First report of Colletotrichum orbiculare causing anthracnose of pointed gourd in India.

Pointed gourd (Trichosanthes dioica Roxb.), of the Cucurbitaceae family, is widely cultivated as a vegetable in many countries such as Bangladesh, India, Pakistan, Myanmar, Nepal and Sri Lanka. Over 800,000 metric tons of pointed gourds are produced annually in India, where cultivation is estimated to occupy over 33,000 hectares of land (MoA & FW, Government of India). In summer 2018, significant losses (approximately 15-20%) occurred in the sub-Himalayan region in West Bengal state of India (21.14-21.30° N, 78.82-79.02°E) due to a disease with typical anthracnose-like symptoms on the fruits. Light yellowish, small sized round to irregular spots were also apparent on the leaves. These spots gradually increased in size and turned into light brown and were surrounded by yellow halo. The lesions on the fruits were circular, yellow-brown, necrotic and sunken. A survey of four fields (1.5 ha) was conducted and a disease incidence of 30-40% was observed. Necrotic tissues from fruit as well as leaves were cut into approximately 5 mm2, surface sterilized with 0.1% HgCl2, plated in potato dextrose agar and incubated at 28ºC for 7 days in the dark. A total of 50 morphologically similar colonies were obtained from 20 sampled fruits and 10 sampled leaves. Fungal colonies were initially white, becoming gray as the cultures aged on PDA. The cultures developed black acervuli around the center of the colony. Setae were brown in colour, 1-5 septate, 40-100 µm long. Conidia were also observed through light and scanning electron microscopy and exhibit as (4-6 ×13-19 µm) hyaline, aseptate, cylindrical to oblong, with one end round and other truncate. The morphological characteristics were found similar to Colletotrichum orbiculare Damm, P.F. Cannon & Crous as reported by Damm et al. (2013). Ten isolates were obtained by transferring hyphal tips to new PDA plates and incubating under the same conditions. To confirm the identity of the pathogen, genomic DNA was extracted from five pure isolates (PG-Pha, PG-Pha-2, PG-Pha-3, PG-Pha-4, PG-Pha-5) with the cetyltrimethylammonium bromide (CTAB). Further, the ITS1-5.8S-ITS2 region, D1/D2 region of the 28S rRNA large subunit (LSU), Actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using specific primers, ITS1/ITS4 (White et al. 1990), NL1/NL4, ACT1/ACT2 and GDF1/GDR1 respectively and PCR conditions described in Damm et al. (2012). A GenBank BLAST search showed 99-100% identity to the Colletotrichum orbiculare (Acc. Nos. KP898988 for ITS1-5.8S-ITS2, Z18997 for 28S rRNA, AB778553 for ACT gene, and KF178482 for GAPDH). All obtained sequences were submitted to the GenBank (Acc. Nos. MN006616, OP811046-OP811049, [ITS1-5.8-ITS2], MN006684, PP391616-PP391619 [28S rRNA], MN168524, PP400822-PP400825 [ACT gene], OP627091, PP400826-PP400829 [GAPDH]). For phylogenetic analysis, MEGA version 11 (Tamura et al. 2021) was used to construct a maximum likelihood tree with 1000 bootstrap replicates, based on a concatenation alignment of three gene sequences (ITS, Actin and GAPDH) of the all the five C. orbiculare isolates as well as sequences of other Colletotrichum species obtained from GenBank. The cluster analysis revealed that, isolate PG-Pha form a cluster with other C. orbiculare isolates. Pathogenicity tests were conducted to confirm Koch's postulates. Pathogenicity tests were performed in mature fruits by inoculating them (n=8) with 10 µl of a 1×106 conidia/ml suspension at needle puncture wound sites. In control set up sterile distilled water was pipetted on fruits. Fruits were placed on sterile trays covered with glasses and incubated at humid chambers at 28±2ºC with 12 h of light. Healthy one-month old potted pointed gourd plants (n=15) were sprayed with conidial suspension until run-off. A set of 15 plants were sprayed with sterile distilled water and maintained as control. The plants were kept in a greenhouse at 25ºC, >75% relative humidity, and a 16/8 h day/night cycle for 15 days. Sterile distilled water was sprayed on the plants at one day interval to maintain the humidity. Inoculated fruits started showing yellowing symptoms one day post inoculation and gradually yellow-brown sunken spots became visible at the place of puncture, whereas control fruits remain symptomless even after 7 days of inoculation. Inoculated leaves showed disease symptoms similar to those observed in the field whereas leaves of control sets were symptomless even after 15 days. The pathogenicity test was repeated thrice under the same conditions mentioned before. C. orbiculare was successfully re-isolated from all the symptomatic tissues of leaves as well as fruits, completing Koch's postulates. Previously, the pathogen has been reported as an important anthracnose pathogen of Cucurbitaceae, especially of cucumber (Cucumis sativus), melons (Cucumis melo), watermelon (Citrullus lanatus), pumpkin (Cucurbita pepo) and squash (Cucurbita maxima) (Farr and Rossman 2020). To our knowledge, this is the first report of C. orbiculare causing anthracnose of pointed gourd. This disease represents a threat to producers in India and central Asia. Further research may contribute to the development of management strategies for this disease.

