Biofabrication of zinc oxide nanoparticles from Melia azedarach and its potential in controlling soybean seed-borne phytopathogenic fungi.

Present study, report the biofabrication of zinc oxide nanoparticles from aqueous leaf extract of Melia azedarach (MaZnO-NPs) through solution combustion method and their novel application in preventing the growth of seed-borne fungal pathogens of soybean (Cladosporium cladosporioides and Fusarium oxysporum). The standard blotter method was employed to isolate fungi and was identified through molecular techniques. The characterization of MaZnO-NPs was carried out by UV-Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FT-IR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) equipped with Energy Dispersive Spectroscopy (EDS) and Transmission Electron Microscopy (TEM). The physicochemical characterization confirmed the particles were of high purity and nano size (30-40 nm) with a hexagonal shape. The synthesized MaZnO-NPs inhibited the growth of C. cladosporioides and F. oxysporum in a dose dependent manner. Biomass, ergosterol, lipid peroxidation, intracellular reactive oxygen species and membrane integrity determination upon MaZnO-NPs treatment offered significant activities there by confirming the mechanism of action against the test pathogens. In conclusion, due to the effectiveness of MaZnO-NPs in controlling the growth of C. cladosporioides and F. oxysporum, the synthesized MaZnO-NPs provides insight towards their potential application in agriculture and food industries.

Fusarium oxysporum f. sp. lycopersici causal agent of vascular wilt disease of tomato: Biology to diversity- A review.

Tomato (Lycopersicon esculentum) is one of the widely grown vegetables worldwide. Fusarium oxysporum f. sp. lycopersici (FOL) is the significant contributory pathogen of tomato vascular wilt. The initial symptoms of the disease appear in the lower leaves gradually, trail by wilting of the plants. It has been reported that FOL penetrates the tomato plant, colonizing and leaving the vascular tissue dark brown, and this discoloration extends to the apex, leading to the plants wilting, collapsing and dying. Therefore, it has been widely accepted that wilting caused by this fungus is the result of a combination of various physiological activities, including the accumulation of fungal mycelia in and around xylem, mycotoxin production, inactivation of host defense, and the production of tyloses; however, wilting symptoms are variable. Therefore, the selection of molecular markers may be a more effective means of screening tomato races. Several studies on the detection of FOL have been carried out and have suggested the potency of the technique for diagnosing FOL. This review focuses on biology and variability of FOL, understanding and presenting a holistic picture of the vascular wilt disease of tomato in relation to disease model, biology, virulence. We conclude that genomic and proteomic approachesare greater tools for identification of informative candidates involved in pathogenicity, which can be considered as one of the approaches in managing the disease.

Specific PCR-based detection of Phomopsis vexans the cause of leaf blight and fruit rot pathogen of Solanum melongena L.

Leaf blight and fruit rot disease caused by Phomopsis vexans is a devastating disease of brinjal. The detection of P. vexans in plant parts and seeds of brinjal can be complicated, mainly when the inoculum is present at low levels and/or overgrown by fast-growing saprophytic fungi or other seed-borne fungi. A PCR-based diagnostic method was developed with specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans. The efficiency and specificity of primer pairs PvexF/PvexR designed were established by PCR analysis of DNA from P. vexans strains isolated from India and fungal isolates of other genera. A single amplification product of 323-bp was detected from DNA of P. vexans isolates. No cross-reaction was observed with any of the other isolates tested. The specific primers designed and employed in PCR detected P. vexans up to 10 pg from DNA isolated from pure culture. This is the first report on the development of species-specific PCR assay for identification and detection of P. vexans. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for the detection of pathogen P. vexans at early stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Phomopsis vexans is an important seed-borne pathogenic fungus responsible for leaf blight and fruit rot in brinjal. Current detection methods, based on culture and morphological identification is time consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans.

Identification and Characterization of Memecylon Species Using Isozyme Profiling.

