Pyrolyzed and unpyrolyzed residues enhance maize yield under varying rates of application and fertilization regimes.

Biochar is increasingly gaining popularity due to its extensive recommendation as a potential solution for addressing the concerns of food security and climate change in agroecosystems, with biochar application for increased carbon sequestration, enhanced soil fertility, improved soil health, and increased crop yield and quality. There have been multiple studies on crop yield utilizing various biochar types and application amounts; however, none have focused on the influence of diverse biochar types at various pyrolysis temperatures with different application amounts and the integration of fertilizer regimes in maize crops. Therefore, a two-year factorial field experiment was designed in a temperate Himalayan region of India (THRI) to evaluate the residual effect of different biochar on maize yield under different pyrolysis temperatures, various application rates and fertilizer regimes. The study included three factors viz., amendment type (factor 1), rate of application (factor 2) and fertilizer regime (factor 3). Amendment type included 7 treatments: No biochar- control (A1), apple biochar @ 400 °C pyrolysis temperature (A2), apple biochar @ 600 °C pyrolysis temperature (A3), apple residue biomass (A4), dal weed biochar @ 400 °C pyrolysis temperature (A5), dal weed biochar @ 600 °C pyrolysis temperatures (A6), and dal weed residue biomass (A7). The rate of application included 3 levels: Low (L- 1 t ha-1), medium (M- 2 t ha-1), and high (H- 3 t ha-1). At the same time, the fertilizer regimes included 2 treatments: No fertilizer (N) and recommended dose of fertilizer (F). The results revealed that among the various amendment type, rate of application and fertilizer regimes, the A3 amendment, H rate of application and F fertilizer regime gave the best maize growth and productivity outcome. Results revealed that among the different pyrolyzed residues used, the A3 amendment had the highest plant height (293.87 cm), most kernels cob-1 (535.75), highest soil plant analysis development (SPAD) value (58.10), greatest cob length (27.36 cm), maximum cob girth (18.18 cm), highest grain cob yield (1.40 Mg ha-1), highest grain yield (4.78 Mg ha-1), higher test weight (305.42 gm), and highest stover yield (2.50 Mg ha-1). The maximum dry weight in maize and the number of cobs plant-1 were recorded with amendments A4 (14.11 Mg ha-1) and A6 (1.77), respectively. The comparatively 2nd year of biochar application than the 1st year, the H level of the rate of application than the L rate and the application and integration of the recommended dose of fertilizer in maize results in significantly higher values of growth and productivity in maize. Overall, these findings suggest that the apple biochar @ 600 °C pyrolysis temperature (A3) at a high application rate with the addition of the recommended dose of fertilizer is the optimal biochar for enhancing the growth and productivity of maize in the THRI.

Proteomic Analysis, Immuno-Specificity and Neutralization Efficacy of Pakistani Viper Antivenom (PVAV), a Bivalent Anti-Viperid Antivenom Produced in Pakistan.

Snakebite envenoming is a neglected tropical disease prevalent in South Asia. In Pakistan, antivenoms are commonly imported from India despite the controversy over their effectiveness. To solve the problem, the locals have developed the Pakistani Viper Antivenom (PVAV), raised against Sochurek's Saw-scaled Viper (Echis carinatus sochureki) and Russell's Viper (Daboia russelii) of Pakistani origin. This study is set to evaluate the composition purity, immuno-specificity and neutralization efficacy of PVAV. Chromatographic and electrophoretic profiling coupled with proteomic mass spectrometry analysis showed PVAV containing high-purity immunoglobulin G with minimum impurities, notably the absence of serum albumin. PVAV is highly immuno-specific toward the venoms of the two vipers and Echis carinatus multisquamatus, which are indigenous to Pakistan. Its immunoreactivity, however, reduces toward the venoms of other Echis carinatus subspecies and D. russelii from South India as well as Sri Lanka. Meanwhile, its non-specific binding activities for the venoms of Hump-nosed Pit Vipers, Indian Cobras and kraits were extremely low. In the neutralization study, PVAV effectively mitigated the hemotoxic and lethal effects of the Pakistani viper venoms, tested in vitro and in vivo. Together, the findings suggest the potential utility of PVAV as a new domestic antivenom for the treatment of viperid envenoming in Pakistan.