Characterization of a hemolytic and antibiotic-resistant Pseudomonas aeruginosa strain S3 pathogenic to fish isolated from Mahananda River in India.

Virulent strain Pseudomonas aeruginosa isolated from Mahananda River exhibited the highest hemolytic activity and virulence factors and was pathogenic to fish as clinical signs of hemorrhagic spots, loss of scales, and fin erosions were found. S3 was cytotoxic to the human liver cell line (WRL-68) in the trypan blue dye exclusion assay. Genotype characterization using whole genome analysis showed that S3 was similar to P. aeruginosa PAO1. The draft genome sequence had an estimated length of 62,69,783 bp, a GC content of 66.3%, and contained 5916 coding sequences. Eight genes across the genome were predicted to be related to hemolysin action. Antibiotic resistance genes such as class C and class D beta-lactamases, fosA, APH, and catB were detected, along with the strong presence of multiple efflux system genes. This study shows that river water is contaminated by pathogenic P. aeruginosa harboring an array of virulence and antibiotic resistance genes which warrants periodic monitoring to prevent disease outbreaks.

Siderophore-producing Bacillus amyloliquefaciens BM3 mitigate arsenic contamination and suppress Fusarium wilt in brinjal plants.

AIM: Arsenic contamination in agricultural soils poses a serious health risk for humans. Bacteria that produce siderophores, primarily for iron acquisition, can be relevant in combating arsenic toxicity in agricultural soils and simultaneously act as biocontrol agents against plant diseases. We evaluated the arsenic bioremediation and biocontrol potential of the rhizosphere isolate Bacillus amyloliquefaciens BM3 and studied the interaction between the purified siderophore bacillibactin and arsenic. METHODS AND RESULTS: BM3 showed high arsenic resistance [MIC value 475 and 24 mM against As(V) and As(III), respectively] and broad spectrum in-vitro antagonism against several phytopathogenic fungi. BM3 was identified by biochemical characterization and 16S rRNA gene sequencing. Scanning electron microscopy (SEM) analysis revealed increased cell size of BM3 when grown in presence of sub-lethal arsenic concentrations. Bioremediation assays showed a 74% and 88.1% reduction in As(V) and As(III) concentrations, respectively. Genetic determinants for arsenic resistance (arsC and aoxB) and antifungal traits (bacAB and chiA) were detected by PCR. Arsenic chelating ability of bacillibactin, the siderophore purified from culture filtrate of BM3 and identified through spectroscopic data analysis, was observed in CAS assay and fluorescence spectrometry. In-vivo application of talc-based formulation of BM3 in brinjal seedlings showed significant reduction in Fusarium wilt disease. CONCLUSION: Strain B. amyloliquefaciens BM3 may be useful in arsenic bioremediation and may be considered for large field trials as an alternative to chemical fungicides by inhibiting soil borne pathogens.

Detection of Virulence-Associated Genes and in vitro Gene Transfer From Aeromonas sp. Isolated From Aquatic Environments of Sub-himalayan West Bengal.

Aeromonas is omnipresent in aquatic environments and cause disease within a wide host range. A total of thirty-four isolates from water samples of small fish farms were identified as Aeromonas based on biochemical characteristics and 16S rRNA gene sequence. A total of six virulent factors were analyzed which indicated 100% of isolates as beta-haemolytic and proteolytic, whereas 44.1, 38.2, and 70.6% of isolates produced DNAse, siderophore, and amylase, respectively. Studies on the occurrence of four genetic determinants of virulence factors revealed that aer/haem (haemolytic toxin) and flaA (polar flagella) genes were present in 44.1% of strains whereas ascV (type 3 secretion system) and aspA (serine protease) genes were detected in 21.5 and 8.82% of strains, respectively. Fish (Anabas testudineus) challenge studies showed that the isolate GP3 (Aeromonas veronii) bearing five virulent factors with the combination of aer/haem + /ascV + /fla + genes induced severe lesions leading to 100% of mortality. In contrast, RB7 possessing four virulence factors and three genes (aer/haem + /ascV + /aspA +) could not produce severe lesions and any mortality indicating the absence of correlation between the virulence factors, its genes, and the pathogenicity in fishes. GP3 was cytotoxic to human liver cell line (WRL-68) in trypan blue dye exclusion assay. The 431 bp aer/haem gene of GP3 was transferable to E. coli Dh5α with a conjugational efficiency of 0.394 × 10 -4 transconjugants per recipient cell. The transfer was confirmed by PCR and by the presence of 23-kb plasmids in both donor and transconjugants. Therefore, the occurrence of mobile genetic elements bearing virulence-associated genes in Aeromonas indicates the need for periodic monitoring of the aquatic habitat to prevent disease outbreaks.

