Jonathan Nimal Selvaraj

Antagonistic Action of Bacillus subtilis Strain SG6 on Fusarium graminearum

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease that leads to extensive yield and quality loss of wheat and barley. Bacteria isolated from wheat kernels and plant anthers were screened for antagonistic activity against F. graminearum. Based on its in vitro effectiveness, strain SG6 was selected for characterization and identified as Bacillus subtilis. B. subtilis SG6 exhibited a high antifungal effect on the mycelium growth, sporulation and DON production of F. graminearum with the inhibition rate of 87.9%, 95.6% and 100%, respectively. In order to gain insight into biological control effect in situ, we applied B. subtilis SG6 at anthesis through the soft dough stage of kernel development in field test. It was revealed that B. subtilis SG6 significantly reduced disease incidence (DI), FHB index and DON (P≤0.05). Further, ultrastructural examination shows that B. subtilis SG6 strain induced stripping of F. graminearum hyphal surface by destroying the cellular structure. When hypha cell wall was damaged, the organelles and cytoplasm inside cell would exude, leading to cell death. The antifungal activity of SG6 could be associated with the coproduction of chitinase, fengycins and surfactins.

Inhibitory effect of essential oils on Aspergillus ochraceus growth and ochratoxin A production.

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains.

Aflatoxin B1 Degradation by a Pseudomonas Strain

Aflatoxin B1 (AFB1), one of the most potent naturally occurring mutagens and carcinogens, causes significant threats to the food industry and animal production. In this study, 25 bacteria isolates were collected from grain kernels and soils displaying AFB1 reduction activity. Based on its degradation effectiveness, isolate N17-1 was selected for further characterization and identified as Pseudomonas aeruginosa. P. aeruginosa N17-1 could degrade AFB1, AFB2 and AFM1 by 82.8%, 46.8% and 31.9% after incubation in Nutrient Broth (NB) medium at 37 °C for 72 h, respectively. The culture supernatant of isolate N17-1 degraded AFB1 effectively, whereas the viable cells and intra cell extracts were far less effective. Factors influencing AFB1 degradation by the culture supernatant were investigated. Maximum degradation was observed at 55 °C. Ions Mn2+ and Cu2+ were activators for AFB1 degradation, however, ions Mg2+, Li+, Zn2+, Se2+, Fe3+ were strong inhibitors. Treatments with proteinase K and proteinase K plus SDS significantly reduced the degradation activity of the culture supernatant. No degradation products were observed based on preliminary LC-QTOF/MS analysis, indicating AFB1 was metabolized to degradation products with chemical properties different from that of AFB1. The results indicated that the degradation of AFB1 by P. aeruginosa N17-1 was enzymatic and could have a great potential in industrial applications. This is the first report indicating that the isolate of P. aeruginosa possesses the ability to degrade aflatoxin.

Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms

Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms.

Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1

Ochratoxin A (OTA), a potentially carcinogenic mycotoxin which contaminates grains, is produced by several Aspergillus species. A comparative sequence analysis of the OTA-producing Aspergillus ochraceus fc-1 strain and other Aspergillus species was performed. Two new OTA-related polyketide synthase (PKS) (AoOTApks) genes were identified. The predicted amino acid sequence of AoOTApks-1 displayed high similarity to previously identified PKSs from OTA-producing A. carbonarius ITEM 5010 (67%; [PI] No. 173482) and A. niger CBS 513.88 (62%; XP_001397313). However, the predicted amino acid sequence of AoOTApks-2 displayed lower homology with A. niger CBS 513.88 (38%) and A. carbonarius ITEM 5010 (28%). A phylogenetic analysis of the β-ketosynthase and acyl-transferase domains of the AoOTApks proteins indicated that they shared a common origin with other OTA-producing species, such as A. carbonarius, A. niger, and A. westerdijkiae. A real-time reverse-transcription PCR analysis showed that the expression of AoOTApks-1 and -2 was positively correlated with the OTA concentration. The pks gene deleted mutants ∆AoOTApks-1 and ∆AoOTApks-2 produced nil and lesser OTA than the wild-type strain, respectively. Our study suggests that AoOTApks-1 could be involved in OTA biosynthesis, while AoOTApks-2 might be indirectly involved in OTA production.

Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.).

MicroRNAs (miRNAs) play important roles in growth, development, and response to environmental changes in plants. Based on the whole-genome shotgun sequencing strategy, more and more wheat miRNAs have been annotated. Now, there is a need for an effective technology to analyse endogenous miRNAs function in wheat. We report here that the modified barley stripe mosaic virus (BSMV)-induced miRNAs silencing system can be utilized to silence miRNAs in wheat. BSMV-based miRNA silencing system is performed through BSMV-based expression of miRNA target mimics to suppress miR159a and miR3134a. The relative expression levels of mature miR159a and miR3134a decrease with increasing transcript levels of their target genes in wheat plants. In summary, the developed approach is effective in silencing endogenous miRNAs, thereby providing a powerful tool for biological function analyses of miRNA molecules in common wheat.