Osteoid Osteoma of Retromolar Trigone: Report of a Rare Case.

Osteoid osteoma is a rare benign bone lesion that is often confused with the osteoblastoma. The osteoid osteoma comprises of 3% of all the primary bone tumours usually found in the long bones and vertebrae with the facial skeleton being the most infrequent site. The lesion usually presents with the swelling and pain that resolves with non-steroidal anti-inflammatory drugs (NSAIDs). Here, we report a case of osteoid osteoma of retromolar trigone, the site which has not been reported in the literature, in a 50-year male patient with a complaint of hard swelling on retromolar trigone associated with the mild intermittent localised pain that aggravated at night and on mastication. Excision of the lesion was carried out with chisel and mallet under local anesthesia with uneventful recovery and no recurrence at the 6 months follow-up. Key Words: Benign, Osteoid osteoma, Retromolar trigone.

Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India.

Plant viruses are a major threat to legume production worldwide. In recent years, new virus strains have emerged with increasing frequencies in various legume cropping systems, which demands the development of cutting-edge virus surveillance techniques. In this study, we surveyed the common bean fields of Kashmir valley for virus infection using a total of 140 symptomatic and non-symptomatic leaf samples collected from different locations. The genetic diversity of viruses was examined by high-throughput sequencing (HTS) with three viruses being identified, namely, Bean Common Mosaic Virus (BCMV), Bean Common Mosaic Necrosis Virus (BCMNV), and Clover Yellow Vein Virus (ClYVV). BCMNV and ClYVV are new reports from India. De novo assembly of transcriptome constructed near-complete genomes of these viruses. RT-PCR results confirmed the presence of these viruses with an emerge incidence of 56. 4% for BCMV, 27.1% for BCMNV and 16.4 for ClYVV in the valley. Several samples were found to contain multiple virus infections with BCMV being the most predominant. Recombination events were detected in the genomes of BCMV and ClYVV, but not BCMNV. Phylogenetic and pairwise identity matrix evidence suggests viral import from multiple countries. Our results demonstrate that HTS followed by multiplex PCR assay is a simple, rapid, and reliable approach for simultaneous diagnosis of plant viruses.

Narrowing down molecular targets for improving phosphorus-use efficiency in maize (Zea mays L.).

Conventional agricultural practices rely heavily on chemical fertilizers to boost production. Among the fertilizers, phosphatic fertilizers are copiously used to ameliorate low-phosphate availability in the soil. However, phosphorus-use efficiency (PUE) for major cereals, including maize, is less than 30%; resulting in more than half of the applied phosphate being lost to the environment. Rock phosphate reserves are finite and predicted to exhaust in near future with the current rate of consumption. Thus, the dependence of modern agriculture on phosphatic fertilizers poses major food security and sustainability challenges. Strategies to optimize and improve PUE, like genetic interventions to develop high PUE cultivars, could have a major impact in this area. Here, we present the current understanding and recent advances in the biological phenomenon of phosphate uptake, translocation, and adaptive responses of plants under phosphate deficiency, with special reference to maize. Maize is one of the most important cereal crops that is cultivated globally under diverse agro-climatic conditions. It is an industrial, feed and food crop with multifarious uses and a fast-rising global demand and consumption. The interesting aspects of diversity in the root system architecture traits, the interplay between signaling pathways contributing to PUE, and an in-depth discussion on promising candidate genes for improving PUE in maize are elaborated.

Prevalence Of Gustatory And Olfactory Impairment In Different Stages Of Sars-Cov-2 Infected Pateints In Isolation Ward.

