Mining the Genome of Bacillus velezensis VB7 (CP047587) for MAMP Genes and Non-Ribosomal Peptide Synthetase Gene Clusters Conferring Antiviral and Antifungal Activity

Chemical pesticides have an immense role in curbing the infection of plant viruses and soil-borne pathogens of high valued crops. However, the usage of chemical pesticides also contributes to the development of resistance among pathogens. Hence, attempts were made in this study to identify a suitable bacterial antagonist for managing viral and fungal pathogens infecting crop plants. Based on our earlier investigations, we identified Bacillus amyloliquefaciens VB7 as a potential antagonist for managing Sclerotinia sclerotiorum infecting carnation, tobacco streak virus infecting cotton and groundnut bud necrosis infecting tomato. Considering the multifaceted action of B. amyloliquefaciens VB7, attempts were made for whole-genome sequencing to assess the antiviral activity against tomato spotted wilt virus infecting chrysanthemum and antifungal action against Fusarium oxysporum f. sp. cubense (Foc). Genome annotation of the isolate B. amyloliquefaciens VB7 was confirmed as B. velezensis VB7 with accession number CP047587. Genome analysis revealed the presence of 9,231,928 reads with an average read length of 149 bp. Assembled genome had 1 contig, with a total length of 3,021,183 bp and an average G+C content of 46.79%. The protein-coding sequences (CDS) in the genome was 3090, transfer RNA (tRNA) genes were 85 with 29 ribosomal RNA (rRNA) genes and 21 repeat regions. The genome of B. velezensis VB7 had 506 hypothetical proteins and 2584 proteins with functional assignments. VB7 genome had the presence of flagellin protein FlaA with 987 nucleotides and translation elongation factor TU (Ef-Tu) with 1191 nucleotides. The identified ORFs were 3911 with 47.22% GC content. Non ribosomal pepide synthetase cluster (NRPS) gene clusters in the genome of VB7, coded for the anti-microbial peptides surfactin, butirosin A/butirosin B, fengycin, difficidin, bacillibactin, bacilysin, and mersacidin the Ripp lanthipeptide. Antiviral action of VB7 was confirmed by suppression of local lesion formation of TSWV in the local lesion host cowpea (Co-7). Moreover, combined application of B. velezensis VB7 with phyto-antiviral principles M. Jalapa and H. cupanioides increased shoot length, shoot diameter, number of flower buds per plant, flower diameter, and fresh weight of chrysanthemum. Further, screening for antifungal action of VB7 expressed antifungal action against Foc in vitro by producing VOC/NVOC compounds, including hexadecanoic acid, linoelaidic acid, octadecanoic acid, clindamycin, formic acid, succinamide, furanone, 4H-pyran, nonanol and oleic acid, contributing to the total suppression of Foc apart from the presence of NRPS gene clusters. Thus, our study confirmed the scope for exploring B. velezensis VB7 on a commercial scale to manage tomato spotted wilt virus, groundnut bud necrosis virus, tobacco streak virus, S. sclerotiorum, and Foc causing panama wilt of banana.

An Ayurvedic approach of constipation

Ayurveda believes in the doshik theory of disease. The balanced state of these doshas is health and imbalanced state of the doshas is called disease. The equilibrium of these three basic substances is responsible for maintaining good health. Similarly disequilibrium of these may cause disintegration of the body or may lead to death. Constipation may be associated with an organic local lesion or generalized systemic disease. In the majority of cases constipation is a functional disorder caused due to delay in the passage of fecal matter or due to an insufficient bulk of the feces.

