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1.
Plant Dis ; 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38568792

RESUMO

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 - 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

2.
Plant Dis ; 2023 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-36947842

RESUMO

Moringa oleifera Lam. (Moringaceae) is one of the important leafy vegetable trees widely spread from India to Africa and widely used as food and medicine. During field investigation (July, 2021) of drumstick fields in Hiriyur (13°95'79" N; 76°64'45" E), Karnataka, fruits of drumstick plants showed a characteristic rot disease with an incidence ranging from 10-12% in an area of 5 hectares surveyed. Initially water soaked lesions turned to small necrotic lesions and later coalesced to form larger areas covered with white mycelial growth leading to softening and later mummification of fruits. Infected fruits were collected (n=5) and infected fruit parts (margins of healthy and infected tissues) were cut into small pieces, surface sterilized with sodium hypochlorite (2%, v/v) and blotter dried after three sterile water washes. An associated fungal species was isolated on PDA medium amended with Streptomycin (40 mg/L) and incubated at 28 ºC for 1 week. The fungal isolate grown on PDA had dense, white, aerial mycelium with light brown coloration on the reverse side of the agar medium. Morphological characteristics of conidia were determined for single-spore cultures grown on water agar media. Microconidia were single-celled, hyaline, non septate, ovoid, and 8.4 - 9.8 × 1.8 - 2.94 µm (n=20). Macroconidia were three- to five-septate, slightly curved, tapered at the apex, and 24.4 - 28 × 2 - 4 µm (n=20). Based on morphological characters' pathogen was identified as Fusarium sp. (Leslie and Summerell, 2006). Further, three representative isolate (SMG, MYS1 & MYS2) were subjected for molecular identification. The genomic DNA was extracted following CTAB method and ITS-rDNA was amplified using ITS1/ITS4 primers (White et al., 1990) and translation elongation factor (tef-1α) gene was amplified using EF1/EF2 primers (O'Donnell et al., 2009) respectively. ITS-rDNA sequence shared 100% sequence similarity (650bp / 650bp) with reference sequence F. incarnatum (ON226997) followed by 100% sequence identity to F. equiseti (KT277307 & MT953927). But, tef-1α gene sequence analysis in nBLAST showed that the sequence shared 100% (123/123bp) identity with F. incarnatum (F. incarnatum NEAU-TG1 MH920853; M2JP3 OP312673; WEH ON456146). Combined phylogenetic analysis revealed that the isolate shared a common clade with reference sequence of F. incarnatum in the Fusarium-incarnatum-equiseti species complex thus confirming the identity of the isolated pathogen as F. incarnatum (Rob. ex Desm.) Sacc. 1886. The sequences obtained in the present study are deposited in GenBank database (ITS: OP508729, OQ159019, OQ159020 and tef-1α: OP477394, OQ176254, OQ176255). To prove Koch's postulates, pathogenicity test was carried out by inoculating healthy drumstick fruits cv. Bhagya (n=10) with spore suspension (105 conidia/ml). Control fruits (n=5) were sprayed with sterile water. The experiments were conducted in triplicates and repeated twice. Inoculated and control fruits were kept in a moist chamber at 26 ± 2°C for 2 days and observed at regular intervals. Development of disease symptoms were recorded on 52 out of 60 inoculated fruits which were identical with symptoms seen in the field and all control fruits remained symptomless. Identity was confirmed after re-isolation by morphology and culture studies. To the best of our knowledge, this is the first report of F. incarnatum causing a fruit rot of drumstick in India. This disease affected the cost of drumstick production and contributed to the decline in production in India.

3.
J Appl Microbiol ; 133(4): 2430-2444, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35809236

RESUMO

AIMS: In the study, seven Plant Growth Promoting Rhizobacteria (PGPR) Azotobacter species were screened against three strains of Fusarium verticillioides to test its antifungal activity. Azotobacter strains were tested for the degradation of fumonisin produced by F. verticillioides. Secondary metabolites were isolated and characterized from the Azotobacter strains for the first time. METHODS AND RESULTS: Potential seven Azotobacter species antifungal activity was tested following the dual culture assay against three strains of Fusarium verticillioides namely FVM-42, FVM-86 and MTCC156 estimating the substantial zone of inhibition. Azotobacter species AZT-31 and AZT-50 strains significantly inhibited the growth of F. verticillioides recording drastic growth enhancement of maize under in-vitro conditions by calculating the infection incidence, vigour index and germination percentage. As confirmation, dereplication studies were conducted for the reconfirmation of Azotobacter strains by isolating from rhizoplane. Azotobacter strains played a key role in the degradation of fumonisin produced by F. verticillioides reporting 98% degradation at 2 h of incubation with the pathogen. Furthermore, in the study first time, we have tried to isolate and characterize the secondary metabolites from the Azotobacter strains exhibiting six compounds from the species AZT-31 (2) and AZT-50 (4). Preliminary in-vitro experiments were carried out using the compounds extracted to check the reduction of infection incidence (90%) and increase in germination percentage upto 50 to 70% when compared to the test pathogen. CONCLUSION: Azotobacter strains referred as PGPR on influencing the growth of plant by producing certain substances that act as stimulators on inhibiting the growth of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The future perspective would be the production of an active combination of carboxamide compound and Azotobacter species for preventively controlling the phytopathogenic fungi of plants and crops and also towards the treatment of seeds.


Assuntos
Azotobacter , Fumonisinas , Fusarium , Antifúngicos/farmacologia , Fumonisinas/metabolismo , Zea mays/microbiologia
4.
Plant Dis ; 2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35471075

RESUMO

Cluster bean (Cyamopsis tetragonoloba (L.) Taub.) is an important vegetable crop cultivated widely in India. During a field survey in November 2021, about 60% of plants exhibited characteristic powdery mildew disease symptoms and signs in a 15 ha field in Northern Karnataka (Raichur), India. Initially, the symptoms and signs appeared as tan lesions, which later became small, circular and chlorotic. The abaxial surface turned yellow and was covered with white mycelial growth. As the disease progressed, white mycelia grew on the adaxial leaf surface, stems and pods as well. In severe infections, drying and premature defoliation of infected leaves were observed. Infected leaf samples with mycelia were collected (n=8) and the fungus was subjected to morphological and molecular observations. Mycelia on leaves was characterized as epiphytic, amphigenous, producing dense, white patches on the upper and lower leaf surfaces, stem and young pods. Hyphae were hyaline, thin-walled, 1.8 to 4.2 µm wide with erect conidiophores consisting of a cylindrical foot-cell, straight flexuous at the base and measured 20 to 36 × 6 to 9 µm (n=30), followed by 1 to 2 shorter cells. Ellipsoid conidia were produced singly and measured 28 to 42 × 12 to 20 µm (n=30) without fibrosin bodies. Chasmothecia were not observed. A reference specimen was deposited at the Institution of Excellence, University of Mysore Herbarium (UOM-IOE 2022_1). The morphology and other characteristics of conidia were consistent with an Erysiphe species (Braun and Cook 2012). Genomic DNA was isolated from a conidial suspension harvested from the powdery mildew affected cluster bean samples. The ITS region was amplified from three samples using powdery mildew-specific primer pair PN23/PN34 and sequenced directly (Chen et al. 2008). nBLAST analysis revealed that the ITS sequence shared 100% similarity with the reference sequence (E. diffusa vouchers HMJAU02177 - KM260363, BRIP 71013 - MW009058) of Erysiphe diffusa (Cooke & Peck) U. Braun & S. Takam. In addition to 100% match to voucher specimens of E. diffusa, there were no vouchers from other species that also had 100% match. The representative sequences were deposited in GenBank with accession numbers OM669776 - OM669778. Koch's postulates were conducted on healthy cluster bean plants grown under greenhouse conditions. Conidia were harvested from infected leaves, suspended in water and sprayed on 40 to 50-day-old cluster bean plants (28 ± 2°C and >70% relative humidity). The development of powdery mildew symptoms was recorded on 22 plants after 10-14 days of post inoculation. Control plants inoculated with sterile water remained healthy without powdery mildew symptoms. Microscopic observation of spores from inoculated plants confirmed the pathogen as E. diffusa. The genus Erysiphe is known to infect many crop plants. E. diffusa has been reported to infect Vigna radiata, Glycine max and Phaseolus mungo in Australia (Kelly et al. 2021). No reports are available at USDA's host-fungus database for cluster bean and E. diffusa (Farr and Rossman 2022). To the best of our knowledge, this is the first report of E. diffusa associated with powdery mildew of cluster bean in India. Further comprehensive investigations will shed a light on the economic impact of powdery mildew disease on the cluster bean in India.

5.
Water Sci Technol ; 86(8): 2008-2019, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36315092

RESUMO

Biojarosite as a replacement for commercial iron catalyst in the oxidative degradation of the dye Rhodamine B was confirmed and established. Investigations on the oxidative degradation by Fenton's oxidation and UV-Fenton's oxidation with EDTA at neutral pH were conducted and degradation of target compound was evaluated. UV-Fenton's oxidation was shown to be efficient over Fenton's oxidation in the degradation of Rhodamine B with removal efficiency of 90.0%. Design of Experiments was performed with Box-Behnken design. Investigation was conducted for the predicted values separately for both Fenton's oxidation and UV-Fenton's oxidation and the Rhodamine B removal was taken as response. Variable parameters biojarosite, H2O2 dosage and EDTA were optimized in the range of 0.1-1 g/L, 2.94-29.4 mM and 10-100 mM, respectively. A quadratic regression model is fitted for both Fenton's and UV-Fenton's oxidation. Analysis of variance (ANOVA) is performed and model fit is discussed.


Assuntos
Peróxido de Hidrogênio , Ferro , Ferro/química , Peróxido de Hidrogênio/química , Ácido Edético , Oxirredução , Concentração de Íons de Hidrogênio
6.
Lett Appl Microbiol ; 73(5): 672-681, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34545601

RESUMO

Linseed commonly called as flaxseed (Linum usitatissimum Linn.) is an important oilseed crop cultivated widely in Northern parts of Karnataka. During, 2019 (January-February), a characteristic disease was noticed with symptoms that resembled phytoplasma or like disease symptoms. The incidence was ranged from 6·5 to 16·5% in the experimental station of Raichur Agricultural University. The typical symptoms observed were virescence of floral parts, fasciation of the inflorescence axis, phyllody, stunted and flattened stem with reduced leaves. Symptomatic and healthy samples were collected and processed for molecular detection of phytoplasma. Total DNA was isolated from four infected plants and two healthy plants. The 16S rDNA region was amplified using P1/P7 followed by R16F2n/R16R2 primer pair which showed the amplification of expected amplicon size from all four infected samples. Furthermore, the SecA gene was amplified using SecA1/SecA3 primers. The PCR amplified products were subjected for direct sequencing from both directions and the consensus sequences were obtained and nBLAST search analysis revealed that the 16Sr RNA and SecA sequences were sharing maximum similarity (100%) with the reference sequence of Ca. P. cynodontis. The sequences were analysed phylogenetically by constructing a Phylogram independently by NJ method along with reference sequence of 16S rRNA region and SecA region retrieved from GenBank database showed that the phytoplasma sequence from linseed phyllody of the present study placed in a distinct clade along with reference sequence of "Ca. P. cynodontis" thus confirming the identity phylogenetically. Furthermore, iPhyClassifier and virtual RFLP proved that the phytoplasma belonged to 16SrXIV (subgroup A) phytoplasma. Previously linseed is known to be associated with 16SrII-D phytoplasma but the association of the 16SrXIV-A group of phytoplasma is not reported so far. Therefore, this is the new host record for Ca. P. cynodontis (16SrXIV-A) phytoplasma associated with linseed stem fasciation, phyllody from India.


Assuntos
Linho , Phytoplasma , DNA Bacteriano/genética , Humanos , Índia , Filogenia , Phytoplasma/genética , Doenças das Plantas , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
7.
J Environ Manage ; 232: 236-242, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30476685

RESUMO

The investigation reports the application of biogenic jarosite, an iron hydroxy sulfate mineral in Fenton's Oxidation process. Ametryn, a herbicide detrimental to aquatic life and also to human is treated by Fenton's oxidation process using synthesized iron mineral, jarosite. The jarosite synthesis was carried out by using an isolated Acidithiobacillus ferrooxidans bacterial strain with ferrous as an iron supplement. The isolated strain was characterized by molecular techniques and biooxidation activity to ferrous to ferric iron was checked. On Fenton's treatment ametryn degradation upto 84.9% and COD removal to the extent of 56.1% was observed within 2 h of treatment and the reaction follows the pseudo first order kinetics with the curve best fit. The slight increase in kinetic rate constant on jarosite loading rate increase from 0.1 g/L to 0.5 g/L with H2O2 dosage of 100 mg/L confirms that jarosite has a catalytic role in the removal of ametryn. Mass spectroscopy analysis of treated synthetic ametryn solution at various intervals reveal the degradation follows dealkylation and hydroxylation pathway with the formation of three major intermediate compounds discussed here.


Assuntos
Acidithiobacillus , Peróxido de Hidrogênio , Ferro , Oxirredução , Triazinas
8.
Virus Genes ; 53(4): 636-642, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28527099

RESUMO

Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.


