Showing papers by "Central Tuber Crops Research Institute published in 2014"

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Abstract: Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R. solanacearum has been classified into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.

Drying kinetics and physico-chemical characteristics of Osmo- dehydrated Mango, Guava and Aonla under different drying conditions

Abstract: Mango (Mangiferra indica L), guava (Psiduim guajava L.) slices and aonla (Emblica officinalis L) segments were osmo-dried under four different dying conditions viz., cabinet drier (CD), vacuum oven drier (VOD), low temperature drier (LTD) and solar drier (SD) to evaluate the best drying condition for the fruits. It was found that vacuum oven drying was superior to other mode of drying as it holds maximum nutrients like acidity, ascorbic acid, sugar and water removal and moisture ratio of products. It was found through regression analysis that drying ratio and rehydration ratio was also superior in vacuum drying followed by cabinet drying. In addition, descriptive analysis on sensory score was also found best with vacuum drying while the Non-enzymatic browning (NEB), which is undesirable character on dried product, was more with solar drier.

In Vitro Synergistic Effect of Curcumin in Combination with Third Generation Cephalosporins against Bacteria Associated with Infectious Diarrhea

Abstract: Diarrhea is one of the leading causes of morbidity and mortality in humans in developed and developing countries. Furthermore, increased resistance to antibiotics has resulted in serious challenges in the treatment of this infectious disease worldwide. Therefore, there exists a need to develop alternative natural or combination drug therapies. The aim of the present study was to investigate the synergistic effect of curcumin-1 in combination with three antibiotics against five diarrhea causing bacteria. The antibacterial activity of curcumin-1 and antibiotics was assessed by the broth microdilution method, checkerboard dilution test, and time-kill assay. Antimicrobial activity of curcumin-1 was observed against all tested strains. The MICs of curcumin-1 against test bacteria ranged from 125 to 1000 μg/mL. In the checkerboard test, curcumin-1 markedly reduced the MICs of the antibiotics cefaclor, cefodizime, and cefotaxime. Significant synergistic effect was recorded by curcumin-1 in combination with cefotaxime. The toxicity of curcumin-1 with and without antibiotics was tested against foreskin (FS) normal fibroblast and no significant cytotoxicity was observed. From our result it is evident that curcumin-1 enhances the antibiotic potentials against diarrhea causing bacteria in in vitro condition. This study suggested that curcumin-1 in combination with antibiotics could lead to the development of new combination of antibiotics against diarrhea causing bacteria.

Preparation and characterization of curcumin loaded cassava starch nanoparticles with improved cellular absorption

Abstract: Objectives: The present study describes loading of curcumin onto cassava starch nanoparticles to improve its cellular absorption. Methods: Cassava starch nanoparticles were prepared by acid hydrolysis. A solution of curcumin in acetone was mixed thoroughly with the starch nanoparticles suspension and the loading efficiency was determined. The loaded nanoparticles were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and fluorescence spectra. Antioxidant activity of pure curcumin and curcumin loaded on nanostarch was studied. Cytotoxicity and cellular uptake of the curcumin were studied using L929 fibroblast cell lines. Results: Curcumin was successfully loaded on starch nanoparticles as revealed by microscopic analyses. Antioxidant assay revealed that antioxidant activity was on par for pure curcumin and loaded curcumin as observed from the EC50 values. The result of cytotoxicity showed that curcumin incorporated starch nanoparticles were non-toxic to normal cells. The cellular absorption of curcumin, which was loaded on starch nanoparticles was significantly higher than that of pure curcumin. Conclusion: Curcumin loaded on starch nanoparticles exhibited improved cellular absorption with non-toxicity and hence it can be potentially used in various pharmaceutical applications.

