Chitinolytic microbes confer Meloidogyne incognita resistance and augment secondary metabolites in Bacopa monnieri (L.) Pennell

Meloidogyne incognita (Kofoid and White) Chitwood a calamitous plant pathogen affects almost all the commercial crops, especially medicinal plants viz. Bacopa monnieri L. In this study, the efficacy of indigenous chitinolytic microbes against M. incognita in B. monnieri was evaluated under sterilised and natural soil conditions. The plant growth was observed for various parameters viz. nutrient uptake, phytochemical activities and secondary metabolites (bacoside). In the natural soil under green house conditions, the isolates significantly (P ≤ 0.05) reduced M. incognita infestation (1.38–2.78-folds) with significant increase in secondary metabolites (1.28–1.88-fold) over the untreated control. The potential strains were identified through 16S-rRNA as Streptomyces sp., Microbacterium arabinogalactanolyticum, Cellulosimicrobium cellulans, Actinomycetales bacterium, Chitiniphilus sp. and Flavobacterium johnsoniae. These findings thus highlight the potential of indigenous chitinolytic microbes that can be utilised for overall fitness of the plant along with the reduced root-knot nematode infestation in B. monnieri.     

Biotechnological interventions in Withania somnifera (L.) Dunal

Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.     

Fungal foliar diseases in Withania somnifera and its effect on secondary metabolites

Withania somnifera is a promising revitalizing medicinal herb. The plant is affected by foliar diseases in Lakkavalli forest region of Bhadra Wildlife Sanctuary. The symptomatology of foliar fungal disease incidence, severity and distribution in the study area was examined during 2006–2009. The seedborne nature and transmission of the causal pathogen and its management with seed dressing fungicides were studied. The results of the study indicated that Alternaria alternata caused severe leaf spot disease, while Myrothecium roridum and Fusarium oxysporum caused minor diseases. Based on the internal transcribed spacer (ITS1 and ITS2) regions of rDNA, the major pathogen was identified as A. alternata. The disease is homogeneously distributed in Lakkavalli forest region and high severity is recorded during November. Alternaria alternata and Fusarium oxysporum were the dominant seedborne pathogens that are transmitted to seedlings. Among the seed dressing fungicides used, Hyzeb was the most effective, followed by Captra, Antracol and Bavistin, in reducing the incidence of A. alternata and other seedborne fungi. The infected W. somnifera foliages had decreased steroids and alkaloids and increased phenolics and flavonoids. Analysis of alkaloids in diseased foliages by high performance thin layer chromatography indicated the occurrence of transformed compounds at R_f = 0.1, 0.77 (254 nm) and 0.2 (366 nm).     

Efficacy of combined applications of antagonist bacteria and chemical resistance inducers for the management of Fusarium solani causing root rot in Withania somnifera

Application of Thiosalicylic acid+Bacillus cereus; O-Acetylsalicylic acid+Pseudomonas fluorescens reduced root rot severity by 85 and 88% and enhanced root yields by 358 and 419%, respectively, against Fusarium solani induced root rot disease in Withania somnifera. Reduction in disease severity was correlated with defence-related enzymes peroxidase, polyphenol oxidase and phenyl ammonium lyase.     

Screening of antagonistic bacterial strains against Meloidogyne incognita using protease activity

Twenty-six bacterial strains that had demonstrated antagonism to some fungal and bacterial pathogens were evaluated for their ability to inhibit Meloidogyne incognita Kofoid & White. The inhibition rates of egg-hatching and second-stage juveniles (J2) mortality of M. incognita by these strains ranged from ?16.5 to 87.4% and from 1.3 to 77.8%, respectively. The 12 strains causing J2 mortality over 40% were chosen for greenhouse experiments in which their biocontrol efficacy reached 33.3–65.6%. On the other hand, among the 26 strains, 20 demonstrated in vitro protease activity and 14 revealed chitinase activity. Significantly, strains Bacillus sp. AR156 and GJ24 in greenhouse tests showed the strongest protease activities. The analyses of the relationships of the efficacy of the 12 strains with their protease and chitinase activities, respectively, indicated that biocontrol efficacy was highly correlated with protease activity (r=0.92, P<0.001) but barely correlated with chitinase activity. The strong positive correlation between protease activity and efficacy suggests that in vitro protease activity could be used as a parameter for selecting biological control agents (BCAs) against root-knot nematodes. Consistently, the biocontrol efficacy of AR156, GJ24, abamectin reached 74.3, 73.4, and 40.9% in the field in Huai-an, Jiangsu; and 71, 69.9, and 37% in Zao-zhuang, Shandong, respectively. The fact that the strains with high protease activities also had significantly higher biocontrol efficacy than abamectin in the field implies that in vitro protease activity may be adopted as a reliable new parameter for speeding up the process of screening the biological control agents (BCAs).     

