Deamination of the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid (ACC) is a key plant‐beneficial trait found in plant growth‐promoting rhizobacteria (PGPR) and phytosymbiotic bacteria, but the diversity of the corresponding gene (acdS) is poorly documented. Here, acdS sequences were obtained by screening putative ACC deaminase sequences listed in databases, based on phylogenetic properties and key residues. In addition, acdS was sought in 71 proteobacterial strains by PCR amplification and/or hybridization using colony dot blots. The presence of acdS was confirmed in established AcdS+ bacteria and evidenced noticeably in Azospirillum (previously reported as AcdS−), in 10 species of Burkholderia and six Burkholderia cepacia genomovars (which included PGPR, phytopathogens and opportunistic human pathogens), and in five Agrobacterium genomovars. The occurrence of acdS in true and opportunistic pathogens raises new questions concerning their ecology in plant‐associated habitats. Many (but not all) acdS+ bacteria displayed ACC deaminase activity in vitro, including two Burkholderia clinical isolates. Phylogenetic analysis of partial acdS and deduced AcdS sequences evidenced three main phylogenetic clusters, each gathering pathogens and plant‐beneficial strains of contrasting geographic and habitat origins. The acdS phylogenetic tree was only partly congruent with the rrs tree. Two clusters gathered both Betaprotobacteria and Gammaproteobacteria, suggesting extensive horizontal transfers of acdS, noticeably between plant‐associated Proteobacteria.

The aim of this study was to isolate, characterize and use phosphate solubilizing bacteria to enhance the bioavailability of insoluble Ca-phosphate for wheat plants. For this purpose, 15 phosphorus solubilizing bacteria (PSB) were isolated from wheat rhizospheric soils of Peshawar and southern Punjab region, Pakistan. These isolates were identified using light microscopy and 16S rRNA gene. Among the isolated bacteria, two strains (Pseudomonas sp. MS16 and Enterobacter sp. MS32) were the efficient P solubilizers based on their P solubilization activity determined qualitatively (solubilization index 3.2–5.8) as well as quantitatively (136–280 μg mL-1). These two strains produced indole-3-acetic acid (25.6–28.1 μg mL-1), gibberellic acid (2.5–11.8), solubilized zinc compounds (SI 2.8–3.3) and showed nitrogenase and 1-Aminocyclopropane-1-carboxylic acid deaminase activity in vitro. Phosphate solubilization activity of Pseudomonas sp. MS16 was further validated by amplification, sequencing and phylogenetic analysis of glucose dehydrogenase (gcd) gene (LT908484) responsible for P solubilization. Response Surface Methodology for large-scale production was used to find optimal conditions (Temperature 22.5°C, pH 7) for P solubilization. Glucose was found to support higher P solubilization in vitro. In an in vitro experiment, PSB treated wheat seedlings improved germination and seedling vigor (11% increases) as compared to un-inoculated control. Rhizoscanning of these seedlings showed an increase in various root growth parameters. Wheat inoculation with selected strain MS16 showed pronounced effect on grain yield in pot (38.5% increase) and field (17–18% increase) experiments compared to non-inoculated control. Root colonization by PSB through Florescent in situ Hybridization and Confocal Laser Scanning Microscopy confirmed their rhizosphere competence in soil. BOX-PCR confirmed the re-isolated colonies of Pseudomonas sp. MS16. The results indicated that gluconic acid producing Pseudomonas sp. MS16 from un-explored soils may be cost effective and environment friendly candidate to improve plant growth and phosphorous uptake by wheat plants.