Cassava Starch Based Superabsorbent Polymer as Soil Conditioner: Impact on Soil Physico-Chemical and Biological Properties and Plant Growth

Abstract: Superabsorbent polymers (SAPs) are macromolecules with the ability to absorb water and release it slowly to the surroundings. SAP hydrogels can be used as soil moisture conditioners which act as water reservoirs and release water depending upon the need of plant roots. In the present study, a cassava starch based superabsorbent hydrogel was evaluated as a soil conditioner and its effect on physical, chemical, and biological properties of the soil as well as on plant growth parameters was studied. Amendment of the soil with the starch based SAP hydrogel significantly altered the physical and chemical properties of the soil. Bulk density of the soil was decreased, whereas water holding capacity and porosity were increased on SAP treatment. There was an improvement in the soil status of major, secondary and micronutrients and organic carbon in the SAP amended soil. An increase in bacteria (16%) and fungi counts (18%) as well as superior plant growth were observed for the treatment with SAP and irrigated at an interval of 3 days, compared to those of control without SAP. Soil amendment with starch based SAP hydrogel was found to be beneficial for maintaining the physicochemical and biological soil properties as well as plant growth.

Isolation and identification of antimicrobial secondary metabolites from Bacillus cereus associated with a rhabditid entomopathogenic nematode

Abstract: The cell-free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain six bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (LCMS, FABMS, 1H NMR, 13C NMR, 1H −1H COSY, 1H −13C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), cyclo (L-Pro-D-Phe), cyclo (L-Pro-L-Val), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene, respectively. Compounds recorded antibacterial activity against all four tested bacteria strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. 3,5-dihydroxy-4-isopropylstilbene recorded activity only against Gram-positive bacteria while cyclo(L-Pro-L-Val) recorded no antibacterial activity. Best antibacterial activity was recorded by 3,5-dihydroxy-4-ethyl-trans-stilbene (4 μg/ml) against Escherichia coli. The six compounds recorded significant antifungal activities against five fungal strains tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and they were more effective than bavistin, the standard fungicide. The activity of cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene against Candida albicans was better than amphotericin B. To the best of our knowledge, this is the first report of antifungal activity of the bioactive compounds against the plant pathogenic fungi Fusarium oxysporum, Rhizoctonia solani, and Penicillium expansum. We conclude that the Bacillus cereus strain associated with entomopathogenic nematode is a promising source of natural bioactive secondary metabolites which may receive great benefit as potential sources of new drugs in the agricultural and pharmacological industry.

Bio-processing of bael (Aegle marmelos L.) fruits into wine with antioxidants

Abstract: Wine, from β-carotene rich tropical bael (Aegle marmelos L.) fruits, widely known for their immense medicinal properties (anti-diarrheic, anti-bacterial and anti-inflammatory) was prepared by fermenting with the wine yeast (Saccharomyces cerevisiae) as starter culture. Biochemical and sensory analyses were conducted to study the proximate and sensory attributes of the wine. The wine had the following proximate composition: Total soluble solids, 2.91 Brix; total sugar of 2.03 g/100 ml; titratable acidity, 0.15 g tartaric acid/100 ml; pH, 4.1; total phenolics, 0.93 g/100 ml; β-carotene, 33 mg/100 ml; ascorbic acid, 80 mg/100 ml; lactic acid, 0.64 mg/100 ml and ethanol content of 7.87% (v/v). The bael wine had a DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity of 48% at a dose of 250 mg/ml. Principal Component Analysis reduced the 10 original analytical and proximate variables (TSS, total sugar, TA, pH, phenol, β-carotene, ascorbic acid, lactic acid, ethanol and DPPH scavenging activity) into three independent components which accounted for 80.78% variation. Sensory evaluation was carried out by 16 selected panelists on various attributes such as taste, aroma, flavor, color/appearance and after taste. The results showed that the bael wine was very much liked by the panelists and there was no significant difference between the two sets (po0.05) in most sensory parameters. Wine prepared from bael fruit pulp is a novel beverage rich in anti-oxidants with an alcoholic concentration of 7.87% (v/v). There is

Cyclo(D-Tyr-D-Phe): a new antibacterial, anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode.

