Abdul Samad

Purification, characterization, and in vitro activity of 2,4-Di-tert-butylphenol from Pseudomonas monteilii PsF84: conformational and molecular docking studies.

A novel strain of Pseudomonas monteilii, PsF84, was isolated from tannery waste soil from Jajmau, Kanpur, India. 16S rRNA gene sequence phylogenetic analysis confirmed the taxonomic affiliation of PsF84 as P. monteilii. An antifungal volatile organic compound (VOC) active against hyphal growth of Fusarium oxysporum (CIMAP-IMI-357464) in vitro was isolated from strain PsF84 by using chromatographic techniques. The molecular formula of the antifungal VOC was deduced to be C₁₄H₂₂O by EI-MS and 1D and 2D NMR spectral analysis. 2,4-Di-tert-butylphenol was found to be effective against an agriculturally important fungus, namely, F. oxysporum, in inhibiting spore germination and hyphal growth. Molecular docking analysis of 2,4-di-tert-butylphenol with β-tubulin further validated the potential of β-tubulin binding in F. oxysporum. Two residues of β-tubulin protein, HIS 118 and THR 117, showed hydrogen binding with ligand. To the authors knowledge, this is the first report of antifungal VOC (2,4-di-tert-butylphenol) produced by P. monteilii PsF84 that can be a potent inhibitor of β-tubulin of F. oxysporum.

Antimicrobial evaluation of mangiferin analogues

The naturally occurring xanthone glycoside mangiferin has been isolated by column chromatography from the ethanol extract of stem bark of Mangifera indica. Mangiferin was further converted to 5-(N-phenylaminomethyleno)mangiferin, 5-(N-p-chlorophenylaminomethyleno) mangiferin, 5-(N-2-methylphenylaminomethyleno) mangiferin, 5-(N-p-methoxyphenylaminomethyleno) mangiferin, 5-(N, N-diphenylaminomethyleno) mangiferin, 5-(N--napthylaminomethyleno) mangiferin and 5-(N-4-methylphenylaminomethyleno) mangiferin. Mangiferin and its analogues were characterized by melting point and Rf value determination and through spectral technique like UV, IR, and NMR spectral analysis. The synthesized compounds were screened for antimicrobial activity.

Occurrence of a clover proliferation (16SrVI) group phytoplasma associated with little leaf disease of Portulaca grandiflora in India.

Portulaca grandiflora (family Portulacaceae), commonly known as moss rose purslane, is a popular ornamental plant widely grown in temperate climates because it blooms all summer. Portulaca is also used for medicinal purposes since it is rich in vitamins A, B1, and C and has antimicrobial and cytotoxic activity. Since March 2005, 30 to 50% of P. grandiflora plants in the ornamental gardens as well as in pots at the Central Institute of Medicinal and Aromatic Plants, Lucknow, India have displayed symptoms resembling phytoplasma infection. Disease symptoms start as a typical bud proliferation, downward curling, and diminishing size of leaves, followed by overall stunted growth and yellowing of the whole plant from April to June. Some plants also formed rosettes and a proliferation of axillary shoots resulting in a witches-broom appearance. Typical pleomorphic bodies, mostly spherical to oval, ranging from 340 to 1,100 nm were observed only in sieve elements of infected plants by transmission electron microscopy (TEM). On the basis of symptoms, TEM observations, PCR, and response to antibiotic treatment, the causal organism was identified as phytoplasma (1). Total genomic DNA from healthy and infected plants was extracted with the CTAB buffer method (2). Of 27 suspected samples screened by PCR, 23 were phytoplasma positive. Presence of phytoplasmas in plants was demonstrated by a nested PCR assay employing primer pair P1/P6 followed by R16F2n/R16R2 that generated rDNA products of 1.5 and 1.2 kb, respectively, only from symptomatic plants. No differences among phytoplasmas in Portulaca plants were detected by restriction fragment length polymorphism (RFLP) analysis of nested rDNA (1.2 kb) products using endonucleases BamHI, RsaI, AluI, HpaII, and EcoRI. Comparative analysis of RFLP patterns with those derived from reference phytoplasmas tentatively identified the Portulaca little leaf (PLL) phytoplasma as a member of 16S rDNA RFLP group 16SrVI (3). A nested PCR product (1.25 kb) was cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. The sequence was deposited in the GenBank database (Accession No. EF651786). Sequence analysis revealed the PLL phytoplasma to be most similar (98%) to Indian brinjal little leaf (Accession No. EF186820) and Candidatus Phytoplasma trifolii (Accession No. AY390261), two 16SrVI group phytoplasmas previously reported from India and Canada, respectively. The status of PLL (EF651786) was also verified by in silico RFLP analysis (4) of the F2n/R2 sequence of six closely related strains (Accession Nos. AF228052, AY390261, AY270156, AY409070, AY409069, and EF186820) of the 16SrVI group using 17 restriction enzymes (AluI, BamHI, BfaI, BsfUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, Sau3AI, RsaI, SspI, and TaqI). In silico restriction digestion and virtual gel plotting showed similar patterns for all enzymes. To our knowledge, this is the first report of a 16SrVI group phytoplasma infecting Portulaca plants in India. References: (1) P. V. Ajayakumar et al. Aust. Plant Dis. Notes 2:67, 2007. (2) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:74, 1999. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) W. Wei et al. Int. J. Syst. Evol. Mic. 57:1855, 2007.

