M. Sajjad Mirza

Nitrogen Fixation with Non-Legumes

Acetobacter diazotrophicus, a diazotrophic endophyte of sugarcane, carries a cluster of nifgenes similar to those characterized in other Proteobacterial diazotrophs. These include, in order, nifA, nifB, nifHDKEN, nifUSVW andfixABC. This order of nifand fix gene organization is most similar to that found in Azospirillum brasilense, although the deduced gene products of each gene are generally more similar to those characterized in species of Rhizobium, Bradyrhizobium or Azorhizobium. Downstream of fixABC is a gene encoding a protein with similarity to MCPA, a methyl-accepting chemotaxis protein which is involved in chemotatic responses to extracellular signals. mcpA gene may play an important role in symbiosis and plant-microbe interaction by allowing colonization of sugarcane tissues far distant from the site of A. diazotrophicus infection or entry.

Isolation, partial characterization, and the effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 106 bacteria per gram dry weight of stem and 107 bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the15N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.

Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.

Analyzing Mosquito (Diptera: Culicidae) Diversity in Pakistan by DNA Barcoding

Background Although 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 Findings Sequence 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. Conclusions As 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.

Effects of Azospirillum brasilense with genetically modified auxin biosynthesis gene ipdC upon the diversity of the indigenous microbiota of the wheat rhizosphere

The phytostimulatory properties of Azospirillum inoculants, which entail production of the phytohormone indole-3-acetic acid (IAA), can be enhanced by genetic means. However, it is not known whether this could affect their interactions with indigenous soil microbes. Here, wheat seeds were inoculated with the wild-type strain Azospirillum brasilense Sp245 or one of three genetically modified (GM) derivatives and grown for one month. The GM derivatives contained a plasmid vector harboring the indole-3-pyruvate/phenylpyruvate decarboxylase gene ipdC (IAA production) controlled either by the constitutive promoter PnptII or the root exudate-responsive promoter PsbpA, or by an empty vector (GM control). All inoculants displayed equal rhizosphere population densities. Only inoculation with either ipdC construct increased shoot biomass compared with the non-inoculated control. At one month after inoculation, automated ribosomal intergenic spacer analysis (ARISA) revealed that the effect of the PsbpA construct on bacterial community structure differed from that of the GM control, which was confirmed by 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). The fungal community was sensitive to inoculation with the PsbpA construct and especially the GM control, based on ARISA data. Overall, fungal and bacterial communities displayed distinct responses to inoculation of GM A. brasilense phytostimulators, whose effects could differ from those of the wild-type.

Coinoculation of chickpea with Rhizobium isolates from roots and nodules and phytohormone-producing Enterobacter strains

The aim of the present study was to isolate plant-beneficial bacteria (both Rhizobium and plant growth promoting rhizobacteria) from roots and nodules of chickpea (Cicer arietinum L.) and to study the effect of coinoculations on growth of two cultivars of chickpea. Four Rhizobium strains were obtained from roots and four from the nodules of field-grown chickpea cv. Parbat and identified on the basis of morphological characteristics, and biochemical and infectivity tests on the host seedlings. Only one type of nitrogen and carbon source utilisation pattern and DNA banding pattern of random amplified polymorphic DNA was observed in all isolates (Rn1, Rn2, Rn3, Rn4) from nodules, while two types of such patterns were detected among the isolates from roots. The isolate Rr1 from roots also exhibited a pattern identical to those of the isolates from nodules, whereas the remaining three isolates (Rr2, Rr3 and Rr4) from roots showed a different pattern. Two strains of plant growth-promoting rhizobacteria belonging to genus Enterobacter were also isolated from chickpea roots. All the Rhizobium strains and Enterobacter strains produced the plant growth hormones indole acetic acid and gibberellic acid in the growth medium. Effects of the bacterial isolates as single- or double-strain inocula were studied on two chickpea cultivars (NIFA 88 and Parbat) grown in sterilised soil. In cultivar NIFA 88, coinoculation of Rhizobium strain Rn1 with Enterobacter strain B resulted in maximum increase in plant biomass and nodulation, as compared with the control treatment (non-inoculated as well as inoculated with Rhizobium strain Rn1 only), whereas the combination of Rhizobium Rn1 with Enterobacter A was more efficient in growth promotion of chickpea cv. Parbat. In non-sterilised soil, the same combinations of the Rhizobium strain Rn1 with Enterobacter strains A and B were found to be the most effective inoculants for cvv. Parbat and NIFA 88, respectively. However, some negative effects on plant growth were also noted in cv. Parbat coinoculated with Rhizobium strain Rr2 and Enterobacter strain B.

