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Fuctional and Genomic Diversity of Agriculturaly Important Bacillus from Rhizosphere of Wheat and Maize

Thesis Info

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Author

Nawaz, Huma

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Microbiology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10796/1/Huma%20Nawaz_Microbiology_%26_Molecular_Genetics_2019_UoPunjab_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726242410

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Rhizosphere of plant is the zone which is directly under the impact of plant roots. The application of microorganisms in the field of agriculture for improving the availability of nutrients has become an important practice nowadays. Different bacterial strains that present in rhizosphere play significant role in crop improvements. These are known as plant growth promoting rhizobacteria (PGPR). These bacteria strains belongs to genus Bacillus, Pseudomonas, Ochrobacterum, Azotobacter, Rhizobium, Stenotrophomonas, Serratia, Azosperillum and Enterobacteria etc. It has already reported by many scientists that genus Bacillus is dominant in the rhizospheric zone because of having external protection of hard cell wall, firm cell membrane, internal system of enzymes and endospore formation, which guards it against physical and chemical risks and having plant growth promoting characteristics. Present study deals with 64 Bacillus strains isolated from different areas of Punjab, Pakistan as 32 strains from Corn rhizosphere and 32 Bacillus strains from Wheat rhizosphere. These strains are identified upto species level by 16S rRNA sequencing. The Corn rhizospheric strains are identified as Bacillus sonorensis-CSH27, Bacillus sp.-CSH23, Bacillus thuringiensis-Cu47, Bacillus axarquiensis-CF18, Bacillus safensis-CU35, Bacillus mojavensis-CU4, Bacillus safensis-CN17, Bacillus subtilis-CN2, Bacillus endophyticus CU33, Bacillus cereus-CMS17, Bacillus pumilus-CU9, Bacillus cereus-CMS7, Bacillus pumilus-CSH4, Bacillus toyonensis-Cu48, Bacillus safensis-CF14, Bacillus tequilensis-CN7, Bacillus anthracis-CMS14, Bacillus cereus-CSH2, Bacillus anthracis-CL13, Bacillus safensis-CF4, Bacillus mycoides-CL2, Bacillus thuringiensis-CMS20, Bacillus pseudomycoides-CN9, Bacillus anthracis-CN13, Bacillus sonorensis-CSH1, Bacillus anthracis-CSH26,Bacillus anthracis-CU7, Bacillus mojavensis-CU14, Bacillus cereus CU46, Bacillus subtilis-CU31, Bacillus anthracis-CL11, Bacillus cereus-CN12. Similarly 32 Bacillus strains isolated from Wheat rhizosphere and identified as Bacillus subtilis-WRY23, Bacillus pumilus-WM3, Bacillus thuringiensis-WSK9,Bacillus thuringiensis-WG5, Bacillus anthracis-WFS6, Bacillus axarquiensis-WSH8, Bacillus anthracis-WUS2,Bacillus thuringiensis-WG1, Bacillussp.-WUS16, Bacillus sp.-WSH5, Bacillus anthracis-WRY1, Bacillus subtilis-WRY11, Bacillus cereus-WFS20, Bacillus subtilis-WL22, Bacillus anthracis-WSH13, Bacillus mojavensis-WSK11, Bacillus pseudomycoides-WSK6, Bacillus flexus-WSH3, Bacillus sonorensis-WG3, Bacillus flexus-WG14, Bacillus cereus-WM13, Bacillus megaterium-WSH10, Bacillus cereus-WSK13, Bacillus thuringiensis-WSK16, Bacillus vallismortis-WUS4, Bacillus anthracis-WM14, Bacillus thuringiensis-WG20, Bacillus pumilus-WM2,Bacillus velezensis-WG4,Bacillus amyloliquefaciens-WG8, Bacillus vallismortis-WG21, Bacillus safensis-WM20. Out of all 32 Bacillus isolates from Corn rhizosphere, most abundant species are Bacillus cereus, Bacillus anthracis and Bacillus subtilis. Similarly isolates from Wheat rhizosphere are Bacillus subtilis, Bacillus cereus and Bacillus thuringiensis. Phylogenetic analysis showed 99-100% homology of all isolated strains to Bacillus genera and all of them showed common evolutionary lineage in their respective phylogenetic trees. These sixty-four strains are gram positive and motile rods with spore forming ability. Majority of the isolates having growth temperature 37ºC and pH range 7, respectively. Similarly majority of Bacillus strains have ability to grow at high concentration of salts (NaCl) provided in the medium. Majority of bacterial isolates are resistant to high concentrations of heavy metals and are involved in catalase production, hydrolysis of starch, lipid and gelatin and use citrate as carbon source and also involved in the nitrification and denitrification process. Plant growth promoting bacteria play important role in nitrogen cycle and acts as ammonifiers, nitrifiers and denitrifiers and plays very important role in transformation of nitrogen and make it available for the plant by the processes of ammonification, nitrification and denitrification, respectively. There is great functional diversity found among these Corn and Wheat Bacillus isolates. All of them are involved in production of auxin. Some strains produces auxin in less quantity whereas some produce more. About 21.8% Corn isolates are phosphate solubilizers and similarly about 21% Wheat isolates are involved in phosphate solubilization. Siderophores are also important contributing factor in plant growth promotion. About 12.5% Corn isolates and 15.6% Wheat isolates are siderophore producers. Majority of Bacillus strains involved in the production of ACC deaminase. 