BACKGROUND: To evaluate the prevalence of gustatory and olfactory impairment and its correlation with the severity of SARS-Cov-2 infection as per WHO guidelines. METHODS: A total of 241 patients of both gender having age from 15-80 years were included in the study. It was cross Sectional study conducted at SARS-COV-2 Isolation wards of Pakistan institute of medical science Islamabad from 15thSeptember 2020 to 15th January 2021. Convenient sampling technique was used to collect data. A proforma was designed for this purpose. Participants were divided into 3 groups on the basis of age. Group-1 (15-30 years), group-2 (31-50 years) and group 3 (51-80 years). Further the participants were graded into mild, moderate and severe stages of Sars-Cov-2 infection according to WHO guidelines. Statistical analysis was performed by using SPSS version 20. RESULTS: In this study 66% were male and 34% population were the female. Regarding the age groups 72% population were from the group-3. Results of study shows 47% in mild stage, 45% in moderate stage and 8% severe stage. The results regarding the Gustatory and Olfactory impairment showed that 126 had gustatory impairment while 130 patients had olfactory impairment. CONCLUSIONS: We found strong relationship of olfactory and gustatory changes associated with SARS-COV-2 patients. It is premature to conclude that taste and smell changes are strongly linked to SARS-COV-2 diagnosis. Further multi center researches are required to find out possible pathophysiological mechanism.

Nutritional Diversity in Native Germplasm of Maize Collected From Three Different Fragile Ecosystems of India.

Native germplasm resources are adapted to specific ecological niches. They have sustained over generations owing to the preference of local communities for their unique taste, the utility to particular dishes, and the low cost of cultivation. They may help eradicate malnutrition and act as a source for trait-linked genes. The present dataset comprises thirty-three native germplasm of maize collected from Rajasthan, Himachal Pradesh, and Andhra Pradesh states of India with an altitudinal variation of 386-2,028 m. They were evaluated for proximate composition, minerals, nutritional attributes, and antioxidant activity and compared with the standard values reported in the Indian Food Composition Table 2017 (IFCT2017). The nutritional profile showed moisture content in the range of 7.16-10.9%, ash 0.73-1.93%, crude protein 8.68-12.0%, crude fat 3.72-8.03%, dietary fiber 5.21-11.2%, and available carbohydrates 60.6-69.8%. Three accessions, namely, Malan 11 (7.06%), Malan 24 (7.20%), and Yellow Chamba Local 02 (8.03%) exhibited almost double the crude fat content as compared with the values notified in IFCT2017 (3.77). Total sugar content obtained was in the range of 5.00-11.3%, whereas the starch content was found between 50.9 and 64.9%. All the germplasm except Yellow Chamba Local reflected a higher protein content than reported values in IFCT2017 (8.80). Sathi, Safed Chamba Local, and Ragal Makka had nearly 12% protein content. Mineral malnutrition, mainly due to iron (Fe) deficiency, is a worldwide issue to science, humanity, and society. The mineral profile revealed that most germplasm had a higher iron content. Accessions with the iron content of nearly three times of IFCT2017 reported value were identified in germplasm belonging to three states. A negative relationship was observed between the altitude of the sample collection site and available carbohydrate content. In contrast, available carbohydrate showed inverse correlations with dietary fiber, protein, and fat content. The information generated in this study can be utilized to promote these germplasm as nutrifood, nutritional surveillance, labeling, and crop improvement programs.

Population Structure Analysis and Association Mapping for Turcicum Leaf Blight Resistance in Tropical Maize Using SSR Markers.