In vitro studies of antagonistic fungi against the root-knot nematode,Meloidogyne incognita

The present study was carried out in vitro to determine the efficacy of indigenous fungi isolated from egg masses of root-knot nematode, Meloidogyne incognita on egg parasitism, egg hatching, mobility and mortality against root-knot nematode, M. incognita. The tested fungi were Acremonium strictum, Aspergillus terreus, A. nidulans, A. niger, Chetomium aubense, Chladosporium oxysporum, Fusarium chlamydosporium, F. dimarum, F. oxysporum, F. solani, Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma viride and T. harzianum. All tested fungi showed varied effects against the nematodes. Culture filtrates of A. strictum was very effective against the nematode in regards to egg parasitism (53%), egg hatching inhibition (86%) and mortality (68%) compared to controls. A. strictum was found to have an advantage over P. lilacinus, P. chlamydosporia, T. viride and T. harzianum in that it caused greater mortality of the second stage juveniles (J₂). A. terreus did not show egg parasitism but was found to be highly toxic against second stage juveniles (J₂) causing high mortality (around 68%). Thus, A. strictum and A. terreus showed good biocontrol potential against root-knot nematode, M. incognita under in vitro conditions.     

蓖麻提取物和淡紫拟青霉对南方根结线虫的防治作用

通过杀线活性测定及盆栽试验,研究了蓖麻提取物和淡紫拟青霉(Paecilomyces lilacinus)对南方根结线虫(Meloidogyne incognita)的杀线活性及防治效果.结果表明:蓖麻碱不影响淡紫拟青霉孢子的萌发.蓖麻碱和淡紫拟青霉均具有较强杀线活性,蓖麻碱处理对南方根结线虫的卵孵化抑制率和二龄幼虫死亡率分别达61.7%和59.2%,显著高于对照处理;蓖麻碱和孢子液复合处理接种南方根结线虫的番茄苗后,植株平均根结数为15±3,显著低于对照的平均根结数37±2,株高、鲜重和根长增长率分别比对照提高38.5%、44.0%和57.0%.说明蓖麻提取物和淡紫拟青霉能减轻线虫危害,对番茄南方根结线虫病控制效果明显.     

Cardioprotection from ischemia and reperfusion injury by Withania somnifera: A hemodynamic,biochemical and histopathological assessment

The efficacy of Withania somnifera (Ws) to limit myocardial injury after ischemia and reperfusion was explored and compared to that of Vit E, a reference standard known to reduce mortality and infarct size due to myocardial infarction. Wistar rats (150–200 g) were divided into six groups and received orally saline (sham, control group), Ws-50/kg (Ws control and treated group) and Vit E-100 mg/kg (Vit E control and treated group) respectively for 1 month. On the 31st day, rats of the control, Vit E and Ws treated groups were anesthetized and subjected to 45 min occlusion of the LAD coronary artery followed by 60 min reperfusion. Hemodynamic parameters: systolic, diastolic and mean arterial pressure (SAP, DAP, MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), left ventricular peak (+) LVdP/dt and (–) LVdP/dt were monitored. Hearts were removed and processed for histopathological and biochemical studies: Myocardial enzyme viz, creatin phosphokinase (CPK), and antioxidant parameters: malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx) were estimated. Postischemic reperfusion produced significant cardiac necrosis, depression of left ventricular functions (MAP, LVEDP, (+) and (–) LVdP/dt) and a significant fall in GSH (p < 0.01), SOD, CAT(p < 0.05), LDH and CPK (p < 0.01) as well as an increase in MDA level (p < 0.05) in the control group rats as compared to sham group. The changes in levels of protein and GPx was however, not significant. Ws and Vit E favorably modulated most of the hemodynamic, biochemical and histopathological parameters though no significant restoration in GSH, MAP (with Vit E) were observed. Ws on chronic administration markedly augmented antioxidants (GSH, GSHPx, SOD, CAT) while Vit E did not stimulate the synthesis of endogenous antioxidants compared to sham. Results indicate that Ws significantly reduced myocardial injury and emphasize the beneficial action of Ws as a cardioprotective agent.     

