Aureobasidium pullulans from the Fire Blight Biocontrol Product, Blossom Protect, Induces Host Resistance in Apple Flowers.

Fire blight, caused by Erwinia amylovora, is a devastating disease of apple. Blossom Protect, a product that contains Aureobasidium pullulans as the active ingredient, is one of the most effective biological controls of fire blight. It has been postulated that the mode of action of A. pullulans is to compete against and antagonize epiphytic growth of E. amylovora on flowers, but recent studies have found that flowers treated with Blossom Protect harbored similar to or only slightly reduced E. amylovora populations compared with nontreated flowers. In this study, we tested the hypothesis that A. pullulans-mediated biocontrol of fire blight is the result of induced host resistance. We found that PR genes in the systemic acquired resistance pathway, but not genes in the induced systemic resistance pathway, were induced in hypanthial tissue of apple flowers after the Blossom Protect treatment. Additionally, the induction of PR gene expression was coupled with an increase of plant-derived salicylic acid in this tissue. After inoculation with E. amylovora, PR gene expression was suppressed in nontreated flowers, but in flowers pretreated with Blossom Protect, the heightened PR expression offset the immune repression caused by E. amylovora, and prevented infection. Temporal and spatial analysis of PR gene induction showed that induction of PR genes occurred 2 days after the Blossom Protect treatment, and required direct flower-yeast contact. Finally, we observed deterioration of the epidermal layer of the hypanthium in some of the Blossom Protect-treated flowers, suggesting that PR gene induction in flowers may be a result of pathogenesis by A. pullulans.

Engineered Nanoparticles, Natural Nanoclay and Biochar, as Carriers of Plant-Growth Promoting Bacteria.

The potential of biochar and nanoparticles to serve as effective delivery agents for beneficial bacteria to crops was investigated. Application of nanoparticles and biochar as carriers for beneficial bacteria improved not only the amount of nitrogen-fixing and phosphorus-solubilizing bacteria in soil, but also improved chlorophyll content (1.2-1.3 times), cell viability (1.1-1.5 times), and antioxidative properties (1.1-1.4 times) compared to control plants. Treatments also improved content of phosphorus (P) (1.1-1.6 times) and nitrogen (N) (1.1-1.4 times higher) in both tomato and watermelon plants. However, the effect of biochars and nanoparticles were species-specific. For example, chitosan-coated mesoporous silica nanoparticles with adsorbed bacteria increased the phosphorus content in tomato by 1.2 times compared to a 1.1-fold increase when nanoclay with adsorbed bacteria was applied. In watermelon, the situation was reversed: 1.1-fold increase in the case of chitosan-coated mesoporous silica nanoparticles and 1.2 times in case of nanoclay with adsorbed bacteria. Our findings demonstrate that use of nanoparticles and biochar as carriers for beneficial bacteria significantly improved plant growth and health. These findings are useful for design and synthesis of novel and sustainable biofertilizer formulations.

Whole genome analysis of Gluconacetobacter azotocaptans DS1 and its beneficial effects on plant growth.

Plant-associated bacteria play an important role in the enhancement of plant growth and productivity. Gluconacetobacter azotocaptans is an exceptional bacterium considering that till today it has been isolated and reported only from Mexico and Canada. It is a plant growth-promoting bacterium and can be used as biofertilizer for different crops and vegetables. The objective of the current study was to evaluate the inoculation effect of Gluconacetobacter azotocaptans DS1, Pseudomonas putida CQ179, Azosprillium zeae N7, Azosprillium brasilense N8, and Azosprillium canadense DS2, on the growth of vegetables including cucumber, sweet pepper, radish, and tomato. All strains increased the vegetables' growth; however, G. azotocaptans DS1 showed better results as compared to other inoculated and control plants and significantly increased the plant biomass of all vegetables. Therefore, the whole genome sequence of G. azotocaptans DS1 was analyzed to predict genes involved in plant growth promotion, secondary metabolism, antibiotics resistance, and bioremediation of heavy metals. Results of genome analysis revealed that G. azotocaptans DS1 has a circular chromosome with a size of 4.3 Mbp and total 3898 protein-coding sequences. Based on functional analysis, genes for nitrogen fixation, phosphate solubilization, indole acetic acid, phenazine, siderophore production, antibiotic resistance, and bioremediation of heavy metals including copper, zinc, cobalt, and cadmium were identified. Collectively, our findings indicated that G. azotocaptans DS1 can be used as a biofertilizer and biocontrol agent for growth enhancement of different crops and vegetables. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02996-1.

