Vishnu Sukumari Nath

Purification and synergistic antibacterial activity of arginine derived cyclic dipeptides, from Achromobacter sp. associated with a rhabditid entomopathogenic nematode against major clinically relevant biofilm forming wound bacteria.

Skin and chronic wound infections caused by various pathogenic bacteria are an increasing and urgent health problem worldwide. In the present investigation ethyl acetate extract of an Achromobacter sp. associated with a Rhabditis entomopathogenic nematode (EPN), displayed promising antibacterial property and was further purified by silica gel column chromatography to get three different cyclic dipeptides (CDPs). Based on the spectral data and Marfeys analyses, the CDPs were identified as cyclo(D-Leu-D-Arg) (1), cyclo(L-Trp-L-Arg) (2), and cyclo(D-Trp-D-Arg) (3), respectively. Three CDPs were active against all the 10 wound associated bacteria tested. The significant antibacterial activity was recorded by CDP 3, and highest activity of 0.5 μg/ml was recorded against Staphylococcus aureus and Pseudomonas aeruginosa. The synergistic antibacterial activities of CDPs and ampicillin were assessed using the checkerboard microdilution method. The results of the current study recorded that the combined effects of CDPs and ampicillin principally recorded synergistic activity. Interestingly, the combination of CDPs and ampicillin also recorded enhanced inhibition of biofilm formation by bacteria. Moreover, CDPs significantly stimulate the production of IL-10 and IL-4 (anti-inflammatory cytokines) by human peripheral blood mononuclear cells. CDPs do not make any significant effect on the production of pro-inflammatory cytokines like TNF-α. The three CDPs have been studied for their effect on intracellular S. aureus in murine macrophages (J774) using 24 h exposure to 0.5X, 1X, and 2X MIC concentrations. Significant decrease in intracellular S. aureus burden was recorded by CDPs. CDPs also recorded no cytotoxicity toward FS normal fibroblast, VERO, and L231 normal lung epithelial cell lines. Antimicrobial activity of the arginine containing CDPs against the wound associated bacteria is reported here for the first. Moreover, this is also the first report on the production of CDPs by Achromobacter sp. Finally, we conclude that the Achromobacter sp. is an incredibly promising source of natural bioactive secondary metabolites especially against wound pathogenic bacteria that may receive significant benefit in the field of human medicine in near future as topical agents.

Rapid and sensitive detection of Phytophthora colocasiae responsible for the taro leaf blight using conventional and real‐time PCR assay

Conventional and real-time PCR assays were developed for sensitive and specific detection of Phytophthora colocasiae, an oomycete pathogen that causes leaf blight and corm rot of taro. A set of three primer pairs was designed from regions of the RAS-related protein (Ypt1), G protein alpha-subunit (GPA1) and phospho-ribosylanthranilate isomerase (TRP1) genes. In conventional PCR, the lower limit of detection was 50 pg DNA, whereas in real-time PCR, the detection limit was 12.5 fg for the primer based on Ypt1 gene. The cycle threshold values were linearly correlated with the concentration of the target DNA (range of R(2) = 0.911-0.999). All the primer sets were successful in detecting P. colocasie from naturally infected leaves and tubers of taro. Phytophthora colocasiae was detected from artificially infested samples after 18 and 15 h of postinoculation in conventional and real-time PCR assay, respectively. The developed PCR assay proved to be a robust and reliable technique to detect P. colocasiae in taro planting material and for assessing the distribution of pathogen within fields, thus aid in mitigating taro leaf blight.

In vitro standardisation of resistance screening methods in cassava against tuber rot disease

Cassava tuber rot caused by Phytophthora palmivora in growing regions of Tamil Nadu and Kerala, is causing yield loss up to 80%. In the present study, resistance reactions of 10 cassava cultivars were analysed on leaf, stem and tuberous roots by artificial inoculation method in search of a suitable in vitro resistant screening method. Leaf and tuber analysis showed positive correlation (0.883) but the stem-based results showed negative correlation with leaf and tuber analysis. The analysis exhibited the susceptibility of the cassava cultivars against P. palmivora. Leaf analysis was superior in discriminating even small variations in resistance reactions than tuber analysis. The cultivar Sree Padmanabha showed higher resistance than other cultivars and the level of resistance in a cultivar is heritable which could be helpful in breeding programme. Based on the results it can be concluded that leaves of cassava could be used for screening resistance in the host and also in analysing the virulence of the isolate. This is the first report on screening the resistance in cassava cultivars against root rot caused by P. palmivora.

Evaluation of fungicides on Indian isolates of Phytophthora colocasiae causing leaf blight of taro

Leaf blight caused by Phytophthora colocasiae is the most destructive disease affecting taro (Colocasia esculenta) worldwide including India. Fungicides (primarily metalaxyl) remain as an important strategy to manage taro leaf blight in India over decades. It is important to monitor isolate sensitivity to identify build-up of fungicide resistance and thereby modify fungicide usage strategies. P. colocasiae isolates representing four different geographical regions of India were evaluated for their sensitivity to metalaxyl and three other commercially available fungicides viz. Samarth, Biofight and Akoton by poisoned media technique. All the isolates tested were sensitive to metalaxyl, nevertheless there is an increase in the effective concentration compared to the previous reports. Among the other fungicides, Samarth was found to be superior in completely inhibiting mycelial growth at 0.05% followed by Biofight at 1%. Metalaxyl and Akoton® shared a common inhibitory concentration at 2%. The most effective fungicide determined by the in vitro method was evaluated in vivo for studying the pattern of inhibition before and after the disease development in detached taro leaf. The results of the study revealed that build-up on metalaxyl resistance in P. colocasiae is in its course and caution should be taken while administering against taro leaf blight. Fungicide Samarth could be used as an alternative to metalaxyl for management of taro leaf blight.