Contact Line Pinning and Depinning Can Modulate the Rod-Climbing Effect.

Our experiments on the rod-climbing effect with an oil-coated rod revealed two key differences in the rod-climbing phenomena compared to a bare rod. First, an enhancement in the magnitude of climbing height for any particular value of the rod rotational speed and second, a decrease in the threshold rod rotational speed required for the appearance of the rod-climbing effect were observed. Observed phenomena are explained by considering the contact line behavior at the rod-fluid interface. Transient evolution of the meniscus at the rod-fluid interface revealed that the three-phase contact line was pinned for a bare rod and depinned for an oil-coated rod. We modeled the subject fluid as a Giesekus fluid to predict the climbing height. The differences in the contact line behavior were incorporated via the contact angle at the rod-fluid interface as a boundary condition. Agreement was found between the observed and predicted climbing height, establishing that contact line behavior may modulate the rod-climbing effect.

Universality in coalescence of polymeric fluids.

A pendant drop merging with a sessile drop and subsequently forming a single daughter drop is known to exhibit complex topologies. But their dynamics are yet to be probed for fluids exhibiting characteristic relaxation time scales while undergoing the deformation process. Here, we unveil a universal temporal evolution of the neck radius of the daughter drop during the coalescence of two polymeric drops. Such a generalization does not rely on the existence of previously explored viscous and inertial dominated regimes for simpler fluids but is fundamentally premised on a unique topographical evolution with essential features of interest exclusively smaller than the dominant scales of the flow. Our findings are substantiated by a theoretical model that considers the drops under coalescence to be partially viscous and partially elastic in nature. These results are substantiated with high-speed imaging experiments on drops of polyacrylamide (PAM), polyvinyl alcohol (PVA), polyethylene oxide (PEO), and polyethylene glycol (PEG). The observations herein are expected to hold importance for a plethora of diverse processes ranging from biophysics and microfluidics to the processing of materials in a wide variety of industrial applications.

Well differentiated papillary mesothelioma of abdomen- a rare case with diagnostic dilemma.

Well-differentiated papillary mesothelioma is a rare tumor occurring predominantly in the peritoneum of young women, a few with history of asbestos exposure. A 28-year-old woman presented with ascites and pain abdomen. Ultrasonography and computed tomography scan of the abdomen revealed a mass in the retroperitoneum measuring 15 cm × 12 cm. Histopathological examination along with immunohistochemistry (IHC) confirmed it to be a papillary mesothelioma in the peritoneum. It is difficult to differentiate from more common malignant mesothelioma and papillary adenocarcinoma, which also have poorer prognosis. The difficulty can be resolved by clinico-radiological correlation along with histopathological examination and IHC.

Evaluation of the biocontrol efficacy of a Serratia marcescens strain indigenous to tea rhizosphere for the management of root rot disease in tea.

The aim of the present study is to evaluate plant growth promoting and biocontrol efficacy of a Serratia marcescens strain ETR17 isolated from tea rhizosphere for the effective management of root rot disease in tea. Isolated bacterial culture ETR17 showed significant level of in vitro antagonism against nine different foliar and root pathogens of tea. The phenotypic and molecular characterization of ETR17 revealed the identity of the bacterium as Serratia marcescens. The bacterium was found to produce several hydrolytic enzymes like chitinase, protease, lipase, cellulase and plant growth promoting metabolites like IAA and siderophore. Scanning electron microscopic studies on the interaction zone between pathogen and antagonistic bacterial isolate revealed severe deformities in the fungal mycelia. Spectral analyses (LC-ESI-MS, UV-VIS spectrophotometry and HPLC) and TLC indicated the presence of the antibiotics pyrrolnitrin and prodigiosin in the extracellular bacterial culture extracts. Biofilm formation by ETR17 on polystyrene surface was also observed. In vivo application of talc-based formulations prepared with the isolate ETR17 in tea plantlets under green house conditions revealed effective reduction of root-rot disease as well as plant growth promotion to a considerable extent. Viability studies with the ETR17 talc formulation showed the survivability of the isolate up to six months at room temperature. The sustenance of ETR17 (concentration of 8-9x108 cfu g-1) in the soil after the application of talc formulation was recorded by ELISA. Safety studies revealed that ETR17 did not produce hemolysin as observed in pathogenic Serratia strains. The biocontrol strain reported in this study can be used for field application in order to minimize the use of chemical fungicides for disease control in tea gardens.