Antimicrobial activity of bacteria associated with the rhizosphere and phyllosphere of Avena fatua and Brachiaria reptans.

Environmental pollution especially heavy metal-contaminated soils adversely affects the microbial communities associated with the rhizosphere and phyllosphere of plants growing in these areas. In the current study, we identified and characterized the rhizospheric and phyllospheric bacterial strains from Avena fatua and Brachiaria reptans with the potential for antimicrobial activity and heavy metal resistance. A total of 18 bacterial strains from the rhizosphere and phyllosphere of A. fatua and 19 bacterial strains from the rhizosphere and phyllosphere of B. reptans were identified based on 16S rRNA sequence analysis. Bacterial genera, including Bacillus, Staphylococcus, Pseudomonas, and Enterobacter were dominant in the rhizosphere and phyllosphere of A. fatua and Bacillus, Marinobacter, Pseudomonas, Enterobacter, and Kocuria, were the dominating bacterial genera from the rhizosphere and phyllosphere of B. reptans. Most of the bacterial strains were resistant to heavy metals (Cd, Pb, and Cr) and showed antimicrobial activity against different pathogenic bacterial strains. The whole-genome sequence analysis of Pseudomonas putida BR-PH17, a strain isolated from the phyllosphere of B. reptans, was performed by using the Illumina sequencing approach. The BR-PH17 genome contained a chromosome with a size of 5774330 bp and a plasmid DNA with 80360 bp. In this genome, about 5368 predicted protein-coding sequences with 5539 total genes, 22 rRNAs, and 75 tRNA genes were identified. Functional analysis of chromosomal and plasmid DNA revealed a variety of enzymes and proteins involved in antibiotic resistance and biodegradation of complex organic pollutants. These results indicated that bacterial strains identified in this study could be utilized for bioremediation of heavy metal-contaminated soils and as a novel source of antimicrobial drugs.

Comparative Study of the Rhizosphere and Root Endosphere Microbiomes of Cholistan Desert Plants.

Microbial communities associated with the rhizosphere and roots of desert halophytes play an important role in plants' growth and development. Very limited information has been available on the microbial diversity of arid environments of Pakistan. Hence in the current study, the microbial diversity of rhizosphere and root endosphere of desert halophytes, Zygophyllum simplex, Haloxylon salicoricum, Aerva javanica, and Capparis decidua was evaluated. The rhizosphere and root endosphere samples of desert halophytes collected from the three geographic sites of Cholistan desert, Punjab, Pakistan were analyzed by using 16S rRNA based Illumina sequencing. The results showed that Proteobacteria were more abundant in the rhizospheric soils while Actinobacteria were more dominant in the root endosphere of halophytes. Bacteroidetes, Firmicutes, and Deinococcus-Thermus were identified from all rhizospheric soils and roots across the three sites, with variable percentage. Bacillus, Kocuria, Pseudomonas, Halomonas, and Flavobacterium were commonly identified from the rhizosphere and root endosphere of halophytes across all the three sites. At the genus level, microbial diversity from Haloxylon showed the greatest variations between the rhizosphere and root endosphere from the site 2. This study revealed that microbial diversity analysis can be used to study how changes in abiotic factors such as soil moisture content and salinity affect the microbial communities associated with the rhizospheric soils and root endosphere of halophytes across the three sites. This study will also help in the discovery of potential inoculants for crops growing in arid and semi-arid regions of Pakistan.

Novel coronavirus disease (COVID-19) pandemic: A recent mini review.

The COVID-19, caused by a novel coronavirus, was declared as a global pandemic by WHO more than five months ago, and we are still experiencing a state of global emergency. More than 74.30 million confirmed cases of the COVID-19 have been reported globally so far, with an average fatality rate of almost 3.0%. Seven different types of coronaviruses had been detected from humans; three of them have resulted in severe outbreaks, i.e., MERS-CoV, SARS-CoV, and SARS-CoV-2. Phylogenetic analysis of the genomes suggests that the possible occurrence of recombination between SARS-like-CoVs from pangolin and bat might have led to the origin of SARS-CoV-2 and the COVID-19 outbreak. Coronaviruses are positive-sense, single-stranded RNA viruses and harbour a genome (30 kb) consisting of two terminal untranslated regions and twelve putative functional open reading frames (ORFs), encoding for non-structural and structural proteins. There are sixteen putative non-structural proteins, including proteases, RNA-dependent RNA polymerase, helicase, other proteins involved in the transcription and replication of SARS-CoV-2, and four structural proteins, including spike protein (S), envelope (E), membrane (M), and nucleocapsid (N). SARS-CoV-2 infection, with a heavy viral load in the body, destroys the human lungs through cytokine storm, especially in elderly persons and people with immunosuppressed disorders. A number of drugs have been repurposed and employed, but still, no specific antiviral medicine has been approved by the FDA to treat this disease. This review provides a current status of the COVID-19, epidemiology, an overview of phylogeny, mode of action, diagnosis, and possible treatment methods and vaccines.