Maize is an important cereal crop in the world for feed, food, fodder, and raw materials of industries. Turcicum leaf blight (TLB) is a major foliar disease that can cause more than 50% yield losses in maize. Considering this, the molecular diversity, population structure, and genome-wide association study (GWAS) for TLB resistance were studied in 288 diverse inbred lines genotyped using 89 polymorphic simple sequence repeats (SSR) markers. These lines werescreened for TLB disease at two hot-spot locations under artificially inoculated conditions. The average percent disease incidence (PDI) calculated for each genotype ranged from 17 (UMI 1201) to 78% (IML 12-22) with an overall mean of 40%. The numbers of alleles detected at a locus ranged from twoto nine, with a total of 388 alleles. The polymorphic information content (PIC) of each marker ranged between 0.04 and 0.86. Out of 89 markers, 47 markers were highly polymorphic (PIC ≥ 0.60). This indicated that the SSR markers used were very informative and suitable for genetic diversity, population structure, and marker-trait association studies.The overall observed homozygosity for highly polymorphic markers was 0.98, which indicated that lines used were genetically pure. Neighbor-joining clustering, factorial analysis, and population structure studies clustered the 288 lines into 3-5 groups. The patterns of grouping were in agreement with the origin and pedigree records of the genotypesto a greater extent.A total of 94.10% lines were successfully assigned to one or another group at a membership probability of ≥0.60. An analysis of molecular variance (AMOVA) revealed highly significant differences among populations and within individuals. Linkage disequilibrium for r2 and D' between loci ranged from 0 to 0.77 and 0 to 1, respectively. A marker trait association analysis carried out using a general linear model (GLM) and mixed linear model (MLM), identified 15 SSRs markers significantly associated with TLB resistance.These 15 markers were located on almost all chromosomes (Chr) except 7, 8, and 9. The phenotypic variation explained by these loci ranged from 6% (umc1367) to 26% (nc130, phi085). Maximum 7 associated markers were located together on Chr 2 and 5. The selected regions identified on Chr 2 and 5 corroborated the previous studies carried out in the Indian maize germplasm. Further, 11 candidate genes were identified to be associated with significant markers. The identified sources for TLB resistance and associated markers may be utilized in molecular breeding for the development of suitable genotypes.

PdO@CoSe2 composites: efficient electrocatalysts for water oxidation in alkaline media.

In this study, we have prepared cobalt selenide (CoSe2) due to its useful aspects from a catalysis point of view such as abundant active sites from Se edges, and significant stability in alkaline conditions. CoSe2, however, has yet to prove its functionality, so we doped palladium oxide (PdO) onto CoSe2 nanostructures using ultraviolet (UV) light, resulting in an efficient and stable water oxidation composite. The crystal arrays, morphology, and chemical composition of the surface were studied using a variety of characterization techniques, including X-ray diffraction (XRD), scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. It was also demonstrated that the composite systems were heterogeneous in their morphology, undergoing a shift in their diffraction patterns, suffering from a variety of metal oxidation states and surface defects. The water oxidation was verified by a low overpotential of 260 mV at a current density of 20 mA cm-2 with a Tafel Slope value of 57 mV dec-1. The presence of multi metal oxidation states, rich surface edges of Se and favorable charge transport played a leading role towards water oxidation with a low energy demand. Furthermore, 48 h of durability is associated with the composite system. With the use of PdO and CoSe2, new, low efficiency, simple electrocatalysts for water catalysis have been developed, enabling the development of practical energy conversion and storage systems. This is an excellent alternative approach for fostering growth in the field.

An experience of mass administration of fractional dose inactivated polio vaccine through intradermal needle-free injectors in Karachi, Sindh, Pakistan.

BACKGROUND: Inactivated Polio Vaccine (IPV) campaign was conducted in February 2019 in Karachi where needle-free injectors were introduced for the administration of the fractional dose of IPV (fIPV) on a large scale. This study aimed to determine the impact of needle-free injectors on vaccination coverage. METHODS: In four towns of Karachi, fIPV was given using needle-free injectors "PharmaJet Tropis ID". Whereas, in six towns full dose of IPV was administered to children of 4-59 months of age. Cluster surveys through rapid convenience assessment method were conducted after the completion of vaccination activity. RESULTS: A total of 33,815 households' data was analyzed. Among these, 27,650 (82.8%) children were vaccinated. In fIPV areas, 85.3% of children were vaccinated compared to 79.5% in full dose IPV areas. A comparison of reasons for unvaccinated showed that 1.6% of parents do not give importance to vaccination in fIPV areas compared to 4.2% in full IPV areas (p-value < 0.0001). More children were not vaccinated due to fear of injection 1.8% in full IPV areas compared to 0.7% in fIPV areas (p-value < 0.0001). The source of campaign information shows that more frequent mobile miking 3.1% was observed in fIPV areas compared to 0.4% in full IPV areas (p-value < 0.0001). CONCLUSIONS: Our analysis supports the fractional dose of IPV in mass campaigns to achieve good vaccination coverage especially using needle-free injectors "PharmaJet Tropis ID" and vigorous social mobilization activities are expedient in accomplishing high coverage.

Hyalomma anatolicum resistance against ivermectin and fipronil is associated with indiscriminate use of acaricides in southwestern Balochistan, Pakistan.

