Robert N.G. Miller

Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerella musicola: gene assembly, annotation and marker development.

BackgroundAlthough banana (Musa sp.) is an important edible crop, contributing towards poverty alleviation and food security, limited transcriptome datasets are available for use in accelerated molecular-based breeding in this genus. 454 GS-FLX Titanium technology was employed to determine the sequence of gene transcripts in genotypes of Musa acuminata ssp. burmannicoides Calcutta 4 and M. acuminata subgroup Cavendish cv. Grande Naine, contrasting in resistance to the fungal pathogen Mycosphaerella musicola, causal organism of Sigatoka leaf spot disease. To enrich for transcripts under biotic stress responses, full length-enriched cDNA libraries were prepared from whole plant leaf materials, both uninfected and artificially challenged with pathogen conidiospores.ResultsThe study generated 846,762 high quality sequence reads, with an average length of 334 bp and totalling 283 Mbp. De novo assembly generated 36,384 and 35,269 unigene sequences for M. acuminata Calcutta 4 and Cavendish Grande Naine, respectively. A total of 64.4% of the unigenes were annotated through Basic Local Alignment Search Tool (BLAST) similarity analyses against public databases.Assembled sequences were functionally mapped to Gene Ontology (GO) terms, with unigene functions covering a diverse range of molecular functions, biological processes and cellular components. Genes from a number of defense-related pathways were observed in transcripts from each cDNA library. Over 99% of contig unigenes mapped to exon regions in the reference M. acuminata DH Pahang whole genome sequence. A total of 4068 genic-SSR loci were identified in Calcutta 4 and 4095 in Cavendish Grande Naine. A subset of 95 potential defense-related gene-derived simple sequence repeat (SSR) loci were validated for specific amplification and polymorphism across M. acuminata accessions. Fourteen loci were polymorphic, with alleles per polymorphic locus ranging from 3 to 8 and polymorphism information content ranging from 0.34 to 0.82.ConclusionsA large set of unigenes were characterized in this study for both M. acuminata Calcutta 4 and Cavendish Grande Naine, increasing the number of public domain Musa ESTs. This transcriptome is an invaluable resource for furthering our understanding of biological processes elicited during biotic stresses in Musa. Gene-based markers will facilitate molecular breeding strategies, forming the basis of genetic linkage mapping and analysis of quantitative trait loci.

Development of expressed sequence tag and expressed sequence tag-simple sequence repeat marker resources for Musa acuminata.

Many varieties of banana (Musa acuminata) lack resistance to biotic stresses. An EST collection was developed, including transcripts expressed in banana-Mycosphaerella fijiensis interactions. Developed polymorphic gene-derived SSR markers are applicable for genetic mapping, diversity characterization and marker assisted breeding.

Development and validation of a novel lateral flow immunoassay device for detection of aflatoxins in soy-based foods

Aflatoxins (AFs) are natural toxins produced as secondary fungal metabolites. Common in diverse commodities, the consumption of aflatoxin-contaminated foodstuffs has harmful effects on human health mainly due to their carcinogenic effects. There is considerable demand for the development of rapid, reliable and cost-effective screening tools, once regulatory authorities employ strict procedures for detection of aflatoxins. In this study, a sensitive and specific strip test assay was developed, based on a competitive format, for the detection of aflatoxins in soybean, a major Brazilian agricultural commodity. A monoclonal antibody (mAb) was produced, named as 3B6, with high specificity to aflatoxins B1, M1, G1, G2 and B2. For the development of a strip test, colloidal gold particles were coated with 3B6 mAb and used as the detector reagent. An AFB1–BSA conjugate was sprayed at the test line of a nitrocellulose membrane to serve as a competitor reagent, with an anti-species-specific antibody used at the control line. The strip test developed was capable of detecting as little as 0.5 μg kg−1 of aflatoxin. To validate the strip test, soybean grains infected and non-infected with the aflatoxin-producing fungus Aspergillus flavus were employed. The applicability of the developed test was demonstrated by screening 12 foodstuff samples (soy protein and soy milk) collected from different local markets. The results indicated that the strip tests developed were capable of accurately qualitatively identified aflatoxin-contaminated samples in less than 10 min. This method offers a promising device for application in routine monitoring during storage, transport, processing and handling of agricultural commodities.

