Insights on Strain-Substrate Interactions and Antioxidant and Anti-Bacterial Properties of the Velvet Foot Medicinal Mushroom Flammulina velutipes (Agaricomycetes).

To study the best substrate for the Indian subcontinent, four different substrates (sawdust + wheat bran, wheat straw + wheat bran + corn cobs, sawdust + corn cobs and wheat straw + wheat bran) were screened for six different Flammulina velutipes strains. The antioxidant and antibacterial properties were studied for these strains. In study it was found that the strain DMRX-767 and DMRX-768 were the most promising for yield and biological efficiency in all substrates and wheat straw + wheat bran being the best with respect to BE. To corroborate the findings, the best strain and best substrate trails were repeated. DMRX-767 and DMRX-768 were the most promising for yield and biological efficiency in all substrates, with wheat straw+wheat bran were again found the best. The methanolic extract of strain DMRX-166 showed highest antibacterial properties as highest inhibition is found for Bacillus subtilis and Pseudomonas syringae. However, DMRO-253 inhibited Ralstonia solanacearum and Xanthomonas campestris. DMRX-768 has the best scavenging ability followed by DMRO-253.

OsRuvBL1a DNA helicase boost salinity and drought tolerance in transgenic indica rice raised by in planta transformation.

RuvBL, is a member of SF6 superfamily of helicases and is conserved among the various model systems. Recently, rice (Oryza sativa L.) homolog of RuvBL has been biochemically characterized for its ATPase and DNA helicase activities; however its involvement in stress has not been studied so far. Present investigation reports the detailed functional characterization of OsRuvBL under abiotic stresses through genetic engineering. An efficient Agrobacterium-mediated in planta transformation protocol was developed in indica rice to generate the transgenic lines and study was focused on optimization of factors to achieve maximum transformation efficiency. Overexpressing OsRuvBL1a transgenic lines showed enhanced tolerance under in vivo salinity stress as compared to WT plants. The physiological and biochemical analysis of the OsRuvBL1a transgenic lines showed better performance under salinity and drought stresses. Several stress responsive interacting partners of OsRuvBL1a were identified using Y2H system revealed to its role in stress tolerance. Functional mechanism for boosting stress tolerance by OsRuvBL1a has been proposed in this study. This integration of OsRuvBL1a gene in rice genome using in planta transformation method helped to achieve the abiotic stress resilient smart crop. This study is the first direct evidence to show the novel function of RuvBL in boosting abiotic stress tolerance in plants.

Identification of Eight High Yielding Strains via Morpho-Molecular Characterization of Thirty-Three Wild Strains of Calocybe indica.

Calocybe indica, generally referred as milky mushroom, is one of the edible mushroom species suitable for cultivation in the tropical and sub-tropical regions of the world. However, lack of potential high yielding strains has limited its wider adaptability. To overcome this limitation, in this study, the germplasms of C. indica from different geographical regions of India were characterized based on their morphological, molecular and agronomical attributes. Internal transcribed spacers (ITS1 and ITS4)-based PCR amplification, sequencing and nucleotide analysis confirmed the identity of all the studied strains as C. indica. Further, evaluation of these strains for morphological and yield parameters led to the identification of eight high yielding strains in comparison to the control (DMRO-302). Moreover, genetic diversity analysis of these thirty-three strains was performed using ten sequence-related amplified polymorphism (SRAP) markers/combinations. The Unweighted Pair-group Method with Arithmetic Averages (UPGMA)-based phylogenetic analysis categorized the thirty-three strains along with the control into three clusters. Cluster I possesses the maximum number of strains. Among the high yielding strains, high antioxidant activity and phenol content was recorded in DMRO-54, while maximum protein content was observed in DMRO-202 and DMRO-299 as compared with the control strain. The outcome of this study will help the mushroom breeders and growers in commercializing C. indica.

Identification and morpho-molecular characterization of low spore strain in oyster mushroom.