Complete genome sequence of Bacillus subtilis SG6 antagonistic against Fusarium graminearum.

Bacillus subtilis SG6 exhibited a high antifungal effect on the mycelium growth, sporulation and DON production of F. graminearum and significantly reduced disease incidence, Fusarium head blight (FHB) index and DON in the field. Here, we present the complete genome sequence of B. subtilis SG6, providing insights into the genomic basis of its effects and facilitating its application in FHB control.

pH-Signaling Transcription Factor AopacC Regulates Ochratoxin A Biosynthesis in Aspergillus ochraceus

In Aspergillus and Penicillium species, an essential pH-response transcription factor pacC is involved in growth, pathogenicity, and toxigenicity. To investigate the connection between ochratoxin A (OTA) biosynthesis and ambient pH, the AopacC in Aspergillus ochraceus was functionally characterized using a loss-of-function mutant. The mycelium growth was inhibited under pH 4.5 and 10.0, while the sporulation increased under alkaline condition. A reduction of mycelium growth and an elevation of sporulation was observed in Δ AopacC mutant. Compared to neutral condition, OTA contents were respectively reduced by 71.6 and 79.8% under acidic and alkaline conditions. The expression of AopacC increased with the elevated pH, and deleting AopacC dramatically decreased OTA production and biosynthetic genes Aopks expression. Additionally, the Δ AopacC mutant exhibited attenuated infection ability toward pear fruits. These results suggest that AopacC is an alkaline-induced regulator responsible for growth and OTA biosynthesis in A. ochraceus and this regulatory mechanism might be pH-dependent.

Novel Aflatoxin-Degrading Enzyme from Bacillus shackletonii L7

Food and feed contamination by aflatoxin (AF)B1 has adverse economic and health consequences. AFB1 degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil samples for AFB1-reducing activity. The higher activity was detected in isolate L7, which was identified as Bacillus shackletonii. L7 reduced AFB1, AFB2, and AFM1 levels by 92.1%, 84.1%, and 90.4%, respectively, after 72 h at 37 °C. The L7 culture supernatant degraded more AFB1 than viable cells and cell extracts; and the degradation activity was reduced from 77.9% to 15.3% in the presence of proteinase K and sodium dodecyl sulphate. A thermostable enzyme purified from the boiled supernatant was designated as Bacillus aflatoxin-degrading enzyme (BADE). An overall 9.55-fold purification of BADE with a recovery of 39.92% and an activity of 3.85 × 103 U·mg−1 was obtained using chromatography on DEAE-Sepharose. BADE had an estimated molecular mass of 22 kDa and exhibited the highest activity at 70 °C and pH 8.0, which was enhanced by Cu2+ and inhibited by Zn2+, Mn2+, Mg2+, and Li+. BADE is the major protein involved in AFB1 detoxification. This is the first report of a BADE isolated from B. shackletonii, which has potential applications in the detoxification of aflatoxins during food and feed processing.

A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence of Aspergillus ochraceus and a Comparative Genomic Analysis

Ochratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the genera Aspergillus and Penicillium are known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor. ABSTRACT Ochratoxin A (OTA) is a toxic secondary metabolite produced by Aspergillus and Penicillium species that widely contaminates food and feed. We sequenced and assembled the complete ∼37-Mb genome of Aspergillus ochraceus fc-1, a well-known producer of OTA. Key genes of the OTA biosynthetic pathway were identified by comparative genomic analyses with five other sequenced OTA-producing fungi: A. carbonarius, A. niger, A. steynii, A. westerdijkiae, and Penicillium nordicum. OTA production was completely inhibited in the deletion mutants (ΔotaA, ΔotaB, ΔotaC, ΔotaD, and ΔotaR1), and OTA biosynthesis was restored by feeding a postblock substrate to the corresponding mutant. The OTA biosynthetic pathway was unblocked in the ΔotaD mutant by the addition of heterologously expressed halogenase. OTA biosynthesis begins with a polyketide synthase (PKS), OtaA, utilizing acetyl coenzyme A (acetyl-CoA) and malonyl-CoA to synthesize 7-methylmellein, which is oxidized to OTβ by cytochrome P450 monooxygenase (OtaC). OTβ and l-β-phenylalanine are combined by a nonribosomal peptide synthetase (NRPS), OtaB, to form an amide bond to synthesize OTB. Finally, OTB is chlorinated by a halogenase (OtaD) to OTA. The otaABCD genes were expressed at low levels in the ΔotaR1 mutant. A second regulator, otaR2, which is adjacent to the biosynthetic gene, could modulate only the expression of otaA, otaB, and otaD. Thus, we have identified a consensus OTA biosynthetic pathway that can be used to prevent and control OTA synthesis and will help us understand the variation and production of the intermediate components in the biosynthetic pathway. IMPORTANCE Ochratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the genera Aspergillus and Penicillium are known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor.