Potential use of Zataria multiflora essential oil to control postharvest Aspergillus flavus fruit rot of strawberry

Postharvest fruit rot is a major problem in strawberry production chain worldwide. Aspergillus sp. is one of the major fungi associated with fruit rot of strawberry. In this study, an Aspergillus flavus was isolated from a rotten strawberry fruit. Based on the nucleotide sequence analysis of the internal transcribed spacer regions of rDNA, the fungus was confirmed as A. flavus. Pathogenicity of the isolated fungus was confirmed by artificially inoculating strawberry fruits under laboratory conditions. This strain was capable of producing aflatoxin B1 in vitro as determined by liquid chromatography-mass spectrometry analysis. Postharvest dip treatment of mature strawberry fruits with Zataria multiflora essential oil (ZEO) (0.1%) completely suppressed A. flavus infection and prevented rotting of fruits. The results of this study suggest that ZEO can be used as a sustainable and safe alternative to chemical fungicides for the control of Aspergillus fruit rot of strawberry

Antagonistic bacterial strains isolated from cabbage rhizosphere release antimicrobial volatile organic compounds against Pythium aphanidermatum

In a previous study, we isolated four antagonistic bacterial strains viz., Pseudomonas aeruginosa B1-SQU, Pseudomonas indica B2-SQU, Serratia marcescens B3-SQU and Pseudomonas brenneri B4-SQU from the rhizosphere of cabbage which suppressed damping-off in cabbage caused by Pythium aphanidermatum. In this study, potential of these bacterial isolates to produce antimicrobial volatile organic compounds (VOCs) against P. aphanidermatum was tested. The results of the two-sealed-base-plates assay revealed that all four bacterial strains produced VOCs against P. aphanidermatum with the maximum inhibition with P. brenneri B4-SQU followed by S. marcescens B3-SQU, P. aeruginosa B1-SQU and P. indica B2-SQU. Solid-phase microextraction coupled with gas chromatography-mass spectrometry was used to profile the VOCs of bacteria. A total of 20 VOCs were detected in P. aeruginosa B1-SQU and the major compounds identified were Carbon dioxide, 1-Butanol, 3-methyl- and Disulfide, dimethyl. The main volatile compounds detected in P. indica B2-SQU were 1-Butanol, 3-methyl-, Disulfide, dimethyl and 1,2-Propanediamine. Disulfide, dimethyl and 1,2-Propanediamine were the predominant compounds identified in S. marcescens B3-SQU among others. The major compounds detected in P. brenneri B4-SQU were 1-Butanol, 3-methyl-, 1,2-Propanediamine and Disulfide, dimethyl. Dimethyl disulfide, a well-known antimicrobial compound, was detected in the volatile profiles of all four antagonistic bacterial isolates. These results suggest that VOCs of antagonistic bacteria may be involved in the suppression of P. aphanidermatum and these antagonistic bacterial strains may be used as biofumigants for controlling damping-off of cucumber

Biological control of downy mildew of maize caused by Peronosclerospora sorghi under environmentally controlled conditions

Downy mildew disease, caused by Peronosclerospora sorghi, is one of the most serious diseases of maize. The disease is currently managed by seed treatment with metalaxyl fungicides. However, problems regarding environmental pollution resulting from the use of fungicides and development of fungicide resistance within populations of P. sorghi are of increasing concern. Assuming that biological control by means of using antagonistic microorganisms may be an alternative for the management of this disease, the efficacy of biocontrol agents viz., Bacillus subtilis G1, Bacillus amyloliquefaciens B2, Brevibacillus brevis 57 and Pseudomonas fluorescens Pf1 for the management of downy mildew of maize and for promoting plant growth was evaluated. The results indicated that seed treatment with B. subtilis G1 and B. amyloliquefaciens B2 significantly (P = 0.05) increased the germination percentage and seedling vigour of maize as assessed by roll towel method. Among them, B. subtilis G1 was the most effective and recorded 9% and 31% increases in germination percentage and seedling vigour of maize respectively, as compared to the control. A talc- based powder formulation of B. subtilis G1 when applied through seed at the rate of 10 g/kg reduced the downy mildew incidence up to 54% under greenhouse conditions. Results of this study suggest that B. subtilis G1 is a promising bioagent for the management of downy mildew of maize and for promoting plant growth. This antagonist could be further exploited for commercial scale up for ecofriendly management of downy mildew of maize under localized climatic conditions

Effect of foliar application of Pseudomonas fluoresencens on activities of phenylalanine ammonia-lyase, chitinase and β-1,3–glucanase and accumulation of phenolics in rice

