Impact of biofertilisers on iron homeostasis and grain quality in the rice variety Uma under Elevated CO2.

The diminishing nutritional quality of rice with increasing concentrations of atmospheric CO2 is currently a major global concern. The present study was designed with the objective of assessing the impact of biofertilisers on grain quality and iron homeostasis in rice under elevated CO2. A completely randomised design with four treatments ([KAU, POP (control), POP+Azolla, POP+PGPR, and POP+AMF]), each replicated three times under ambient and elevated CO2 conditions, was followed. The analysed data revealed that yield, grain quality, and iron uptake and translocation were modified in an unfavourable manner under elevated CO2, which was reflected in the lower quality and iron content of the grains. The response of iron homeostasis in the experimental plants to the application of biofertilisers, especially plant-growth-promoting rhizobacteria (PGPR), under elevated CO2 strongly suggests the possibility of utilising them for designing iron management strategies for achieving higher quality in rice.

Silver nanoparticles green synthesized with leaf extract of disease-resistant amaranthus genotypes effectively suppress leaf blight (Rhizoctonia solani Kühn) disease in a susceptible red amaranthus cultivar.

Silver nanoparticles (AgNPs) were green synthesized using leaf extract of the leaf blight disease (Rhizoctonia solani) susceptible red amaranthus (Amaranthus tricolor L.) and the disease-resistant green (A. dubius) and the wild amaranthus (A. viridis) genotypes, physically characterized, and assessed for their anti-fungal effects against R. solani. The green synthesized nanostructures showed an absorption maximum typical of silver nanoparticles in spectroscopy, and face-centered cubic structures in X-ray diffraction. Field Emission Scanning Electron Microscopic analysis and High-Resolution Transmission Electron Microscopy revealed the size range to be 35-45 nm for all the samples. In vitro mycelial growth inhibition of the pathogen occurred with 500 and 750 ppm concentrations of the nanoparticles in a poisoned-food assay. Further, detached leaves of red amaranthus variety were sprayed with the nanoparticles, and then challenged with the pathogen. There was significant difference in lesion development on leaves sprayed with Ad-AgNPs and Av-AgNPs compared to those treated with At-AgNPs. In the in vivo assay, challenging with the pathogen after spraying the foliage of the leaf blight susceptible red amaranthus variety with Ad-AgNPs at 750 ppm concentration recorded the lowest disease index (7.40) followed by that received Av-AgNPs spray at the same concentration (17.69), five days after inoculation. At-AgNPs treated plants had a disease index of 49.38. Our findings suggest that application of AgNPs green synthesized with leaf extract of disease-resistant genotypes of amaranthus effectively suppressed leaf blight disease incidence in a susceptible amaranthus genotype. To our knowledge, this is the first report on the improved plant pathogen-suppressive activity of any metal nanoparticle when biogenically synthesized using extracts from a disease-resistant plant genotype.

Root colonization by the endophytic fungus Piriformospora indica shortens the juvenile phase of Piper nigrum L. by fine tuning the floral promotion pathways.

Piriformospora indica, the mutualistic biotrophic root colonizing endosymbiotic fungus belonging to the order Sebacinales, offers host plants various benefits and enhances its growth and performance. The effect of colonization of P. indica in Piper nigrum L. cv. Panniyur1 on growth advantages, floral induction and evocation was investigated. Growth and yield benefits are credited to the alteration in the phytohormone levels fine-tuned by plants in response to the fungal colonization and perpetuation. The remarkable upregulation in the phytohormone levels, as estimated by LC- MS/MS and quantified by qRT-PCR, revealed the effectual contribution by the endophyte. qRT-PCR results revealed a significant shift in the expression of putative flowering regulatory genes in the photoperiod induction pathway (FLOWERING LOCUS T, LEAFY, APETALA1, AGAMOUS, SUPPRESSOR OF CONSTANS 1, GIGANTEA, PHYTOCHROMEA, and CRYPTOCHROME1) gibberellin biosynthetic pathway genes (GIBBERELLIN 20-OXIDASE2, GIBBERELLIN 2-OXIDASE, DELLA PROTEIN REPRESSOR OF GA1-3 1) autonomous (FLOWERING LOCUS C, FLOWERING LOCUS VE, FLOWERING LOCUS CA), and age pathway (SQUAMOSA PROMOTER LIKE9, APETALA2). The endophytic colonization had no effect on vernalization (FLOWERING LOCUS C) or biotic stress pathways (SALICYLIC ACID INDUCTION DEFICIENT 2, WRKY family transcription factor 22). The data suggest that P. nigrum responds positively to P. indica colonization, affecting preponement in floral induction as well as evocation, and thereby shortening the juvenile phase of the crop.

Novel in vitro methods for simultaneous screening of two antagonistic bacteria against multiple fungal phytopathogens in a single agar plate.