BACKGROUND: Sporocarps of oyster mushroom liberate enormous spores and cause allergic reactions to workers involved in its cultivation. These spore-related allergies include stiffness or pain in the forearms, limbs, itchy throat, grogginess, and respiratory problems and are major problems during oyster mushroom cultivation. METHODS AND RESULTS: In this study, we have generated seven hybrids using single-spore isolates (SSIs) of Pleurotus ostreatus var. florida (DMRP-49) and P. ostreatus (DMRP-30). Chimera was observed during cultivation trial of these hybrids and led to the development of low spore-producing/sporeless strain (DMRP-395) as evident from spore print and microscopic analysis. Further, the cultivation trial of this sporeless strain revealed a bunchy fruiting pattern and required 20-24 °C temperature for fruiting. At par yield was observed in sporeless strain. Notably, a prominent infundibuliform-shaped pileus along with central attachment of stipe was observed in the sporeless strain. Moreover, genetic diversity and principal component biplot analysis revealed resemblance of sporeless strain with one of the parental strain, i.e., P. ostreatus var. florida (DMRP-49). CONCLUSIONS: The developed sporeless strain (DMRP-395) contains high protein and at par yield as compared with the control (DMRP-136). This sporeless strain will be helpful to reduce spore-related allergic responses in mushroom growers.

Identification of Key Regulatory Pathways of Basidiocarp Formation in Pleurotus spp. Using Modeling, Simulation and System Biology Studies.

Pleurotus (Oyster mushroom) is an important cultivated edible mushroom across the world. It has several therapeutic effects as it contains various useful bio-molecules. The cultivation and crop management of these basidiomycete fungi depends on many extrinsic and intrinsic factors such as substrate composition, growing environment, enzymatic properties, and the genetic makeup, etc. Moreover, for efficient crop production, a comprehensive understanding of the fundamental properties viz. intrinsic-extrinsic factors and genotype-environment interaction analysis is required. The present study explores the basidiocarp formation biology in Pleurotus mushroom using an in silico response to the environmental factors and involvement of the major regulatory genes. The predictive model developed in this study indicates involvement of the key regulatory pathways in the pinhead to fruit body development process. Notably, the major regulatory pathways involved in the conversion of mycelium aggregation to pinhead formation and White Collar protein (PoWC1) binding flavin-chromophore (FAD) to activate respiratory enzymes. Overall, cell differentiation and higher expression of respiratory enzymes are the two important steps for basidiocarp formation. PoWC1 and pofst genes were participate in the structural changes process. Besides this, the PoWC1 gene is also involved in the respiratory requirement, while the OLYA6 gene is the triggering point of fruiting. The findings of the present study could be utilized to understand the detailed mechanism associated with the basidiocarp formation and to cultivate mushrooms at a sustainable level.

De novo genome sequencing of mycoparasite Mycogone perniciosa strain MgR1 sheds new light on its biological complexity.

Mycogone perniciosa is a mycoparasite causing Wet Bubble Diseases (WBD) of Agaricus bisporus. In the present study, the whole genome of M. perniciosa strain MgR1 was sequenced using Illumina NextSeq500 platform. This sequencing generated 8.03 Gb of high-quality data and a draft genome of 39 Mb was obtained through a de novo assembly of the high-quality reads. The draft genome resulted into prediction of 9276 genes from the 1597 scaffolds. NCBI-based homology analysis revealed the identification of 8660 genes. Notably, non-redundant protein database analysis of the M. perniciosa strain MgR1 revealed its close relation with the Trichoderma arundinaceum. Moreover, ITS-based phylogenetic analysis showed the highest similarity of M. perniciosa strain MgR1 with Hypomyces perniciosus strain CBS 322.22 and Mycogone perniciosa strain PPRI 5784. Annotation of the 3917 genes of M. perniciosa strain MgR1 grouped in three major categories viz. biological process (2583 genes), cellular component (2013 genes), and molecular function (2919 genes). UniGene analysis identified 2967 unique genes in M. perniciosa strain MgR1. In addition, prediction of the secretory and pathogenicity-related genes based on the fungal database indicates that 1512 genes (16% of predicted genes) encode for secretory proteins. Moreover, out of 9276 genes, 1296 genes were identified as pathogenesis-related proteins matching with 51 fungal and bacterial genera. Overall, the key pathogenic genes such as lysine M protein domain genes, G protein, hydrophobins, and cytochrome P450 were also observed. The draft genome of MgR1 provides an understanding of pathogenesis of WBD in A. bisporus and could be utilized to develop novel management strategies.