Ivermectin and fipronil have been used regularly to control the hard tick Hyalomma anatolicum (Acari: Ixodidae) in domestic ruminants for more than a half-decade in Balochistan, Pakistan. Inappropriate and indiscriminate use of these acaricides has resulted in the development of resistances in tick species. In this work, acaricides (ivermectin and fipronil) resistance was evaluated in H. anatolicum through in vitro and in vivo bioassays in a horse farm of Quetta, Balochistan province, Pakistan. A participatory epidemiological survey was conducted to assess potential risk factors associated with the development of acaricide resistance in H. anatolicum. The results of the epidemiological survey revealed that the horse keepers did not follow the manufacturer's instructions for the use of acaricides and applied indiscriminate doses of acaricides. The results of in vitro bioassays (adult immersion test and larval immersion test) showed that fipronil and ivermectin have protective efficacy against H. anatolicum. The results of in vivo bioassay (adult-tick mortality assay) revealed that fipronil had a higher efficacy (78.16%) than ivermectin (49.94%). More than 80% of tick mortality was not achieved in any bioassays, even for the highest acaricide concentration (100 ppm), which suggests the development of acaricide resistance against fipronil and ivermectin. This study highlights the urgency to implement a country-wide awareness about resistance monitoring and effective tick control. Graphical abstract.

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production.

BACKGROUND: Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F1-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation. RESULTS: In contrast to genomic integration, we found that episomal expression of the αßγ subunits of the F1-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the ß subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli's F1-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain. CONCLUSIONS: Our study shows that expression of genes of the F1-portion of E. coli's ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.

Genome-wide association studies in tropical maize germplasm reveal novel and known genomic regions for resistance to Northern corn leaf blight.

Northern Corn Leaf Blight (NCLB) caused by Setosphaeria turcica, is one of the most important diseases of maize world-wide, and one of the major reasons behind yield losses in maize crop in Asia. In the present investigation, a high-resolution genome wide association study (GWAS) was conducted for NCLB resistance in three association mapping panels, predominantly consisting of tropical lines adapted to different agro-ecologies. These panels were phenotyped for disease severity across three locations with high disease prevalence in India. High density SNPs from Genotyping-by-sequencing were used in GWAS, after controlling for population structure and kinship matrices, based on single locus mixed linear model (MLM). Twenty-two SNPs were identified, that revealed a significant association with NCLB in the three mapping panels. Haplotype regression analysis revealed association of 17 significant haplotypes at FDR ≤ 0.05, with two common haplotypes across three maize panels. Several of the significantly associated SNPs/haplotypes were found to be co-located in chromosomal bins previously reported for major genes like Ht2, Ht3 and Htn1 and QTL for NCLB resistance and multiple foliar disease resistance. Phenotypic variance explained by these significant SNPs/haplotypes ranged from low to moderate, suggesting a breeding strategy of combining multiple resistance alleles towards resistance for NCLB.

Serial population based serosurvey of antibodies to SARS-CoV-2 in a low and high transmission area of Karachi, Pakistan

Serial household antibody sero-surveys informs the pandemic where testing is non-uniform. Young populations with intergenerational co-residence may have different transmission dynamics. We conducted two serial cross-sectional surveys in April and June 2020 in low- and high-transmission neighborhoods of Karachi, Pakistan, using random sampling. Symptoms were assessed and blood tested for antibody using chemiluminescence. Seroprevalence was adjusted using Bayesian regression and post stratification. CRI with 95% confidence intervals was obtained. We enrolled 2004 participants from 406 households. In June 8.7% (95% CI 5.1-13.1) and 15.1% (95% CI 9.4-21.7) were infected in low- and high-transmission-areas respectively compared with 0.2% (95% CI 0-0.7) and 0.4% (95% CI 0-1.3) in April. Conditional risk of infection was 0.31 (95% CI 0.16-0.47) and 0.41(95% CI 0.28-0.52) respectively with only 5.4% symptomatic. Rapid increase in seroprevalence from baseline is seen in Karachi, with a high probability of infection within household. Article Summary LineRapid increase in seroprevalence of antibodies against SARS-CoV-2 was seen in Karachi, Pakistan from April to June 2020 with a high conditional risk of infection within the household

Elemental Characterization of Medicinal Plants and Soils from Hazarganji Chiltan National Park and Nearby Unprotected Areas of Balochistan, Pakistan.

