unintended consequences for human and environmental health. To minimize nematicides, eco-friendly integrated nematode management is required. Trichoderma, an antagonistic fungus has been explored to control root-knot nematode. The fungal bio-control strain FbMi6 was identified as Trichoderma asperellum (accession no. MT529846.1). T. asperellum FbMi6 showed substantial nematicidal activity in the laboratory, with egg hatch suppression (96.6%) and juvenile mortality (90.3%) of M. incognita. T. asperellum FbMi6 was examined under pot and field  conditions (after neem cake enrichment), both alone and in combination, and compared with controls. Application of T. asperellum FbMi6 enriched neem cake (1-ton ha-1) increased (28.3%) the okra yield and decreased (57.1%) nematode population as compared with control. T. asperellum FbMi6 enriched neem cake had higher polyphenol content (resistance enhancer) in okra compared with inoculated check.

Integrated Management of Meloidogyne Incognita and Soilborne Fungi Infecting Cucumber Under Protected Cultivation.

Relative efficacy of various approaches for management of Meloidogyne incognita and the soilborne fungus Fusarium oxysporum f. sp. cucumerinum has been tested in cucumber under protected cultivation conditions for two seasons. Management practices, namely, chemicals (fumigant, nonfumigant, and fungicide), organic amendments (neem cake, leaves, and oil opted as soil and seed treatment), and biocontrol agents (egg-parasitic fungus and Purpureocillium lilacinum), were combined for the management of the disease complex in a randomized block design. Two significant parameters were measured: plant growth parameters (shoot length, dry shoot weight, dry root weight, and yield) and disease parameters (galls per plant, final nematode population, egg masses per plant, and fungal incidence). All treatments significantly improved plant growth parameters and reduced nematode reproduction as compared to untreated check. The integration of formalin and neem oil seed treatment favors the low root galling index compared to all other treatments in both the seasons. Formalin and neem oil seed treatment reduced the nematode population and fungal incidence, and increased the yield of cucumber during both the seasons.

Management of root-knot nematode, Meloidogyne incognita and soil borne fungus, Fusarium oxysporum in cucumber using three bioagents under polyhouse conditions.

Complex diseases caused by Meloidogyne incognita and Fusarium fungus in cucumber is the most destructive disease under polyhouses. The experiment was conducted in the polyhouse of the Department of Horticulture, CCS HAU, Hisar, Haryana, India during summer season (2015-16) to evaluate the potential of bacterial and fungal biocontrol agents against Meloidogyne incognita and Fusarium oxysporum f. sp. cucumerinum in cucumber. Bioagents - Trichoderma viride (Tv), Pseudomonas fluorescence (Pf), Purpureocillium lilacinum (Pl) were taken 10 and 20 g kg-1 seed and bioagents liquid formulation, 10- and 15-ml kg-1 seed, were mixed with the potted soil. Chemical as well as untreated check were also maintained. All the treatments significantly improved the plant growth parameter, viz., shoot length (SL), root length (RL), fresh shoot weight (FSW), fresh root weight (FRW), dry shoot weight (DSW) and dry root weight (DRW) as compared to untreated check. However, significant reduction in nematode population and maximum improvement in plant growth parameter was recorded with carbofuran followed by higher dose of bioagents liquid formulation. Among the bioagents, bioagents liquid formulation was most effective in suppressing root knot nematode galling (43 / root system) and final population in soil (131 J2s / 200 cc soil) and fungus wilt incidence (25 %) at 30th day of after germination and significantly improved the plant growth parameters - shoot length (147.3 cm), fresh shoot weight (55.6 g), dry shoot weight (22.51 g) and dry root weight (4.50 g) from other bioagents. Bioagents liquid formulation was effective in suppression of root-knot nematode and fungus complex disease than the powder formulations of bioagents. More studies should be needed in future to evaluate the efficacy of bioagents as seed treatments and soil applications under field conditions.

Serotype and genotype diversity of dengue viruses circulating in India: a multi-centre retrospective study involving the Virus Research Diagnostic Laboratory Network in 2018.

OBJECTIVES: A retrospective study was undertaken to investigate the circulating dengue virus (DENV) serotypes and genotypes in India in 2018. METHODS: In total, 4963 samples referred to virus research diagnostic laboratories (n=21), the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV) and ICMR-NIV field units (n=2) for diagnosis of dengue in 2018 were tested using a real-time reverse transcription polymerase chain reaction assay for the presence of DENV serotypes. Representative samples were sequenced for the envelope (E) gene. RESULTS: Regional diversity was observed with regard to the dominant circulating serotypes. DENV-2 was found to be the most common serotype in many states. Thrombocytopenia, petechiae and malaise were associated with DENV-2 infection. Phylogenetic analyses of DENV E gene sequences revealed the circulation of genotypes I and V of DENV-1, two lineages of DENV-2 genotype IV, DENV-3 genotype III and DENV-4 genotype I. CONCLUSIONS: This study found regional differences in the prevalence of circulating DENV serotypes in India, and provides baseline data for continuous molecular surveillance. Molecular surveillance may have implications for predicting large-scale outbreaks of dengue if regional shifts in the predominantly circulating serotypes and genotypes are detected during the early phase of the dengue season.