Nanofomulation of zinc oxide and chitosan zinc sustain oxidative stress and alter secondary metabolite profile in tobacco.

Advancement in nanotechnology has improved ways for large-scale production and characterization of nanoparticles of physiologically important metals. The current study explores the impact of Zinc Oxide Nanoparticles (ZnO-NP) and Chitosan-Zinc oxide nano-bioformulation (CH-ZnO) in tissue culture raised callus of Nicotiana benthamiana. Results indicated augmented biomass in CH-ZnO treated callus, while a reduced biomass was observed in ZnO-NP treated callus, at all the concentrations tested. Higher chlorophyll and carotenoid content were recorded in callus treated with 800 ppm CH-ZnO as compared to ZnO-NP treated callus. A higher accumulation of proline was observed in CH-ZnO treated callus when compared to ZnO-NP treatment, which was significantly higher at 50, 200 and 400 ppm CH-ZnO treatment. A maximum reduction in malondialdehyde (MDA) content was recorded at 800 ppm, for both the nano-formulations tested. Likewise, a significant reduction in the H2O2 levels was observed in all the treatments, while the callus treated with 400 ppm ZnO-NP and 800 ppm CH-ZnO recorded the highest reduction. Phenylalanine Ammonia-Lyase (PAL), activity increased significantly in callus treated with 400 ppm concentration for both ZnO-NP and CH-ZnO with respect to control. An increased level of tannin and nicotine were recorded in callus supplemented with 50, 200 and 400 ppm CH-ZnO. Notably, a significant decline of 94 and 52% in tannin content and 25 and 50% in nicotine content was recorded in the callus treated with 800 ppm CH-ZnO and ZnO-NP, respectively. The findings of this study suggest that an optimized dosage of these nano-bioformulations could be utilized to regulate the nicotine content and stress tolerance level.

Biopriming with Piriformospora indica ameliorates cadmium stress in rice by lowering oxidative stress and cell death in root cells.

Piriformospora indica is known for plant growth promotion and abiotic stress alleviation potential in several agricultural crops. However, a systemic analysis is warranted to explore potential application of this important fungus to augment heavy metal tolerance in rice. The present study explores potential of P. indica in ameliorating the effect of cadmium (Cd) stress in rice cultivars N22 and IR64. Seedlings inoculated with P. indica recorded significantly higher root-shoot length and biomass as compared to non-inoculated plants under control and Cd stress, respectively. Moreover, P. indica inoculated stressed roots accumulated more Cd as compared to non-inoculated stressed roots in both the varieties. Interestingly, cell death and reactive oxygen species (ROS) accumulation were significantly lower in the inoculated plant roots as compare with non-inoculated roots under Cd stress. The results emphasized significantly higher accumulation of Cd in fungal spores could reduce ROS accumulation in root cells resulting in lower cell death.

An EMS-induced new sequence variant, TEMS5032, in the coding region of SRS3 gene leads to shorter grain length in rice (Oryza sativa L.).

Grain shape and size influence yield and consumer preferences in rice. In the present study, we characterized and mapped a short and bold grained mutant and named it as TEMS5032, as the mutant is a result of EMS-induced transition from C to T at the 5032nd bp of SRS3 gene, which is known to affect grain size in rice. The substitution led to creation of a stop codon in the motor domain of SRS3, a kinesin 13 family gene, translating into a truncated protein product. However, transcription of this gene remained unaffected in TEMS5032 compared to the wild type, N22. Further, the mutation was found to affect 13 of the 25 cell cycle-related genes as they showed differential expression with respect to N22. Based on rate of grain filling, dry matter accumulation in the endosperm and histological studies, the effect of mutation in TEMS5032 was found to be similar to a known variant, TCM758, but less severe than sar1 mutant. Sequencing of 88 rice germplasm lines in the kinesin motor domain region did not reveal the presence of this mutation, establishing it as a new variant of SRS3 gene.

Reactive Oxygen Species Generation-Scavenging and Signaling during Plant-Arbuscular Mycorrhizal and Piriformospora indica Interaction under Stress Condition.