Molecular identification of three isolates of Jatropha mosaic India virus associated with mosaic disease of Withania somnifera in India

The association of begomovirus with mosaic disease of Withania somnifera was detected at three locations in India by PCR using begomovirus-specific degenerate primers. The resulting amplicons of ～1.2 kb from three locations (Aligarh, Lucknow and Hindaun City) were sequenced. The begomovirus isolates of three locations shared 96–97% identities among them and 91% identities with Jatropha mosaic India virus (JMIV). Based on highest sequence identities and close phylogenetic relationships with JMIV, these begomovirus isolates associated with mosaic disease of W. somnifera were identified as isolates of JMIV.     

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as “Indian Ginseng”, is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and the secondary metabolites produced by this plant can be exploited further for the benefit of human health in a sustainable way.     

Mode of action and efficacy of iprodione against the root‐knot nematode Meloidogyne incognita

The ban and restriction on the use of several synthetic chemicals for controlling plant parasitic nematodes, and concern about their side effects necessitate the availability of effective methods of control with low toxicity to humans and non‐target organisms. Therefore, efficacy and mode of action of iprodione, a dicarboximide fungicide, was evaluated against the root‐knot nematode Meloidogyne incognita, in vitro and in vivo conditions, in comparison with the nematicides fenamiphos, fosthiazate and oxamyl at 7.00, 1.66 and 1.66 mL/5 L water, respectively. In vitro, iprodione showed nematostatic rather than nematicidal activity against second‐stage juveniles of M. incognita in contrast to fenamiphos, fosthiazate and oxamyl which were nematicidal. In the in vivo experiment with tomato, iprodione controlled M. incognita less than fenamiphos, fosthiazate and oxamyl. No visual symptoms of phytotoxicity were observed. Therefore, iprodione can be a useful chemical for controlling nematode populations if included in an Integrated Pest Management program.     

Pre-cold acclimation promotes cryoprotectant accumulation and enhances freezing tolerance of Meloidogyne incognita

Strong evidence suggests that cryoprotectant accumulation during pre-cold acclimation protects cells against freezing injuries caused by cellular dehydration. In this study, the concentrations of trehalose and glycerol were measured in Meloidogyne incognita and it was found that both cryoprotectants were significantly accumulated in second-stage juveniles (J2) of M. incognita after acclimation at 4°C. However, compared with non-acclimated samples, only a higher level of trehalose was induced in the egg masses of M. incognita in response to cold treatment. Further characterizations indicated that pre-cold acclimation efficiently accelerated the speed of larvae hatching from egg masses that were subjected to freezing at −1°C. In addition, the survival rate and pathogenicity of M. incognita J2 that had been acclimated prior to freezing were significantly enhanced when compared with non-acclimated J2 individuals. As far as we know, this is the first time that this phenomenon has been reported in M. incognita.     

银胶菊叶和花提取物对南方根结线虫的毒杀活性比较

为进一步明确银胶菊(Parthenium hysterophorus L.)的杀线虫活性,对银胶菊叶和花的不同溶剂提取物、甲醇提取物的不同萃取物以及甲醇提取物碱水层的不同极性组分对南方根结线虫(Meloidogyne incognita Chitwood)的杀虫活性进行了检测,并对不同提取物、萃取物和萃取组分进行了生物碱的定性分析.结果表明:银胶菊叶和花的蒸馏水、甲醇、乙酸乙酯和石油醚提取物的得率分别为24.5％和20.3％、19.6％和10.9％、6.8％和7.7％、2.0％和2.7％,其中,叶和花的蒸馏水和甲醇提取物的杀线虫活性均较强,而石油醚提取物的杀线虫活性最弱.用质量体积分数1.0％和0.5％的叶和花蒸馏水提取物分别处理24和48 h后试虫的校正死亡率均达到100.00％;用质量体积分数1.0％和0.5％的叶和花甲醇提取物处理48 h,试虫的校正死亡率均大于90％.叶和花甲醇提取物的碱水层、三氯甲烷Ⅰ层和Ⅱ层萃取物均具有一定的杀线虫活性,其中,用质量体积分数1.0％的花和叶碱水层萃取物以及花的三氯甲烷Ⅰ层萃取物分别处理48 h,试虫的校正死亡率均为100.00％,而三氯甲烷Ⅱ层萃取物的杀线虫活性最弱.银胶菊叶和花甲醇提取物碱水层的11个不同极性组分(A1～A11)也表现出不同程度的杀线虫活性,其中,用质量体积分数0.2％和0.1％花的A2[溶剂为V(三氯甲烷)∶V(甲醇)=10∶1]和A7[溶剂为V(三氯甲烷)∶v甲醇)=1∶1]组分以及叶的A2和A6[溶剂为V(三氯甲烷)∶V(甲醇)=2∶1]组分处理48 h后,试虫的校正死亡率均达100.00％,显著高于其他组分.定性实验结果表明:银胶菊叶和花中具有杀线虫活性的提取物、萃取物和萃取组分中均含有生物碱.研究结果说明:银胶菊花的杀线虫活性高于叶片,其毒杀活性不仅与提取部位及溶剂的种类和极性有关,还与提取物浓度及作用时间等因素有关.     