Use of EDTA in the treatment of local tissue damage caused by the Bothrops alternatus venom

ABSTRACT Twelve adult rabbits were distributed in three groups and received on the femoral biceps region, via intradermal injection (ID), 25µg of Bothrops alternatus venom dissolved in NaCl 0.9% and diluted in 0.25mL of phosphate buffered saline (PBS). Thirty minutes later, the group G1 received 0.25mL of phosphate buffered saline (PBS) ID while to G2 and G3 25mg of ethylenediamine tetraacetic acid (EDTA) dissolved in 0.25mL of PBS were administered via intramuscular (IM) and intravenous (IV) injection, respectively. Evaluations included local lesion and blood profile of all animals, before (time zero) and at 1, 2, 3, 4, 5, 6, 12, 18 and 24h after venom administration. All animal treated with PBS (G1) and EDTA IV (G3) presented increase of nociceptive stimuli at the site of inoculation of the venom, followed by moderate edema that persisted for 24h. Animals treated with IM EDTA (G2) only manifested increase of nociceptive stimuli at the site of injection 1h after treatment with discrete local edema between 12 and 24h. In relation to the local hemorrhagic halo no differences were found amongst the studied groups. Blood profile revealed significant decrease of segmented neutrophils in all groups. There was also increase in triglycerides and decrease in total protein and albumin in all groups. The local lesion was not altered by the treatments.

Integrating Local Lesion Assays with Conventional RT-PCR for Detection of Interspecies Tospovirus Reassortants and Mixed Tospovirus Infections

Tomato spotted wilt virus (TSWV) has historically been the major tospovirus present in North America. Recent emergence of Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) in Florida and the Caribbean has complicated reliable identification of tospoviruses in this region. Field symptoms of these three tospoviruses are indistinguishable in most host plants, and commercially available TSWV lateral-flow immunoassay reagents cross react with GRSV and TCSV, leading to incorrect diagnoses of GRSV or TCSV as TSWV. Reliable diagnosis of TSWV, GRSV, and TCSV is further confounded by the fact that all currently known isolates of GRSV in the United States are reassortants containing one genomic RNA segment derived from TCSV. To address these practical challenges, we developed and validated genome segment-specific primers for conventional reverse-transcription polymerase chain reaction (RT-PCR) detection of the large, medium, and small RNA segments of TSWV, GRSV, and TCSV. When used in conjunction with local lesion-passaged virus isolates, the genome segment-specific RT-PCR assays developed in this study will facilitate high-throughput screening of plant or thrips samples for interspecies reassortants in epidemiological studies and reliable identification of these three tospoviruses in mixed infections commonly observed in the field.

Induction and Maintenance of Systemic Acquired Resistance by Acibenzolar-S-Methyl in Three Cultivated Tobacco Types

Induction and maintenance of systemic acquired resistance (SAR) in ‘N’ gene containing burley, flue-cured, and oriental tobacco cultivars were assessed by monitoring decreases in the number of local lesions caused by Tobacco mosaic virus (TMV) following treatment with acibenzolar-S-methyl (ASM). Leaf samples were collected from lower, middle, and top positions on seedlings at 3-day intervals over 21 days following ASM treatment and subsequent inoculation with TMV under laboratory conditions. Local lesion number for each leaf was recorded 7 days postinoculation. Reductions in TMV local lesion numbers on ASM-treated versus nontreated tobacco varied over time, and differed for each tobacco type. Based on reduced local lesion numbers, SAR was induced in burley and flue-cured tobacco by 3 and 6 days postinoculation, respectively, while oriental tobacco responded by 9 days. SAR was maintained in burley tobacco from 3 to 9 days after ASM application, and from 9 to 15 days after application in oriental tobacco. ASM treatment reduced local lesion numbers in flue-cured tobacco significantly at 6, 12, and 21 days postapplication, but not at 15 and 18 days after treatment. The SAR response was similar among lower, middle, and top leaves with no effect of ASM on response by leaf position, although TMV local lesion numbers were greater on lower leaves than on middle and top leaves 6 days after treatment, but significantly less on lower leaves 18 days after treatment compared to middle and top leaves.

Measuring Root Disease Suppression in Response to a Compost Water Extract

Commercial application of compost to prevent plant disease is hindered by variable performance. Here, we describe the use of a growth pouch assay to measure impact of a compost water extract (CWE) on root infection under controlled conditions. Most pea roots (≥95%) inoculated with Fusarium solani or Phoma pinodella spores rapidly develop a single local lesion in the region of elongation. In the presence of CWE, infection of pea roots grown in pouches was reduced by 93 to 100%. CWE used as a drench on pea seedlings grown in sand also resulted in 100% protection but, in a heavy clay soil, infection was reduced by <50%. CWE filtered to remove microorganisms did not inhibit frequency of F. solani infection, and resulted in increased local lesion development on individual roots. CWE inhibited mycelial growth of both pea- and cucumber-infecting isolates of F. solani in culture but exerted <40% protection against cucumber root infection. CWE treatment of pea but not cucumber was associated with retention of a sheath of border cells interspersed with bacteria covering the region of elongation. Growth pouch assays may provide a system to monitor effects of specific compost mixtures on root–rhizosphere interactions, and to identify variables influencing disease control.

First Report of Tomato ringspot virus Infecting Pepper in Iran

Chili pepper (Capsicum frutescens) represents an important crop in Iran and is under cultivation in different regions in Northern Iran. In spring 2012, commercially grown tabasco (Capsicum frutescens) peppers in Varamin, Shahriar, and Karaj districts of Tehran province developed an undescribed disease. Symptoms observed were mosaic, leaf malformations, and stunting. Fruit symptoms included chlorosis and distortion. To verify the identity of the disease, six fields were surveyed and 72 symptomatic leaves were collected and screened by double antibody sandwich (DAS)-ELISA using specific antibodies to Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), Pepper mild mottle virus (PMMV), Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), and Arabis mosaic virus (ArMV). ToRSV was found in 23% of the samples collected. None of the samples had a positive reaction to other tested viruses. The ToRSV-positive peppers were used for mechanical transmission to Chenopodium quinoa, local lesion host, and after two cycles of single local lesion isolation, they were transferred to Cucumis sativus, Solanum esculentum, and Capsicum fructescens. Inoculations resulted in systemic mosaic and chlorotic local lesion on C. sativus; leaf distortion and mosaic on S. esculentum; and mosaic, mottle, and stunting on C. fructescens. All inoculated plants were positive for ToRSV with DAS-ELISA. To further verify ToRSV infection, reverse transcription (RT)-PCR was conducted. Two primers were designed on the basis of the highly conserved sequences of the putative viral polymerase gene available in the GenBank. RT-PCR of total RNA extract from infected peppers and inoculated plants with the designed primers RdR-R (5′-CGCCTGGTAATTGAGTAGCCC-3′) and RdR-F (5′-GAAGAGCTAGAGCCTCAACCAGG-3′), consistently amplified the 411-bp product, while no amplification products were obtained from noninfected control (healthy plants). The fragment from tabasco pepper was cloned into pTZ57R/T (Ins T/A clone PCR Cloning kit, Fermentas, St. Leon-Rot, Germany) and sequenced in both directions of three clones. The resulting nucleotide sequence (GenBank Accession No. JQ972695) had the highest identity (94%) with the polymerase gene of a ToRSV isolate from blueberry cv. Patriot (Accession No. GQ141528) and had lower identity (91%) with that of a ToRSV isolate from blueberry cv. Bluecrop (Accession No. GQ141525). Tomato ringspot virus (ToRSV) is reported to infect Capsicum spp. in the United States (1,2). Our results confirm the natural infection of pepper plants in Tehran by ToRSV. To our knowledge, this is the first report of ToRSV infection of pepper in Iran. The finding of this disease in Tehran confirms further spread of the virus within northern regions of Iran and prompts the need for research to develop more effective management options to reduce the impact of ToRSV on pepper crops. Beside, primers designed on the basis of putative viral polymerase gene sequences may improve the detection of ToRSV isolates by RT-PCR in Iran. References: (1) S. K. Green and J. S. Kim. Technical Bulletin. No.18, 1991. (2) G. P. Martelli and A. Quacquarelli. Acta Hortic. 127:39, 1983.