BACKGROUND: The protein/isozyme fingerprint is useful in differentiating the species and acts as a biochemical marker for identification and systematic studies of medicinal plant species. OBJECTIVE: In the present study, protein and isozyme profiles for peroxidase, esterase, acid phosphatase, polyphenol oxidase, alcohol dehydrogenase, and alkaline phosphatase of five species of Memecylon (Melastomataceae), Memecylon umbellatum, Memecylon edule, Memecylon talbotianum, Memecylon malabaricum, and Memecylon wightii were investigated. MATERIALS AND METHODS: Fresh leaves were used to prepare crude enzyme extract for analyzing the five enzymes isozyme variations. Separation of isozymes was carried out using polyacrylamide gel electrophoresis (PAGE) and the banding patterns of protein were scored. Pair-wise comparisons of genotypes, based on the presence or absence of unique and shared polymorphic products, were used to regenerate similarity coefficients. The similarity coefficients were then used to construct dendrograms, using the unweighted pair group method with arithmetic averages. RESULTS: A total of 50 bands with various Rf values and molecular weight were obtained through PAGE analysis. Among the five Memecylon species, more number of bands was produced in M. wightii and less number of bands was observed in M. edule. The results of similarity indices grouped M. malabaricum and M. wightii in one cluster with 98% similarity and M. umbellatum, M. edule, and M. talbotianum are grouped in another cluster with 79% similarity showing close genetic similarities which is in accordance with the morphological identification of Memecylon species. CONCLUSION: The protein/isozyme fingerprint is useful in differentiating the species and acts as a biochemical marker for identification of Memecylon species. SUMMARY: Biochemical characterization of Memecylon species was evaluated by SDS-PAGE of extracted protein and isozyme profiling on native PAGE.After electrophoresis, each gel was stained with specific stains. Genetic distance relationships were evaluated based on the banding patterns of protein on isozymes.Unique banding pattern of esterase, peroxidase, acid phosphatase, alcohol dehydrogenase and polyphenol oxidase are observed in all the five species of Memecylon, which represent the fingerprint of Memecylon species.SDS-PAGE and isozyme profiling of five Memecylon species revealed that M. malabaricum and M. wightii grouped in one cluster and M. umbellatum, M. edule and M. talbotianum grouped in another cluster showing close genetic similarities which is in accordance with the morphological identification of Memecylon species.This is the first report on the comparison of protein and isozyme profile of five different Memecylon species. Abbreviations Used: SDS-PAGE: Sodium docecyl sulfate polyacrylamide gel electrophoresis; NTSYS PC2: Numerical taxonomy system, version 2.2 for Windows XP, Vista, Win7, Win 8 and Win10 including 64 bit.

Molecular phylogeny, pathogenicity and toxigenicity of Fusarium oxysporum f. sp. lycopersici.

The present study aimed at the molecular characterization of pathogenic and non pathogenic F. oxysporum f. sp. lycopersici strains isolated from tomato. The causal agent isolated from symptomatic plants and soil samples was identified based on morphological and molecular analyses. Pathogenicity testing of 69 strains on five susceptible tomato varieties showed 45% of the strains were highly virulent and 30% were moderately virulent. Molecular analysis based on the fingerprints obtained through ISSR indicated the presence of wide genetic diversity among the strains. Phylogenetic analysis based on ITS sequences showed the presence of at least four evolutionary lineages of the pathogen. The clustering of F. oxysporum with non pathogenic isolates and with the members of other formae speciales indicated polyphyletic origin of F. oxysporum f. sp. lycopersici. Further analysis revealed intraspecies variability and nucleotide insertions or deletions in the ITS region among the strains in the study and the observed variations were found to be clade specific. The high genetic diversity in the pathogen population demands for development of effective resistance breeding programs in tomato. Among the pathogenic strains tested, toxigenic strains harbored the Fum1 gene clearly indicating that the strains infecting tomato crops have the potential to produce Fumonisin.

Delftia tsuruhatensis WGR-UOM-BT1, a novel rhizobacterium with PGPR properties from Rauwolfia serpentina (L.) Benth. ex Kurz also suppresses fungal phytopathogens by producing a new antibiotic-AMTM.