Impact of plant growth promoting Pseudomonas monteilii PsF84 and Pseudomonas plecoglossicida PsF610 on metal uptake and production of secondary metabolite (monoterpenes) by rose-scented geranium (Pelargonium graveolens cv. bourbon) grown on tannery sludge amended soil

Bacterial strains PsF84 and PsF610 were isolated from tannery sludge polluted soil, Jajmau, Kanpur, India. 16S rRNA gene sequence and phylogenetic analysis confirmed the taxonomic affiliation of PsF84 as Pseudomonas monteilii and PsF610 as Pseudomonas plecoglossicida. A greenhouse study was carried out with rose-scented geranium (Pelargonium graveolenscv. bourbon) grown in soil treated with tannery sludge in different proportions viz. soil: sludge ratio of 100:0, 25:75, 50:50, 75:25 and 0:100 to evaluate the effects of bacterial inoculation on the heavy metal uptake. The isolates solubilized inorganic phosphorus and were capable of producing indole acetic acid (IAA) and siderophore. The isolate PsF84 increased the dry biomass of shoot by 44%, root by 48%, essential oil yield 43% and chlorophyll by 31% respectively over uninoculated control. The corresponding increase with the isolate PsF610 were 38%, 40%, 39% and 28%, respectively. Scanning electron microscopic (SEM) studies reveal that the Cr(VI) accumulation resulted in breakdown of vascular bundles and sequesters Cr(VI) in roots. The glandular trichomes (GT) were investigated using SEM studies as these glands are probably the main site of essential oil synthesis. Owing to its wide action spectrum, these isolates could serve as an effective metal sequestering and bioinoculants due to the production of IAA, siderophore and solubilization of phosphate for geranium in metal-stressed soil. The present study has provided a new insight into the phytoremediation of metal-contaminated soil.

First Report of Chilli leaf curl India virus Infecting Mentha spicata (Neera) in India

