Short protocols in molecular biology

Nanotechnology can offer green and eco-friendly alternatives for plant disease management. Apart from being eco-friendly, fungi are used as bio-manufacturing units, which will provide an added benefit in being easy to use, as compared to other microbes. The non-pathogenic nature of some fungal species in combination with the simplicity of production and handling will improve the mass production of silver nanoparticles. Recently, a diverse range of fungi have been screened for their ability to create silver nanoparticles. Mycosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, usnic acid, magnetite, cadmium telluride and uraninite nanoparticles has also been reported by various researchers. Nanotechnological application in plant pathology is still in the early stages. For example, nanofungicides, nanopesticides and nanoherbicides are being used extensively in agriculture practices. Remote activation and monitoring of intelligent nano-delivery systems can assist agricultural growers of the future to minimize fungicides and pesticides use. Nanoparticle-mediated gene transfer would be useful for improvement of crops resistant to pathogens and pest. This review critically assesses the role of fungi in the synthesis of nanoparticles, the mechanism involved in the synthesis, the effect of different factors on the reduction of metal ions in developing low-cost techniques for the synthesis and recovery of nanoparticles. Moreover, the application of nanoparticles in plant disease control, antimicrobial mechanisms, and nanotoxicity on plant ecosystem and soil microbial communities has also been discussed in detail.

Myconanoparticles: synthesis and their role in phytopathogens management

Nanotechnology can offer green and eco-friendly alternatives for plant disease management. Apart from being eco-friendly, fungi are used as bio-manufacturing units, which will provide an added benefit in being easy to use, as compared to other microbes. The non-pathogenic nature of some fungal species in combination with the simplicity of production and handling will improve the mass production of silver nanoparticles. Recently, a diverse range of fungi have been screened for their ability to create silver nanoparticles. Mycosynthesis of gold, silver, gold–silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, usnic acid, magnetite, cadmium telluride and uraninite nanoparticles has also been reported by various researchers. Nanotechnological application in plant pathology is still in the early stages. For example, nanofungicides, nanopesticides and nanoherbicides are being used extensively in agriculture practices. Remote activation and monitoring of intelligent nano-delivery systems can assist agricultural growers of the future to minimize fungicides and pesticides use. Nanoparticle-mediated gene transfer would be useful for improvement of crops resistant to pathogens and pest. This review critically assesses the role of fungi in the synthesis of nanoparticles, the mechanism involved in the synthesis, the effect of different factors on the reduction of metal ions in developing low-cost techniques for the synthesis and recovery of nanoparticles. Moreover, the application of nanoparticles in plant disease control, antimicrobial mechanisms, and nanotoxicity on plant ecosystem and soil microbial communities has also been discussed in detail.

ARTICLE; AGRICULTURE AND ENVIRONMENTAL BIOTECHNOLOGY Myconanoparticles: synthesis and their role in phytopathogens management

该文通过添加不同浓度的植酸于黄曲霉菌(Aspergillus flavus)培养基中,用不同添加方式,于不同生长时刻测定黄曲霉菌糖化力,研究植酸对黄曲霉菌糖化力的影响.结果表明加入植酸后黄曲霉菌糖化力均有很大提高,可为工业生产带来可观的经济效益.植酸最佳作用浓度范围是0.20%～0.25%,发酵24h糖化力最大可提高44.32%.

植酸对黄曲霉菌（Aspergillus flavus）糖化力的影响

Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.

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Mycofabricated biosilver nanoparticles interrupt Pseudomonas aeruginosa quorum sensing systems.

A total of 75 isolates belonging to five different species of Trichoderma viz., T. asperellum, T. harzianum, T. longibrachiatum, T. pseudokoningii and T. virens were screened for the production of silver nanoparticles. Although all the isolates produced nanoparticles, T. virens VN-11 could produce maximum nanoparticles as evident from the UV-Vis study. The highest Plasmon band was observed at 420 nm at every 24 h that attained maximum intensity at 120 h (0.543). The high resolution transmission electron microscopy (HRTEM) further provided the morphology of the nanoparticles. These nanoparticles were found single or aggregated with round and uniform in shape and 8-60 nm in size. The nitrate reductase activity of VN-11 was found to be 150 nmol/h/mL which confirmed the production of silver nanoparticles through reduction of Ag+ to Ag0.

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Biosynthesis of silver nanoparticles from Trichoderma species.