Nematicidal effect of cruciferous bio-fumigants against the root-knot nematode, Meloidogyne incognita infesting okra.

In Haryana, India, only carbofuran is registered for the management of root-knot nematodes. The objective of this study was to investigate the potential of cruciferous bio-fumigants for the management of root-knot nematodes (Meloidogyne incognita) in okra. The experiments were conducted at research area Department of Nematology in 2017 to 2018 and 2018 to 2019. During this investigation, cruciferous bio-fumigants such as cabbage leaves and cauliflower leaves were used as bio-fumigant sources to protect Okra cv. Hisar Unnat. Fresh and chopped leaf mass of cabbage and cauliflower was incorporated uniformly into a naturally infested field. The initial nematode population in both years was 224 and 256 J2/200 cc soil, respectively. The results of our investigation showed that in both the years okra yield was enhanced significantly by the measures of nematode management. In addition, both of the tested bio-fumigant plants leaves proved to be potentially promising for the management of root-knot nematodes. Among the bio-fumigants, the highest decrease in nematode population, root gall index and increase in yield was observed in cabbage leaves @ 50 t/ha in both years, consecutively.In Haryana, India, only carbofuran is registered for the management of root-knot nematodes. The objective of this study was to investigate the potential of cruciferous bio-fumigants for the management of root-knot nematodes (Meloidogyne incognita) in okra. The experiments were conducted at research area Department of Nematology in 2017 to 2018 and 2018 to 2019. During this investigation, cruciferous bio-fumigants such as cabbage leaves and cauliflower leaves were used as bio-fumigant sources to protect Okra cv. Hisar Unnat. Fresh and chopped leaf mass of cabbage and cauliflower was incorporated uniformly into a naturally infested field. The initial nematode population in both years was 224 and 256 J2/200 cc soil, respectively. The results of our investigation showed that in both the years okra yield was enhanced significantly by the measures of nematode management. In addition, both of the tested bio-fumigant plants leaves proved to be potentially promising for the management of root-knot nematodes. Among the bio-fumigants, the highest decrease in nematode population, root gall index and increase in yield was observed in cabbage leaves @ 50 t/ha in both years, consecutively.

Clinical evaluation of an in-house-developed real-time RT-PCR assay for serotyping of dengue virus.

In the present study, an in-house-developed real-time RT-PCR (rRT-PCR) for serotyping of dengue virus (DENV) was evaluated for its performance, using 612 clinical samples. Compared to the composite reference standard, the in-house-developed rRT-PCR had an overall sensitivity of 97.5% and a specificity of 100%. The assay had a sensitivity of 100%, 95.6%. 96.9% and 100% for detecting DENV-1, DENV-2, DENV-3 and DENV-4, respectively. We recommend periodic evaluation of real-time RT-PCR assays for detecting DENV serotypes with a large number of samples and the use of at least two assays that target different regions of DENV genomes.

Serotype-specific differences in the laboratory parameters among hospitalized children with dengue and genetic diversity of dengue viruses circulating in Tamil Nadu, India during 2017.

In 2017, Tamil Nadu, a southern state, had the second highest number of dengue cases from India. In the present study, the serotype-specific differences in the clinical manifestations and laboratory parameters among hospitalized children with dengue were investigated and molecular characterization of the circulating dengue virus (DENV) serotypes during 2017 in Tamil Nadu was performed. Eighty children with dengue-like symptoms consecutively admitted to a tertiary care hospital and positive for DENV NS1 antigen were investigated for DENV serotype utilizing a real-time reverse transcriptase based polymerase chain reaction assay. Complete envelope (E) gene sequencing of the DENV strains was performed. Seventy samples were positive for serotyping (25 DENV-1, 17 DENV-2, six DENV-3, and 22 DENV-4). DENV-4 infections were associated with elevated levels of liver enzymes; Alanine aminotransferase (P = .021) and aspartate aminotransferase (P = .001). However, none of the serotype was associated with any specific clinical features and severe dengue. Asian and American/African genotypes of DENV-1 were cocirculating. The circulating genotype was cosmopolitan for DENV-2 with multiple lineages, genotype III for DENV-3 and genotype I for DENV-4. Unique mutations were present in the 2017 DENV-4 isolates. The present study suggests the association of DENV-4 with elevated liver enzymes in children hospitalized for dengue. Further, the study reports the genetic diversity of DENV circulating in Tamil Nadu during 2017. The study calls for continuous monitoring of the circulating serotypes and genotypes at regional level in India which might result in a region wise database useful in predicting future outbreaks.