A defined balance between the generation and scavenging of reactive oxygen species (ROS) is essential to utilize ROS as an adaptive defense response of plants under biotic and abiotic stress conditions. Moreover, ROS are not only a major determinant of stress response but also act as signaling molecule that regulates various cellular processes including plant-microbe interaction. In particular, rhizosphere constitutes the biologically dynamic zone for plant-microbe interactions which forms a mutual link leading to reciprocal signaling in both the partners. Among plant-microbe interactions, symbiotic associations of arbuscular mycorrhizal fungi (AMF) and arbuscular mycorrhizal-like fungus especially Piriformospora indica with plants are well known to improve plant growth by alleviating the stress-impacts and consequently enhance the plant fitness. AMF and P. indica colonization mainly enhances ROS-metabolism, maintains ROS-homeostasis, and thereby averts higher ROS-level accrued inhibition in plant cellular processes and plant growth and survival under stressful environments. This article summarizes the major outcomes of the recent reports on the ROS-generation, scavenging and signaling in biotic-abiotic stressed plants with AMF and P. indica colonization. Overall, a detailed exploration of ROS-signature kinetics during plant-AMF/P. indica interaction can help in designing innovative strategies for improving plant health and productivity under stress conditions.

EcoTILLING-Based Association Mapping Efficiently Delineates Functionally Relevant Natural Allelic Variants of Candidate Genes Governing Agronomic Traits in Chickpea.

The large-scale mining and high-throughput genotyping of novel gene-based allelic variants in natural mapping population are essential for association mapping to identify functionally relevant molecular tags governing useful agronomic traits in chickpea. The present study employs an alternative time-saving, non-laborious and economical pool-based EcoTILLING approach coupled with agarose gel detection assay to discover 1133 novel SNP allelic variants from diverse coding and regulatory sequence components of 1133 transcription factor (TF) genes by genotyping in 192 diverse desi and kabuli chickpea accessions constituting a seed weight association panel. Integrating these SNP genotyping data with seed weight field phenotypic information of 192 structured association panel identified eight SNP alleles in the eight TF genes regulating seed weight of chickpea. The associated individual and combination of all SNPs explained 10-15 and 31% phenotypic variation for seed weight, respectively. The EcoTILLING-based large-scale allele mining and genotyping strategy implemented for association mapping is found much effective for a diploid genome crop species like chickpea with narrow genetic base and low genetic polymorphism. This optimized approach thus can be deployed for various genomics-assisted breeding applications with optimal expense of resources in domesticated chickpea. The seed weight-associated natural allelic variants and candidate TF genes delineated have potential to accelerate marker-assisted genetic improvement of chickpea.

PDH45 transgenic rice maintain cell viability through lower accumulation of Na(+), ROS and calcium homeostasis in roots under salinity stress.

Salinity severely affects the growth/productivity of rice, which is utilized as major staple food crop worldwide. PDH45 (pea DNA helicase 45), a member of the DEAD-box helicase family, actively provides salinity stress tolerance, but the mechanism behind this is not well known. Therefore, in order to understand the mechanism of stress tolerance, sodium ion (Na(+)), reactive oxygen species (ROS), cytosolic calcium [Ca(2+)]cyt and cell viability were analyzed in roots of PDH45 transgenic-IR64 rice lines along with wild-type (WT) IR64 rice under salinity stress (100mM and 200 mM NaCl). In addition, the roots of salinity-tolerant (FL478) and susceptible (Pusa-44) rice varieties were also analyzed under salinity stress for comparative analysis. The results reveal that, under salinity stress (100mM and 200 mM NaCl), roots of PDH45 transgenic lines accumulate lower levels of Na(+), ROS and maintain [Ca(2+)]cyt and exhibit higher cell viability as compared with roots of WT (IR64) plants. Similar results were also obtained in the salinity-tolerant FL478 rice. However, the roots of WT and salinity-susceptible Pusa-44 rice accumulated higher levels of Na(+), ROS and [Ca(2+)]cyt imbalance and lower cell viability during salinity stress, which is in contrast to the overexpressing PDH45 transgenic lines and salinity-tolerant FL478 rice. Further, to understand the mechanism of PDH45 at molecular level, comparative expression profiling of 12 cation transporters/genes was also conducted in roots of WT (IR64) and overexpressing PDH45 transgenic lines (L1 and L2) under salt stress (24h of 200 mM NaCl). The expression analysis results show altered and differential gene expression of cation transporters/genes in salt-stressed roots of WT (IR64) and overexpressing transgenic lines (L1 and L2). These observations collectively suggest that, under salinity stress conditions, PDH45 is involved in the regulation of Na(+) level, ROS production, [Ca(2+)]cyt homeostasis, cell viability and cation transporters in roots of PDH45 transgenic-IR64 rice and consequently provide salinity tolerance. Elucidating the detailed regulatory mechanism of PDH45 will provide a better understanding of salinity stress tolerance and further open new ways to manipulate genome to achieve higher agricultural production under stress.

