Kanniah Rajasekaran

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+‐ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1-expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and preparatory physiological responses. In addition to the increased accumulation of its own transcript, SaVHAc1 expression led to increased accumulation of messages of other native genes in rice, especially those involved in cation transport and ABA signalling. The SaVHAc1-expressing plants maintained higher relative water content under salt stress through early stage closure of the leaf stoma and reduced stomata density. The increased K(+) /Na(+) ratio and other cations established an ion homoeostasis in SaVHAc1-expressing plants to protect the cytosol from toxic Na(+) and thereby maintained higher chlorophyll retention than the WT plants under salt stress. Besides, the role of SaVHAc1 in cell wall expansion and maintenance of net photosynthesis was implicated by comparatively higher root and leaf growth and yield of rice expressing SaVHAc1 over WT under salt stress. The study indicated that the genes contributing toward natural variation in grass halophytes could be effectively manipulated for improving salt tolerance of field crops within related taxa.

Developing resistance to aflatoxin in maize and cottonseed.

At this time, no “magic bullet” for solving the aflatoxin contamination problem in maize and cottonseed has been identified, so several strategies must be utilized simultaneously to ensure a healthy crop, free of aflatoxins. The most widely explored strategy for the control of aflatoxin contamination is the development of preharvest host resistance. This is because A. flavus infects and produces aflatoxins in susceptible crops prior to harvest. In maize production, the host resistance strategy has gained prominence because of advances in the identification of natural resistance traits. However, native resistance in maize to aflatoxin contamination is polygenic and complex and, therefore, markers need to be identified to facilitate the transfer of resistance traits into agronomically viable genetic backgrounds while limiting the transfer of undesirable traits. Unlike maize, there are no known cotton varieties that demonstrate enhanced resistance to A. flavus infection and aflatoxin contamination. For this reason, transgenic approaches are being undertaken in cotton that utilize genes encoding antifungal/anti-aflatoxin factors from maize and other sources to counter fungal infection and toxin production. This review will present information on preharvest control strategies that utilize both breeding and native resistance identification approaches in maize as well as transgenic approaches in cotton.

Chemical Composition, Antifungal and Insecticidal Activities of Hedychium Essential Oils

The antimicrobial properties of essential oils have been documented, and their use as “biocides” is gaining popularity. The aims of this study were to analyze the chemical composition and assess the biological activities of Hedychium essential oils. Oils from 19 Hedychium species and cultivars were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) techniques. The antifungal and insecticidal activities of these oils were tested against Colletotrichum acutatum, C. fragariae, and C. gloeosporioides, and three insects, the azalea lace bug (Stephanitis pyrioides), the yellow fever mosquito (Aedes aegypti), and the red imported fire ant (Solenopsis invicta). Hedychium oils were rich in monoterpenes and sesquiterpenes, especially 1,8-cineole (0.1%–42%), linalool (<0.1%–56%), α-pinene (3%–17%), β-pinene (4%–31%), and (E)-nerolidol (0.1%–20%). Hedychium oils had no antifungal effect on C. gloeosporioides, C. fragariae, and C. acutatum, but most Hedychium oils effectively killed azalea lace bugs. The oils also show promise as an adult mosquito repellent, but they would make rather poor larvicides or adulticides for mosquito control. Hedychium oils acted either as a fire ant repellent or attractant, depending on plant genotype and oil concentration.

RNA Interference (RNAi) as a Potential Tool for Control of Mycotoxin Contamination in Crop Plants: Concepts and Considerations

Mycotoxin contamination in food and feed crops is a major concern worldwide. Fungal pathogens of the genera Aspergillus. Fusarium, and Penicillium are a major threat to food and feed crops due to production of mycotoxins such as aflatoxins, 4-deoxynivalenol, patulin, and numerous other toxic secondary metabolites that substantially reduce the value of the crop. While host resistance genes are frequently used to introgress disease resistance into elite germplasm, either through traditional breeding or transgenic approaches, such resistance is often compromised by the evolving pathogen over time. RNAi-based host-induced gene silencing of key genes required by the pathogen for optimal growth, virulence and/or toxin production, can serve as an alternative, pre-harvest approach for disease control. RNAi represents a robust and efficient tool that can be used in a highly targeted, tissue specific manner to combat mycotoxigenic fungi infecting crop plants. Successful transgenic RNAi implementation depends on several factors including (1) designing vectors to produce double-stranded RNAs (dsRNAs) that will generate small interfering RNA (siRNA) species for optimal gene silencing and reduced potential for off-target effects; (2) availability of ample target siRNAs at the infection site; (3) efficient uptake of siRNAs by the fungus; (4) siRNA half-life and (5) amplification of the silencing effect. This review provides a critical and comprehensive evaluation of the published literature on the use of RNAi-based approaches to control mycotoxin contamination in crop plants. It also examines experimental strategies used to better understand the mode of action of RNAi with the aim of eliminating mycotoxin contamination, thereby improving food and feed safety.