Identification of plasmid encoded osmoregulatory genes from halophilic bacteria isolated from the rhizosphere of halophytes.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3 M salt concentrations. All the strains showed optimum growth at 1.5-3.0 M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5-4 M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.

Isolation and characterization of bacteria associated with the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola) for production of hydrolytic enzymes.

Microbes from hypersaline environments are useful in biotechnology as sources of novel enzymes and proteins. The current study aimed to characterize halophilic bacteria from the rhizosphere of halophytes (Salsola stocksii and Atriplex amnicola), non-rhizospheric, and brine lake-bank soils collected from Khewra Salt Mine and screening of these bacterial strains for industrially important enzymes. A total of 45 bacterial isolates from the rhizosphere of Salsola, 38 isolates from Atriplex, 24 isolates from non-rhizospheric, and 25 isolates from lake-bank soils were identified by using 16S rRNA gene analysis. Phylogenetic analysis showed that bacterial strains belonging to Bacillus, Halobacillus, and Kocuria were dominant in the rhizosphere of halophytes (Salsola and Atriplex), and Halobacillus and Halomonas were dominating genera from non-rhizospheric and lake-bank soils. Mostly identified strains were moderately halophilic bacteria with optimum growth at 1.5-3.0 M salt concentrations. Most of the bacterial exhibited lipase, protease, cellulase, amylase, gelatinase, and catalase activities. Halophilic and halotolerant Bacilli (AT2RP4, HL1RS13, NRS4HaP9, and LK3HaP7) identified in this study showed optimum lipase, protease, cellulase, and amylase activities at 1.0-1.5 M NaCl concentration, pH 7-8, and temperature 37 °C. These results indicated that halophilic and halotolerant bacteria can be used for bioconversion of organic compounds to useful products under extreme conditions.

Impact of soil salinity on the microbial structure of halophyte rhizosphere microbiome.

The rhizosphere microbiome plays a significant role in the life of plants in promoting plant survival under adverse conditions. However, limited information is available about microbial diversity in saline environments. In the current study, we compared the composition of the rhizosphere microbiomes of the halophytes Urochloa, Kochia, Salsola, and Atriplex living in moderate and high salinity environments (Khewra salt mines; Pakistan) with that of the non-halophyte Triticum. Soil microbiomes analysis using pyrosequencing of 16S rRNA gene indicated that Actinobacteria were dominant in saline soil samples whereas Proteobacteria predominated in non-saline soil samples. Firmicutes, Acidobacteria, Bacteriodetes and Thaumarchaeota were predominant phyla in saline and non-saline soils, whereas Cyanobacteria, Verrucomicrobia, Gemmatimonadetes and the unclassified WPS-2 were less abundant. Sequences from Euryarchaeota, Ignavibacteriae, and Nanohaloarchaeota were identified only from the rhizosphere of halophytes. Dominant halophilic bacteria and archaea identified in this study included Agrococcus, Armatimonadetes gp4, Halalkalicoccus, Haloferula and Halobacterium. Our analysis showed that increases in soil salinity correlated with significant differences in the alpha and beta diversity of the microbial communities across saline and non-saline soil samples. Having a complete inventory of the soil bacteria from different saline environments in Pakistan will help in the discovery of potential inoculants for crops growing on salt-affected land.

Diversity of Bacillus-like bacterial community in the rhizospheric and non-rhizospheric soil of halophytes (Salsola stocksii and Atriplex amnicola), and characterization of osmoregulatory genes in halophilic Bacilli.