Assuntos
Chrysanthemum/virologia , Variação Genética , Doenças das Plantas/virologia , Viroides/genética , Viroides/isolamento & purificação , Sequência de Bases , Índia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Viral/química , RNA Viral/genética , Viroides/química , Viroides/classificação
10.
Indian J Exp Biol ; 54(5): 322-31, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27319051

RESUMO

Azotobacter strains were isolated by serial dilution method and colonies were viscous, smooth, glistening, and brown to black colour on Jenson's N-free agar. Morphological and biochemical tests showed characteristic features of Azotobacter. Further, molecular analyses revealed the presence of different Azotobacter species viz., A. armeniacus, A. chroococcum, A. salinestris, A. tropicalis and A. vinelandii. The isolates were tested for their ability of nitrogen fixation, indole acetic acid (IAA), gibberllic acid production and phosphate solubilization. Four isolates (GVT-1, GVT-2 KOP-11 and SND-4) were efficient in fixation of highest amount of N2 (29.21 µg NmL(-1) day(-1)), produced IAA (25.50 µg mL(-1)), gibberllic acid (17.25 µg 25 mL(-1)) and formed larger P solubilizing zone (13.4 mm). Some of the Azotobacter strains were produced siderophores, hydrogen cyanide and were positive for ammonia production with respect to antifungal activity of Azotobacter was tested with dual culture method and A. tropicalis inhibited the growth of Fusarium, Aspergillus and Alternaria species. Azotobacter isolates were tested against salt (0-10%), temperature (4-55 degrees C), pH (5.0-10) and insecticide chloropyrifos (0-3%) tolerance study. Among them, A. chroococcum was found tolerant to a maximum of 6% NaCl with a temperature of 35-45 degrees C and to a pH up to 8. All the 4 strains showed effective growth against 3% chloropyrifos concentration. The studies revealed that the Azotobacter strains not only produced plant growth promoting substances but are also tolerant to abiotic stresses such as temperature, pH and insecticides.


Assuntos
Alternaria/crescimento & desenvolvimento , Aspergillus/crescimento & desenvolvimento , Azotobacter/metabolismo , Fusarium/crescimento & desenvolvimento , Desenvolvimento Vegetal , Microbiologia do Solo , Estresse Fisiológico , Azotobacter/classificação , Azotobacter/efeitos dos fármacos , Azotobacter/isolamento & purificação , Clorpirifos/farmacologia , Giberelinas/metabolismo , Concentração de Íons de Hidrogênio , Ácidos Indolacéticos/metabolismo , Inseticidas/farmacologia , Fixação de Nitrogênio , Fosfatos/metabolismo , Filogenia , Plantas/metabolismo , Sideróforos/metabolismo , Solubilidade , Temperatura
11.
World J Microbiol Biotechnol ; 30(1): 1-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23813305

RESUMO

A total of 14 Azotobacter strains were isolated from different paddy cultivating soils with pH ranging from 6.5 to 9.5 by using serial dilution agar plate method. The strains were Gram negative, rod shaped, cyst forming and developed brown to black colored colonies, which were glistening, smooth, slimy on Ashby's agar plates. Biochemically they were positive for biochemical tests namely, indole production, citrate, catalase, carbohydrate fermentation and Voges-Proskauer test. Further, sequence analysis of PCR amplicons obtained from these cultures revealed the presence of five different Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris, Azotobacter sp., Azotobacter nigricans subsp. nigricans and Azotobacter tropicalis. Phylogenetically these strains were grouped into two distinct clusters. These strains were tested for their ability to grow on a media containing four different pesticides such as pendimethalin, glyphosate, chloropyrifos and phorate, which are commonly used for the paddy. Out of 14 strains tested, 13 strains were able to grow on a media containing herbicides such as pendimethalin, glyphosate and insecticides like chloropyrifos and phorate. However, five Azotobacter strains were able to grow at higher concentration of 5% pesticides, without affecting their growth rate. Further, the effect of pesticides on the indole acetic acid (IAA) production by Azotobacter strains was also estimated. Azotobacter-16 strain was found to produce 34.4 µg ml(-l) of IAA in a media supplemented with 1,000 mg of tryptophan and 5% of pendimethalin. Present study reveals that species of Azotobacter are able to grow and survive in the presence of pesticides and no significant effects were observed on the metabolic activities of Azotobacter species.


Assuntos
Azotobacter/classificação , Azotobacter/efeitos dos fármacos , Tolerância a Medicamentos , Praguicidas/metabolismo , Praguicidas/toxicidade , Microbiologia do Solo , Azotobacter/isolamento & purificação , Azotobacter/metabolismo , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Genótipo , Índia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA
12.
Artigo em Inglês | MEDLINE | ID: mdl-39180663