Spatio-temporal distribution analysis of circulating genotypes of dengue virus type 1 in western and southern states of India by a one-step real-time RT-PCR assay.

Dengue virus type 1 (DENV-1) Asian and American/African (AM/AF) genotypes were reported to be co-circulating in southern and western states of India based on envelope (E) gene sequencing of few representative samples. The objective of the present study was to develop a one-step real-time RT-PCR to discriminate between Asian and AM/AF genotypes of DENV-1 and investigate the spatio-temporal distribution of the DENV-1 genotypes in southern and western states of India. A one-step real-time RT-PCR to discriminate the Asian and AM/AF genotypes of DENV-1 was developed and validated using 40 samples (17 Asian and 23 AM/AF), for which the envelope (E) gene sequence data was available. DENV-2, DENV-3 and DENV-4 isolates, one each and DENV negative samples (n = 17) were also tested by the assay. Additional 296 samples positive for DENV-1 from selected Southern and Western states of India were genotyped using the real-time RT-PCR assay. Among the samples used for validation, the genotyping results were concordant with sequencing results for 39 samples. In the one discordant sample which was positive for AM/AF by sequencing, the genotyping assay tested positive for both Asian and AM/AF genotype. DENV-2, DENV-3 and DENV-4 isolates were not reactive in the assay. None of the DENV negative samples were positive (sensitivity 100% and specificity 98.2%). A total of 336 samples (40 samples with sequence data and 296 samples without sequence data) were used for spatio-temporal distribution analysis. The results revealed that the Asian genotype was the predominant genotype in Tamil Nadu and Kerala, the southern states. The AM/AF genotype was the predominant genotype in Maharashtra, a western state of India. In Nashik district of Maharashtra, Asian genotype was observed in 32.6% of DENV-1 samples during 2017 while the same decreased to 7.3% during 2018. In Pune district, Asian genotype was observed in 40.0% of DENV-1 samples during 2018 only. To conclude, a one step real-time RT-PCR has been developed for discriminating Asian and AM/AF genotypes of DENV-1. This assay can act as a complement to sequencing but not a substitute and can be utilized in resource limited settings for molecular surveillance of DENV-1. DENV-1 Asian genotype was the dominant genotype in South India while, AM/AF genotype was dominant in Western India.

Emergence of dengue virus type 1 and type 3 as dominant serotypes during 2017 in Pune and Nashik regions of Maharashtra, Western India.

India witnessed dengue outbreaks during 2017 in different parts with more than 180000 cases. There is no data on the serotypes/genotypes of dengue virus (DENV) associated with the 2017 outbreak season. The present study investigated DENV circulating in Pune and Nashik regions of Maharashtra, Western India at molecular level. IgM negative samples that were collected before 6th post onset days of illness were tested for DENV RNA and serotyped by real time RT-PCR based methods. Representative samples of each serotype were processed for virus isolation and envelope (E) gene sequencing. Among the 472 samples tested for DENV serotypes from Nashik, DENV-1 was observed in 36.2%, DENV-2 in 12.9%, DENV-3 in 35.4%, DENV-4 in 8.0%, and multiple serotypes in 7.4% of the samples respectively. In Pune region, among the 109 samples tested for DENV serotypes, DENV-1 was observed in 27.5%, DENV-2 in 11.0%, DENV-3 in 52.3%, DENV-4 in 4.6%, and multiple serotypes in 4.6% of the samples respectively. Comparison of serotype distribution from 2009 to 2017 from the Pune region revealed the emergence of DENV-3 as the dominant serotype followed by DENV-1 in 2017. In the Nashik region, both DENV-1 and DENV-3 were predominant in 2017. Phylogenetic analyses revealed co-circulation of American African (AM/AF) and Asian genotypes of DENV-1. DENV-1 Asian genotype was detected for the first time in the region. No genotype changes were observed for DENV-2 (cosmopolitan genotype), DENV-3 (genotype III) and DENV-4 (genotype I). For DENV-3, a unique amino acid substitution (I380T) was observed in the domain III of E protein of 2017 isolates and was not observed in earlier DENV-3 genotype III isolates. To conclude, the results suggest the emergence of DENV-1 with circulation of both Asian and AM/AF genotypes and DENV-3 with unique amino acid substitutions in Pune and Nashik regions. The study underscores the need for continuous molecular monitoring at a large scale to detect the changes in DENV serotypes/genotypes that might have implications for earlier prediction of dengue outbreaks and designing dengue vaccines and predicting its efficacy.