UNLABELLED: The bacterial strain designated as WGR-UOM-BT1 isolated from rhizosphere of Rauwolfia serpentina exhibited broad-spectrum antifungal activity and also improved early plant growth. Based on morphological, biochemical and 16S rRNA gene sequence analyses, the strain BT1 was identified as Delftia tsuruhatensis (KF727978). Under in vitro conditions, the strain BT1 suppressed the growth of wide range of fungal phytopathogens. Purified antimicrobial metabolite from the strain BT1 was identified as nitrogen-containing heterocyclic compound, 'amino(5-(4-methoxyphenyl)-2-methyl-2-(thiophen-2-yl)-2,3-dihydrofuran-3-yl)methanol' (AMTM), with molecular mass of 340•40 and molecular formula of C17 H19 NO3 S. The strain BT1 was positive for rhizosphere colonization (tomato), IAA production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and phosphate solubilization. Under laboratory and greenhouse conditions, the strain BT1 promoted plant growth and suppressed foliar and root fungal pathogens of tomato. Therefore, antimicrobial and disease protection properties of strain BT1 could serve as an effective biological control candidate against devastating fungal pathogens of vegetable plants. Besides, the production of IAA, P solubilization and ACC deaminase activity enhance its potential as a biofertilizer and may stabilize the plant performance under fluctuating environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we reported that Delftia tsuruhatensis WGR-UOM-BT1 strain has the plant growth promotion activities such as rhizosphere colonization (tomato), IAA production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and phosphate solubilization. This bacterial strain was found producing an antimicrobial nitrogen-containing heterocyclic compound identified as 'amino(5-(4-methoxyphenyl)-2-methyl-2-(thiophen-2-yl)-2,3-dihydrofuran-3-yl)methanol' [C17 H19 NO3 S] (AMTM), which is new to the bacterial world.

Analysis of genetic and aflatoxin diversity among Aspergillus flavus isolates collected from sorghum seeds.

Thirty-four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive-ELISA (ic-ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using ß-tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and ß-tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0-0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin-producing potential in-vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India.

Attenuation of reactive oxygen/nitrogen species with suppression of inducible nitric oxide synthase expression in RAW 264.7 macrophages by bark extract of Buchanania lanzan.

BACKGROUND: Oxidative stress is one of the most critical factors implicated in disease conditions. Buchanania lanzan Spr. (Anacardiaceae) bark powder preparation has been reported for treating an inflammatory condition in the Ayurvedic Pharmacopoeia of India. OBJECTIVE: In the present study, we investigate the effect of the bark methanol extract (BLM) on reactive oxygen/nitrogen species (ROS/RNS), the expression of protein and mRNA of inducible nitric oxide synthase (iNOS) in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) and sodium nitroprusside (SNP) to provide scientific validation of the above said medicinal property. MATERIALS AND METHODS: The capacity to quench ROS and RNS was evaluated by 5-(and-6) chloromethyl-20,70-dichlorodihydrofluorescein diacetate acetyl ester fluorescence and nitrite estimations in LPS/SNP-stimulated macrophages respectively. The protein and transcript expression of iNOS was evaluated through Western Blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis respectively. RESULTS: Macrophages pretreated with BLM (>100 µg/mL) for 24 h, stimulated with LPS for the last 18 h of experimental duration recorded a significantly (P < 0.05) reduced levels of ROS (3.45-fold) against LPS-stimulated conditions (5.7-fold). SNP-stimulation resulted in increased NO accumulation (17-fold) which was neutralized by BLM at >100 µg/ml (1.6-fold) credited to a reduced protein and mRNA expression of iNOS as recorded by Western blots and RT-PCR results respectively. The reversed-phase liquid chromatography-diode array detection analysis identified the presence of 4-hydroxybenzoic acid, quercetin and p-coumaric acid (Rt values 5.444, 5.569 and 9.580 respectively). CONCLUSIONS: The potential of BLM inhibiting ROS/RNS production validates the medical use of bark, could find beneficial application under conditions of immune stimulation and/or bacterial infection.