Abstract: A new microbial cyclic dipeptide (diketopiperazine), cyclo(d-Tyr-d-Phe) was isolated for the first time from the ethyl acetate extract of fermented modified nutrient broth of Bacillus sp. N strain associated with rhabditid Entomopathogenic nematode. Antibacterial activity of the compound was determined by minimum inhibitory concentration and agar disc diffusion method against medically important bacteria and the compound recorded significant antibacterial against test bacteria. Highest activity was recorded against Staphylococcus epidermis (1 µg/ml) followed by Proteus mirabilis (2 µg/ml). The activity of cyclo(d-Tyr-d-Phe) against S. epidermis is better than chloramphenicol, the standard antibiotics. Cyclo(d-Tyr-d-Phe) recorded significant antitumor activity against A549 cells (IC50 value: 10 μM) and this compound recorded no cytotoxicity against factor signaling normal fibroblast cells up to 100 μM. Cyclo(d-Tyr-d-Phe) induced significant morphological changes and DNA fragmentation associated with apoptosis in A549 cells. Acridine orange/ethidium bromide stained cells indicated apoptosis induction by cyclo(d-Tyr-d-Phe). Flow cytometry analysis showed that the cyclo(d-Tyr-d-Phe) did not induce cell cycle arrest. Effector molecule of apoptosis such as caspase-3 was found activated in treated cells, suggesting apoptosis as the main mode of cell death. Antioxidant activity was evaluated by free radical scavenging and reducing power activity, and the compound recorded significant antioxidant activity. The free radical scavenging activity of cyclo(d-Tyr-d-Phe) is almost equal to that of butylated hydroxyanisole, the standard antioxidant agent. We also compared the biological activity of natural cyclo(d-Tyr-d-Phe) with synthetic cyclo(d-Tyr-d-Phe) and cyclo(l-Tyr-l-Phe). Natural and synthetic cyclo(d-Tyr-d-Phe) recorded similar pattern of activity. Although synthetic cyclo(l-Tyr-l-Phe) recorded lower activity. But in the case of reducing power activity, synthetic cyclo(l-Tyr-l-Phe) recorded significant activity than natural and synthetic cyclo(d-Tyr-d-Phe). The results of the present study reveals that cyclo(d-Tyr-d-Phe) is more bioactive than cyclo(l-Tyr-l-Phe). To the best of our knowledge, this is the first time that cyclo(d-Tyr-d-Phe) has been isolated from microbial natural source and also the antibacterial, anticancer, and antioxidant activity of cyclo(d-Tyr-d-Phe) is also reported for the first time. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Biocontrol of Aspergillus species on peanut kernels by antifungal diketopiperazine producing Bacillus cereus associated with entomopathogenic nematode.

Abstract: The rhabditid entomopathogenic nematode associated Bacillus cereus and the antifungal compounds produced by this bacterium were evaluated for their activity in reducing postharvest decay of peanut kernels caused by Aspergillus species in in vitro and in vivo tests. The results showed that B. cereus had a significant effect on biocontrol effectiveness in in vitro and in vivo conditions. The antifungal compounds produced by the B. cereus were purified using silica gel column chromatography and their structure was elucidated using extensive spectral analyses. The compounds were identified as diketopiperazines (DKPs) [cyclo-(L-Pro-Gly), cyclo(L-Tyr-L-Tyr), cyclo-(L-Phe-Gly) and cyclo(4-hydroxy-L-Pro-L-Trp)]. The antifungal activities of diketopiperazines were studied against five Aspergillus species and best MIC of 2 µg/ml was recorded against A. flavus by cyclo(4-hydroxy-L-Pro-L-Trp). To investigate the potential application of cyclo(4-hydroxy-L-Pro-L-Trp) to eliminate fungal spoilage in food and feed, peanut kernels was used as a food model system. White mycelia and dark/pale green spores of Aspergillus species were observed in the control peanut kernels after 2 days incubation. However the fungal growth was not observed in peanut kernels treated with cyclo(4-hydroxy-L-Pro-L-Trp). The cyclo(4-hydroxy-L-Pro-L-Trp) was nontoxic to two normal cell lines [fore skin (FS) normal fibroblast and African green monkey kidney (VERO)] up to 200 µg/ml in MTT assay. Thus the cyclo(4-hydroxy-L-Pro-L-Trp) identified in this study may be a promising alternative to chemical preservatives as a potential biopreservative agent which prevent fungal growth in food and feed. To the best of our knowledge, this is the first report demonstrating that the entomopathogenic nematode associated B. cereus and cyclo(4-hydroxy-L-Pro-L-Trp) could be used as a biocontrol agents against postharvest fungal disease caused by Aspergillus species.