RESUMO

Though numerous bacteria have been used as probiotics by industries, at present, Saccharomyces boulardii and Saccharomyces cerevesiae are the only yeast probiotics which are industrially exploited. In view of this, yeast probiotics were isolated from traditional fermented foods and products collected from different parts of Karnataka, India. In this work, we have studied the probiotic attributes of ten yeast isolates isolated from different traditionally fermented foods and products. About 73 yeast isolates were initially isolated by serially diluting the samples and plating on the Potato Dextrose Agar (PDA) plates. The spot assay was performed to screen the yeast isolates against test pathogens. Ten isolates were selected based on their significant antimicrobial activity. These isolates were subjected to biochemical characterization and then assessed for probiotic properties. The ability of probiotics to endure at pH 2.0 and tolerate bile conditions (0.3%) are crucial attributes for the survival in the gastrointestinal tract (GIT). The yeast isolates were also assessed for cell surface hydrophobicity and autoaggregation capabilities. All the ten isolates showed endurance in GIT tract and > 40% of adhesion. The study further examined cholesterol assimilation, antioxidant and antagonistic properties of the yeasts. Subsequently, the molecular characterization was performed by isolating the DNA of yeast isolates by phenol-chloroform method and identified molecularly through sequencing of D1/D2 regions. The isolates tested negative for gelatinase and DNase and were non-haemolytic indicating they are safe for consumption. Among ten isolates, Meyerozyma guillermondii (MYSY23), Meyerozyma caribbica (MYSY22) and Meyerozyma guillermondii (MYSY19) showed significant results for all probiotic and functional characteristics with greater than 65% survivability in GIT tract and > 50% of antagonistic activity against test pathogens and also proved non-cytotoxic and safe. These findings suggest that yeasts with significant probiotic attributes could be recommended for various probiotic application.

13.
Front Microbiol ; 15: 1322758, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38404595

RESUMO

Alternaria alternata, a notorious phytopathogenic fungus, has been documented to infect several plant species, leading to the loss of agricultural commodities and resulting in significant economic losses. Lactic acid bacteria (LAB) hold immense promise as biocontrol candidates. However, the potential of LABs derived from fruits remains largely unexplored. In this study, several LABs were isolated from tropical fruit and assessed for their probiotic and antifungal properties. A total of fifty-five LABs were successfully isolated from seven distinct fruits. Among these, seven isolates showed inhibition to growth of A. alternata. Two strains, isolated from fruits: Ficus benghalensis, and Tinospora cordifolia exhibited promising antifungal properties against A. alternata. Molecular identification confirmed their identities as Lactiplantibacillus plantarum MYSVB1 and MYSVA7, respectively. Both strains showed adaptability to a wide temperature range (10-45°C), and salt concentrations (up to 7%), with optimal growth around 37 °C and high survival rates under simulated gastrointestinal conditions. Among these two strains, Lpb. plantarum MYSVB1 demonstrated significant inhibition (p < 0.01) of the growth of A. alternata. The inhibitory effects of cell-free supernatant (CFS) were strong, with 5% crude CFS sufficient to reduce fungal growth by >70% and complete inhibition by 10% CFS. Moreover, the CFS was inhibitory for both mycelial growth and conidial germination. CFS retained its activity even after long cold storage. The chromatographic analysis identified organic acids in CFS, with succinic acid as the predominant constituent, with lactic acid, and malic acid in descending order. LAB strains isolated from tropical fruits showed promising probiotic and antifungal properties, making them potential candidates for various applications in food and agriculture.

