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Potential of Phytohormones Producing Endophytic Fungi As Biofertilizers

Thesis Info

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Author

Lubna

Program

PhD

Institute

Abdul Wali Khan University

City

Mardan

Province

KPK

Country

Pakistan

Thesis Completing Year

2018

Thesis Completion Status

Completed

Subject

Botany

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10836/1/Lubna_Botany_2018_AWKU_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726979241

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Being a good source of phytohormones fungal endophytes have been considered as potent plant growth promoters. The present report elucidates the isolation of endophytic fungi from medicinal as well as crop plants. In current study 46 endophytic fungal isolates were isolated from medicinal (Cannabis sativa, Chenopodium album, Oxalis corniculata, Euphorbia helioscopia, Amaranthus viridis, And Parthenium hysterophorus) and crop (Triticum asativum, Brassica compestris) plants. Culture filtrated (CF) and biomass was initially screened on maize in order to find the plant growth promoting endophytic fungi and for indole acetic acid. Among these isolate culture filtrate of 13 strains showed promotary effect on maize seedling. The isolates produced IAA via L-tryptophan-independent pathway in rage of 1.254ug/mL to 26.73ug/mL and tryptophan dependent pathway in the range of 0.301ug/mL to 35.434ug/mL. Other important secondary metabolites including phenolic, flavonoid and sugars were also detected in the culture media of these endophytes. The isolated endophytes also supported growth of maize seedlings grown in fungal biomass supplemented soil. Based on their plant growth promoting potential, the endophytes CSR1, CSR3, TS2, CSL1, CHS1, EUR1, TS1 and BRL1 were selected for further study. Culture filtrates of the selected endophytes were screened for gibberellins (GAs) by using GAs deficient mutant rice Waito-C. The results revealed that different plant growth characteristic such as chlorophyll content, root-shoot length, and biomass production of Waito-C rice was significantly promoted during endophyts treatment showing the presence of GAs in their culture filtrate. Majority of these endophytes produced iron chelating siderophores and solubilized phosphate which is among the known phytostimulant tools of the endophytes. These morphologically representative strains were identified further by means of molecular phylogenetic analysis based on ITS and D1/D2 regions. Molecular identification using ITS and partial 28S ribosomal DNA (D1/D2) sequences from the isolates revealed 4 genera including Aspergillus, Fusarium, Bioplaris and Curvularia. These fungi are curvularia lunata TS2, Aspergillus fumigatus TS1, Aspergillus terreus EUR1, Aspergillus flavus CHS1, Aspergillus niger CSR3, Fusarium proliferatum x BRL1, Fusarium oxysporum CSR1 and Bipolaris sp CSL1. The isolated strains belonged to division Ascomycetes and among these 2 species belong to order Pleosporales, 2 belong to hypocreals and 4 belong to eurotiales. The selected endophytes were further characterized by determining phytohormones including IAA and gibberellins (GAs) using GC/MS SIM. This study is the first report related to curvularia lunata, Aspergillus terreus and Fusarium proliferatum to produce IAA. This growth promotion was due to various types of GAs and presence of IAA in endophyte culture filtrate. The gas chromatography/mass spectrometry (GC/MS) analysis showed the presence of different gibberellins in various quantities (ng/ml). Nine kinds of GAs tested were biologically active: GA1, GA3, GA4, GA7, GA8, GA9, GA12, GA20 and GA24. Biologically active gibberellins include GA1, GA3, GA4, and GA7 were identified in almost all fungi. Moreover, GA1 was detected in high quantity in CSR1 (0.638±0.019 ng/mL), followed by BRL1 (0.392±0.007ng/mL), CSL1 (0.753±0.005 ng/mL) and CHS1 (0.133±0.012ng/mL) respectively. Similarly, GA3 was found in high concentration in two fungi EUR1 (0.352±0.012ng/mL) and TS1 (0.324±0.077ng/mL). Notably, GA4, important bioactive GA, was detected in almost all fungal cultures in significant amount, However, the highest concentrations were found in CSL1 (0.943±0.081ng/mL), CSR3 (0.479±0.01ng/mL) and CHS1 (0.435±0.016ng/mL). Similarly, the fungal strains CSL1, CHS1, EUR1, and BRL1 produced 0.638±0.012, 0.476±0.082, 0.423±0.003 and 0.492±0.005 ng/mL of GA7. Furthermore, Inactive types of GA present in the FCF were GA8, GA9, GA12, GA20 and GA24. The ABA contents were also checked in these isolates and among these fungal strains, CSR1 had the highest concentration of ABA (0.0903ng/ml) in its CF. Upon inoculation, these endophytes contributed significant amount of GAs to the endogenous pool of Waito-C rice. Among these endophytes, CSL1 increased GA1, GA3, GA4 and GA12 concentration up to several fold peaking at 18.37ng/g DW, 11.37ng/g DW, 33.23ng/g DW and 25.534ng/g DW respectively. The isolate CSR3 enhanced endogenous level of GA4, GA7 and GA12 by up to 20.34ng/g DW, 28.264ng/g DW and 34.23ng/g DW as compared to the non-endophytes seedlings. Similarly, TS2 significantly increased the concentration of endogenous GA4 and GA12 of the mutant rice. Along with GAs, endogenous ABA level was also significantly enhanced in Wiato-C rice inoculated with the isolated endophytes than the control. The percent increase by different strains TS2, TS1, BRL1, CSR1, EUR1, CHS1, CSL1 and CSR3 is, 14%, 46.6%, 6.5%, 14%, 67%, 39%, 60.7%, 77.9% respectively. Contrary to this, endogenous concentration of JA dropped significantly in the endophytes associated seedlings indicating involvement of fungal GA and IAA. The percent decrease of JA in treated seedlings as compared to non-treated Waito-C rice seedlings is TS2 (27%), TS1 (53.8%), BRL1 (23.9%), CSR1 (33.5%), EUR1 (61%), CHS1 (49.9%), CSL1 (56%), CSR3 (68%). Furthermore, RT-PCR confirmed the presence of GA and IAA pathways genes (P50-1, P450-4, ggs2, des and iaaH) in the selected endophytic strains by positive expression. Moreover, the application of these fungal spore suspensions with uniconazole and yucasin on maize seedling revealed that like exogenous GA3 and IAA, the endophytic fungal strains CF application mitigated the inhibitory effect of both yucasin and uniconazole and promote growth attributes of maize seedling. Also, secondary metabolites of maize seedlings associated with the endophytic fungi were significantly enhanced recovering the seedlings from the suppressing effects of both uniconazole and yucasin. Besides, the selected strains were checked on cucumber cotyledon to check its cytokine like activity. In these strains some increase chlorophyll contents as compare to control which indicate the presence of cytokinin like compound in them. These finding suggest that these gibberellins and IAA producing endophytic strains plays important roles in plant growth promotion, which could be used for the improvement of crop growth under diverse environmental condition to mitigate the stresses.
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سر آسو توش مکر جی