Two novel and rapid in vitro agar plate methods for simultaneous screening of two bacterial isolates for antagonism against multiple fungal phytopathogens in a single agar plate are described. In the modified plus streak method (MPS), in a 10 cm diameter PDA agar plate, two different bacterial isolates were screened against four fungal pathogens. The second method used 20 cm diameter PDA agar plate surface and was termed as the parallel multiple streak (PMS) method. Here, two bacterial isolates were screened for antagonism against ten fungal phytopathogens on a single agar plate surface. Our new methods are rapid and more economical, and could reduce the number of agar plates and the medium required for in vitro screening by several fold, compared to that used in the conventional dual culture plate assay.

Farm typology of smallholders integrated farming systems in Southern Coastal Plains of Kerala, India.

Adoption of an integrated farming system (IFS) is essential to achieve food and nutritional security in small and marginal holdings. Assessment of IFS to know the resource availability and socio-economic condition of the farm household, farm typology plays a critical role. In this regard, a sample survey of 200 marginal households practicing mixed crop-livestock agriculture was conducted during 2018-2019 at Southern Coastal Plains, which occupies 19,344 ha in Thiruvananthapuram district, Kerala, India. Farming system typology using multivariate statistical techniques of principal component analysis and cluster analysis characterized the diverse farm households coexisting within distinct homogenous farm types. Farming system typology identified four distinct farm types viz. resource constrained type-1 households with small land owned, high abundance of poultry, very low on-farm income, constituted 46.5%; resource endowed type-2 households oriented around fruit and vegetable, plantation crop, with a moderate abundance of large ruminant and poultry, high on-farm income, constituted 12.5%; resource endowed type-3 household oriented around food grain, extensive use of farm machinery, with a moderate abundance of large ruminant, low on-farm income, constituted 21.5%; and resource endowed type-4 household oriented around fodder, with high abundance of large ruminant, medium on-farm income, constituted 19.5% of sampled households. Constraint analysis using constraint severity index assessed the severity of constraints in food grain, horticulture, livestock, complementary and supplementary enterprises in each farm type, which allowed targeted farming systems interventions to be envisaged to overcome soil health problems, crops and animal production constraints. Farming system typology together with constraint analysis are therefore suggested as a practical framework capable of identifying type-specific farm households for targeted farming systems interventions.

Improved transformation of Agrobacterium assisted by silver nanoparticles.

In transgenic plant development, the low transformation efficiency of Agrobacterium with exogenous DNA is the major constraint, and hence, methods to improve its transformation efficiency are needed. Recently, nanoparticlemediated gene transfer has evolved as a key transformational tool in genetic transformation. Since silver nanoparticles (AgNPs) can induce pores on the cell membrane, their efficacy in the improvement of conventional calcium chloride freeze-thaw technique of transformation of Agrobacterium was explored in this study. Agrobacterium cells in the exponential growth phase were exposed to different concentrations of AgNPs (0.01, 1, 5, 10, and 20 mg/l), and the half-maximal effective concentration (EC50) was determined via Probit analysis using the SPSS software. Transformation efficiency of AgNPs alone and in combination with calcium chloride was compared with that of the conventional calcium chloride freeze-thaw technique. AgNPs at a concentration of 0.01 mg/l in combination with calcium chloride (20 mM) showed a ten fold increase in the transformation efficiency (3.33 log CFU (colony-forming unit/microgram of DNA) of Agrobacterium tumefaciens strain EHA 105 with plasmid vector pART27 compared with the conventional technique (2.31 log CFU/µg of DNA). This study indicates that AgNPs of size 100 nm can eliminate the freeze-thaw stage in the conventional Agrobacterium transformation technique, with a 44% improvement in efficiency. The use of AgNPs (0.01 mg/l) along with 20 mM calcium chloride was found to be an economically viable method to improve the transformation of Agrobacterium with exogenous plasmid DNA.

Silver nanoparticles for biolistic transformation in Nicotiana tabacum L.

The present study reports the use of silver nanoparticles as a gene carrier, substituting gold microcarrier for biolistic gene delivery in Nicotiana tabacum L. Efficiency of biolistic transformation using silver nanoparticles (100 nm) was compared with that of gold microcarriers (0.6 micron) under varying helium pressure (450 psi, 650 psi, 900 psi and 1100 psi) and target distance (6 cm and 9 cm). Among the different concentrations (0.01-100 mgL-1) of silver nanoparticles tried, 10 mgL-1 produced the highest number of transient GUS expression (30) with statistical significance. Helium pressure of 650 and target distance of 9 cm, and 900 psi pressure and 6 cm distance resulted in the highest GUS expression with gold microcarriers and silver nanoparticles, respectively. Transformation efficiency was significantly higher with silver nanoparticles than gold microparticles as carriers resulting in a reduction up to 37.5-fold on the cost of consumables. Regeneration efficiencies of tissues bombarded with gold microcarriers and silver nanoparticles were 62.5% and 70.83%, respectively.

Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates' antagonism against multiple fungal phytopathogens.

Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay.

A novel approach for increasing transformation efficiency in E. coli DH5α cells using silver nanoparticles.