Fermentation of sapota (Achras sapota Linn.) fruits to functional wine

Abstract: Wine from sapota (Achras sapota Linn.) fruit was prepared by fermenting with wine yeast (Saccharomyces cerevisiae) to preserve the nutritional, antioxidative and functional properties of the fruits. The wine had the following proximate compositions: total soluble solids, 2.38°Brix; total sugar, 3.28 g/100 ml; titratable acidity (TA), 1.29 g tartaric acid/100 ml; pH, 3.02; total phenolics, 0.21 g/100 ml; β-carotene, 22 μg/100 ml; ascorbic acid, 1.78 mg/100 ml; lactic acid, 0.64 mg/100 ml; and ethanol content, 8.23% (v/v). The sapota wine had a 2,2-diphenyl-1picryl hydrazyl (DPPH)-scavenging activity of 46% at a dose of 250 μg/ml. Infrared spectroscopy revealed the presence of alcohols, phenols, anhydrides, amides, esters and alkenes in the sapota wine. Principal component analysis (PCA) reduced the 10 original analytical and proximate variables (total soluble solids, total sugar, TA, pH, phenol, β-carotene, ascorbic acid, lactic acid, ethanol and DPPH-scavenging activity) into four independent components, which accounted for 87.55% variations. Sensory evaluation was carried out by 16 panellists on various attributes like taste, aroma, flavour, colour/appearance and aftertaste. The results showed that the flavour, taste and aroma of sapota wine was strongly liked by the panellists and there was no significant difference (p<0.05) between the two replicates for most sensory parameters.

Rheological and thermal properties of saponified cassava starch‐g‐poly(acrylamide) superabsorbent polymers varying in grafting parameters and absorbency

Abstract: Cassava starch-graft-poly(acrylamide) superabsorbent polymers (SAPs) with varying absorbencies were synthesized. Weight average molecular weight (Mw) of the hydrolyzed starch-graft-copolymers ranged from 1.6 × 106 to 2.8 × 106 g/mol, the largest being shown by the sample with highest percentage grafting. The storage (G′) and loss modulus (G″) of hydrogels were determined as a function of frequency. G″ was larger than G′ for the hydrogels with higher absorbencies and exhibited a liquid-like behavior. However, hydrogels with lower absorbencies showed a reverse viscoelastic behavior. The viscosity of hydrogels determined using a Brookfield viscometer at different shear rates was found to be larger for the hydrogels with higher absorbencies. The melting temperature (Tm) and enthalpy change of fusion (ΔHf) of the SAPs ranged from 149.7 to 177.7°C and 65 to 494.9 J/g, respectively and showed a positive correlation with grafting parameters and Mw. Heavy metal ion removal capacity of hydrogel followed the order Cu2+ > Pb2+ > Zn2+. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40368.

Antifungal Activity of Diketopiperazines and Stilbenes Against Plant Pathogenic Fungi In Vitro