NPKS uptake, sensing, and signaling and miRNAs in plant nutrient stress.

Sessile nature of higher plants consequently makes it highly adaptable for nutrient absorption and acquisition from soil. Plants require 17 essential elements for their growth and development which include 14 minerals (macronutrients: N, P, K, Mg, Ca, S; micronutrients: Cl, Fe, B, Mn, Zn, Cu, Ni, Mo) and 3 non-mineral (C, H, O) elements. The roots of higher plants must acquire these macronutrients and micronutrients from rhizosphere and further allocate to other plant parts for completing their life cycle. Plants evolved an intricate series of signaling and sensing cascades to maintain nutrient homeostasis and to cope with nutrient stress/availability. The specific receptors for nutrients in root, root system architecture, and internal signaling pathways help to develop plasticity in response to the nutrient starvation. Nitrogen (N), phosphorus (P), potassium (K), and sulfur (S) are essential for various metabolic processes, and their deficiency negatively effects the plant growth and yield. Genes coding for transporters and receptors for nutrients as well as some small non-coding RNAs have been implicated in nutrient uptake and signaling. This review summarizes the N, P, K, and S uptake, sensing and signaling events in nutrient stress condition especially in model plant Arabidopsis thaliana and involvement of microRNAs in nutrient deficiency. This article also provides a framework of uptake, sensing, signaling and to highlight the microRNA as an emerging major players in nutrient stress condition. Nutrient-plant-miRNA cross talk may help plant to cope up nutrient stress, and understanding their precise mechanism(s) will be necessary to develop high yielding smart crop with low nutrient input.

PDH45 overexpressing transgenic tobacco and rice plants provide salinity stress tolerance via less sodium accumulation.

Salinity stress negatively affects the crop productivity worldwide, including that of rice. Coping with these losses is a major concern for all countries. The pea DNA helicase, PDH45 is a unique member of helicase family involved in the salinity stress tolerance. However, the exact mechanism of the PDH45 in salinity stress tolerance is yet to be established. Therefore, the present study was conducted to investigate the mechanism of PDH45-mediated salinity stress tolerance in transgenic tobacco and rice lines along with wild type (WT) plants using CoroNa Green dye based sodium localization in root and shoot sections. The results showed that under salinity stress root and shoot of PDH45 overexpressing transgenic tobacco and rice accumulated less sodium (Na(+)) as compared to their respective WT. The present study also reports salinity tolerant (FL478) and salinity susceptible (Pusa-44) varieties of rice accumulated lowest and highest Na(+) level, respectively. All the varieties and transgenic lines of rice accumulate differential Na(+) ions in root and shoot. However, roots accumulate high Na(+) as compared to the shoots in both tobacco and rice transgenic lines suggesting that the Na(+) transport in shoot is somehow inhibited. It is proposed that the PDH45 is probably involved in the deposition of apoplastic hydrophobic barriers and consequently inhibit Na(+) transport to shoot and therefore confers salinity stress tolerance to PDH45 overexpressing transgenic lines. This study concludes that tobacco (dicot) and rice (monocot) transgenic plants probably share common salinity tolerance mechanism mediated by PDH45 gene.

Physiological, anatomical and transcriptional alterations in a rice mutant leading to enhanced water stress tolerance.