Changes in activities of phenylalanine ammonia-lyase,chitinase,ß-1,3-glucanase and phenolic content in rice leaves were measured at different times after treatment of leaves with Pseudomonas fluorescens strain Pf1.When rice leaves were sprayed with P.fluorescens,substantial increase in the phenylalanine ammonia-lyase activity was observed 1 day after treatment.Following increase of the first enzyme of the phenylpropanoid pathway,phenolic content of rice leaves also increased to a maximum at 4 days after P.fluorescens treatment.Chitinase activity increased in rice leaves in response to application of P.fluorescens and the maximum enzyme activity was observed 3 days after treatment.ß-1,3-Glucanase activity also increased significantly from 1 day after P.fluorescens treatment and continued to increase through 7 days.A five-fold increase in glucanase activity was observed 7 days after P.fluorescens treatment

Analysis of aflatoxin B1 and aflatoxigenic mold in commercial poultry feeds in Tamil Nadu, India

A total of 48 commercial poultry feed samples collected from different poultry feed manufactures in Tamil Nadu, India were examined for the contamination of aflatoxin B1 (AFB1) and Aspergillus flavus. AFB1 in the samples was estimated by sandwich ELISA and the presence of A. flavus was detected by Real-Time PCR assay. Real-Time PCR analysis using A. flavus- specific omt primers confirmed the presence of A. flavus in all the samples tested. ELISA results indicated that the AFB1 contents in the poultry feeds ranged from 1.0 to18.7 ppb, which were below the permissible safe limits for poultry bird consumption and health. The results suggest adoption of good man-ufacturing practices by the commercial poultry feed manufacturers during procurement of feed ingredients, handling, storage and processing which might have suppressed the growth of A. flavus and aflatoxin contamination

Occurrence of aflatoxin contamination in maize kernels and molecular characterization of the producing organism, Aspergillus

Aflatoxins are toxic metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1) is a potent carcinogen, teratogen and mutagen. 660 pre- and post- harvest maize samples were collected from major maize growing areas in Tamil Nadu, India. Aflatoxin contamination was observed in 40.22% of the samples tested of which, 22.97% of pre-harvest and 53.93% post-harvest maize samples were found to be infected with AFB1 and 12.05% of the total samples exceeded WHO permissible limit of 20 μg/kg. AFB1 contamination ranged from 0 to 149.32 μg/kg. 28 A. flavus isolates were isolated and grouped into three sets based on aflatoxin producing potential viz., highly aflatoxin producing isolates, medium producing isolates and no aflatoxin producer or traces of aflatoxin producing isolates. The genetic coefficient matrix analysis using random amplified polymorphic DNA (RAPD) with ten random primers revealed minimum and maximum percent similarities among the tested A. flavus strains ranging from 35 to 89%. Cluster analysis separated the three sets of isolates into two groups (groups I and II) with each two subgroup confirming the genetic diversity among the A. flavus isolates from maize.Keywords: Maize, survey, Aspergillus flavus, aflatoxin, random amplified polymorphic DNA (RAPD).African Journal of Biotechnology Vol. 12(40), pp. 5839-584

Analysis of defense genes expression in maize upon infection with Peronosclerospora sorghi

Downy mildew, caused by Peronosclerospora sorghi is one of the important diseases affecting maize (Zea mays L.) production worldwide. Several downy mildew resistant maize lines have been identified. However, variability in the degree of resistance among maize genotypes to P. sorghi has been reported. In the present study the molecular basis of resistance of maize to P. sorghi was studied by using differential-display reverse transcription PCR (DDRT-PCR) technique. Maize seedlings of downy mildew resistant (MAI 756) and susceptible (CM 500) cultivars at two-leaf stage were inoculated with P. sorghi and leaf samples were collected at 0, 3 and 5 days after inoculation and analyzed for differentially expressed cDNAs using cDNA-RAPD approach. A total of 17 cDNA fragments corresponding to transcripts that showed alterations during the defence response of maize to P. sorghi were identified. Genes involved in signal transduction and several genes with unknown functions were found to be upregulated in maize after infection by P. sorghi. Among 35 random primers tested, OPD-05 has identified a differentially expressed cDNA coding for serine/threonine kinase protein in resistant maize genotype. Constitutive and high level expression of serine/threonine kinase gene was observed in the uninoculated plants of resistant genotype, whereas no expression of this gene was observed in uninoculated plants of susceptible genotype. However, the transcript level was induced 3 days after inoculation in the susceptible genotype and slightly reduced 5 days after inoculation. This study represents the first identification of maize serine/threonine kinase gene that is upregulated following infection by P. sorghi