Abstract: The present study aimed to investigate antifungal activity of a stilbene and diketopiperazine compounds against plant pathogenic fungi, including Phytophthora capsici, P. colocasiae, Botrytis cinerea and Colletotrichum gloeosporioides. Minimal inhibition concentrations (MIC) and minimal fungicidal concentrations (MFC) of stilbenes and diketopiperazines for each fungus were determined using microplate method. Best activity was recorded by stilbenes against P. capsici and P. colocasiae. All four test compounds were effective in inhibiting different stages of the life cycle of test fungi. Stilbenes were more effective than diketopiperazines in inhibiting mycelial growth and inhibiting different stages of the life cycle of P. capsici and P. colocasiae. Rupture of released zoospores induced by stilbenes was reduced by addition of 100 mM glucose. The effects of stilbenes on mycelial growth and zoospore release, but not zoospore rupture, were reduced largely when pH value was above 7. In addition, stilbenes were investigated for its antifungal stability against Phytophthora sp. The results showed that stilbenes maintained strong fungistatic activity over a wide pH range (pH 4–9) and temperature range (70–120 °C). The compound stilbenes exhibited strong and stable broad-spectrum antifungal activity, and had a significant fungicidal effect on fungal cells. Results from prebiocontrol evaluations performed to date are probably useful in the search for alternative approaches to controlling serious plant pathogens.

Implications of organic management on yield, tuber quality and soil health in yams in the humid tropics

Abstract: Global consciousness of food safety, health and environmental issues has stimulated interest in alternative agricultural systems like organic farming. Since information on organic farming of tuber crops is meagre, a field experiment was conducted in split plot design over a five-year period at Central Tuber Crops Research Institute, India. The aims were to evaluate the impact of organic, conventional and traditional production systems on yield, proximate composition and mineral content of tubers and soil physico-chemical and biological properties in three species of Dioscorea (white yam: D. rotundata, greater yam: D. alata and lesser yam: D. esculenta). The production systems were assigned to main plots and species to subplots. Organic farming (20.34 t ha -1 ) produced significantly higher yield over conventional practice (18.64 t ha -1 ) by 9%. All the species responded well to organic management, which lowered the bulk density and particle density slightly and improved the water holding capacity (by 15%) of soil. Tuber quality was improved with significantly higher Ca (72.67 mg 100g -1 ), slightly higher dry matter, crude protein, K and Mg contents. Organic plots showed significantly higher available K, by 34% and pH, by 0.46 unit and higher soil organic matter by 14%. The dehydrogenase enzyme activity (1.174 µg TPF formed g -1 soil h -1 ), population of bacteria, fungi and P solubilizers were promoted by 14%, 23%, 17% and 22% respectively. Thus organic farming was found to be an eco-friendly management strategy in yams for sustainable yield of quality tubers besides maintaining soil health. Technology involving farmyard manure, green manuring, neem cake, biofertilizers and ash was standardized.

Endosymbiotic Bacteria Associated with the Mealy Bug, Rhizoecus amorphophalli (Hemiptera: Pseudococcidae)

Abstract: The mealy bug, Rhizoecus amorphophalli, is a menace to the aroid farmers due to the intensive infestation on stored tubers. Spraying of pesticides was able to control this pest but it always left a chance for fungal growth. Bacterial endosymbionts associated with the insects provide several benefits to their host. Since such endosymbionts play a vital role even in the physiology of their host, revealing the types of bacteria associated with mealy bug will give basic information, which may throw light on the management of this noxious pest. The present study is the first to identify bacterial endosymbionts associated with R. amorphophalli employing phenotypic characterization and 16S rDNA sequencing. Three culturable bacteria, namely, Bacillus subtilis, Staphylococcus gallinarum, and Staphylococcus saprophyticus, were isolated from R. amorphophalli. Moreover, the antibiotic susceptibility tests against the isolated bacteria showed that all the isolates were susceptible to the three antibiotics tested, except cephalexin. Recently, endosymbionts are used as effective biocontrol agents (BCAs) and the present study will stand as a connecting link in identification and effective utilization of these endosymbionts as BCAs for management of R. amorphophalli.

