Muhammad Nadeem Hassan

Biological control of red rot in sugarcane by native pyoluteorin-producing Pseudomonas putida strain NH-50 under field conditions and its potential modes of action

BACKGROUND Rhizobacteria have a good potential to suppress soilborne diseases, but their efficacy against sugarcane pests is rarely reported. Bacterial strains isolated from the rhizosphere of sugarcane were evaluated for their potential to suppress red rot disease on two susceptible varieties, Co-1148 and SPF-234, under field conditions. The strains were also characterised for the production of secondary metabolites associated with their antagonistic activity. RESULTS One out of four strains, the Pseudomonas putida strain NH-50 (EU627168), reduced disease severity by 44-60% in different field trials. This potent antagonistic strain produced pyoluteorin antibiotic, as confirmed by high-performance liquid chromatography (HPLC). The PltB gene involved in pyoluteorin synthesis was amplified from the P. putida strain NH-50 and sequenced. The extracellular metabolites and volatile and diffusible antibiotics secreted by the tested strains inhibited mycelial growth of Glomerella tucumensis (Speg.) Arx & E Mull in vitro by 7-55%. CONCLUSION The pyoluteorin-producing bacteria P. putida strain NH-50 significantly reduced disease severity on both sugarcane varieties, irrespective of fungal inoculation, i.e. either inoculated through stem or through soil. This strain also possesses other plant growth characteristics and can be used as a biopesticide for sugarcane crop.

Root Associated Bacillus sp. Improves Growth, Yield and Zinc Translocation for Basmati Rice (Oryza sativa) Varieties

Plant associated rhizobacteria prevailing in different agro-ecosystems exhibit multiple traits which could be utilized in various aspect of sustainable agriculture. Two hundred thirty four isolates were obtained from the roots of basmati-385 and basmati super rice varieties growing in clay loam and saline soil at different locations of Punjab (Pakistan). Out of 234 isolates, 27 were able to solubilize zinc (Zn) from different Zn ores like zinc phosphate [Zn3 (PO4)2], zinc carbonate (ZnCO3) and zinc oxide (ZnO). The strain SH-10 with maximum Zn solubilization zone of 24 mm on Zn3 (PO4)2ore and strain SH-17 with maximum Zn solubilization zone of 14–15 mm on ZnO and ZnCO3ores were selected for further studies. These two strains solubilized phosphorous (P) and potassium (K) in vitro with a solubilization zone of 38–46 mm and 47–55 mm respectively. The strains also suppressed economically important rice pathogens Pyricularia oryzae and Fusarium moniliforme by 22–29% and produced various biocontrol determinants in vitro. The strains enhanced Zn translocation toward grains and increased yield of basmati-385 and super basmati rice varieties by 22–49% and 18–47% respectively. The Zn solubilizing strains were identified as Bacillus sp. and Bacillus cereus by 16S rRNA gene analysis.

Plant Growth-Promoting Rhizobacteria as Zinc Mobilizers: A Promising Approach for Cereals Biofortification

As cereal crops are the main source of micronutrients such as Zn, subsequently Zn-deficient food crops are responsible for causing hidden hunger and malnutrition in people living in developing countries. Moreover, crop plants such as wheat, rice, maize, and sorghum show reduced photosynthetic carbon metabolism due to Zn deficiency. Different approaches are in progress to improve the Zn insufficiency in cereal. Strategies for the improvement of the Zn contents of food crops like chemical fertilization, agronomic practices, and transgenic plants development appear to have potential; however, these engaged practices have increased the environmental pollution, high-cost, socioeconomic, and political issues. The use of plant growth-promoting rhizobacteria (PGPR) offers an attractive approach to substitute chemical fertilizer, pesticides, and supplements. In particular, in vivo trials of Zn-mobilizing PGPR result in cereal biofortification.

Bacillus spp., a bio-control agent enhances the activity of antioxidant defense enzymes in rice against Pyricularia oryzae

Plant growth promoting rhizobacteria (PGPR) are found to control the plant diseases by adopting various mechanisms. Induced systemic resistance (ISR) is an important defensive strategy manifested by plants against numerous pathogens especially infecting at aerial parts. Rhizobacteria elicit ISR by inducing different pathways in plants through production of various metabolites. In the present study, potential of Bacillus spp. KFP-5, KFP-7, KFP-17 was assessed to induce antioxidant enzymes against Pyricularia oryzae infection in rice. The antagonistic Bacillus spp. significantly induced antioxidant defense enzymes i-e superoxide dismutase (1.7–1.9-fold), peroxidase (3.5–4.1-fold), polyphenol oxidase (3.0–3.8-fold), phenylalanine ammonia-lyase (3.9–4.4-fold), in rice leaves and roots under hydroponic and soil conditions respectively. Furthermore, the antagonistic Bacillus spp significantly colonized the rice plants (2.0E+00–9.1E+08) and secreted multiple biocontrol determinants like protease (1.1–5.5 U/mg of soil or U/mL of hydroponic solution), glucanase, (1.0–1.3 U/mg of soil or U/mL of hydroponic solution), siderophores (6.5–42.8 μg/mL or mg) in the rhizosphere of different rice varieties. The results showed that treatment with Bacillus spp. enhanced the antioxidant defense activities in infected rice, thus alleviating P. oryzae induced oxidative damage and suppressing blast disease incidence.