38% of Corn and 35% of Wheat isolates were involved in production of hydrogen cyanide. Majority of isolates showed antibacterial activity against pathogens as among Corn isolates, about 50% showed activity against S. aureus, 15.6% against E. coli, 62.5% against Klebsiella, 56.3% against Pseudomonas, 50% against Proteus, 21.8% against Enterobacter and 78% showed activity against Acinetobacter. Similarly among Wheat isolates 18.8% showed activity against S.aureus, 3% against E.coli, 21.8% against Klebsiella, 21.8% against Pseudomonas, 12.5% against Proteus 6.25% against Enterobacter, 12.5% against Acinetobacter. These Corn rhizospheric strains were used for the growth promotion of Corn in the field experiments. It was observed that majority of bacterial strains induced positive growth promoting attributes in vegetative growth of Corn. Strain CU9 showed significant, (49%) increase in shoot length of plants. Inoculation with bacterial isolates significantly increased the cob length in all Corn plants except CSH23 inoculated plant as compared to control plant. Inoculated plants also exhibited increment in dry weight of plant except CN17 and CSH4. Among all inoculated plants strain CU47 showed significantly high peroxidase activity (163%) whereas CSH23, Comb 4A inoculated plants also showed increase of about 107% and 94%, respectively, as compared to control plants. Comb 4B showed remarkable high content of acid phosphatase (811%) as compared to control plants. Bacteria isolated from Wheat rhizosphere were also used for growth promotion of Wheat plant both in laboratory as well as in field trials. In laboratory trials, there is increment in shoot length and root length of Wheat seedling by majority of strains. Comb 5A and MIX inoculated seedlings exhibited increase in shoot length, root length and number of lateral roots. In pot experiments, there is an increase in plant height by all strains whereas combinations of strains (5A and 5B) and mixture of strains (MIX) also produced good results. About 18% to 19% increase in grain yield is observed in case of Comb 5A and Comb 5B inoculated plants as compared to control. WG1 showed about 71% increase in peroxidase activity than control whereas WUS2 showed remarkable increase of about 172% in acid phosphatase content. In another field trial where the impact of bacterial strains along with chemical fertilizer was applied, four different treatments were given (a) Bacterial inoculation and its respective uninoculated control (b) Bacterial inoculation along with DAP and its respective uninoculated control (c) Bacterial inoculation along with Urea and its respective uninoculated control (d) Bacterial inoculation along with Urea and DAP and its respective uninoculated control. Comb 5B showed maximum enhancement (27%) in shoot length without addition of any chemical fertilizer. The significant increase in spike length is also observed in inoculated plants supplemented with DAP, Urea and mixture of these two fertilizers in comparison to their respective uninoculated plants used as control. MIX inoculated plants showed 29% increase in spike length of Wheat with DAP supplementation and 18% in case of Urea addition. Similarly increase in weight of spikes in WSK11 is 81% in Urea supplemented plant. There is an increase in weight of 100 grains per plant in all inoculated plants but Urea supplemented WRY23 and Comb 5B showed 70% increase in weight of grains. Majority of inoculated plants supplemented with Urea showed increase in peroxidase activity whereas Comb 5B inoculated plant treated with Urea and DAP showed increment of 846%. DAP treated WM3 showed 356% and MIX showed 842% increase in acid phosphatase activity. Some species of Bacillus (Bacillus thuringiensis, Bacillus safensis, Bacillus pumilus, Bacillus subtilis, Bacillus axarquiensis, Bacillus anthracis, Bacillus tequiliensis, Bacillus cereus, Bacillus sonorensis) were present in both Wheat and Corn rhizosphere whereas some species were host specific as only in Corn (Bacillus toyoensis, Bacillus endophyticus, Bacillus mycoides) and only in Wheat (Bacillus flexus, Bacillus vallismortis, Bacillus amyloliquiefaciens, Bacillus megaterium,Bacillus velezensis). The species also varies from sample to sample taken from same crop. Similarly plant growth promoting attributes also varies from strain to strain. Majority of them involved in auxin production and ACC deaminase activity. Phosphate solubilization, HCN production, Siderophore production, Phytase activity, Antibacterial activities varies in all strains. These differences may be due to several reasons which may affect the presence of bacterial community in rhizospheric soil which involves root exudation, use of fertilizers in certain fields, pH of Soil, presence of organic matter, phosphorus and potassium present in soil etc. Majority of Bacillus species have remarkable plant growth promoting attributes and showed significant effects on growth of Corn and Wheat plants. These plant growth promoting strains can helpful in future for the formulations of biofertilizer. The objective of this study is to isolate different species of Bacillus from Wheat and Corn rhizosphere to check their functional and genomic diversity. Rhizospheric bacterial strains from Corn and Wheat rhizosphere will be screened for different plant growth promoting attributes and selected strains will be used to study the growth promoting effects on Corn and Wheat plant. The interaction of chemical fertilizers and bacterial strains effect on Wheat will also be studied." xml:lang="en_US
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58. Al-Mujadilah/The Pleading Woman

58. Al-Mujadilah/The Pleading Woman

I/We begin by the Blessed Name of Allah

The Immensely Merciful to all, The Infinitely Compassionate to everyone.