Cytotoxic effect of p-Coumaric acid on neuroblastoma, N2a cell via generation of reactive oxygen species leading to dysfunction of mitochondria inducing apoptosis and autophagy.

p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. In the present study, p-CA was evaluated for its capacity to induce cytotoxic effect to neuroblastoma N2a cells and we report here the possible mechanism of its action. p-CA at a concentration of 150 µmol/L, upon exposure for 72 h, stimulated 81.23 % of cells to apoptosis, as evidenced by flow cytometer studies mediated through elevated levels of ROS (7.5-fold over control). Excess ROS production activated structural injury to mitochondrial membrane, observed as dissipation of its membrane potential and followed by the release of cytochrome c (8.73-fold). Enhanced generation of intracellular ROS correlated well with the decreased levels (~60 %) of intracellular GSH. Sensitizing neuroblastoma cells for induction of apoptosis by p-CA identified p53-mediated upregulated accumulation of caspase-8 messenger RNA (2.8-fold). Our data report on autophagy, representing an additional mechanism of p-CA to induce growth arrest, detected by immunoblotting and fluorescence, correlated with accumulation of elevated levels (1.2-fold) of the LC3-II protein and acridine orange-stained autophagosomes, both autophagy markers. The present study indicates p-CA was effective in production of ROS-dependent mitochondrial damage-induced cytotoxicity in N2a cells.

Structural and functional diversity of rhizobacteria associated with Rauwolfia spp. across the Western Ghat regions of Karnataka, India.

The present study carried out with denaturing gradient gel electrophoresis of DNA extracted from rhizosphere soils of Rauwolfia spp. collected from Western Ghat (WG) regions of Karnataka indicated that Pseudomonas sp. was prevalently found followed by Methylobacterium sp., Bacillus sp. and uncultured bacteria. A total of 200 rhizobacteria were isolated from 58 rhizosphere soil samples comprising of 15 different bacterial genera. The Shannon Weaver diversity index (H') and Simpson's diversity index (D) were found to be 2.57 and 0.91 for cultivable bacteria, respectively. The total species richness of cultivable rhizobacteria was high in Coorg district comprising 15 bacterial genera while in Mysore district, four bacterial genera were recorded. Rarefaction curve analysis also indicated the presence of higher species richness in samples of Shimoga and Coorg. All the rhizobacteria were screened for their multiple plant growth promotion and disease suppression traits. The results revealed that 70% of the isolates colonized tomato roots, 42% produced indole acetic acid, 55% solubilized phosphorus, while 43, 22, 27, 19, 40, 15 and 44% produced siderophore, salicylic acid, hydrogen cyanide, chitinase, phytase, cellulase and protease, respectively. Rhizobacterial isolates showing antagonistic activity against Fusarium oxysporum and Aspergillus flavus were 53 and 33%, respectively. Plant growth promotion studies revealed that most of the isolates increased percent germination with significantly higher vigour index as compared to untreated control. Most predominant rhizobacteria found in the rhizospheres of Rauwolfia spp. of WG regions are potential PGPR which can serve as biofertilizers and biopesticides.

Mechanisms of plant growth promotion and disease suppression by Pseudomonas aeruginosa strain 2apa.

A new Pseudomonas strain, designated as 2apa was isolated from tomato rhizosphere and identified as a member of species Pseudomonas aeruginosa based on its morphology, conventional, biochemical, cell wall fatty acid methyl ester analysis, and 16S rRNA gene sequence analysis. The strain 2apa was positive for root colonization, indole acetic acid (IAA), salicylic acid and siderophore production and inhibited the growth of wide range of microorganisms. Antimicrobial substances produced by this strain with further purification and structure elucidation proved to be phenazine. Under laboratory and greenhouse conditions the strain promoted plant growth and suppressed a wide range of foliar and root pathogens in tomato. The protection offered by strain 2apa to foliar pathogens is considered as induced systemic resistance and was further confirmed by enhanced accumulation of phenolics, elicitation of lipoxygenas activity, and jasmonic acid levels. The broad-spectrum antimicrobial and induced systemic resistance exhibiting strain P. aeruginosa 2apa can be used as an effective biological control candidate against devastating fungal and bacterial pathogens, which attack both root and foliar portions of tomato plant. Production of other functional traits such as IAA and siderophore may enhance its potential as biofertilizer.

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower.

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers-ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.

Prospects of molecular markers in Fusarium species diversity.