DNA Barcoding of Bemisia tabaci Complex (Hemiptera: Aleyrodidae) Reveals Southerly Expansion of the Dominant Whitefly Species on Cotton in Pakistan

Background Although 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 Findings Sequence 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. Conclusions DNA 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.

PCR-amplified 16S rRNA sequence analysis to confirm nodulation of Datisca cannabina L. by the endophyte of Coriaria nepalensis Wall.

Different Frankia strains and crushed nodule suspensions were tested for their ability to nodulate Coriaria nepalensis and Datisca cannabina. Datisca cannabina seedlings were nodulated effectively by both crushed nodule suspension from Coriaria nepalensis and Datisca cannabina. The origin of the endophyte in Datisca nodules induced by crushed nodules of Coriaria was confirmed by comparing partial PCR-amplified 16S rRNA sequences with those of the endophytes of both plants. Coriaria seedlings could only be nodulated by crushed nodule suspensions of Coriaria nepalensis. All pure cultures of Frankia used as a single inoculum source or in combinations with a nodule filtrate, failed to induce nodulation on Coriaria. Two atypical Frankia strains Cn3 and Cn7 isolated from Coriaria nodules showed no acetylene reduction activity and did not induce nodulation on the host seedlings.

Identification of plant growth hormones produced by bacterial isolates from rice, wheat and kallar grass

Identification and quantification of the plant growth hormones indoleacetic acid and gibberellic acid, produced by plant growth-promoting rhizobacteria (PGPR), was carried out by using high-pressure liquid chromatography (HPLC). The PGPR strains were isolated from roots of rice, wheat and kallar grass and belonged to the genera Azoarcus, Azospirillum, Enterobacter, Pseudomonas and Zoogloea. For these studies, bacteria were grown in liquid nitrogen free malate (NFM) or combined carbon medium (CCM) containing tryptophan and combined nitrogen. Some Azospirillum strains produced both indoleacetic acid and gibberellic acid, while none of the Enterobacter spp. tested produced these growth hormones. Azoarcus strain K-1 produced higher amounts of gibberellic acid and Azospirillum strain ER-2 produced higher amounts of indoleacetic acid. Indoleacetic acid production increased with the age of bacterial cultures while a decrease in the production of gibberellic acid was noted at later growth stages. Pure indoleacetic acid and gibberellic acid in the concentration range 1–2 µg/ml increased root area and plant biomass of rice and wheat. Among PGPR strains tested, Pseudomonas 96–51 and its extract containing growth hormones increased root area, root length and plant biomass of rice and wheat.

Cultivation-Based and Molecular Assessment of Bacterial Diversity in the Rhizosheath of Wheat under Different Crop Rotations.

A field study was conducted to compare the formationand bacterial communities of rhizosheaths of wheat grown under wheat-cotton and wheat-rice rotation and to study the effects of bacterial inoculation on plant growth. Inoculation of Azospirillum sp. WS-1 and Bacillus sp. T-34 to wheat plants increased root length, root and shoot dry weight and dry weight of rhizosheathsoil when compared to non-inoculated control plants, and under both crop rotations. Comparing both crop rotations, root length, root and shoot dry weight and dry weight of soil attached with roots were higher under wheat-cotton rotation. Organic acids (citric acid, malic acid, acetic acid and oxalic acid) were detected in rhizosheaths from both rotations, with malic acid being most abundant with 24.8±2 and 21.3±1.5 μg g-1 dry soil in wheat-cotton and wheat-rice rotation, respectively. Two sugars (sucrose, glucose) were detected in wheat rhizosheath under both rotations, with highest concentrations of sucrose (4.08±0.5 μg g-1and 7.36±1.0 μg g-1) and glucose (3.12±0.5 μg g-1 and 3.01± μg g-1) being detected in rhizosheaths of non-inoculated control plants under both rotations. Diversity of rhizosheath-associated bacteria was evaluated by cultivation, as well as by 454-pyrosequencing of PCR-tagged 16S rRNA gene amplicons. A total of 14 and 12 bacterial isolates predominantly belonging to the genera Arthrobacter, Azospirillum, Bacillus, Enterobacter and Pseudomonaswere obtained from the rhizosheath of wheat grown under wheat-cotton and wheat-rice rotation, respectively. Analysis of pyrosequencing data revealed Proteobacteria, Bacteriodetes and Verrucomicrobia as the most abundant phyla in wheat-rice rotation, whereas Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria were predominant in wheat-cotton rotation. From a total of 46,971 sequences, 10.9% showed ≥97% similarity with 16S rRNA genes of 32 genera previously shown to include isolates with plant growth promoting activity (nitrogen fixation, phosphate-solubilization, IAA production). Among these, the most predominant genera were Arthrobacter, Azoarcus, Azospirillum, Bacillus, Cyanobacterium, Paenibacillus, Pseudomonas and Rhizobium.