The present study is the first report on the application of silver nanoparticles for efficient bacterial transformation. EC50 value of 100 nm silver nanoparticles against E. coli DH5α cells was recorded as 4.49 mg L-1 in toxicity assay. Competency induction in E. coli DH5α cells by treatment with 100 nm silver nanoparticles at a concentration of 1 mg L-1 for 60 min and transformation using three plasmid vectors of different sizes, viz. pUC18, pBR322 and pCAMBIA resulted in tenfold increase in the bacterial transformation efficiency, i.e. 8.3 × 104, 8.0 × 104 and 7.9 × 104 cfu ng-1 of DNA, respectively, even without heat shock compared to the conventional chemical method using 0.1 M calcium chloride (2.3 × 103 cfu ng-1 of DNA).

Piriformospora indica cell wall extract as the best elicitor for asiaticoside production in Centella asiatica (L.) Urban, evidenced by morphological, physiological and molecular analyses.

Vascular plants synthesise a multitude of organic molecules or phytochemicals, referred to as "secondary metabolites". These molecules are involved in a variety of roles in the life span of plants, ranging from structural ones to protection. Centella asiatica (L.) Urban has probably been used since prehistoric times and has been reported to have been used for various medicinal and cosmetic purposes. The plant contains several active constituents, of which the most important is asiaticoside, a triterpenoid. Asiaticoside content in C. asiatica can be enhanced by the use of biotic elicitors like Piriformospora indica. P. indica has been used as a model to study the mechanisms and evolution of mutualistic symbiosis. P. indica is similar to Arbuscular Mycorrhizal (AM) fungi in terms of plant growth promotional effects. The autoclaved fraction from P. indica (PiCWE) was found to be the most active fraction in promoting the plant biomass and asiaticoside content. To date, there are no reports on the potential role of PiCWE in enhancement of asiaticoside over the control and P. indica colonized plants, which was evidenced by the differential expression of key genes involved and final asiaticoside content along with the determination of phytohormones. Moreover, differential expression of selected miRNAs in PiCWE - C. asiatica root interactions over the control and P. indica treated C. asiatica leaf samples was also scrutinized. The important consequence of induction with PiCWE was the significant enhancement of asiaticoside in the PiCWE induced plants in comparison with the asiaticoside content in control and P. indica-C. asiatica interaction. In addition, the role of miRNAs in C. asiatica - PiCWE would enable more in-depth studies for deciphering the molecular and physiological mechanisms of the association and regulation of PiCWE - C. asiatica interactions.

Efficacy of Plant Growth-Promoting Rhizobacteria, Acibenzolar-S-Methyl, and Soil Amendment for Integrated Management of Bacterial Wilt on Tomato.

Greenhouse experiments were conducted to study the effect of plant growth promoting rhizobacteria (PGPR; Bacillus pumilus SE 34, Pseudomonas putida 89B61, BioYield, and Equity), acibenzolar-S-methyl (Actigard), and a soil amendment with S-H mixture (contains agricultural and industrial wastes such as bagasse, rice husk, oyster shell powder, urea, potassium nitrate, calcium super phosphate, and mineral ash) on bacterial wilt incidence caused by Ralstonia solanacearum (race 1, biovar 1) in susceptible tomato (Lycopersicon esculentum cv. Solar Set). In experiments with PGPR, Pseudomonas putida 89B61 significantly reduced bacterial wilt incidence when applied to the transplants at the time of seeding and 1 week prior to inoculation with Ralstonia solanacearum. BioYield, a formulated PGPR that contained two Bacillus strains, decreased disease significantly in three experiments. Equity, a formulation containing more than 40 different microbial strains, did not reduced wilt incidence compared with the untreated control. With inoculum at low pathogen densities of 1 × 105 and 1 × 106 CFU/ml, disease incidence of Actigard-treated plants was significantly less than with nontreated plants. This is the first report of Actigard-mediated reduction of bacterial wilt incidence in a susceptible tomato cultivar. When PGPR and Actigard applications were combined, Actigard plus P. putida 89B61 or BioYield reduced bacterial wilt incidence compared with the untreated control. Incorporation of S-H mixture into infested soil 2 weeks before transplanting reduced bacterial wilt incidence in one experiment. Combination of Actigard with the S-H mixture significantly reduced bacterial wilt incidence in tomato in two experiments.

Screening of antagonistic bacteria for biological control of nursery wilt of black pepper (Piper nigrum).

Bacterial antagonists of Phytophthora capsici were isolated from underground shoot portions of rooted cuttings of black pepper. Initially isolates were screened by dual culture on potato dextrose agar and carrot agar. Further, a screening was done on black pepper shoots for supression of lesion caused by the pathogen. Most of the antagonists showed varying levels of antagonism in the dual culture and the shoot assay. Isolate PN-026, showing the highest suppression of lesion development in the shoot assay was found to be the most efficient antagonist in reducing Phytophthora capsici induced nursery wilt of black pepper. This screening involving the host, pathogen, and the antagonist, performed on black pepper shoot (the planting material for this vegetatively propagated crop), could be used as a rapid and reliable method for the isolation of efficient bacterial antagonists of P. capsici.