Characterization of three depside compounds from a Western Ghat lichen Parmelia erumpens Kurok with special reference to antimicrobial and anticancer activity

Abstract: The aim of this study was to investigate the major chemical compositions of the chloroform extract of lichen Parmelia erumpens from Western Ghats, Kerala, India and its antimicrobial and anticancer activities. Chloroform extract was purified by silica gel column chromatography to obtain three major compounds and their chemical structures were characterized by 1H-NMR, 13C-NMR, UV and HR-MS spectroscopic methods as atranorin (1), (+)-usnic acid (2) and 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid (3). The minimal inhibitory concentration (MIC) by the broth micro dilution and agar disc diffusion methods was used to record the antimicrobial activity. Out of three compounds tested, 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid recorded excellent antimicrobial activity especially against medically important bacteria and fungi and the MIC values ranged from 0.06 to 4 μg ml−1 against test bacteria and 0.12 to 16 μg ml−1 against test fungi. The best MIC of 0.06 μg ml−1 by 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid was recorded against Vibrio cholera, a human pathogenic bacterium responsible for causing life threatening diseases like profuse watery diarrhea. Anti cancer activity was initially screened by MTT assay in A549, B16F10, Caski and HepG2 cell lines. MTT assay results showed that the growth of cancer cells was suppressed by 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid in both dose- and time-dependent manners. A549, B16F10 and Caski cells treated with 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid showed typical apoptotic morphology when stained with acridine orange–ethidium bromide and hoechst staining. Cell cycle analysis clearly indicated that cell death was due to apoptosis. Enhancement in the proliferation of lymphocytes suggested immunomodulatory activity of this compound. To our best knowledge anticancer activity of 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid was reported here for the first time. Thus the results of the present study suggest that 2-hydroxy-4-methoxy-3,6-dimethylbenzoic acid has a strong potential to be developed as an antimicrobial and anticancer drug target after further clinical evaluation.

A protocol for micropropagation of Aloe vera L. (Indian Aloe) – a miracle plant

Abstract: Emerging axillary shoots of Aloe vera (Indian Aloe) produced new plants and roots simultaneously when cultured in MS medium supplemented with BAP 1.5 mg l -1 . Each explant produced on average 14 shoots and roots simultaneously within 8 weeks. Explant multiplication could be continued even after a year by transferring each divided shoot explant to the same medium. Regenerated plantlets could be successfully transferred to the soil where they grew well within 4-6 weeks with 83% survival.

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities

Abstract: The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, 1H NMR, 13C NMR, 1H–1H COSY, 1H–13C HMBC) and Marfey’s method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(l-Pro-l-Trp), cyclo(l-Leu-l-Val), cyclo(d-Pro-d-Met), and cyclo(d-Pro-d-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(l-Leu-l-Val). Cyclo(l-Leu-l-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(l-Pro-l-Trp) against S. aureus (4 μg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 μg/ml by cyclo(l-Pro-l-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete’s foot, jock itch, and ringworm. The activity of cyclo(l-Pro-l-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 μg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial secondary metabolites, which may receive greater benefit as potential sources of new drugs in the pharmaceutical industry.

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

Abstract: 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 ( Ypt 1), G protein alpha-subunit ( GPA 1) and phospho-ribosylanthranilate isomerase ( TRP 1) 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 Ypt 1 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.

Purification and identification of two antifungal cyclic dipeptides from Bacillus cereus subsp. thuringiensis associated with a rhabditid entomopathogenic nematode especially against Fusarium oxysporum

Abstract: The cell-free culture filtrate of Bacillus cereus subsp. thuringiensis associated with an entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain two cyclic dipeptides (CDPs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic analyses (FABMS, (1)H NMR, (13)C NMR, (1)H-(1)H COSY, (1)H-(13)C HMBC) and Marfey's method. The compounds were identified as cyclo(D-Pro-L-Met) and cyclo(D-Pro-D-Tyr). CDPs showed significantly higher activity than the standard fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum. The highest activity of 2 µg/ml by cyclo(D-Pro-D-Tyr) was recorded against F. oxysporum, a plant pathogen responsible for causing fusarium wilt followed by R. solani, a pathogen that causes root rot and P. expansum. To our knowledge, this is the first report on the isolation of these compounds from Rhabditis EPN bacterial strain Bacillus cereus subsp. thuringiensis.