سرآسوتوش مکرجی
گذشتہ ماہ کا سب سے بڑا علمی اور تعلیمی حادثہ سر آسوتوش مکر جی کی وفات ہے، بنگال کا یہ سپوت فرزند گو ایک نامور بیرسٹر، ایک قابل جج ہائیکورٹ، ایک بڑا مصنف، ایک مشہور ریاضی دان تھا، تاہم اس کی ناموری، قابلیت، بڑائی اور شہرت کا سب سے بڑا مظہر یہ تھا کہ اس نے تقریباً بیس برس تک ہندوستان کی سب سے بڑی درسگاہ کلکتہ یونیورسٹی پر بہ حیثیت وائس چانسلر سب سے عمدہ اور بہتر حکمرانی کی ان کی اس تعلیمی فرمان روائی کا زمانہ بنگال کی تعلیمی ترقی، اور امتحانات کی وسعت اور یونیورسٹی کے انتظامات کی خوبی اور معاملات تعلیمی میں حکومت کے مقابلہ میں پوری قوت کے ساتھ اپنے حقوق کی حفاظت کے لحاظ سے ہندوستان کا تعلیمی عہد زریں کہا جاسکتا ہے، موصوف نے اپنے بست سالہ عہد فرمان روائی میں یہ ثابت کردیاکہ جہاں تک یونیورسٹی کا تعلق ہے بنگال حکومت کی بے جا قید سے آزاد اور خود مختار ہے، ۲۹؍ مئی ۱۹۲۴؁ء ان کی وفات کا دن بنگال کے دائرہ تعلیم کے لیے ایک سانحہ عظیم ہے۔ (’’س‘‘، جون ۱۹۲۴ء)