Nematicidal activity of powder and extracts of Melia azedarach fruits against Meloidogyne incognita

Pulverised lyophilised (dehydrated) Melia azedarach (Meliaceae) fruits (PMF) were tested in a dose–response pot experiment against juveniles (J2) of the root-knot nematode Meloidogyne incognita. Successively, with different extraction procedures, a polar (melia methanol extract, MME) and a non-polar (melia oil, MO) fragments were obtained and their effects were tested on nematode motility and development, in dose and time response bioassays and pot experiments. An EC₅₀ value was calculated for all experiments. A dose–response effect was found in pot bioassays using PMF and, after an incubation period of 24 and 48 h, the EC₅₀ values were calculated at 0.41% and 0.34% w/w, respectively. Motility bioassays revealed a dose and time dependent response effect, after exposure to MME, but not to MO. Doses of MME higher than 0.08% were nematicidal, whereas lower ones were nematostatic (the loss of motility as a result of the presence of the substance was reversible). In a pot experiment, MME doses higher than 2.5% w/w caused 100% nematode control with EC₅₀ value of 0.916% w/w.     

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha^?1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha^?1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisation of M. incognita eggs (75%). Under field trials also, the best protection of root-knot disease of tomato (Lycopersicon esculentum L.), in terms of reduction of galls (61%) and reproductive factor (P_f/P_i (RF) = 0.2) was achieved through application of HYBDPL-04 + FYM compared to control and other tested isolates. It also enhanced marketable yield of tomato up to 43%. It is concluded that the HYBDPL-04 strain of P. lilacinum is highly effective for management of root-knot disease of tomato under naturally infested field conditions. It is the isolate which produced the maximum number of metabolites which were extracted through high pressure liquid chromatography.     

Effect of certain organic materials in controlling Meloidogyne incognita root-knot nematode infesting banana

Efficacy of certain plant wastes as onion bulb envelope, dry leaves of sugar beet, fleabane and jojoba, filter cake or mud as sugar cane industrial residue and nile fertile mineral bio-fertilisers were studied under field conditions for managing Meloidogyne incognita on banana Cv. Williams. All the tested treatments significantly (P ≤ 0.05 and 0.01) proved to be effective in reducing the studied nematode criteria during the growing season of banana. The highest percentage reductions of 87.5 and 85.5% were recorded in the number of second-stage juveniles caused by fleabane at vegetative and harvest stages, respectively. As for galls, the highest percentage reductions of 80.4 and 79.6% were achieved at harvest stage by sugar beet waste and filter cake residue, respectively. Also, sugar beet waste was the best at increasing banana fruit yield per feddan (77.0%), followed by jojoba (53.1%) and fleabane (50.4%). The number of fingers and hands per bunch increased by the different materials at various degrees.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

Nemato-toxic analysis of several chopped plant leaves against Meloidogyne incognita affecting tomato In vitro and In pots

Tomato plant is affected by several pathogens, including root-knot nematodes (RKNs), belonging to the genus Meloidogyne. Meloidogyne incognita is among the most potent pests infecting tomato roots. Therefore, it is of interest to discuss the management of Meloidogyne incognita using selected botanicals such as Cammelina benghalensis, Evolvulus nummularius, Gomphrena celosioides, Lindenbergia indica, Scoparia dulcis and Vernonia cinerea. The second-stage juveniles (J2s) of M. incognita were directly treated with the aqueous extracts of the botanicals at varied concentration ranging from 10-100%. 100% concentration of Lindenbergia indica was found to be the most toxic against the survival of J2s of M. incognita as compared to other concentrations. In vitro tests also showed the maximum inhibition in egg hatching at 100% concentration after seven days in the extract of Lindenbergia indica. Moreover, botanicals significantly reduced the infestations in relation to number of root galls, eggmasses/root and nematode population/250 g soil in pots. The plant treated with Scoparia dulcis leaves showed the highest nematicidal efficacy with maximum reductions in all the pathological parameters as compared to the untreated control. All treatments resulted in increased growth, physiological parameters and decreased pathological parameters of tomato.     

On the action and mechanism of withaferin-A from Withania somnifera,a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.