Rapid and Sensitive Detection of Sclerotium rolfsii Associated with Collar Rot Disease of Amorphophallus paeoniifolius by Species-Specific Polymerase Chain Reaction Assay

Abstract: Collar rot disease caused by Sclerotium rolfsii is an economically important disease prevailing in all Amorphophallus growing areas. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of S. rolfsii in soil and planting material. The PCR detection limit was 10 pg in conventional assay whereas 0.1 pg in nested assay. The primers designed were found to be highly specific and could be used for accurate identification of pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.

Analysis of genetic diversity in Phytophthora colocasiae causing leaf blight of taro ( Colocasia esculenta ) using AFLP and RAPD markers

Abstract: 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. Molecular and cultural techniques were employed for assessing and exploiting the genetic variability among isolates of P. colocasiae obtained from different geographical regions of India. Analysis of the 5.8-ITS region revealed detectable intraspecific variation among isolates. Ten random amplified polymorphic DNA (RAPD) and eight amplified fragment length polymorphism (AFLP) primers produced 198 and 510 reproducible fragments, respectively. AFLP produced 100 % polymorphism, whereas RAPD showed 93.5 % polymorphism. The average value of the number of observed alleles, the number of effective alleles, mean Nei’s genetic diversity, and Shannon’s information index were 2.00–1.94, 1.53–1.36, 0.31–0.24, and 0.47–0.40, respectively, for two DNA markers used. Analysis of molecular variance (AMOVA) for both markers produced similar results with the majority (85 %, AFLP; 89 %, RAPD) of the diversity present within population of P. colocasiae. Dendrograms based on two molecular data using the unweighted pair group method with arithmetic mean (UPGMA) was incongruent and classified the P. colocasiae isolates into one and two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r = 0.904) and AFLP (r = 0.825). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed.

Cyclo(L-Pro-D-Arg): a new antibacterial and antitumour diketopiperazine from Bacillus cereus associated with a rhabditid entomopathogenic.

Abstract: In continuation of our search for new antimicrobial secondary metabolites from Bacillus cereus associated with rhabditid entomopathogenic nematode, a new microbial diketopiperazine, cyclo(L-Pro-D-Arg), was isolated from the ethyl acetate extract of fermented modified nutrient broth. The chemical structures of the isolated compounds were identified based on their 1D, 2D NMR and high-resolution electrospray ionisation-mass spectroscopy data. Antibacterial activity of the compound was determined by minimum inhibitory concentration and disc diffusion method against medically important bacteria, and the compound was recorded to have significant antibacterial activity against test bacteria. The highest activity was recorded against Klebsiella pneumoniae (1 μg/mL). Cyclo(L-Pro-D-Arg) was recorded to have significant antitumor activity against HeLa cells (IC50 value 50 μg/mL), and this compound was recorded to have no cytotoxicity against normal monkey kidney cells (VERO) up to 100 μg/mL). To the best of our knowledge, this is the first time that cyclo(L-Pro-D-Arg) has been isolated from a microbial natural source.

Comparative evaluation of manual cassava harvesting techniques in Kerala, India

Abstract: In India, cassava is consumed as a secondary staple along with the main staple, rice, and many rural poor consume it as the staple in different forms of preparations. Though harvesting is known to be one of the most difficult and cost - intensive field operation in cassava cultivation, mechanisation of cassava harvesting is still very low in most cassava growing areas of India due to topographic constraints, method s and scale of cultivation. The most viable solution to overcome these constraints is to promote the use of more efficient manual harvesting tools. Thus, the main objective of this study was to field evaluate the efficiency of four manual cassava harvesting techniques under different land preparation methods in terms of field capacity, level of drudgery and root tuber damage or breakage. The study also sought to investigate the effect of cassava agronomic parameters on uprooting force requirement. Field study was carried out at the Central Tuber Crops Research Institute (CTCRI) research field (under upland mound method) and at Chenkal village on farmers’ fields (under lowland flat method); both in the Kerala state of India. Harvesting was done using the CTCRI lever, prototype harvester, hoe and manual uprooting (control) techniques. Results from the study showed that the use of manual harvesting tools is preferable on relatively dryer soils, whereas manual uprooting technique is best suited for soils with relatively higher moisture contents. However, best efficiency of manual harvesting is achieved when cassava plants are coppiced before harvesting. Also, cassava uprooting force requirement, to a greater extent is influenced by root tuber yield, root depth and number of root tubers per plant, especially under upland mound land preparation method. It is however recommended that a user performance assessment and economic feasibility analysis of the prototype harvester and CTCRI lever be conducted with farmers to facilitate future design modifications, where necessary and to support future adoption. As a design recommendation, the pressure at the fulcrum for both the CTCRI lever and prototype harvester should be reduced to avoid sinking during harvesting in soils with relatively higher moisture contents. Keywords: cassava, field capacity, drudgery, coppiced, efficiency