Co-inoculation of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels

A currently utilized pre-harvest biocontrol method involves field inoculations with non-aflatoxigenic Aspergillus flavus strains, a tactic shown to strategically suppress native aflatoxin-producing strains and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic A. flavus strain (AF70), labeled with green fluorescent protein (GFP), in the presence of a non-aflatoxigenic A. flavus biocontrol strain (AF36), to better understand the competitive interaction between these two strains in seed tissue of corn (Zea mays). Corn kernels that had been co-inoculated with GFP-labeled AF70 and wild-type AF36 were cross-sectioned and observed under UV and blue light to determine the outcome of competition between these strains. After imaging, all kernels were analyzed for aflatoxin levels. There appeared to be a population difference between the co-inoculated AF70-GFP+AF36 and the individual AF70-GFP tests, both visually and with pixel count analysis. The GFP allowed us to observe that AF70-GFP inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 inhibited progression of AF70-GFP. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70-GFP. The suppressed growth of AF70-GFP was reflected in a corresponding (upto 73%) suppression in aflatoxin levels. Our results indicate that the decrease in aflatoxin production correlated with population depression of the aflatoxigenic fungus by the biocontrol strain supporting the theory of competitive exclusion through robust propagation and fast colonization by the non-aflatoxigenic fungus.

Transgenic approaches for pre-harvest control of mycotoxin contamination in crop plants

Mycotoxins are fungal metabolites that can contaminate food and feed crops worldwide and are responsible for toxic effects in animals and humans that consume contaminated commodities. Regulatory guidelines and limits for mycotoxins have been set by the US Food and Drug Administration (FDA) and food safety agencies of other countries for both import and export of affected commodities. Mycotoxin contamination of foods and feeds can also cause serious economic hardships to producers, processors, and the consumer. Therefore, there has been a concerted effort by researchers worldwide to develop strategies for the effective control of mycotoxin contamination of crops, particularly at the pre-harvest stage. Strategies currently being utilised to combat pre-harvest mycotoxin contamination include: (1) use of non-toxigenic biocontrol strains; (2) improved agricultural practices; (3) application of agrochemicals; (4) plant breeding for resistance; and (5) genetic engineering of resistance genes into crop plants. This article highlights research on the genetic engineering of plants for resistance to invasion by mycotoxigenic fungi as well as detoxification of mycotoxins. Emphasis is placed on the most economically relevant fungi and the mycotoxins they produce. These include aflatoxins produced mainly by Aspergillus flavus and A. parasiticus, trichothecenes produced mainly by Fusarium graminearum, and to a lesser extent, fumonisins produced by F. verticillioides. Information is also presented on the use of genomics and proteomics technologies as a means of identifying genes and proteins that can be utilised in transgenic approaches to control the growth of mycotoxigenic fungi and the mycotoxins that they produce in food and feed crops.

Aflatoxin control through transgenic approaches.

Control of preharvest aflatoxin contamination of susceptible crops such as corn, cotton, peanut, and tree nuts is possible through time-consuming and expensive agronomic practices. Breeding for disease-resistant crops is also very time consuming and does not lend itself readily to combat the evolution of new virulent fungal races. Moreover, availability of known genotypes with natural resistance to mycotoxin-producing fungi is a prerequisite for the successful breeding program. While it is possible to identify a few genotypes of corn or peanuts that are naturally resistant to Aspergillus, we do not know whether these antifungal factors are specific to Aspergillus flavus. In crops like cotton, there are no known varieties naturally resistant to Aspergillus spp. So far, the best options available to us are through biocontrol and/or genetic engineering. Availability of transgenic varieties with antifungal traits is extremely valuable as a breeding tool. Use of fungicides or chemicals can add to the cost of production. Moreover, the growing concerns regarding environmental safety and groundwater quality demand less dependence on agrochemicals. Disease-resistant transgenic crops would not only control mycotoxin-producing organisms such as A. flavus, A. parasiticus and Fusarium spp. but also other microbial (fungal, bacterial, and viral) diseases that cause significant economic losses in crop production. Above all, transgenic crops resistant to aflatoxin-producing fungi offer the promise of negating the adverse effects caused by the toxin on immunocompromised humans and animals. This review explores recent advances regarding genetic engineering approaches towards the control of aflatoxin contamination using native and heterologous genes.