Computer Aided Screening of Phytochemicals from Garcinia against the Dengue NS2B/NS3 Protease.

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy.

Homology modeling of an antifungal metabolite plipastatin synthase from the Bacillus subtilis 168

Lipopeptides have a widespread role in different pathways of Bacillus subtilis; they can act as antagonists, spreader and immunostimulators. Plipastatin, an antifungal antibiotic, is one of the most important lipopeptide nonribosomly produced by Bacillus subtilis. Plipastatin has strong fungitoxic activity and involve in inhibition of phospholipase A2 and biofilm formation. For better understanding of the molecule and pathway by which lipopeptide plipastatin is synthesized, we present a computationally predicted structure of plipastatin using homology modeling. Primary and secondary structure analysis suggested that ppsD is a hydrophilic protein containing a significant proportion of alpha helices, and subcellular localization predictions suggested it is a cytoplasmic protein. The tertiary structure of protein (plipastatin synthase subunit D) was predicted by homology modeling. The results suggest a flexible structure which is also an important characteristic of active enzymes enabling them to bind various cofactors and substrates for proper functioning. Validation of 3D structure was done using Ramachandran plot ProsA-web and QMEAN score.This predicted information will help in better understanding of mechanisms underlying plipastatin synthase subunit D synthesis. Plipastatin can be used as an inhibitor of various fungal diseases in plants.

Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management.

Molecular Identification and Genetic Characterization of Macrophomina phaseolina Strains Causing Pathogenicity on Sunflower and Chickpea

Macrophomina phaseolina is the most devastating pathogen which causes charcoal rot and root rot diseases in various economically important crops. Three strains M. phaseolina 1156, M. phaseolina 1160, and M. phaseolina PCMC/F1 were tested for their virulence on sunflower (Helianthus annuus L.) and chickpea (Cicer arietinum L.). The strains showed high virulence on both hosts with a disease score of 2 on chickpea and sunflower. The strains also increased the hydrogen per oxide (H2O2) content by 1.4- to 1.6-fold in root as well as shoot of chickpea and sunflower. A significant increase in antioxidant enzymes was observed in fungal infected plants which indicated prevalence of oxidative stress during pathogen propagation. The M. phaseolina strains also produced hydrolytic enzymes such as lipase, amylase, and protease with solubilization zone of 5–43 mm, 5–45 mm, and 12–35 mm, respectively. The M. phaseolina strains were identified by 18S rRNA and analyzed for genetic diversity by using random amplified polymorphic DNA (RAPD) markers. The findings based on RAPD markers and 18S rRNA sequence analysis clearly indicate genetic variation among the strains collected from different hosts. The genetically diverse strains were found to be pathogenic to sunflower and chickpea.

Utilization of plant associated rhizobacteria to improve plant nutrition and protection: a cost effective and ecofriendly strategy

Plant associated bacteria are beneficial microbes which inhabit various parts of plants such as rhizosphere, endosphere and polysphere. These small little wonders improve plant health either directly by providing nutrients or indirectly by suppressing disease causing pathogens. We isolated more than 1000 rhizobacteria from the rhizosphere and endosphere of various field crops and screened for biofertilizer and biopesticide characteristics. About 100 isolates were selected on the basis of best N-fixation (3624 nmole C2H4/mg protein/h), P-solubilization (320 µg /mL), Silicate solubilization (35 mm zone), Indole acetic acid production (62 µg /mL), and in vivo biocontrol activity (30-60%) under field conditions. These effective PGPR belonged to different genera such as Acetobacter, Pseudomonas, Azospirillum, Rhizobium, Azotobacter, Bacillus, Enterobacter, Aeromonas, Burkholderia and Serratia as identified by 16S rDNA. Biocontrol activity of the antagonistic strains was found to be associated with the production of siderophores, antibiotics such as surfactin, pyoluteorin and hydrolytic enzymes like protease, glucanase and chitinase. The potent strains were preserved at Pakistan Collection of Microbial Cells (PCMC). The some efficient strains capable to solubilize phosphorus and survive in humic acid with a population of 10 7 -10

BIOLOGICAL SUPPRESSION OF SUGARCANE RED ROT BY BACILLUS spp. UNDER FIELD CONDITIONS

Yield of sugarcane (Saccharum officinarum L.) is critically limited by the red rot disease caused by Colletotrichum falcatum Went. Four native antagonistic bacteria Bacillus subtilis strains NH-100, B. subtilis NH- 160, Bacillus sp. NH-217 and NH-69, that successfully suppressed the red rot disease under greenhouse conditions were evaluated to determine their efficacy as biological control agents in the field. Performance of the antagonistic strains was assessed on two different sugarcane varieties SPF-234 and Co-1148 under field conditions for three consecutive years. Two types of trials were conducted in which the red rot pathogen was inoculated by different methods to observe its direct suppression as well as the induction of systemic resistance. Three strains of the genus Bacillus reduced disease incidence by 45-49% in sugarcane plants challenged by pathogen inoculation in the stem and by 48-56% in the plants inoculated in the soil near the roots. The results of present study suggest a potential use of these strains in the development of commercial inoculants to be applied for the control of red rot disease.