Characterization of Trichoderma spp. antagonistic to Phytophthora colocasiae associated with leaf blight of taro

Abstract: Eighteen Trichoderma strains isolated from soils of different regions of India were analyzed for their biocontrol potential against the taro leaf blight pathogen Phytophthora colocasiae. The strains were characterized using a combination of phenotypic and molecular approaches, viz., in vitro antagonism, extracellular enzyme production, ITS and tef1 gene sequencing and AFLP analysis. Eight isolates exhibited more than 75 % inhibition of the pathogen P. colocasiae. Differential antagonistic activity was evident for different strains. Assay of lytic enzyme production suggested the role of glucanase in mycoparasitism of P. colocasiae. Sequencing and phylogenetic analysis of the Trichoderma strains showed that isolates belong to three species: Trichoderma asperellum, Trichoderma longibrachiatum and Trichoderma harzianum. AFLP analysis revealed profound genetic diversity among the isolates. The most potent strain overall (TR7) identified as T. harzianum was able to control P. colocasiae in vivo also. This isolate could be used as a promising candidate for biological control of taro leaf blight disease.

Aroids and Water Relations: An Overview

Abstract: Major edible aroids, elephant foot yam, taro and tannia constitute staple food for the low income group and tribal people in India. As such their yield potential has not been well realized. Limited studies have been conducted on the scientific water management, especially water relations and water requirement of aroids. These crops are mostly rainfed. However, they are known to have high water requirement due to great transpiration loss and long duration. Their large leaves are extensive transpiring surfaces through which large quantities of water are transpired. Effects of water deficit stress during various phases of these crops are reported to affect the productivity, particularly, if stress occurs during the tuber bulking phase rather than tuber initiation or maturation phases. Elephant foot yam produces large corms when water supply is adequate. Continuous flooding adversely affects the performance. The initiation of sprouts from seed corms to initiation of senescence is decided by the availability of soil moisture. Taro comes up well in moist environments. Tuber bulking phase has been considered as the most critical phase in taro. Tannia is relatively more tolerant to dry conditions and is sensitive to water-logging, but performs better under irrigation especially when rainfall is irregular. Thus water can be considered as the most critical input in aroid cultivation. Limited studies conducted on water management and water relations of major edible aroids are reviewed here.

Is PCR‐DGGE an Innovative Molecular Tool for the Detection of Microbial Plant Pathogens?

Abstract: Historically, it was necessary to perform time-consuming indexing for microbial detection or to cultivate the microorganisms for one or more days at a certain temperature on the appropriate medium in order to identify microbial communities, using appropriate stains as well as their biochemical and physiological characteristics. This process was obviously not suited to routine analysis of a large number of samples. Unfortunately, the use of new detection techniques in phytopathology has traditionally lagged behind developments in other fields. In the past, it has taken several years from the development of a new technique for clinical diagnosis before it has been applied to detect phytopathogenic viruses and bacteria. Three decades ago, the detection and diagnosis techniques available for bacteria were based on microscopy, isolation, biochemical characterization, serological techniques (mainly IF), bioassays and pathogenicity tests, and for viruses on biological indexing,