Zahid Ali

Flavobacterium rivuli sp. nov., Flavobacterium subsaxonicum sp. nov., Flavobacterium swingsii sp. nov. and Flavobacterium reichenbachii sp. nov., isolated from a hard water rivulet

Strains WB 3.3-2(T), WB 3.2-61(T), WB 4.1-42(T) and WB 2.3-68(T) were isolated from the Westerhöfer Bach hard water rivulet, North Germany. The strains were Gram-staining-negative and catalase-, aminopeptidase- and oxidase-positive. The novel strains lacked flagella and only strain WB3.2-61(T) showed gliding motility. Isolates WB 3.2-61(T), WB 4.1-42(T) and WB 2.3-68(T) produced flexirubin pigments. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strains showed <98.2 % similarity to the type strains of all recognized species of the genus Flavobacterium. Strains WB 3.3-2(T) and WB 4.1-42(T) shared 96.3 % sequence similarity and were only distantly related to the type strains of all of the members of the genus Flavobacterium. Strain WB 3.2-61(T) branched adjacent to Flavobacterium limicola DSM 15094(T) (98.0 %), while strain WB 2.3-68(T) was a neighbour of Flavobacterium psychrophilum DSM 3660(T) (97.1 %). On R2A medium, iso-C(15 : 0) was the common major fatty acid; fatty acids C(15 : 0), C(16 : 0), iso-C(15 : 0) 3-OH, iso-C(17 : 1)omega9c, iso-C(17 : 0) 3-OH and summed feature 3 (comprising C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH) occurred in all strains though sometimes in low amounts. Metabolic properties revealed clear differences between the four isolates as well as between the isolates and their nearest phylogenetic neighbours. The lack of close relatedness was confirmed by Riboprinter and MALDI-TOF analyses of cell extracts. On the basis of a high number of phenotypic differentiating properties and phylogenetic uniqueness, four novel Flavobacterium species are proposed with the following names: Flavobacterium rivuli (type strain WB 3.3-2(T)=DSM 21788(T)=CIP 109865(T)), Flavobacterium subsaxonicum (type strain WB 4.1-42(T)=DSM 21790(T)=CIP 109867(T)), Flavobacterium swingsii (type strain WB 2.3-68(T)=DSM 21789(T)=CIP 109868(T)) and Flavobacterium reichenbachii (type strain WB 3.2-61(T)=DSM 21791(T)=CIP 109866(T)).

Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures.

The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.

Dicistronic binary vector system—A versatile tool for gene expression studies in cell cultures and plants

Dicistronic binary vector constructs based on pGreenII vectors for Agrobacterium mediated gene transfer alleviate the translational expression monitoring of a target gene in plants. The functionality of the transformation vectors was proven by marker gene constructs containing a mannopine synthase promoter (p-MAS) fused to a beta-glucuronidase (gus) gene followed by an internal ribosome entry site and a firefly luciferase (luc) gene. The cap-dependent translation of a physically independent target protein can be monitored by the cap-independently co-translated luciferase, because both mRNAs are located on the same strand. Among three different IRES elements, the tobamo IRES element showed highest activity in transient expression. As a proof of principle for physiological studies the gus gene was replaced by a sodium antiporter gene (Atnhx1). Comparative studies with Atnhx1 transgenic luc expressing tobacco cell cultures and pea plants (Pisum sativum L.) showed improved salt tolerance in relation to their wild type counterparts grown under corresponding conditions. A coincidence of the luc gene expression and increased sodium chloride tolerance is demonstrated by measurement of luminescence and cell growth.

Strigolactones Biosynthesis and Their Role in Abiotic Stress Resilience in Plants: A Critical Review

Strigolactones (SLs), being a new class of plant hormones, play regulatory roles against abiotic stresses in plants. There are multiple hormonal response pathways, which are adapted by the plants to overcome these stressful environmental constraints to reduce the negative impact on overall crop plant productivity. Genetic modulation of the SLs could also be applied as a potential approach in this regard. However, endogenous plant hormones play central roles in adaptation to changing environmental conditions, by mediating growth, development, nutrient allocation, and source/sink transitions. In addition, the hormonal interactions can fine-tune the plant response and determine plant architecture in response to environmental stimuli such as nutrient deprivation and canopy shade. Considerable advancements and new insights into SLs biosynthesis, signaling and transport has been unleashed since the initial discovery. In this review we present basic overview of SL biosynthesis and perception with a detailed discussion on our present understanding of SLs and their critical role to tolerate environmental constraints. The SLs and abscisic acid interplay during the abiotic stresses is particularly highlighted. Main Conclusion: More than shoot branching Strigolactones have uttermost capacity to harmonize stress resilience.

Bioefficacy of Rhizobacterial Isolates against Root Infecting Fungal Pathogens of Chickpea (Cicer arietinum L.)