14.
Plant Dis ; 97(1): 149, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30722272

RESUMO

Members of the genus Coleviroid (Coleus blumei viroid [CbVd]), family Pospiviroidae have been reported to infect Coleus (Solenostemon sp.). CbVd-1 was first reported from Brazil, CbVd-2, -3, and -4 were first reported from Germany, whereas CbVd-5 and -6 were recently identified in China (2). In India, Coleus is extensively cultivated as an ornamental plant in home gardens. In March to June 2012, Coleus leaf samples with irregular chlorotic spots/patches were collected from home gardens of two different districts of Karnataka (Kodagu and Mysore districts), India, suspecting the presence of Coleus blumei viroids (CbVd 1 to 6). Low molecular weight RNAs were extracted using 2% CTAB buffer containing 1.4 M NaCl, 20 mM EDTA, pH 8.0 and 100 mM Tris-Cl, pH 8.0 (1). Viroid-like RNA was enriched by fractionation 2M LiCl soluble nucleic acids (4). A DNA fragment with the expected size of CbVd-1 was detected in 10 (including both districts) of 14 analyzed (incident rate of 71%) from reverse transcription (RT)-PCR assay using Coleviroid specific primers (forward 5'-TGGATCCAGCGCTGCAACGGAATCCA-3' and reverse 5'-TTGGATCCGCCAGGGAACCCAGGTAAG-3'). RT was performed at 37°C for 60 min in 25 µl reaction mix containing 5 µl RNA extracts, 1 µl reverse primer, 1× first strand buffer, 10 mM dNTPs, and 200U M-MuLV-RT (Invitrogen, USA). For PCR, 2 µl RT was mixed in 25 µl PCR mix containing 0.2 µM each forward and reverse primers and 2U LA Taq (Takara-Bio, Japan) according to the manufacturer's instruction. PCR parameter was one cycle at 94°C for 2 min, 35 cycles at 94°C for 45 s, 53°C for 30 s, and 72°C for 60 s, followed by final extension at 72°C for 10 min (4). Sequence analysis of cloned amplicons detected CbVd-1 in India. To confirm the sequence of the primer regions, an additional set of tail-tail primers were designed, CbVd1-136F (5'-CTTCGTGGAACGGCTCCGCG-3') and CbVd1-136Rev (5'-GAAGAAGCCGAAGCAACTCTC-3') and were used for RT-PCR. Amplified products were cloned, sequenced and compared with previously obtained data. Further, the presences of CbVd-1 in Coleus samples was confirmed by a RNA gel blot assay using digoxigenin-labeled CbVd-1 cRNA probe (3). Alignment of 19 sequences obtained from four representative Coleus samples found the presence of two sequence variants of CbVd-1, namely Ind-1 (GenBank Accession No. AB740017) and Ind-2 (AB740018). Ind-1 was found to differ from Ind-2 by two nucleotide substitutions at position 40 (C to T) and 211 (T to C). BLAST analysis of Ind-1 showed 100% sequence similarity with CbVd-1 isolates from China (DQ178399) and South Korea (EU 410620), whereas Ind-2 was 99% identical to these two Chinese and Koreans isolates. Furthermore, 97% and 96% sequence identity with CbVd 1-RL RNA (Accession no. X95366) was observed for Ind-1 and Ind-2, respectively. Isolates from India were 88% similar with Coleus blumei viroid 1-RG (X95291). To the best of our knowledge, this is the first molecular evidence for the presence of CbVd-1 infecting Coleus in India. Coleus harbors various viroid species and CbVd-1, reported widely, can transmit efficiently through seed and also could infect the other herbaceous plants (3). This report from India will contribute further understanding of a potential risk of Coleus viroids in ornamental species. References: (1) J. J. Doyle and J. L. Doyle. Phytochem. Bull. 19:11, 1987. (2) F. H. Fu et al. Plant Dis. 95:494, 2011. (3) Ishiguro et al. Ann. Phytopathol. Soc. Jpn. 62:84, 1996. (4) S.-F. Li et al., Plant Pathol. 55:565, 2006.

15.
Plant Dis ; 97(11): 1517, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30708486

RESUMO

During March through July 2012, 10 to 15% of the Vitis vinifera cultivars Thompson Seedless and Anab-e-Shahi exhibited yellow leaf spots and flecks, shortened internodes, and tiny yellow leaves in vineyards of the Bijapur, Doddaballapur, and Kolar districts of Karnataka State, India. These are the major grapevine cultivation regions in India. Samples were collected from four different plants from each district (12 samples in total) and RNA was extracted using 2X CTAB buffer (1). Presence of Grapevine yellow speckle viroid1 (GYSVd-1, genus Apscaviroid) was tested by reverse transcription (RT)-PCR with primer pair PBCVd100C/194H (4) for the amplification of a 220-bp region of the genome. In agarose gel electrophoresis, five samples showed amplicons of the expected size. These amplicons were cloned and sequenced. BLAST analysis confirmed the presence of GYSVd-1. Based on this data, the full-length genome of GYSVd-1 was amplified by RT-PCR using primer pair 341M (5'-CACTCGCGGGGCGCGTTGGA-3') and 342P (5'-CAATCCCCGGAACCCCCGCT-3') and the amplicons were cloned and sequenced. Sequence analysis revealed two sequence variants namely Kar-1 (GenBank Accession No. AB742222) and Kar-2 (AB742223) with 98% and 99% identity to GYSVd-1 variants IXc (X87913) and II (X87906), respectively. GYSVd-1 variants Kar-1 and Kar-2 clustered in two distinct phylogenetic sub-clades. All 12 samples also tested positive for Hop stunt viroid (HpSVd, genus Hostuviroid) in two separate sets of RT-PCR using HSV-78P (5'-AACCCGGGGCAACTCTTCTC-3') and HSV-83M (5'-AACCCGGGGCTCCTTTCTCA-3'); and HSV-7P (5'-AATTCTCGAGTTGCCGC-3') and HSV-220M (5'-CGAACCGAGAGGTGATGCCA-3'), with the expected size of 303 and 213 bp, respectively (3). Sequence analysis of the amplicons confirmed the presence of HpSVd. Alignment of HpSVd nucleotide sequences obtained from the 12 samples showed the presence of a single type of sequence variant, namely Ind-2 (AB742225). BLAST analysis showed 99% sequence identity of Ind-2 with a HpSVd variant isolated from a 100-year-old grapevine in China. All 12 grapevine samples were also tested for the presence of Australian grapevine viroid (AGVd, genus Apscaviroid), Grapevine yellow speckle viroid 2 (GYSVd 2, genus Apscaviroid), and Citrus exocortis viroid (CEVd, genus Pospiviroid) by RT-PCR as described previously (2). None of the samples showed any positives. Northern blot assay using appropriate digoxigenin-labeled riboprobes performed as described previously (2) further confirmed RT-PCR results. Positive controls for RT-PCR and Northern blot were obtained from viroid-infected grapevines maintained in the greenhouse. GYSVd-1 and HpSVd were detected in symptomatic and symptomless plants. Hence, the symptoms observed in the vineyard cannot be attributed to viroid infection. More work is needed to identify the causal agent(s) of the decline of Thompson Seedless and Anab-e-Shadi cultivars. References: (1) C. R. Adkar-Purushothama et al. Plant Dis. 97:149, 2013. (2) D. Jiang et al. Virus Res, 169:237, 2012. (3) Y. Kawaguchi-Ito et al. PLoS One 4:e8386, 2009. (4) L. I. Ward et al. Plant Dis. 95:617, 2011.

16.
Front Microbiol ; 14: 1192449, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37389341

RESUMO

The primary objective of this study was to assess the probiotic attributes and antifungal activity of lactic acid bacteria (LAB) against the fungus, Trichophyton tonsurans. Among the 20 isolates screened for their antifungal attributes, isolate MYSN7 showed strong antifungal activity and was selected for further analysis. The isolate MYSN7 exhibited potential probiotic characteristics, having 75 and 70% survival percentages in pH3 and pH2, respectively, 68.73% tolerance to bile, a moderate cell surface hydrophobicity of 48.87%, and an auto-aggregation percentage of 80.62%. The cell-free supernatant (CFS) of MYSN7 also showed effective antibacterial activity against common pathogens. Furthermore, the isolate MYSN7 was identified as Lactiplantibacillus plantarum by 16S rRNA sequencing. Both L. plantarum MYSN7 and its CFS exhibited significant anti-Trichophyton activity in which the biomass of the fungal pathogen was negligible after 14 days of incubation with the active cells of probiotic culture (106 CFU/ml) and at 6% concentration of the CFS. In addition, the CFS inhibited the germination of conidia even after 72 h of incubation. The minimum inhibitory concentration of the lyophilized crude extract of the CFS was observed to be 8 mg/ml. Preliminary characterization of the CFS showed that the active component would be organic acids in nature responsible for antifungal activity. Organic acid profiling of the CFS using LC-MS revealed that it was a mixture of 11 different acids, and among these, succinic acid (9,793.60 µg/ml) and lactic acid (2,077.86 µg/ml) were predominant. Additionally, a scanning electron microscopic study revealed that CFS disrupted fungal hyphal structure significantly, which showed scanty branching and bulged terminus. The study indicates the potential of L. plantarum MYSN7 and its CFS to control the growth of T. tonsurans. Furthermore, in vivo studies need to be conducted to explore its possible applications on skin infections.

17.
Biotechnol Rep (Amst) ; 38: e00800, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37215743

RESUMO

During past twenty years the opportunistic fungal infections have been emerging, causing morbidity and mortality. The fungi belonging to Aspergillus, Mucor, Rhizopus, Candida, Fusarium, Penicillium, Dermatophytes and others cause severe opportunistic fungal infections. Among these Aspergillus and Candida spp cause majority of the diseases. The continuum of fungal infections will prolong to progress in the surroundings of the growing inhabitants of immunocompromised individuals. Presently many chemical-based drugs were used as prophylactic and therapeutic agents. Prolonged usage of antibiotics may lead to some severe effect on the human health. Also, one of the major threats is that the fungal pathogens are becoming the drug resistant. There are many physical, chemical, and mechanical methods to prevent the contamination or to control the disease. Owing to the limitations that are observed in such methods, biological methods are gaining more interest because of the use of natural products which have comparatively less side effects and environment friendly. In recent years, research on the possible use of natural products such as probiotics for clinical use is gaining importance. Probiotics, one of the well studied biological products, are safe upon consumption and are explored to treat various fungal infections. The antifungal potency of major groups of probiotic cultures such as Lactobacillus spp, Leuconostoc spp, Saccharomyces etc. and their metabolic byproducts which act as postbiotics like organic acids, short chain fatty acids, bacteriocin like metabolites, Hydrogen peroxide, cyclic dipeptides etc. to inhibit these opportunistic fungal pathogens have been discussed here.