BackgroundAlthough they are important disease vectors mosquito biodiversity in Pakistan is poorly known. Recent epidemics of dengue fever have revealed the need for more detailed understanding of the diversity and distributions of mosquito species in this region. DNA barcoding improves the accuracy of mosquito inventories because morphological differences between many species are subtle, leading to misidentifications.Methodology/Principal FindingsSequence variation in the barcode region of the mitochondrial COI gene was used to identify mosquito species, reveal genetic diversity, and map the distribution of the dengue-vector species in Pakistan. Analysis of 1684 mosquitoes from 491 sites in Punjab and Khyber Pakhtunkhwa during 2010–2013 revealed 32 species with the assemblage dominated by Culex quinquefasciatus (61% of the collection). The genus Aedes (Stegomyia) comprised 15% of the specimens, and was represented by six taxa with the two dengue vector species, Ae. albopictus and Ae. aegypti, dominant and broadly distributed. Anopheles made up another 6% of the catch with An. subpictus dominating. Barcode sequence divergence in conspecific specimens ranged from 0–2.4%, while congeneric species showed from 2.3–17.8% divergence. A global haplotype analysis of disease-vectors showed the presence of multiple haplotypes, although a single haplotype of each dengue-vector species was dominant in most countries. Geographic distribution of Ae. aegypti and Ae. albopictus showed the later species was dominant and found in both rural and urban environments.ConclusionsAs the first DNA-based analysis of mosquitoes in Pakistan, this study has begun the construction of a barcode reference library for the mosquitoes of this region. Levels of genetic diversity varied among species. Because of its capacity to differentiate species, even those with subtle morphological differences, DNA barcoding aids accurate tracking of vector populations.

Xanthomonas oryzae pv. oryzae (Xoo) is widely prevalent and causes Bacterial Leaf Blight (BLB) in Basmati rice grown in different areas of Pakistan. There is a need to use environmentally safe approaches to overcome the loss of grain yield in rice due to this disease. The present study aimed to develop inocula, based on native antagonistic bacteria for biocontrol of BLB and to increase the yield of Super Basmati rice variety. Out of 512 bacteria isolated from the rice rhizosphere and screened for plant growth promoting determinants, the isolate BRp3 was found to be the best as it solubilized 97 μg/ mL phosphorus, produced 30 μg/mL phytohormone indole acetic acid and 15 mg/ L siderophores in vitro. The isolate BRp3 was found to be a Pseudomonas aeruginosa based on 16S rRNA gene sequencing (accession no. HQ840693). This bacterium showed antagonism in vitro against different phytopathogens including Xoo and Fusarium spp. Strain BRp3 showed consistent pathogen suppression of different strains of BLB pathogen in rice. Mass spectrometric analysis detected the production of siderophores (1-hydroxy-phenazine, pyocyanin, and pyochellin), rhamnolipids and a series of already characterized 4-hydroxy-2-alkylquinolines (HAQs) as well as novel 2,3,4-trihydroxy-2-alkylquinolines and 1,2,3,4-tetrahydroxy-2-alkylquinolines in crude extract of BRp3. These secondary metabolites might be responsible for the profound antibacterial activity of BRp3 against Xoo pathogen. Another contributing factor toward the suppression of the pathogen was the induction of defense related enzymes in the rice plant by the inoculated strain BRp3. When used as an inoculant in a field trial, this strain enhanced the grain and straw yields by 51 and 55%, respectively, over non-inoculated control. Confocal Laser Scanning Microscopy (CLSM) used in combination with immunofluorescence marker confirmed P. aeruginosa BRp3 in the rice rhizosphere under sterilized as well as field conditions. The results provide evidence that novel secondary metabolites produced by BRp3 may contribute to its activity as a biological control agent against Xoo and its potential to promote the growth and yield of Super Basmati rice.

The rhizosphere is undoubtedly the most complex microhabitat, comprised of an integrated network of plant roots, soil, and a diverse consortium of bacteria, fungi, eukaryotes, and archaea. The rhizosphere conditions have a direct impact on crop growth and yield. Nutrient-rich rhizosphere environments stimulate plant growth and yield and vice versa. Extensive cultivation exhaust most of the soils which need to be nurtured before or during the next crop. Chemical fertilizers are the major source of crop nutrients but their uncontrolled and widespread usage has posed a serious threat to the sustainability of agriculture and stability of an ecosystem. These chemicals are accumulated in the soil, drained in water, and emitted to the air where they persist for decades causing a serious threat to the overall ecosystem. Plant growth-promoting rhizobacteria (PGPR) present in the rhizosphere convert many plant-unavailable essential nutrients e.g., nitrogen, phosphorous, zinc, etc. into available forms. PGPR produces certain plant growth hormones (such as auxin, cytokinin, and gibberellin), cell lytic enzymes (chitinase, protease, hydrolases, etc.), secondary metabolites, and antibiotics, and stress alleviating compounds (e.g., 1-Aminocyclopropane-1- carboxylate deaminase), chelating agents (siderophores), and some signaling compounds (e.g., N-Acyl homoserine lactones) to interact with the beneficial or pathogenic counterparts in the rhizosphere. These multifarious activities of PGPR improve the soil structure, health, fertility, and functioning which directly or indirectly support plant growth under normal and stressed environments. Rhizosphere engineering with these PGPR has a wide-ranging application not only for crop fertilization but developing eco-friendly sustainable agriculture. Due to severe climate change effects on plants and rhizosphere biology, there is growing interest in stress-resilient PGPM and their subsequent application to induce stress (drought, salinity, and heat) tolerance mechanism in plants. This review describes the three components of rhizosphere engineering with an explicit focus on the broader perspective of PGPM that could facilitate rhizosphere engineering in selected hosts to serve as an efficient component for sustainable agriculture.