A study was intended to corroborate the positive relatedness of molecular and morphological characters with antagonistic ability of Trichoderma species. On the basis of morphological and cultural characteristics, the Trichoderma isolates were identified as T. virens (11 isolates), T. asperellum (15), T. harzianum (14) and T. longibrachiatum (32). This result was in concordance with the result obtained from the DNA sequence data analysis of internal transcribed spacer 1 and 2 region (ITS1 and ITS2) and the elongation factor 1-alpha gene ( tef1 ). The phylogenetic analyses of the above two marker loci sequences were done. Trichoderma isolates were not fairly separated by ITS 1 and 2 regions analysis whereas these were clearly separated with tef1 sequences analysis into different clusters. Comparative sequence analyses suggest that tef1 is a better marker to distinguish Trichoderma species because of greater transition/transversion ratio (1.32) and evolutionary divergence (1.965). T. virens (Vn) and T. harzianum (Th) were assessed for their mycoparasitic effect on soil borne plant pathogens, Rhizoctonia solani , Pythium aphanidermatum , Fusarium udum , F. solani , Sclerotium rolfsii and Macrophomina phaseolina . The percent inhibitory effect among T. harzianum isolates was between 70 to 90% and T. virens isolates ranged from 50 to 80%. However, Vn09 and Th-12 were distinguishable in exhibiting higher degree of antagonism. Key words: Biocontrol, ITS, tef1 , Trichoderma .

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Characterization of Indian native isolates of Trichoderma spp. and assessment of their bio-control efficiency against plant pathogens

A set of 72 Trichoderma isolates from India were morphologically and molecularly characterized and confirmed as Trichoderma virens, T. harzianum, T. longibrachiatum and T. asperellum. ITS1 and ITS2 regions were amplified using universal ITS primers. The amplicon size varied from 550 to 600 bp in the isolates studied. Cluster analysis based on amplicon sequence of ITS region could not clearly differentiate the Trichoderma species into distinct group. Thus tef1 was used for differentiating the Trichoderma sp. The sequence analysis based on tef1 gene primers could differentiate T. longibrachiatum, T. virens, T. harzianum and T. asperellum species and the phylogenetic analyses were in conformity with the morphological observations. Species specific tef1primer sets T1F/T1R and T2F/T2R were designed for T. virens and T. longibrachiatum isolates respectively and validated against other species of Trichoderma. T1F/T1R specifically amplified in T. virens giving fragment size of 330 bp and T2F/T2R gave amplification of 452 bp only in T. longibrachiatum.

Development of Species Specific Markers For Detection of Trichoderma Species

Aspergillus flavus is the causal agent of corns and peanuts mold known to produce aflatoxin. A quick and reliable PCR-based diagnostic assay has been developed to detect A. flavus using a fungus-specific marker derived from genomic DNA. An amplified RAPD product of 600 bp obtained in A. flavus isolates using a random primer OPB-11 was cloned in pGEMT easy vector and sequenced. Based on sequences, six primers were designed, out of which a primer pair Asp f1 (CCCGTGAAGTTGCCCAGGT) and Asp r2 (GTCGTTTGGTGAGTGGGAA) amplified a sequence of 490 bp which was specific toA. flavus. The specificity of the marker when tested against 31 isolates of Aspergillus flavus, 7 isolates of Aspergillus clavatus, 4 each isolates of Aspergillus terreus, Aspergillus oryaze, Aspergillus tamari, 1 each isolate of A. parasiticus and A. kambarensis and 4 isolates of 2 different Cheatomium species, 5 isolates of 3 differentTrichoderma species and 5 isolates of 5 different Fusarium species showed a specific band of 600 bp only in A. flavus. With the optimized PCR parameters, this sequence characterized amplified region (SCAR) marker was sensitive and could detect small quantities of A. flavus DNA as low as 10 to 25 ng with high efficiency. This marker could also clearly distinguish A. flavus from other fungal plant pathogens, including differentAspergillus spp. The utilization of this diagnostic PCR assay in analysis of post harvest samples will be a strong step towards aflatoxin detection in animal feed and export commodity.

	 

	Key words: Aspergillus flavus, OPB11, random amplified polymorphic DNA (RAPD), sequence characterized amplified region (SCAR) marker

Development of SCAR marker for specific detection of Aspergillus flavus

The study of morphology and bioefficacy of Trichoderma was undertaken to select the effective isolates against soil-borne pathogens. Fifty one (51) isolates (23 isolates of Trichoderma virens and 28 isolates of Trichoderma harzianum) were morphologically characterised based on the growth characteristics on PDA medium, the size and shape of phialides and conidia.These isolates were screened for bioefficacy against soil borne plant pathogens (Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii) based on their percent inhibition observed during dual culture, volatile and non-volatile methods. Eight T. virens isolates and 12 T. harzianum isolates were proven to be potential isolates against the soil-borne pathogens tested. No correlation was found between bioefficacy and morphology in both species isolates.

	Key words: Trichoderma virens, Trichoderma harzianum, morphology, dual culture, biocontrol and bioefficacy.

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Jyoti Lekha Borah

Papers

Biosynthesis of silver nanoparticles from Trichoderma species.

Characterization of Indian native isolates of Trichoderma spp. and assessment of their bio-control efficiency against plant pathogens

Development of Species Specific Markers For Detection of Trichoderma Species

Development of SCAR marker for specific detection of Aspergillus flavus

Bioefficacy of Trichoderma isolates against soil-borne pathogens