Chickpea is considered to be food for the poor in Pakistan. Its yield is much lower than expected due to infestation of a number of fungal pathogens. The present study was designed to determine the effect of rhizobacterial isolates against fungal pathogens infecting chickpea roots. RH-31, RH-32 and RH-33 were isolated from groundnut rhizosphere. Antifungal activities of these isolates were tested by seed treatment and soil application methods against three root fungal pathogens. Data on disease incidence, bio-control efficiency and root biomass was recorded. Phylogenetic analysis indicated that sequences of RH-31, RH-32 and RH-33 showed >99% identity with Paenibacillus illinoisensis, Bacillus subtilis, and Pseudomonas psychrotolerans respectively. RH-33 was effective against Fusarium oxysporum and Macrophomina phaseolina with highest levels of inhibition, whereas RH-32 inhibited Fusarium solani. However, RH-31 showed best activity against F. oxysporum. Disease incidence and bio-control efficiency revealed that all isolates reduced disease severity and increased overall plant biomass as compared to control treatment. Present findings show potential of bacterial isolates from rhizosphere of Pakistan. Application of selected rhizobacteria through seed treatment method might be a promising strategy to lower damage caused by root pathogens in chickpea. This could be efficient, economical, environment friendly and might serve as a biocontrol agent.

Efficient and reproducible somatic embryogenesis and micro propagation in tomato via novel structures -Rhizoid Tubers.

An improved and highly reproducible system for invitro regeneration via somatic embryogenesis (S.E), applicable to several varieties of tomato (cv. Riogrande, cv. Roma grande, hybrid 17905 and model cv. M82) has been developed. First, we developed a conventional indirect organogenesis for all four varieties used in this study. The cotyledons and hypocotyls of 6-day-old tomato were used as explants (1-2 cm) for callus induction (CI) on different callus induction media (CIM) T0 – T12 (6-Benzylaminopurine BAP, NAA Naphthalene acetic acid, ZEA Zeatin, IAA Indole-3-acetic acid, KIN Kinetin). Maximum CI response was seen on CIMT6 (0.5 mg/L NAA, 1 mg/L BAP) and CIMT7 (2 mg/L IAA, 2 mg/L NAA, 2 mg/L BAP, 4mg/L KIN) in a period of 2 weeks for commercial varieties cvs. Riogrande and Roma. However, cv. M82 responded after 4 weeks to a combination of treatments [CIMT6 (0.5 mg/L NAA + 1 mg/L BAP) and CIMT8 (2 mg/L IAA + 2 mg/L NAA + 2 mg/L BAP + 4 mg/L ZEA)] for the production of calli. The Riogrande, being the most responsive commercial variety, was selected for invitro morphogenesis via S.E. During S.E. young cotyledons and hypocotyls explants were tested on media with different ranges of pH (3 – 7) supplemented with 0.5 and 2 mg/L NAA. Resultantly, numerous rhizoids (~38) were produced from each explant at pH4 in dark conditions. Further incubation of each rhizoid under light conditions led to the formation of a novel structure - rhizoid tubers (RTBs) on MS media supplemented with 5 mg/L TDZ/BAP at pH4. We observed that only lower pH-induced rhizoids and RTBs regenerated into multiple individual shoots on media at normal pH (5.8). The RTBs led to a complete plantlets regeneration in 45 days compared to the conventional invitro morphogenesis (60 days).

Computational modeling and functional characterization of a GgChi: A class III chitinase from corms of Gladiolus grandiflorus

The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI‐QTOF‐MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4‐methylumbelliferyl β‐d‐N,N′,N′′‐triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (β/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 μg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 μg/well Similarly, insect toxicity bioassays of GgChi (50 μg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase.

Structure–activity relationships of strigolactones via a novel, quantitative in planta bioassay

The biological activity of natural and novel strigolactone D-lactam analogues is assessed using a novel bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to firefly luciferase.

SERODIAGNOSIS OF Xanthomonas campestris pv. sesami CAUSING BACTERIAL LEAF BLIGHT DISEASE IN SESAME

Sesame is an important crop that has potential to add to edible oil production of Pakistan but bacterial blight incited by Xanthomonas campestris pv. sesami (Xcs) is most serious and devastating disease of sesame responsible for colossal losses. For decision of effective management strategy, proper identification of pathogen is pre-requisite. Present study was designed to evaluate ELISA with polyclonal antibodies (PAbs) for detection of Xcs with a fact that ELISA is cost effective and can be used for screening of large samples. PAbs were prepared in rabbits against pure isolate of Xcs. Direct antigen coated (DAC)ELISA was used for the analysis of pathogen in sesame germplasm obtained from NARC (Islamabad). Pathogen from 29 sesame varieties, categorized from resistant to susceptible, was isolated and used as an antigen. After incubation, OD was calculated at 405 nm and results revealed that lowest reaction (0.068) was observed in highly resistant sesame genotypes i.e. SG-22 and SG-55. While varieties SG-34, SG-33 and SG-72 were categorized as resistant based on reactivity (0.073). Thus it is concluded that ELISA with PAbs should be preferred for detection of Xcs because of the fact that they are more stable, target multiple epitopes on single antigen and are easy to produce.