Mint (Mentha spp.; family Lamiaceae) is an important essential oil-bearing crop cultivated on the Indian subcontinent as a cash crop for the international market and industrial purposes. Since May 2010, typical symptoms such as yellow vein, leaf yellowing, mosaic, crinkling, and cupping were observed, which led to significant yield loss in spearmint (M. spicata var. Neera) at CIMAP experimental fields and farmers fields of Badaun, Rampur, and Moradabad regions of Uttar Pradesh province, India. Disease incidence was recorded in the range of 40 to 50%. Mentha spp. has been reported to be affected by many viral diseases (3). Due to the absence of fungal/bacterial infection, lack of mechanical transmission of the pathogen, and presence of whiteflies in the fields, the causal pathogen was suspected to be a begomovirus. Total genomic DNA was extracted from the leaves of naturally infected and healthy samples of Mentha by the CTAB protocol. Eighteen symptomatic samples were collected from different location of fields and screened for the presence of begomovirus. DNA from these samples was used as PCR template to amplify a 771-bp fragment using begomovirus coat protein (CP) gene specific primers. Eleven of 18 (61.1%) samples were found positive. PCR products were cloned into the pGEM-T Easy (Promega) and sequenced using the universal M13F/M13R primers showed sequence similarity with Chilli leaf curl India virus. To amplify the full-length DNA-A/B and a possible β-satellite, a second detection method was used: rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA products were digested independently with various restriction enzymes: BamHI, EcoRI, EcoRV, HincII, HindIII, SacI, and KpnI. Digested products were resolved on 1% agarose gel and the bands corresponding to ~2.7 and ~1.3 kb were purified using Nucleospin Gel and PCR Clean-up Kit and cloned into the respective sites of pGreen0029 vector. The sequence of full-length DNA-A (2,749 bp) and β-satellite component (1,347-bp) were obtained and deposited in NCBI GenBank with accession nos. KF312364 and KF364485, respectively. The sequence analysis showed maximum nucleotide identity (99%) with Chilli leaf curl India virus (FM877858) and distant affinities (≤88%) with other begomoviruses. The sequence analysis of isolated β-satellite showed 93% identity with Ageratum yellow vein virus satellite (AJ252072.1). No presence of DNA-B was detected using the universal primer PBL1v2040/PCRc1 (2), thus confirming it to be a monopartite begomovirus (1). Viruliferous whiteflies (Bemisia tabaci) proved Kochs postulation by inducing similar symptoms on healthy plants while aphids (Myzus persicae) failed to transmit the virus. To our knowledge, this is the first report of Chilli leaf curl India virus infecting M. spicata var. Neera in India. Mint is widely grown together with other reported hosts of begomoviruses, and thus could pose a serious threat as future expansion of begomovirus to new crops. Hence, the development of resistant varieties coupled with the implementation of adapted integrated pest management strategies would be essential for successful production of mint crops. References: (1) Y. Kumar et al. Plant Pathol. 60:1040, 2011. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) I. E. Tzanetakis et al. Plant Dis. 94:4, 2010.

In vitro and in silico antifungal efficacy of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani.

We have investigated in vitro antifungal efficiency of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani (R. solani) plant pathogenic fungi. NCNH with size of 50–60 nm and concentrations of 10, 50, 100, and 150 μg mL−1 were used. The results showed that growth of fungi in the presence of NCNH was significantly (p > .05) inhibited at 150 μg mL−1 (85.13 ± .97) after 72 h. The results were validated through computational approaches. Molecular docking analysis of NCNH with endochitinase protein of R. solani was performed to validate the potential of antifungal activity of NCNH. Docking results showed different conformations of interaction of NCNH with endochitinase enzyme. The conformation with least binding energy −13.54 kcal/mol was considered further. It is likely that NCNH interacts with the pathogens by mechanically wrapping, which may be one of the major toxicity actions of NCNH against R. solani. The analysis showed that NCNH might interwinds to endochitinase of R. solani leading to the deactivation of the enzyme. To best of our knowledge, this is the first report of antifungal efficacy of NCNH against R. solani and provides useful information about the application of NCNH in resisting crop disease.

The Interaction Pattern between a Homology Model of 40S Ribosomal S9 Protein of Rhizoctonia solani and 1-Hydroxyphenaize by Docking Study

1-Hydroxyphenazine (1-OH-PHZ), a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases.

First report on the molecular identification of phytoplasma (16SrII-D) associated with witches' broom of Kalmegh (Andrographis paniculata) in India.

