A. C. Udayashankar

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.

Chemical and Molecular Methods for Detection of Toxigenic Fungi and Their Mycotoxins from Major Food Crops

Mycotoxins are the secondary metabolites produced by certain molds on a wide range of agricultural commodities and are closely related to human and animal food chains. Mycotoxins are capable of causing disease in humans and other animals, and their detection is largely dependent on the sample matrix and the type of fungus causing their contamination. The strict regulations on trade of contaminated grains and seeds and other produce in industrial countries lead to economic burdens on farmers. In developing countries, the situation is aggravated where regulations may be nonexistent or not enforced and where consumption of home-grown cereals leads to a wide exposure to toxins. Important mycotoxins that occur quite often in food are deoxynivalenol/nivalenol, trichothecenes, zearalenone, ochratoxin A fumonisins, and aflatoxins. High concentrations of mycotoxins such as aflatoxins are consumed by humans in areas of the world with higher-than-average levels of liver cancer, childhood malnutrition, and disease. This chapter introduces rapid, robust, and user-friendly protocols currently applied in the identification of toxigenic fungi and important mycotoxins.

Comparative analysis of activities of vital defence enzymes during induction of resistance in pearl millet against downy mildew

Pearl millet [Pennisetum glaucum (L.) R. Br.] has the seventh largest annual production in the world giving it significant economic importance. Although generally well adapted to the growing conditions in arid and semi-arid regions, major constraints to yields are susceptibility to downy mildew disease caused by the oomycete Sclerospora graminicola (Sacc.) Schroet. Induction of resistance against downy mildew disease of pearl millet has been well established using various biotic and abiotic inducers. The present study demonstrated the comparative analysis of the involvement of the important defence enzymes like β-1,3-Glucanase, chitinase, phenylalanine ammonia-lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and lipoxygenase (LOX) during induced systemic resistance (ISR) mediated by inducers like Benzo(1,2,3)-thiadiazole-7-carbothionic acid-S-methyl ester (BTH), Beta amino butyric acid (BABA), Chitosan and Cerebroside against pearl millet downy mildew disease. Native-PAGE showed six POX isozymes in all categories of uninoculated pearl millet seedlings and maximum intensity of bands was noticed in resistant seedlings. After inoculation in Cerebroside-treated seedlings, there were seven isoforms, POX-4 was not present in any other seedlings. Native-PAGE analysis showed the presence of five PPO isozymes in all categories of uninoculated pearl millet seedlings and after inoculation seven isoforms of PPO-7 were noticed, and the intensity of banding was more in resistant and Cerebroside-treated seedlings. The isoforms PPO-3 were present as an extra band after inoculation in all seedlings. Isoform PPO-7, though found in all seedlings, was very prominent in Chitosan- and Cerebroside-treated seedlings. β-1,3-Glucanase Native-PAGE analysis showed the presence of only one isozyme in all categories of uninoculated/inoculated pearl millet seedlings. Glu-1 isozyme was very prominent in all seedlings including resistant and susceptible seedlings. Among the induced resistant seedlings, highest intensity was observed in Cerebroside-treated seedlings. Native-PAGE analysis showed the presence of three LOX isozymes in all categories of uninoculated pearl millet seedlings, and the intensity of banding pattern was very low in BTH-treated seedlings. LOX-1 and LOX-2 were very prominent in resistant, Chitosan- and Cerebroside-treated seedlings. Upon inoculation, one extra band, LOX-3, was exclusively noticed in Cerebroside-treated seedlings. In inoculated seedlings, LOX-1, LOX-2 and LOX-4 were very prominent in Chitosan Cerebroside-treated seedlings compared to other seedlings.

ACUTE ORAL TOXICITY, DERMAL IRRITATION AND EYE IRRITATION STUDY OF ECLIPTA ALBA AQUEOUS EXTRACT IN SPRAGUE DAWLEY RATS AND NEWZEALAND WHITE RABBITS

Eclipta alba is used as a medicinal herb in many herbal preparations including most of hair oils produced in India. The E. alba extracts are part of many Ayurvedic/herbal medicines with hepatoprotective, anti-diabetic, anti-inflammatory and anti-microbial properties. Aqueous extract, hydroalcoholic extracts and methanolic extract are reported to possess hepatoprotective, anti-diabetic, anti-inflammatory and anti-microbial properties. Studies in Burkina Faso have demonstrated that soaking seeds with 2.5% E. alba aqueous extract for 6 h improved seed quality parameters followed by increased yield in sorghum. Accordingly, it has been proposed to use E. alba for seed treatment on a larger scale. Hence it was relevant to test the toxicity of E. alba. The purpose of this study was to test the acute oral toxicity, dermal irritation and eye irritation of aqueous extract of E. alba dried leaves in Sprague Dawley rats and New Zealand white rabbits. The acute toxicity studies were carried out based on OECD guidelines 423. The highest dose administered at 2000 mg/kg body weight did not produce mortality or changes in general behaviour of the test animals indicating safety of the oral administration of aqueous E. alba extract in Sprague Dawley rats. The acute dermal irritation study in Newzealand white rabbits was investigated according to OECD test guideline No. 404. The E. alba fine powder applied to the intact left flank of female rabbit did not elicit any skin reactions at the application site of animal at any of the observation time points and hence ‘Non Irritant’ to the rabbit skin. The acute eye irritation study on Newzealand white rabbits did not cause corneal opacity, iris and conjunctivae in any of the treated animals and did not cause staining of the treated eye and is termed as ‘not irritating’ to the rabbit eyes / eye mucosa. The toxicological studies prove that the E. alba aqueous extract are safe to be used as seed treatment and can be handled safely by humans under field conditions.

Immunocapture RT-PCR detection of Bean common mosaic virus and strain blackeye cowpea mosaic in common bean and black gram in India

The strains of Bean common mosaic virus (BCMV) and blackeye cowpea mosaic (BICM), genus Potyvirus, were detected from 25 common bean and 14 black gram seeds among 142 seed samples collected from different legume-growing regions of India. The samples were subjected to a growing-on test, an indicator plant test, an electron microscopic observations, an enzyme linked immunosorbent assay and an immunocapture RT-PCR. The incidence of the two tested viruses in common bean and black gram seed samples was 1–6% and 0.5–3.5%, respectively in growing-on test evaluations. Electron microscopic observations revealed filamentous virion particles from the leaves of plants showing characteristic virus disease symptoms in growing-on and host inoculation tests. The identity of the strains was confirmed by immunocapture RT-PCR, with a final amplification product of approximately 700 bp for BCMV and BCMV–BICM. The complete identity of the two viruses was further confirmed by nucleotide sequencing of the partial coat protein and 3′-UTR regions. The sequences of the four BCMV and BCMV–BICM isolates each consisted of 583–622 and 550–577 nucleotides. The present report confirms the widespread nature of these two serious potyviruses in the two most important legume crops in India.