Rapid detection of Ganoderma lucidum and assessment of inhibition effect of various control measures by immunoassay and PCR

Molecular and immunological methods have been applied for detecting the Ganoderma disease of coconut. Polyclonal antibodies (PAbs) raised against basidiocarp protein of Ganoderma were used for detection. For polymerase chain reaction (PCR) test, the primer generated from the internal transcribed spacer region one (ITS 1) of ribosomal DNA gene of Ganoderma, which produced a PCR product of 167 bp in size is used for early detection. Ganoderma disease in apparently healthy palms in two coconut gardens was tested by ELISA test using basidiocarp protein antiserum. Field trials were laid out in these early-diagnosed palms for the management of the disease. Based on the ELISA results,Pseudomonas fluorescens + Trichoderma viride with chitin amended treatments arrested the multiplication of the pathogen and showed below the infection level of optical density (O.D) within six months. Integrated disease management (IDM) and fungicide tridemorph treated palms showed below infection level (O.D value) within seven months and T. harzianum and P. fluorescens + T. viride treatedpalms showed below infection level (OD value) of the disease in eighth months

Assessment of aflatoxin B1 content and aflatoxigenic molds in imported food commodities in Muscat, Oman

Aflatoxins, mainly produced by Aspergillus flavus and A. parasiticus are considered as serious food safety and human health issues due to their hepatotoxic effects. In the present study, the occurrence of aflatoxin B1 (AFB1), the most potent human liver carcinogen, and prevalence of toxigenic isolates of Aspergillus spp. were assessed in 140 food commodities in Muscat markets, Oman, and the 95 quarantined imported food commodities. These samples consisted of rice, corn, peanut, red chilli powder, soybean, dried dates and tree nuts. AFB1 was analyzed using competitive ELISA/LC-MS and the aflatoxigenic fungi were detected using plating technique followed by molecular identification. No AFB1 was detected in 89 (63.6%) samples collected from local markets, while 44 (31.4%) samples contained 1-5 ppb and the remaining 7 (5%) samples (red chili powder) contained 6-10 ppb. None of the samples exceeded the maximum permissible limit of 10 ppb set for foods by Oman legislation. Of the 95 quarantined samples, only 17 (17.9%) samples were positive and contained AFB1 at concentrations ranging from 1-3.4 ppb. Four isolates of Aspergillus pp. were isolated from the collected samples and were identified as Aspergillus flavus (A14, A16 and A23) and A. chevalieri (A46) on the basis of internal transcribed spacer (ITS) sequences of ribosomal DNA. Among them, A. flavus strain A14 alone produced AFB1 (7.6 ppb), while A16, A23, and A46 were non-toxigenic. This is the first detailed report on the occurrence of AFB1 in food commodities imported into Oman

Antifungal Activity of Shirazi Thyme (Zataria multiflora Boiss.) Essential Oil against Hypomyces perniciosus, a causal agent of wet bubble disease of Agaricus bisporus

Wet bubble disease (WBD) caused by Hypomyces perniciosus is a major constraint of button mushroom (Agaricus bisporus) cultivated worldwide. A few synthetic chemical fungicides are used to control WBD. In our study, the potential of essential oil (EO) from Zataria multiflora in inhibition of H. perniciosus was evaluated as an alternative to chemical fungicides. An isolate of H. perniciosus was isolated from wet bubble diseased A. bisporus and pathogenicity of the mycoparasite was determined under artificially inoculated conditions. The mycoparasitic fungus was identified using sequences of the internal transcribed spacer (ITS) region of ribosomal DNA. The EO was extracted from the aerial parts of Z. multiflora by microwave extraction method and evaluated in vitro for its antifungal activity against H. perniciosus. The EO of Z. multiflora (ZEO) at the tested concentrations (50% and 100%) inhibited the growth of H. perniciosus in the agar diffusion test. The minimum inhibitory concentration (MIC) of ZEO was 0.04% as assessed by the poisoned food technique. The chemical composition of ZEO was determined by gas chromatography-mass spectrometry analysis. A total of 23 compounds were identified. Among them, the most abundant compounds were Linalool (20.3%) and Bornyl acetate (15.5%). Linalool at the tested concentrations of 0.25% and 0.125% completely inhibited the mycelial growth of H. perniciosus in an in vitro assay. These results suggest that ZEO can be exploited for control of WBD