Salinity is one of the major abiotic stresses; a total of 3% of the world's land mass is affected by salinity. Approximately 6.3 million hectares of land in Pakistan is affected by salinity to varying degrees, and most of the areas are arid to semiarid with low annual precipitation. The aim of the present study is to identify and characterize Bacillus and Bacillus-derived bacterial genera from the rhizospheric and non-rhizospheric soil samples from the Khewra Salt Mine, Pakistan, by using culture-independent and -dependent methods. Seven Bacillus-like bacterial genera, Bacillus, Halobacillus, Virgibacillus, Brevibacillus, Paenibacillus, Tumebacillus, and Lysinibacillus, were detected by using pyrosequencing analysis, whereas only four genera, Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus, were identified by culture-dependent methods. Most of the Bacillus-like isolates identified in this study were moderately halophilic, alkaliphilic, and mesophilic bacteria and were considered a good source of hydrolytic enzymes because of their ability to degrade proteins, carbohydrates, and lipids. Eight Bacillus-like strains from the genera Bacillus, Halobacillus, Oceanobacillus, and Virgibacillus showed positive results for the presence of ectABC gene cluster (ectoine), six strains could synthesize betaine from choline, and six strains tested positive for the synthesis of proline from either glutamate or ornithine by using proline dehydrogenase enzyme.

Assessment of two carrier materials for phosphate solubilizing biofertilizers and their effect on growth of wheat (Triticum aestivum L.).

Biofertilizers are usually carrier-based inoculants containing beneficial microorganisms. Incorporation of microorganisms in carrier material enables easy-handling, long-term storage and high effectiveness of biofertilizers. Objective of the present study was to assess enriched biogas sludge and soil as biofertilizer carriers on growth and yield of wheat. Six phosphate solubilizing strains were used in this study. Three phosphate solubilizing strains, 77-NS2 (Bacillus endophyticus), 77-CS-S1 (Bacillus sphaericus) and 77-NS5 (Enterobacter aerogenes) were isolated from the rhizosphere of sugarcane, two strains, PSB5 (Bacillus safensis) and PSB12 (Bacillus megaterium) from the rhizosphere of wheat and one halophilic phosphate solubilizing strain AT2RP3 (Virgibacillus sp.) from the rhizosphere of Atriplex amnicola, were used as bioinoculants. Phosphate solubilization ability of these strains was checked in vitro in Pikovskaya medium, containing rock phosphate (RP) as insoluble P source, individually supplemented with three different carbon sources, i.e., glucose, sucrose and maltose. Maximum phosphate solubilization; 305.6µg/ml, 217.2µg/ml and 148.1µg/ml was observed in Bacillus strain PSB12 in Pikovskaya medium containing sucrose, maltose and glucose respectively. A field experiment and pot experiments in climate control room were conducted to study the effects of biogas sludge and enriched soil based phosphorous biofertilizers on growth of wheat. Bacillus strain PSB12 significantly increased root and shoot dry weights and lengths using biogas sludge as carrier material in climate control room experiments. While in field conditions, significant increase in root and shoot dry weights, lengths and seed weights was seen by PSB12 and PSB5 (Bacillus) and Enterobacter strain 77-NS5 using biogas sludge as carrier. PSB12 also significantly increased both root and shoot dry weights and lengths in field conditions when used as enriched soil based inoculum. These results indicated that bacterial isolates having plant beneficial traits such as P solubilization are more promising candidates as biofertilizer when used with carrier materials.

Comparison of Microbial Communities Associated with Halophyte (Salsola stocksii) and Non-Halophyte (Triticum aestivum) Using Culture-Independent Approaches.

Halophyte microbiome contributes significantly to plant performance and can provide information regarding complex ecological processes involved in osmoregulation of these plants. The objective of this study is to investigate the microbiomes associated with belowground (rhizosphere), internal (endosphere) and aboveground (phyllosphere) tissues of halophyte (Salsola stocksii) through metagenomics approach. Plant samples were collected from Khewra Salt Mines. The metagenomic DNA from soil, root and shoot samples was isolated with the help of FastDNA spin kit. Through PCR, the 16S rRNA gene from four different Salsola plants and wheat plants was amplified and cloned in InsTAclone PCR cloning kit. Metagenomic analyses from rhizosphere, endosphere and phyllosphere of Salsola showed that approximately 29% bacteria were uncultured and unclassified. Proteobacteria and Actinobacteria were the most abundant phyla in Salsola and wheat. However, Firmicutes, Acidobacteria, Bacteriodetes, Planctomycetes, Cyanobacteria, Thermotogae, Verrucomicrobia, Choroflexi and Euryarchaeota were predominant groups from halophyte whereas Actinobacteria, Proteobacteria, Firmicutes, Cyanobacteria, Acidobacteria, Bacteriodetes, Planctomycetes and Verrucomicrobia were predominant phyla of wheat samples. Diversity and differences of microbial flora of Salsola and wheat suggested that functional interactions between plants and microorganisms contribute to salt stress tolerance.