BackgroundAlthough whiteflies (Bemisia tabaci complex) are an important pest of cotton in Pakistan, its taxonomic diversity is poorly understood. As DNA barcoding is an effective tool for resolving species complexes and analyzing species distributions, we used this approach to analyze genetic diversity in the B. tabaci complex and map the distribution of B. tabaci lineages in cotton growing areas of Pakistan.Methods/Principal FindingsSequence diversity in the DNA barcode region (mtCOI-5′) was examined in 593 whiteflies from Pakistan to determine the number of whitefly species and their distributions in the cotton-growing areas of Punjab and Sindh provinces. These new records were integrated with another 173 barcode sequences for B. tabaci, most from India, to better understand regional whitefly diversity. The Barcode Index Number (BIN) System assigned the 766 sequences to 15 BINs, including nine from Pakistan. Representative specimens of each Pakistan BIN were analyzed for mtCOI-3′ to allow their assignment to one of the putative species in the B. tabaci complex recognized on the basis of sequence variation in this gene region. This analysis revealed the presence of Asia II 1, Middle East-Asia Minor 1, Asia 1, Asia II 5, Asia II 7, and a new lineage “Pakistan”. The first two taxa were found in both Punjab and Sindh, but Asia 1 was only detected in Sindh, while Asia II 5, Asia II 7 and “Pakistan” were only present in Punjab. The haplotype networks showed that most haplotypes of Asia II 1, a species implicated in transmission of the cotton leaf curl virus, occurred in both India and Pakistan.ConclusionsDNA barcodes successfully discriminated cryptic species in B. tabaci complex. The dominant haplotypes in the B. tabaci complex were shared by India and Pakistan. Asia II 1 was previously restricted to Punjab, but is now the dominant lineage in southern Sindh; its southward spread may have serious implications for cotton plantations in this region.

The present study was conducted to evaluate the potential of rice rhizosphere associated antagonistic bacteria for growth promotion and disease suppression of bacterial leaf blight (BLB). A total of 811 rhizospheric bacteria were isolated and screened against 3 prevalent strains of BLB pathogen Xanthomonas oryzae pv. oryzae (Xoo) of which five antagonistic bacteria, i.e., Pseudomonas spp. E227, E233, Rh323, Serratia sp. Rh269 and Bacillus sp. Rh219 showed antagonistic potential (zone of inhibition 1–19 mm). Production of siderophores was found to be the common biocontrol determinant and all the strains solubilized inorganic phosphate (82–116 μg mL-1) and produced indole acetic acid (0.48–1.85 mg L-1) in vitro. All antagonistic bacteria were non-pathogenic to rice, and their co-inoculation significantly improved plant health in terms of reduced diseased leaf area (80%), improved shoot length (31%), root length (41%) and plant dry weight (60%) as compared to infected control plants. Furthermore, under pathogen pressure, bacterial inoculation resulted in increased activity of defense related enzymes including phenylalanine ammonia-lyase and polyphenol oxidase, along with 86% increase in peroxidase and 53% increase in catalase enzyme activities in plants inoculated with Pseudomonas sp. Rh323 as well as co-inoculated plants. Bacterial strains showed good colonization potential in the rice rhizosphere up to 21 days after seed inoculation. Application of bacterial consortia in the field resulted in an increase of 31% in grain yield and 10% in straw yield over non-inoculated plots. Although, yield increase was statistically non-significant but was accomplished with overall saving of 20% chemical fertilizers. The study showed that Pseudomonas sp. Rh323 can be used to develop dual-purpose inoculum which can serve not only to suppress BLB but also to promote plant growth in rice.

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