Inhibitory activity of plant growth regulators on Phytophthora palmivora causing cassava tuber rot

Auxins and cytokinins are implicated in a wide variety of developmental and physiological processes in plants. Phytophthora palmivora causes tuber rot in cassava growing regions of Tamil Nadu and Kerala, South India. The in vitro effect of cytokinin, benzyl amino purine (BAP) and auxins, naphthalene acetic acid (NAA) and indole acetic acid (IAA) on P. palmivora mycelium growth was investigated. The inhibitory activity varied among the growth regulators and complete inhibition of the pathogen was observed at 50, 2000 and 2500 ppm by the BAP, IAA and NAA, respectively. The effective growth regulator, BAP was also analysed on tubers before and after the invasion of the pathogen to observe its effect in tuber. Further, it was also checked against the bio-control agent Trichoderma harzianum. The study indicates that the use of BAP could be an important approach in controlling tuber rot pathogen, P. palmivora.

Morphological, pathological and molecular characterization of Phytophthora colocasiae responsible for taro leaf blight disease in India

The oomycetous fungus Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro and is widely distributed in India. A combination of morphological (colony morphology, mating type, pathogenicity, metalaxyl sensitivity) and molecular techniques (rDNA ITS sequencing and Start codon targeted polymorphism, ScoT analysis) was used to characterize 50 isolates of P. colocasiae obtained from different locations in India. Considerable differences in morphological parameters were observed. ScoT analysis revealed high polymorphism among the isolates. This study confirms that isolates of P. colocasiae are highly dynamic in nature and a considerable degree of diversity exists among them. A detailed knowledge of the morphological and molecular characters of P. colocasiae will help in developing suitable control strategies against the taro leaf blight disease.

Incidence and identification of Cassava tuber rot caused by Phytophthora palmivora

The occurrence of Cassava tuber rot in regions of Kolli hills, Kollam, and Kottayam of South India, causes major economic loss up to 70% in Cassava production. The disease tuber is characterised by brown watery lesions with foul smell, making it unfit for further use. The sporangia of the pathogen were oval and ellipsoid with a short pedicle. Identification of the isolate from these regions was also confirmed by ribosomal internal transcribed spacer (ITS) of rDNA region. The pathogen was highly aggressive when pathogenicity was tested. Based on morphological, pathogenicity and ITS sequences, the pathogen was identified as Phytophthora palmivora. Development of integrated disease management practices is essential to combat the disease. This is the first report recording the spread of Cassava tuber rot disease in regions of Kolli hills of Tamil Nadu and Kollam and Kottayam, of Kerala.

Molecular diagnosis of Colletotrichum gloeosporioides causing Anthracnose/Dieback disease in Greater Yam (Dioscorea alata L.)

The Greater Yam (Dioscorea alata L.), a significant tropical tuber crop is highly affected by the anthracnose/dieback disease caused by Colletotrichum gloeosporioides, which greatly reduces the yield as well as market acceptability of the tubers. The different methods that could be used for proper identification of the pathogen by PCR were investigated. The studies indicate that species specific polymerase chain reaction assay based on highly conserved regions in ITS in the genome of the pathogen can be the best strategy for detection of this pathogen at species level. The use of genus specific primers was also successful in detection of Colletotrichum spp. The cloning and sequencing of evolutionarily conserved regions such as ITS, PelB and paralleling them with the available sequences in NCBI database is also costly but reliable approach. The various methods are elaborately tested and their use in diagnosis is discussed in detail.

Genetic and Phenotypic characterization of Phytophthora colocasiae inTaro Growing Areas of India

Phenotypic and molecular methods were used for characterizing 40 Phytophthora colocasiae isolates obtained from Andhra Pradesh, Assam, Kerala, and Odisha regions of India over a period of five years. Phenotypic parameters such as virulence, colony morphology and mating type varied among isolates collected from different regions over the years. No correlation was observed between phenotypic parameters of the isolates and their geographical origins. Considerable inter and intra specific variation were detected by random amplified microsatellites (RAMS) analysis with 100% polymorphism among the isolates. Dendrogram constructed based on RAMS data using the unweighted pair group method with arithmetic mean (UPGMA) grouped the P. colocasiae isolates into two major clusters. No relationship was obtained between RAMS groups of the isolates and phenotypic characters/geographical origin. Population genetic analysis showed that P. colocasiae isolates were highly diverse among different regions. Analysis of molecular variance (AMOVA) showed that most of the genetic variability in P. colocasiae was confined to within a population (93.21%). These results indicate that P. colocasiae populations in India are highly diverse and care should be taken in developing disease management programmes or in breeding resistant cultivars.

Rapid isolation of DNA from Dioscorea species suitable for PCR, restriction digestion and pathogen screening

The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too.