 

بیوی کے نفقے کاقضیہ: شرعی اور عصری (پاکستانی) قوانین کے تناظر میں ایک علمی جائزہ

In Islamic point of view, the family is an institution that starts from the legal bond of marriage. The marriage is a contract that confirms the mutual rights of husband and wife. Including the other rights, one is the provision of maintenance to wife. The wife having leaving  her family and making a life time compromise to live with her husband reserves the right to be exempted from all kind of financial obligations. In Islamic family system, the husband is responsible both in legal and moral angles, to support his wife and provide the maintenance according to his financial status. Likewise, the wife has the right to demand the provision of maintenance from her husband. In time of none Provision, she can take this right through court. The wife reserves this right only if she is willing to live with her husband and does not disobey her husband’s reasonable orders. If it is so, then the stand for provision of maintenance shall be treated as invalid. In this paper, the matter of maintenance provision and its related problems have been discussed in contrast with the Pakistan family Laws which will provide a profound knowledge to the readers.

Identification of Differentially Expressed Proteins in Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer and a major public health issue worldwide. Survival rate of patients with CRC depends upon early diagnosis of the disease. In that CRC is mostly asymptomatic at an early stage and typically diagnosed only at advanced stages, implementation of screening strategies for early detection is highly desirable. Notwithstanding the fact that continuous development in screening protocols and available treatment options has resulted in a considerable decline in the CRC mortality rate in the developed nations, the scope of benefits so far has not extended to the developing nations like Pakistan where most of the patients present at the advanced stage with resultant poor survival rates. Currently available invasive, semi-invasive and non-invasive diagnostic/screening methods [e.g., carcinoembryonic antigen (CEA), faecal occult blood test (FOBT), colonoscopy, sigmoidoscopy, computed tomography (CT) scan or barium enema, etc.] have significantly improved the patient survival rates for CRC. However, some of the blood- and stool-based clinical tests, currently in practice, lack sufficient level of sensitivity and/or specificity. Hence, there remains a compelling need for developing more reliable, sensitive and specific non-invasive methods and the molecular markers for earlystage diagnosis CRC, to ensure increased survival rate of the patients and better disease management. Proteomic strategies to identify markers for the diagnosis of cancers (such as lung, liver, pancreas breast and ovarian cancers) at an early stage have been employed with noteworthy results. To extend these studies, we have utilized two dimensional gel electrophoresis (2D-PAGE) and mass spectrometry (MS) for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls) with an aim of identifying novel CRC associated proteins. Based on the anatomic/Duke’s staging, the vi collected CRC tissue samples (n=12) were grouped as 1) Stage B, 2) Stage C, and 3) Stage D samples. Equal amounts of the protein from tissue lysates of tumor and adjacent normal mucosa were resolved by 2D-PAGE followed by colloidal coomassie blue staining. On average, 505±216 spots in normal and 497±221 in tumor tissue lysate appeared when the colloidal-coomassie stained gels of the three CRC patient groups were compared. To select statistically significant, differentially stained gel spots, the quantification of individual spots was performed using ImageMaster 2D platinum (v. 7.0) software program. During the initial screening, 49 gel spots were found to have at-least one fold change in staining with p-value ≤0.05. False discovery rate (FDR) estimation of the data was performed with value set as ≤0.1 reflecting that more than 90% of the findings are accurate. The selected spots were subjected to identification by MALDI-TOF and/or liquid chromatography-based tandem mass spectrometry (LC-MS/MS). In the case of MALDITOF- MS data analysis, the threshold value for positive hits of peptide mass finger printing (PMF) score was set as ≥79 whereas in the case of LC-MS/MS, only the proteins identified with >95% probability were considered for further analysis. These analyses led to the identification of 21 distinct proteins. In silico characterization of the proteins identified by MS analysis (MALDI-TOFMS and LC-MS/MS) analysis