Andrographis paniculata (family Acanthaceae), also known as King of Bitters or Kalmegh, is an important medicinal plant used for the treatment of various diseases. It has antimicrobial, antiviral, anti-inflammatory, hepatoprotective, antidiabetic, antihyperglycemic, and antioxidant properties (1). During June 2014, while performing a routine survey of the commercial trial fields of Kalmegh at Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, typical phytoplasma disease symptoms such as virescence, proliferation, and witches broom along with little leaf and stunted growth were observed. The disease incidence was estimated to be approximately 7 to 10%. To ascertain the presence of phytoplasma, 16 samples of leaves were collected from nine different field sites, and total genomic DNA was extracted from the symptomatic and symptomless Kalmegh plants by the CTAB method. Direct and nested PCR assays were performed targeting the 16S rDNA using generic phytoplasma primer pairs P1/P6 followed by R16F2n/R16R2 (2). Resulting bands of the expected size (1.5 kb and 1.2 kb, respectively) were amplified from symptomatic plants. No amplification was observed with DNA from asymptomatic plant samples. The purified nested PCR products were cloned into E. coli DH5α, using the pGEM-T Easy vector (Promega, United States) and sequenced with primers M13For/M13Rev using an automatic sequencer (ABI Prism, Perkin Elmer) at CIMAP. The sequence was analyzed by BLASTn and found to share 99% similarity with Echinacea witches-broom phytoplasma and Sesame phyllody phytoplasma strain (GenBank Accession Nos. JF340080 and KF612966, respectively), which belong to the 16SrII-D group. The sequence was deposited in NCBI as GenBank Accession No. KM359410. A phylogenetic tree using MEGA v5.0 (4) was constructed with 16S rDNA; consensus sequences of phytoplasmas belonging to distinct groups revealed that the present phytoplasma clustered with the 16SrII group. iPhyClassifier software was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile (5). On the basis of iPhyClassifier, the 16S rDNA sequence analysis of our isolate showed 99.2% similarity with that of the Candidatus Phytoplasma australasiae reference strain (GenBank Accession No. Y10097), which belongs to 16Sr group II. The virtual RFLP pattern of F2n/R2 fragment was most similar to the 16SrII-D subgroup (similarity coefficient of 0.91) but showed a difference in profile with HpaI, HhaI, and MseI enzymes. Several bacterial/fungal and viral diseases have been reported on A. paniculata (3); however, to our knowledge, this is the first report of witches broom disease in India and the first record of a 16SrII-D group phytoplasma on Kalmegh. Its presence in Kalmegh is of great significance due to its commercial interest. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) D. E. Gundersen and M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

New Record of Catharanthus yellow mosaic virus and a Betasatellite Associated with Lethal Leaf Yellowing of Kalmegh (Andrographis paniculata) in Northern India

Andrographis paniculata (Family Acanthaceae), also called Kalmegh, is a medicinal herb in India well-known for its various pharmaceutical properties (1). In August 2012, during a survey in the northern parts of India, several Kalmegh plants in Barabanki District of Uttar Pradesh Province showed typical virus-like symptoms along with prominent lethal leaf yellowing. The infected plants initially showed some chlorotic streaks, which later turned completely yellow, ultimately leading to premature death. Mechanical/sap inoculation failed to transmit the pathogen. Based on the symptomology, a heavy infestation of whiteflies (Bemisia tabaci) in the infected fields, and lack of mechanical transmission, the association of a begomovirus was suspected. The disease incidence was calculated to be about 15 to 20% on the basis of plant population. Twenty samples from naturally infected plants of A. paniculata were collected from various field locations. Total genomic DNA from the symptomatic and non-symptomatic samples was isolated by the modified CTAB method (4). The initial PCR-based detection was performed using begomovirus coat protein gene specific primers (forward 5-ATGGCGAAGCGACCAG-3 and reverse 5-TTAATTTGTGACCGAATCAT-3), which generated an amplicon of 771 bp in most of the (17/20) symptomatic samples. No amplification was obtained in healthy or non-symptomatic plant samples. The full-length genome was amplified via rolling-circle amplification (RCA) according to the manufacturers instructions using random hexamer primers and φ29 DNA polymerase. A portion of the RCA product (1 μl) was subjected to digestion with different restriction enzymes, out of which BamHI yielded DNA fragments of approximately 2.7 and 1.3 kb, corresponding to DNA-A and β satellite molecules, respectively. These fragments were eluted from the gel and cloned into the suitable restriction site of pGreen0029 vector. The positive clones were checked by restriction digestion. Twelve out of 20 clones were found to be positive and sequenced. The complete genome sequences of DNA A (2,754 bp) and β (1,366 bp) satellites were deposited in the GenBank database with the accession numbers KM359406 and KM359407, respectively. The absence of DNA-B molecule was ascertained, as no PCR amplification was detected with DNA-B-specific primers. Sequence analysis showed highest nucleotide identity (90%) with Catharanthus yellow mosaic virus (CYMV) (HE580234) and ≤85% identity with other begomoviruses of the database. Sequence analysis of the associated betasatellite showed 96% identity with Andrographis yellow vein leaf curl betasatellite (KC967282). CYMV was first reported on Catharanthus roseus with no associated betasatellite from Pakistan (2). However, this is the first report of CYMV along with a betasatellite infecting A. paniculata in India. Recently a begomovirus (Eclipta yellow vein virus) infection was reported on A. paniculata in association with Andrographis yellow vein leaf curl betasatellite from India for the first time (3); now the crop has also become a host of CYMV. Thus, this study highlights the spread of CYMV from its preliminary host to a new host plant (A. paniculata), across the South Asian countries. Therefore, it is important to take measures for the management of its transmitting vector so as to curtail the spread of the virus to new economically and commercially important crops. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) M. Ilyas et al. Arch. Virol. 158:505, 2013. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:1, 1999.