Recent developments in genomics have opened up for newer opportunities to study the diversity and classification of fungi. The genus Fusarium contains many plant pathogens that attack diverse agricultural crops. Fusarium spp. are not only pathogenic to plants but are also known as toxin producers that negatively affect animal and human health. The identification of Fusarium species still remains one of the most critical issues in fungal taxonomy, given that the number of species recognized in the genus has been constantly changing in the last century due to the different taxonomic systems. This review focuses of various molecular-based techniques employed to study the diversity of Fusarium species causing diseases in major food crops. An introduction of fusarial diseases and their mycotoxins and molecular-marker-based methods for detection introduce the concept of marker application. Various well-known molecular techniques such as random amplified polymorphic DNA, amplification fragment length polymorphism, etc. to more modern ones such as DNA microarrays, DNA barcoding, and pyrosequencing and their application form the core of the review. Target regions in the genome which can be potential candidates for generation of probes and their use in phylogeny of Fusarium spp. are also presented. The concluding part emphasizes the value of molecular markers for assessing genetic variability and reveals that molecular tools are indispensable for providing information not only of one Fusarium species but on whole fungal community. This will be of extreme value for diagnosticians and researchers concerned with fungal biology, ecology, and genetics.

First Report of Bean common mosaic virus Infecting Lablab purpureus in India.

Lablab bean (Lablab purpureus L. Sweet) is a widely cultivated, highly drought tolerant legume vegetable crop grown in diverse environmental conditions worldwide. In India and elsewhere, the young pods are consumed as a fresh vegetable and mature dry seeds are important in the diet of people preferring vegetarian food (2). Small-holding farmers use their own saved seeds for sowing. During October 2008, L. purpureus exhibiting symptoms of stunting, mosaic, vein-banding, vein-clearing, mottling, and blisters suggestive of a viral infection were observed in and around the Mysore District of Karnataka State, India. Incidence of the disease ranged from 1 to 10% in different fields. Symptomatic leaves were collected from fields of Daripura Village, Mysore District, Karnataka. Viruses that were tested by indirect ELISA included Cucumber mosaic virus, Tobacco mosaic virus, Cowpea aphid-borne mosaic virus, Cowpea mosaic virus, Cowpea mottle virus, Southern bean mosaic virus, and Bean common mosaic virus (BCMV). Results of the ELISA tests indicated that all 28 samples collected from different fields were infected with BCMV. Examination of tissue sap from symptomatic plants by electron microscopy revealed flexuous rod-shaped particles (~750 nm long). An immunocapture-reverse transcription (IC-RT)-PCR assay employing degenerate primers for amplifying partial coat protein (CP) and 3'-UTR of potyviruses (1) yielded a ~700-bp product that was cloned and sequenced (GenBank Accession No. HM776637). Sequence identity at the nucleotide level was 96% with BCMV strain NL-7n (GenBank Accession No. GQ456169) infecting common bean from Himachal Pradesh, India. RTPCR was performed with a virus-specific primer pair (FW3-5'-GCAGTAGCACAGATGAAGGCA-3': Rv3-5'-GGTTCTTCCGGCTTACTCATAAACAT-3') designed to amplify 340 bp, the partial coat protein gene of BCMV. All symptomatic L. purpureus field samples and screenhouse-grown seedlings manually inoculated with infected sap were positive for BCMV infection in RT-PCR assay employing specific primers with amplification of a 340-bp product. To our knowledge, this is the first report of BCMV infecting L. purpureus in India. BCMV has also been reported in L. purpureus in Uganda (4) and Nigeria (3). Plants that were confirmed by ELISA to be infected were tagged, and from these plants, seeds were collected and pooled. Four hundred seeds were germinated and a rate of 6.5% seed transmission was determined based on symptoms, ELISA, and PCR. From December 2008 to December 2010, different L. purpureus plantings were monitored for BCMV incidence. Plants infected at different growth stages were tagged and pods were harvested from infected and healthy plants. Data from at least 100 BCMV-infected L. purpureus plants from each of 12 different fields were recorded for yield loss analysis. In terms of number of pods per plant, number of seeds per pod, and seed weight, an average as much as 40% yield loss was recorded from 12 different fields. Because seeds collected from these plants are used for subsequent plantings, these plants may act as virus reservoirs or foci of infection. References: (1) A. S. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (2) M. N. Maruthi et al. Ann. Appl. Biol. 149:187, 2006. (3) O. O. Odedara et al. J. Gen. Virol. 74:322, 2008. (4) T. N. Sengooba et al. Plant Pathol. 46:95, 1997.