Cultivation of Pleurotus mushrooms in substrates obtained by short composting and steam pasteurization

This paper presents results of two experiments for cultivation of Pleurotus ostreatus , Pleurotus pulmonarius and Pleurotus eryngii grown with different formulations of grass and straw mixtures derived from agro-industrial residues. Cultivation was prepared through a number of approaches, such as short composting/pasteurization and axenic culture. In the first experiment, P. pulmonarius was grown on two formulations of different grasses, with no significant differences observed for either productivity or biological efficiency, with values close to 20 and 60%, respectively. The second experiment revealed similar productivity and biological efficiency between P. pulmonarius and P. ostreatus for both forms of substrate treatment (short composting/pasteurization vs. axenic culture), with similar values to those observed in the first experiment. P. eryngii did not produce mushrooms in the composting treatment and showed lower productivity (17.5%) than the other two species (20.5 and 20.8%, respectively) when the substrates were autoclaved (axenic culture). The preparation for short composting and steam pasteurization was described in illustrative figures in order to provide expertise to small producers who wish to initiate economic and sustainable mushroom cultivation making use of regional lignocellulosic residues. Keywords: Steam pasteurization, lignocellulosic biomass, straw mixtures, mushrooms

A novel immunochromatographic strip test for rapid detection of Cry1Ac and Cry8Ka5 proteins in genetically modified crops

The cultivation of genetically modified (GM) crops has grown rapidly worldwide. This has led to regulatory authorities implementing strict procedures to monitor and verify the presence and abundance of GM varieties in agricultural crops. Immunochromatographic strip tests have been employed for the detection of transgenic proteins expressed in GM crops as rapid, reliable and cost-effective screening tools. In this study, we developed a novel and sensitive strip test assay, based on a sandwich format, for the identification of Cry1Ac and Cry8Ka5 transgenic proteins. We generated two monoclonal antibodies (mAb), namely 1B1 and 5H4, that bind with high specificity to Cry1Ac and Cry8Ka5 proteins. For the development of strip tests, colloidal gold particles were coated with 1B1 mAb and used as the detector reagent. The 5H4 mAb was sprayed at the test line of a nitrocellulose membrane to serve as a capture reagent and anti-species specific antibodies were used at the control line. The strip test developed was capable of detecting 0.06 μg of Cry1Ac and Cry8Ka5 proteins. For validation of the strip test, GM and non-GM cotton leaf samples were employed. The results indicated that the strip test was capable of distinguishing between GM and non-GM cotton samples, offering potential for use as a rapid and cost-effective screening tool for insect-resistant GM crops expressing Cry1Ac and Cry8Ka5 proteins to control lepidopterans and coleopteran pests, respectively.

Cellulase Systems in Trichoderma : An Overview

Abstract Filamentous fungi secrete a large amount of cellulose-degrading enzymes. Among them, fungi from Trichoderma genus are described as producers and secretors of a wide spectrum of cellulose-degrading enzymes. The biological deconstruction of cellulose within the biomass up to glucose monom is achieved through the synergistic action of multiple enzymes, which can associate into complexes. The enzymatic degradation of cellulose is divided into two hydrolysis steps. The primary hydrolysis takes place on the cellulose surface. It is primarily dependent on endoenzymes (endoglucanases) that cleave accessible intramolecular β-1,4-glucosidic bonds of cellulose chains randomly in a nonprocessive manner with formation of new chain ends. Furthermore, exoenzymes (exoglucanases) cleave cellulose chains in a processive way at the reducing or nonreducing ends to release cellobiose or glucose. To date, the carbohydrate-active enzyme database contains reports of 60 cellulases or cellulase fragments in different Trichoderma species.

Current Strategies for the Detoxification of Jatropha curcas Seed Cake: A Review

Jatropha curcas is an important oilseed plant, with considerable potential in the development of biodiesel. Although Jatropha seed cake, the byproduct of oil extraction, is a residue rich in nitrogen, phosphorus, potassium, and carbon, with high protein content suitable for application in animal feed, the presence of toxic phorbol esters limits its application in feed supplements and fertilizers. This review summarizes the current methods available for detoxification of this residue, based upon chemical, physical, biological, or combined processes. The advantages and disadvantages of each process are discussed, and future directions involving genomic and proteomic approaches for advancing our understanding of biodegradation processes involving microorganisms are highlighted.

Trichoderma : A Dual Function Fungi and Their Use in the Wine and Beer Industries

The genus Trichoderma was first described in 1794 and presents a link to the sexual state Hypocrea . Whilst classification in the genus Trichoderma or Hypocrea using morphological characters is limited, an online identification tool was developed for accurate identification of all species within the genus Trichoderma. A group of Trichoderma species are described which share interesting features, including adaptation to different environmental conditions, metabolism of different biomolecules or compounds, and fast growth rate. These metabolic features place these Trichoderma species as the principal decomposers of the ecosphere and also raise attention of researchers for exploitation of their metabolism for industrial purposes. Indeed, Trichoderma species have been applied in different biotechnological applications such as a biological control, biofertilizers, as a source of industrial enzymes and as protein producers. The present chapter will present an introduction to industrial Trichoderma species and their application in wine and beer industries.