Emergence of the Asian genotype of DENV-1 in South India.

A large outbreak of dengue occurred in Tamil Nadu, South India in 2012 with 12,000 cases and CFR of 0.5%. Molecular characterization of virus present in the sera of dengue patients was undertaken to determine if there were changes in the virus population. All four serotypes were circulating but DENV-1 was dominant, present in 52% of the serotyped samples. Furthermore, the genotype of only DENV-1 had changed; the Asian genotype had displaced the American/African. Phylogenetic analysis revealed that the Asian genotype was introduced from Singapore and shared 99% similarity with viruses, associated with large outbreaks in Singapore and Sri Lanka. We report for the first time the emergence of the Asian genotype of DENV-1 in southern India causing an extensive and severe outbreak. The study proves how movement of DENV can affect dengue outbreaks and underscores the need for close molecular monitoring of DENV.

Influence of evolutionary events on the Indian subcontinent on the phylogeography of dengue type 3 and 4 viruses.

During 1960-80 dengue disease profile in India was mild despite circulation of all four serotypes of dengue virus (DENV). Increase in disease severity with a concomitant change in the population of DENV-1 and 2 have been reported since then. To determine population dynamics of DENV-3 and 4, the envelope (E) gene sequence was determined for 16 Indian isolates of DENV-3 and 11 of DENV-4 and analyzed together with 97 DENV-3 and 43 DENV-4 global sequences. All Indian DENV-3 isolates belonged to genotype III, lineages C, D, E and F. Lineage F was newly identified and represented non-circulating viruses. Three non-conservative amino acid changes in domain I, II & III were identified during the transition from lineages F/E, associated with mild disease, to A-D, associated with severe disease. For DENV-4, the current viruses clustered in genotype I, lineage C, whilst the isolates from 1960s formed the new genotype V. A 1979 Indian isolate of DENV-4 was found to be an inter-genotypic recombinant of Sri Lankan isolate (1978) of genotype I and Indian isolate (1961) of genotype V. The rates of nucleotide substitution and time to the most recent common ancestor (tMRCA) estimated for DENV-3 (1782-1934) and DENV-4 (1719-1931) were similar to earlier reports. However, the divergence time for genotype III of DENV-3, 1938-1963, was a more accurate estimate with the inclusion of Indian isolates from the 1960s. By phylogeographical analysis it was revealed that DENV-3 GIII viruses emerged from India and evolved through Sri Lanka whilst DENV-4 emerged and dispersed from India. The present study demonstrates the crucial role that India/Sri Lanka have played in the evolution and dispersion of the major genotypes, GIII of DENV-3 and GI of DENV-4 which are more virulent and show higher dissemination potential.

Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962-2005).

Dengue is a major health problem in India with all four serotypes represented. Recently there has been an increase in the occurrence of dengue-1 outbreaks. It is possible that there have been changes in the genetics of dengue virus-1 (DENV-1), either by fresh introductions or by evolution in situ. The studies on DENV-1 evolution so far have no Indian sequences included. To gain insight into the dynamics of DENV-1 in India, the envelope (E) gene of thirteen virus isolates representative of the period 1962-2005 were sequenced and analyzed together with the available sequences of 40 globally representative isolates. All the Indian DENV-1 isolates were found to belong to the American African (AMAF) genotype. With the addition of 13 Indian isolates, the AMAF genotype can now be called Cosmopolitan. The Indian isolates were distributed into four lineages, India I, II, III and the Africa lineage, now called Afro-India. Of these, India III was the oldest and extinct lineage; the Afro-India was a transient lineage while India I, imported from Singapore and India II, evolving in situ, were the circulating lineages. Despite the extinction and introduction of lineages, no specific codon site was observed to be under selection pressure. The rate of nucleotide substitution estimated for DENV-1 was 6.5 × 10(-4) substitutions/site/year, and the time to the most recent common ancestor (tMRCA) was estimated to be 78-180 years (1825-1925), similar to previous estimates. The tMRCA for the AMAF/Cosmopolitan genotype was 56-98 years (1907-1949), a period that covers World War I and II. The two imports from Africa (1953-1968) and Singapore (1964-1975) and an export to the Americas (1955-1965) prove that there have been changes in the lineage of the DENV-1 viruses circulating in India which has contributed to the global dynamics of DENV-1 evolution and perhaps to the changing epidemiology of dengue in India.

Development of real time PCR for detection and quantitation of Dengue Viruses.

BACKGROUND: Dengue virus (DENV), a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop an improved real time RT-PCR (qRT-PCR) for DENV in this study. RESULTS: The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE), West Nile (WN), Chikungunya (CHK) viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples. CONCLUSION: We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.