Antifungal Traits of a 14 kDa Maize Kernel Trypsin Inhibitor Protein in Transgenic Cotton

ABSTRACT Transgenic cotton plants expressing the maize kernel trypsin inhibitor protein (TIP) were produced and evaluated for antifungal traits. This 14 kDa trypsin inhibitor protein has been previously associated with resistance to aflatoxin-producing fungus Aspergillus flavus. Successful transformation of cotton (Gossypium hirsutum L.) and expression of trypsin inhibitor was demonstrated by PCR and Northern analysis, respectively. Proteins extracted from cottonseed and leaf tissues of transgenic plants were separated using SDS-PAGE, and it indicated the presence of a 15–16 kDa protein in transgenic tissues as compared to control. Only transgenic cottonseed tissue reacted with the TIP antibody, indicating the expression in cottonseed. No cross-reaction to the TIP antibody was detected from leaf extracts, indicating the TIP was either not expressed or expressed at a level too low to be detected by Western blot. Crude leaf extracts from transgenic cotton plants did not show significant control of colonies from pre-germinated spores of Aspergillus flavus or Verticillium dahliae; however, extracts from transgenic cottonseed tissue showed about 60% reduction of V. dahliae colonies, indicating the antifungal nature of the maize TIP by itself. Cotton bolls inoculated with a green fluorescent protein (GFP) expressing A. flavus strain showed no difference among controls or transgenic cotton plants indicating that the expression of TIP in cottonseed is not high enough to prevent A. flavus colonization.

Peanuts that keep aflatoxin at bay: a threshold that matters

Summary Aflatoxin contamination in peanuts poses major challenges for vulnerable populations of sub‐Saharan Africa and South Asia. Developing peanut varieties to combat preharvest Aspergillus flavus infection and resulting aflatoxin contamination has thus far remained a major challenge, confounded by highly complex peanut–Aspergilli pathosystem. Our study reports achieving a high level of resistance in peanut by overexpressing (OE) antifungal plant defensins MsDef1 and MtDef4.2, and through host‐induced gene silencing (HIGS) of aflM and aflP genes from the aflatoxin biosynthetic pathway. While the former improves genetic resistance to A. flavus infection, the latter inhibits aflatoxin production in the event of infection providing durable resistance against different Aspergillus flavus morphotypes and negligible aflatoxin content in several peanut events/lines well. A strong positive correlation was observed between aflatoxin accumulation and decline in transcription of the aflatoxin biosynthetic pathway genes in both OE‐Def and HIGS lines. Transcriptomic signatures in the resistant lines revealed key mechanisms such as regulation of aflatoxin synthesis, its packaging and export control, besides the role of reactive oxygen species‐scavenging enzymes that render enhanced protection in the OE and HIGS lines. This is the first study to demonstrate highly effective biotechnological strategies for successfully generating peanuts that are near‐immune to aflatoxin contamination, offering a panacea for serious food safety, health and trade issues in the semi‐arid regions.

Antifungal Activities of Hedychium Essential Oils and Plant Extracts Against Mycotoxigenic Fungi

Plant-derived antifungal compounds are preferred to chemicals to reduce the risk of toxic effects on humans, livestock, and the environment. Essential oil extracted from rhizomes and plant extracts of ornamental ginger lily (Hedychium spp.) were evaluated for their antifungal activity against two fungi, Aspergillus flavus and Fusarium verticillioides, that produce two major classes of mycotoxins, aflatoxin, and fumonisin, respectively, on several crops. Essential oil or leaf extracts from several Hedychium species and varieties totally inhibited the growth of pre-germinated spores of A. flavus and F. verticillioides. Control of these two fungi by aqueous extracts of rhizome was not effective, although a reduced number of colonies was observed. In view of these results, essential oils or crude leaf extracts from Hedychium spp. need to be considered for the control of mycotoxin-producing fungi under storage conditions.