18.
Biotechnol Rep (Amst) ; 34: e00716, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35257004

RESUMO

Probiotics are vital and beneficial organisms which offers the health benefits to the host organisms. The fungal probiotic field is one of the developing fields nowadays. Yeast has an enormous and diverse group of microorganisms that is attracting and expanding the attention from researchers and industries. Saccharomyces boulardii, the only patented strain belonging to yeast genera for the human use, has been broadly evaluated for its probiotic effect. Yeasts belonging to the genera Debaryomyces, Pichia, Yarrowia, Meyerozyma, Kluyveromyces etc.., have attained more interest because of their beneficial and probable probiotic features. These yeast probiotics produce VOCs (Volatile organic compounds), mycocins and antimicrobials which shows the antagonistic effect against pathogenic fungi and bacteria. Additionally, those yeasts have been recorded as good plant growth promoting microorganisms. Yeast has an important role in environmental applications such as bioremediation and removal of metals like chromium, mercury, lead etc., from waste water. Probiotic yeasts with their promising antimicrobial, antioxidant, anticancer properties, cholesterol assimilation and immunomodulatory effects can also be utilized as biotherapeutics. In this review article we have made an attempt to address important yeast probiotic attributes.

19.
J Fungi (Basel) ; 8(5)2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35628729

RESUMO

Antifungal efficacy of Azotobacter salinestris against trichothecene-producing Fusarium spp. was investigated in maize, sorghum, and wheat. The three cereals were subjected to four treatments as control (T1), Fusarium alone (T2), combination of Fusarium and A. salinestris treatment (T3), and only A. salinestris (T4). All the treatments were evaluated for total mass of seedlings, root and shoot length, seed germination, and vigor index (VI), and extent of rhizoplane colonization by A. salinestris was investigated. Further, greenhouse studies were conducted to learn the efficacy of A. salinestris in vivo conditions. Antifungal efficacy was tested by the dual-culture method which resulted in significant reduction in Fusarium growth. Infection by Fusarium was reduced up to 50% in treated cereals such as maize, sorghum, and wheat, and there was also significant increase in seedling mass in the three hosts. Maize showed the highest VI (1859.715), followed by sorghum (1470.84), and wheat (2804.123) with A. salinestris treatment. In addition, seed germination was enhanced to 76% in maize, 69% in sorghum, and 68% in wheat, respectively. Efficacy of rhizoplane colonization showed successful isolation of A. salinestris with high CFU rate, and furthermore, significant colonization inhibition by Fusarium spp. was observed. In the greenhouse conditions, on the 45th day of the experimental set-up, the highest shoot length/root length recorded in maize was 155.70/70.0 cm, in sorghum 165.90/48.0 cm, and in wheat 77.85/56.0 cm, and the maximum root mass recorded was 17.53 g in maize, 4.52 g in sorghum, and 1.90 g in wheat. Our present study showed that seed treatment by A. salinestris, may be used as an alternate biocontrol method against Fusarium infection in maize, sorghum, and wheat.

20.
Front Microbiol ; 13: 911243, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35774469

RESUMO

Fermented foods are sources of functionally salient microbes. These microbes when ingested can regulate biomolecule metabolism which has a plethora of health benefits. Lactic acid bacteria species (LABs) isolated from fermented beetroot were biochemically characterized and validated using 16s rRNA sequence. Also, an in vitro assay was conducted to confirm the probiotic activity of the isolates. The cell-free supernatant (CS), cell-free extract (CE), and intact cell (IC) were evaluated for α-glucosidase and α-amylase inhibition. The six isolates RAMULAB01-06 were categorized to be Lactobacillus spp. by observing phenotypic and biochemical characters. Molecular validation using 16S rDNA sequencing, followed by homology search in NCBI database, suggested that the isolates are >95% similar to L. paracasei and L. casei. Also, isolates exhibited probiotic potential with a high survival rate (>96%) in the gastrointestinal condition, and adherence capability (>53%), colonization (>86%), antibacterial, and antibiotic activity. The safety assessments expressed that the isolates are safe. The α-glucosidase and α-amylase inhibition by CS, CE, and IC ranged from 3.97 ± 1.42% to 53.91 ± 3.11% and 5.1 ± 0.08% to 57.15 ± 0.56%, respectively. Hence, these species have exceptional antidiabetic potential which could be explicated to its use as a functional food and health-related food products.

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