The aim of this work was to evaluate the variability in elemental composition of seven medicinal plants and their respective soils belonging to protected and nearby unprotected sites of the Hazarganji Chiltan National Park. The medical plants under study were Achillea wilhelmsii C. Koch, Peganum harmala Linn, Sophora mollis (Royle) Baker, Perovskia atriplicifolia Benth, Seriphidium quettense (Podlech.) Ling, Hertia intermedia (Bioss) O. Ktze, and Nepeta praetervisa Rech. F. Macro (C, H, N, S, K, Ca), micro (Cl, Cu, Fe, Mn, and Zn), beneficial (Al, Co, Na), others (As, Br, Cr, Cs, Hf, Pb, Rb, Sb, Sr, Sn, V and Th) and rare earth elements (Ce, Eu, La, Lu, Nd Sc, Sm, Tb and Yb) were characterized by means of standard organic elemental and instrumental neutron activation methodologies and by flame atomic absorption spectroscopy. Results showed that, among macro nutrients, carbon concentration was the highest element in both plant and soil samples followed by H and K. Elements such as Cl, Na and Fe were detected in considerably good amounts; all the other elements were found in trace quantities. Principal component analysis (PCA) was applied to identify spatial variation in elemental composition of medicinal plants, in which 80-90% of the total variance in whole set of data was found. In particular, the findings highlighted the presence of essential and beneficial elements such as C, H, N, K, Ca, Fe, Mn and Na, in samples from protected sites, while potentially dangerous elements such as Al, As, Br and Cr were detected in samples from unprotected sites. These results emphasized on the need for rational exploitation of valuable medicinal plants and supporting protected areas as an excellent source of biodiversity conservation.

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli.

The targeted increase of cellular adenosine triphosphate (ATP) turnover (enforced ATP wasting) has recently been recognized as a promising tool for metabolic engineering when product synthesis is coupled with net ATP formation. The goal of the present study is to further examine and to further develop the concept of enforced ATP wasting and to broaden its scope for potential applications. In particular, considering the fermentation products synthesized by Escherichia coli under anaerobic conditions as a proxy for target chemical(s), i) a new genetic module for dynamic and gradual induction of the F1 -part of the ATPase is developed and it is found that ii) induction of the ATPase leads to higher metabolic activity and increased product formation in E. coli under anaerobic conditions, and that iii) ATP wasting significantly increases substrate uptake and productivity of growth-arrested cells, which is vital for its use in two-stage processes. To the best of the authors' knowledge, the glucose uptake rate of 6.49 mmol gCDW-1 h-1 achieved with enforced ATP wasting is the highest value reported for nongrowing E. coli cells. In summary, this study shows that enforced ATP wasting can be used to improve yield and titer (in growth-coupled processes) as well as volumetric productivity (in two-stage processes) depending on which of the performance measures is more crucial for the process and product of interest.

Evaluation of pyruvate decarboxylase-negative Saccharomyces cerevisiae strains for the production of succinic acid.

Dicarboxylic acids are important bio-based building blocks, and Saccharomyces cerevisiae is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase-negative S. cerevisiae strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of GPD1 and FUM1 and multi-copy integration of the native PYC2 gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade-off between redox cofactor supply and energy availability in the form of ATP.

Mortierella alpina CS10E4, an oleaginous fungal endophyte of Crocus sativus L. enhances apocarotenoid biosynthesis and stress tolerance in the host plant.