was also performed. More specifically, the proteins of data sets were uploaded in Protein ANalysis THrough Evolutionary Relationships (PANTHER) database that categorized all proteins into molecular functions, biological processes and cellular components. Gene ontology (GO) data search for molecular functions classified these proteins based on of their binding, receptor activity, structural molecule activity, catalytic activity, antioxidant activity and transporter activity. In the context of biological processes, these proteins were found to be involved in ten major categories of biological processes viz., cellular component organization or biogenesis, cellular processes, vii localization, biological regulation, response to stimulus, developmental process, multicellular organismal process, biological adhesion, metabolic process and immune system process. Data analysis using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was also performed for an overview of the functionally connected proteins. It was found that two proteins PSME1 (proteasome activator subunit 1) and FABP5 functionally separated proteins than rest of proteins in our data set. So we performed individual network analysis of these two proteins and our protein of interest, ACTBL2. Collectively ACTBL2 appears to interact with proteins in cells that have potential to arrest cell growth, elicit tumorigenesis and promote cell migration. PSME1 directly involved in regulation of proteasome activity and FABP5 is important in cellular lipid catabolic process. Further for differentially expressed proteins, disease assessment was performed by Ingenuity Pathways Analysis (IPA). Most proteins showed up to be important in cancer. These prediction analyses by different tools all support together that experimentally identified proteins are vital in health and disease and could be beneficial if carefully studied in disease assessment. In the present study about 50% of the identified proteins were represented by more than one spots, generally 2-5 which could be due to difference in PTMs or alternative splicing. Therefore, the intensities of multiple gel spots that corresponded to the same protein were pooled/summed up and their statistical significance was re-calculated. Four gel spots that qualified four tier criteria (fold change >1.5, p-value ≤ 0.05, PMF score ≥79 and FDR analysis ≤0.05), were selected for further validation analysis. Two gel spots showed greater staining in the CRC samples compared to the matched samples from the normal tissue region while two showed decreased staining for the same comparison. These four spots include six proteins; beta-tropomyosin (TPM2), ACTBL2, PSME1, prohibitin viii (PHB), annexin A2 (ANXA2) and myosin light chain 9 (MYL9). Out of these six proteins five had previously been reported to be associated with colorectal cancer. In that ACTBL2 was identified in CRC at greater abundance than in normal tissue and had not previously been associated with CRC, we verified the mass spectrometry data by performing immunohistochemistry (IHC) for ACTBL2 in six paraffin embedded formalin fixed (FFPE) CRC patients samples and respective controls which were obtained from the Biorepository and Tissue Research Facility, University of Virginia, USA for validation of selected proteins.. From the mass spectrometry experiments above, PSME1 also appeared to be in high abundance in CRC samples compared to normal tissue. Given that PSME1 has previously been associated with CRC in some patients, we decided to perform IHC on another panel of CRC patient samples as well. For ACTBL2, significantly higher staining was observed in all six patient samples with p-value ≤0.005 and fold change +3.5. These results are similar to those obtained from the 2DE/MS data. However, in the case of PSME1, the IHC results showed marginally higher staining only in three out of six CRC patient samples when compared with the normal epithelium. This suggested that PSME1 is not upregulated in CRC patients. The higher abundance observed in 2DE/MS data for PSME1 could be due to the greater staining of spot CRC-05 containing two other upregulated proteins i.e., ANXA2 and PHB. Thus, ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer. Uncovering the differentially expressed proteins in colorectal cancer observed in this study will be important to understand the CRC carcinogenesis. However for more precise statistically significant proteins, much larger number of samples are needed along with testing the samples in serum/plasma samples. Respective studies have been started in our laboratory." xml:lang="en_US