First report of Tagetes erecta damping off caused by Ceratobasidium sp. from India.

The Mexican marigold (Tagetes erecta) is cultivated commercially in India for medicinal, ceremonial, and decorative purposes. It is native to Mexico and the United States. The natural phytochemical thiophene extracted from T. erecta has been shown to have antibacterial activity. It is also grown to extract lutein, a common yellow/orange food color. During winter of 2011, approximately 15% marigold seedlings exhibited damping off symptoms at CSIR-CIMAP, Lucknow, India, and its adjoining areas. Infected seedlings initially produced water-soaked lesions on the stem at the soil level that later turned pinkish with a brownish halo in the center. The infected seedlings were cut into small pieces, surface disinfected with 1% sodium hypochlorite, rinsed thrice with sterile distilled water, and placed onto potato dextrose agar (PDA) plates. The plates were incubated at 25°C for 3 days. The isolation yielded whitish fungal growth that later turned tan brown. The mycelium was binucleate, septate, sub branching at right angles, with distinct constriction at the origin of branching. Bisbenzamide (Hoechst 33258; Chemical Abstracts no. 23491-45-4) was used as fluorescent dye for the staining of nuclei. Based on cultural as well as morphological characteristic features, the fungus was identified as Ceratobasidium sp. (1,2). The molecular identification was on the basis of internal transcribed spacer (ITS) region sequence. Amplification of the ITS of rDNA using primers ITS1/ITS4 yielded a ~700 bp band and sequenced data were deposited in the NCBI GenBank (KC193238). The ITS region (700 bp) was 100% identical to Ceratobasidium sp. AG-F strain BAGF101 isolated from Musa spp. in Georgia, United States (GenBank Accession No. HQ168370). The pathogenicity of the fungus was tested under glasshouse conditions. The inoculum of the fungus was prepared on sterile maize seeds in Erlenmeyer flasks by inoculating seeds with three disks (1 mm) of 5-day-old culture, and kept at 25 ± 2 °C for 14 days in the dark. The healthy, 5 to 7 day old seedlings were inoculated with five artificially infested maize seeds per pot. The uninoculated seedlings served as control. Both inoculated and uninoculated seedlings were kept at 28 ± 2°C in a humidity (95%) chamber for 3 days and thereafter placed in the glasshouse at 28 ± 2°C for development of disease symptoms. Initial symptoms developed as water-soaked lesions on the infected seedlings in 2 to 3 days, while typical disease symptoms appeared after 4 to 5 days of inoculation. Uninoculated seedlings were free from infection. The fungus was reisolated from the artificially infected seedlings on PDA and its identification as Ceratobasidium sp. was confirmed by morphological and molecular characteristics. Recently, Ceratobasidium sp. was reported as causal organism of root rot on Atractylodes macrocephala and banana (3,4). To the best of our knowledge, marigold damping off disease caused by Ceratobasidium sp. has not been reported so far on T. erecta. Hence, it is the first report from India. During fungal disease management for marigold, association of Ceratobasidium sp. should not be ignored for better crop protection. References: (1) R. T. Moore. Mycotaxon 29:91, 1987. (2) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN. 1991. (3) J. Yin et al. Plant Dis. 95:490, 2011. (4) J. M. You et al. Plant Dis.97:139, 2013.