Water stress is one of the most severe constraints to crop productivity. Plants display a variety of physiological and biochemical responses both at the cellular and whole organism level upon sensing water stress. Leaf rolling, stomatal closure, deeper root penetration, higher relative water content (RWC) and better osmotic adjustment are some of the mechanisms that plants employ to overcome water stress. In the current study, we report a mutant, enhanced water stress tolerant1 (ewst1) with enhanced water stress tolerance, identified from the ethyl methanesulfonate-induced mutant population of rice variety Nagina22 by field screening followed by withdrawal of irrigation in pots and hydroponics (PEG 6000). Though ewst1 was morphologically similar to the wild type (WT) for 35 of the 38 morphological descriptors (except chalky endosperm/expression of white core, decorticated grain colour and grain weight), it showed enhanced germination in polyethylene glycol-infused medium. It exhibited increase in maximum root length without any significant changes in its root weight, root volume and total root number on crown when compared with the WT under stress in PVC tube experiment. It also showed better performance for various physiological parameters such as RWC, cell membrane stability and chlorophyll concentration upon water stress in a pot experiment. Root anatomy and stomatal microscopic studies revealed changes in the number of xylem and phloem cells, size of central meta-xylem and number of closed stomata in ewst1. Comparative genome-wide transcriptome analysis identified genes related to exocytosis, secondary metabolites, tryptophan biosynthesis, protein phosphorylation and other signalling pathways to be playing a role in enhanced response to water stress in ewst1. The possible involvement of a candidate gene with respect to the observed morpho-physiological and transcriptional changes and its role in stress tolerance are discussed. The mutant identified and characterized in this study will be useful for further dissection of water stress tolerance in rice.

A substitution mutation in OsCCD7 cosegregates with dwarf and increased tillering phenotype in rice.

Dwarf plant height and tillering ability are two of the most important agronomic traits that determine the plant architecture, and have profound influence on grain yield in rice. To understand the molecular mechanism controlling these two traits, an EMS-induced recessive dwarf and increased tillering1 (dit1) mutant was characterized. The mutant showed proportionate reduction in each internode as compared to wild type revealing that it belonged to the category of dn-type of dwarf mutants. Besides, exogenous application of GA3 and 24-epibrassinolide, did not have any effect on the phenotype of the mutant. The gene was mapped on the long arm of chromosome 4, identified through positional candidate approach and verified by cosegregation analysis. It was found to encode carotenoid cleavage dioxygenase7 (CCD7) and identified as an allele of htd1. The mutant carried substitution of two nucleotides CC to AA in the sixth exon of the gene that resulted in substitution of serine by a stop codon in the mutant, and thus formation of a truncated protein, unlike amino acid substitution event in htd1. The new allele will facilitate further functional characterization of this gene, which may lead to unfolding of newer signalling pathways involving plant development and architecture.

Comparative evaluation of genetic diversity using RAPD, SSR and cytochrome P450 gene based markers with respect to calcium content in finger millet (Eleusine coracana L. Gaertn.).

Genetic relationships among 52 Eleusine coracana (finger millet) genotypes collected from different districts of Uttarakhand were investigated by using randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and cytochrome P450 gene based markers. A total of 18 RAPD primers, 10 SSR primers, and 10 pairs of cytochrome P450 gene based markers, respectively, revealed 49.4%, 50.2% and 58.7% polymorphism in 52 genotypes of E. coracana. Mean polymorphic information content (PIC) for each of these marker systems (0.351 for RAPD, 0.505 for SSR and 0.406 for cyt P450 gene based markers) suggested that all the marker systems were effective in determining polymorphisms. Pair-wise similarity index values ranged from 0.011 to 0.999 (RAPD), 0.010 to 0.999 (SSR) and 0.001 to 0.998 (cyt P450 gene based markers) and mean similarity index value of 0.505, 0.504 and 0.499, respectively. The dendrogram developed by RAPD, SSR and cytochrome P450 gene based primers analyses revealed that the genotypes are grouped in different clusters according to high calcium (300-450 mg/100 g), medium calcium (200-300 mg/100 g) and low calcium (100-200 mg/100 g). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.95 for all the three marker systems. The dendrograms and principal coordinate analysis (PCA) plots derived from the binary data matrices of the three marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. Comparison of RAPD, SSR and cytochrome P450 gene based markers, in terms of the quality of data output, indicated that SSRs and cyt P450 gene based markers are particularly promising for the analysis of plant genome diversity. The genotypes of finger millet collected from different districts of Uttarakhand constitute a wide genetic base and clustered according to calcium contents. The identified genotypes could be used in breeding programmes and amajor input into conservation biology of cereal crops.