Phylogeny and synonymous codon usage pattern of Papaya ringspot virus coat protein gene in the sub-Himalayan region of north-east India.

Sub-Himalayan West Bengal is favorable for the production of several fruits and vegetables. Papaya is one of the common plants cultivated in the area. Most of the papaya plants of the area are susceptible to Papaya ringspot virus (PRSV). Coat protein genes of 6 PRSV isolates of the area were sequenced following RT-PCR. Phylogenetic study of the PRSV isolates showed about 80%-90% similarity with Cuban isolates. The codon usage pattern of our isolates was also analyzed, along with several other isolates. PRSV isolates of our study showed a preference for 8 putative optimal codons. Correspondence analysis of the genes of different isolates along the first 2 major axes were done, as the first 2 axes contributed more in shaping codon usage pattern. In the phylogenetic tree constructed by the neighbour-joining method, our isolates clustered together with the east Indian, north Indian, and Bangladeshi isolates. The diversity and codon usage pattern of the PRSV isolates of different regions were studied, and it was observed that the codon usage pattern of PRSV isolates is probably influenced by translational selection along with mutational bias.

Fanconi anemia with incidental haemoglobin e trait: the first reported case in literature.

Fanconi anemia (FA), commonly inherited as an autosomal recessive trait, is the commonest cause of inherited bone marrow failure syndrome often accompanied by skeletal deformities, genitourinary abnormalities and an increased risk of malignancies especially acute myeloid leukemia. Haemoglobin E ((ß26 Glu→Lys)) is common haemoglobin variant in the north-eastern part of India and the northern part of West Bengal. Here, we report a case of FA with concomitant haemoglobin E-trait in a 5-year-old child who presented with weakness, epistaxis, malaena and intermittent fever. She had a hyper-pigmented patch over the nape of neck and polydactyly. Complete blood counts revealed pancytopenia. The bone marrow aspiration and trephine biopsy showed marrow hypoplasia. Chromosome analysis showed chromatid and chromosome breaks in 22 % cells suggestive of FA. The results were corroborated with MMC stress test. Thus, we present the first reported case of FA with haemoglobin E-trait.

Molecular detection and partial characterization of a begomovirus causing leaf curl disease of potato in sub-Himalayan West Bengal, India.

The characteristic disease symptoms of apical leaf curl, crinkled leaves and conspicuous mosaic were observed in potato plants grown in areas of Coochbehar, one of the distinct agroclimatic zones of sub-Himalayan West Bengal. Whitefly (Bemisia tabaci) population was also observed in and around the infected plants. The characteristic disease symptoms and presence of whitefly (Bemisia tabaci) population indicated the possibility of begomovirus infection. Total DNA was extracted from infected samples and PCR was carried out using begomovirus specific primers. PCR product of 1539 nucleotide long containing pre-coat protein, coat protein, AC5, AC3, AC2 and partial AC1 gene of DNA-Asegment and 1001 nucleotide long DNA-B containing BV1 and BC1 genes were submitted to the GenBank (accession numbers HQ597033 for DNA-A and JN390432 for DNA-B). The amplified DNA-A and DNA-B segment showed highest 98% and 99% nucleotide sequence similarity respectively with Tomato leaf curl New Delhi virus (ToLCNDV). In phylogenetic analysis also the virus sequence clustered with ToLCNDV isolates. The disease was successfully transmitted to healthy tomato plants using both whitefly vector, B. tabaci and mechanical sap inoculation using sap of infected potato leaves. It is the first record of begomovirus infection of cultivated potato in sub-Himalayan West Bengal of Eastern India.

A new antifungal coumarin from Clausena excavata.

A new γ-lactone coumarin, named as excavarin-A, showing antifungal activity was isolated from the leaves of Clausena excavata by bioassay guided fractionation method. The structure was elucidated by spectroscopic data analysis and identified as 7((2E)-4(4,5-dihydro-3-methylene-2-oxo-5-furanyl)-3-methylbut-2-enyloxy) coumarin. Minimum inhibitory concentration (MIC) was determined against fifteen fungal strains pathogenic against plants and human. The least MIC was recorded against the human pathogen, Candida tropicalis and the plant pathogens Rhizoctonia solani and Sclerotinia sclerotiorum. Antifungal activities against the human pathogens, Aspergillus fumigatus and Mucor circinelloides and plant pathogens, Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Fusarium oxysporum and Rhizopus stolonifer were stronger than that of the standard antimicrobials.