Crocus sativus is the only plant species which produces apocarotenoids like crocin, picrocrocin and safranal in significant amounts. These compounds impart organoleptic properties to saffron (dried stigmas of Crocus flower) making it world's costliest spice. Crocus apocarotenoids have tremendous medicinal properties as well. Effect of endophytes on Crocus apocarotenoid production and the molecular mechanism involved has not been reported so far. Here we studied the effect of an oleaginous fungal endophyte, Mortierella alpina CS10E4 on Crocus growth, apocarotenoid metabolism and tolerance to corm rot disease. The results demonstrated that there was a significant improvement in many morphological and physiological traits in endophyte treated Crocus plants including total biomass and size of corms, stigma biomass, number of apical sprouting buds, and number of adventitious roots. The endophyte also shifted metabolic flux towards enhanced production of apocarotenoids by modulating the expression of key pathway genes. Further, M. alpina CS10E4 enhanced tolerance to corm rot disease by releasing arachidonic acid which acts as conserved defense signal and induces jasmonic acid production in endophyte treated Crocus corms. This is first report on effect of a fungal endophyte on Crocus apocarotenoid metabolism and stress tolerance.

Molecular phylogeny, diversity, community structure, and plant growth promoting properties of fungal endophytes associated with the corms of saffron plant: An insight into the microbiome of Crocus sativus Linn.

A total of 294 fungal endophytes were isolated from the corms of Crocus sativus at two stages of crocus life cycle collected from 14 different saffron growing sites in Jammu and Kashmir (J & K) State, India. Molecular phylogeny assigned them into 36 distinct internal transcribed spacer (ITS) genotypes which spread over 19 genera. The diversity of endophytes was higher at the dormant than at the vegetative stage. The Saffron microbiome was dominated by Phialophora mustea and Cadophora malorum, both are dark septate endophytes (DSEs). Some endophytes were found to possess antimicrobial properties that could be helpful for the host in evading the pathogens. These endophytes generally produced significant quantities of indole acetic acid (IAA) as well. However, thirteen of the endophytic taxa were found to cause corm rot in the host with different levels of severity under in vitro as well as in vivo conditions. This is the first report of community structure and biological properties of fungal endophytes associated with C. sativus, which may eventually help us to develop agro-technologies, based on plant-endophyte interactions for sustainable cultivation of saffron. The endophytes preserved ex situ, in this study, may also yield bioactive natural products for pharmacological and industrial applications.

Comprehensive transcriptome analysis of Crocus sativus for discovery and expression of genes involved in apocarotenoid biosynthesis.

BACKGROUND: Crocus sativus stigmas form rich source of apocarotenoids like crocin, picrocrocin and saffranal which besides imparting color, flavour and aroma to saffron spice also have tremendous pharmacological properties. Inspite of their importance, the biosynthetic pathway of Crocus apocarotenoids is not fully elucidated. Moreover, the mechanism of their stigma specific accumulation remains unknown. Therefore, deep transcriptome sequencing of Crocus stigma and rest of the flower tissue was done to identify the genes and transcriptional regulators involved in the biosynthesis of these compounds. RESULTS: Transcriptome of stigma and rest of the flower tissue was sequenced using Illumina Genome Analyzer IIx platform which generated 64,604,402 flower and 51,350,714 stigma reads. Sequences were assembled de novo using trinity resulting in 64,438 transcripts which were classified into 32,204 unigenes comprising of 9853 clusters and 22,351 singletons. A comprehensive functional annotation and gene ontology (GO) analysis was carried out. 58.5 % of the transcripts showed similarity to sequences present in public databases while rest could be specific to Crocus. 5789 transcripts showed similarity to transcription factors representing 76 families out of which Myb family was most abundant. Many genes involved in carotenoid/apocarotenoid pathway were identified for the first time in this study which includes zeta-carotene isomerase and desaturase, carotenoid isomerase and lycopene epsilon-cyclase. GO analysis showed that the predominant classes in biological process category include metabolic process followed by cellular process and primary metabolic process. KEGG mapping analysis indicated that pathways involved in ribosome, carbon and starch and sucrose metabolism were highly represented. Differential expression analysis indicated that key carotenoid/apocarotenoid pathway genes including phytoene synthase, phytoene desaturase and carotenoid cleavage dioxygenase 2 are enriched in stigma thereby providing molecular proof for stigma to be the site of apocarotenoid biosynthesis. CONCLUSIONS: This data would provide a rich source for understanding the carotenoid/apocarotenoid metabolism in Crocus. The database would also help in investigating many questions related to saffron biology including flower development.