58:01
a. Allah has heard the woman who was pleading with you, concerning the issue of her husband, and was complaining to Allah.
b. And Allah was hearing the conversation between you.
c. For Allah is All-Listening, All-Watching.

58:02
a. Those among you who estrange their women/wives by declaring them to be as their mothers;
b. should know that they can never be made as their mothers, for their mothers are only those who gave birth to them.
c. Indeed, they utter what is highly contemptuous, baseless and false.
d. Yet, assuredly, Allah will be Pardoning, Forgiving to the one who repudiates by zihaar through an atonement.

58:03
a. Those of the husbands who thus estrange their women/wives, then they wish to retract what they had uttered, let them free a captive before any skin-to-skin cohabitation between them.
b. Thus you are being warned so that you will never utter such contemptuous words again.
c. And Allah is Well-Aware of what you do.

58:04
a. And whoever cannot find any captive to set free or does not have the means of doing so, then let him keep Fast for two consecutive months, without any interruption, before any skin-to-skin cohabitation between them.
b. And whoever is unable for reasons of ill health or old age, then let him feed sixty poor people.
c. All this is decreed so that you may believe in Allah, HIS Injunctions and HIS Messenger.
d. And these are the limits set by Allah.
e. And the disbelievers/disobedient - those who will not comply...

Prognostic Significance of Cellular Iron Metabolism in Breast Cancer

Breast carcinoma is among the most common malignancy in women. Objective: Aim of the present study was to evaluate the prognostic significance of iron expression in the biopsies of patients with breast cancer. Methods: 24 breast biopsies were studied. 19 cases were poorly differentiated, 5 cases were moderately differentiated and there was no well differentiated case. Iron, Estrogen receptor (ER), Progesterone receptor (PR), HER2 and Ki-67 immunohistochemical staining was performed for all these cases. Results: Among the 5 moderately differentiated cases, 3 (60%) were positive for iron staining and among 19 poorly differentiated cases, 11 cases (57.89%) were positive. More iron positive cases (7 out of 14) were triple positive belonging to Luminal B class. Out of 14 iron positive cases, 11 were positive for HER2, 10 for ER, 9 for PR and all positive for Ki-67. Conclusions: Iron deficiency in premenopausal and overload in post-menopausal women can contribute to the development of breast carcinoma. So, iron can be considered as a cheap and effective marker for the prognosis of breast cancer. Association between a risein iron levels and HER2 expression may providenewstrategy for breast cancer treatment.

Prevalence of Nasal Carriage of Methicillin-Resistant Staphylococcus Aureus in Healthcare Workers at Aga Khan University Hospital, Nairobi

Objective: To establish the prevalence of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in healthcare workers (HCW) at Aga Khan University Hospital, Nairobi (AKUHN). Background: Healthcare workers are a major reservoir of MRSA and may play a role in the spread of nosocomial MRSA infections. MRSA can cause significant morbidity and mortality. There is both molecular and epidemiological evidence linking HCW to MRSA outbreaks. Method: A cross sectional study conducted between July and December 2010. Nasal swabs were taken from 246 randomly selected HCW from various departments.MRSA was identified using both phenotypic and genotypic methods. Antibiotic susceptibility testing of all Staphylococcus aureus isolates was done using the broth dilution MIC technique. Results: The prevalence of nasal carriage of MRSA was 0% (95% CI:0.0%-1.5%) while that of methicillin sensitive Staphylococcus aureus (MSSA) was 18.3% (95% CI: 14.0%-23.6%). Carriage of MSSA was significantly higher in males (p=0.001, OR 5.74:2.09-15.77). Carriage was highest among HCW in the intensive care/high dependency units at 37%. Resistance to more than one class of antibiotics was seen in 37.7% of isolates. Prevalence of Panton Valentine Leukocidin (PVL) was 24.4%. Conclusion: The absence of nasal carriage of MRSA by HCW working in AKUHN suggests that they are unlikely to be the source of infections caused by MRSA in patients attending AKUHN. Screening of HCW for nasal carriage of MRSA at AKUHN should be limited to an outbreak setting. The presence of the exotoxin PVL as well as the presence of multidrug resistant strains of Staphylococcus aureus shows that HCW can still be a potential source of infections caused by MSSA.