Proceedings of 1st International Electronic Conference on Toxins, 2021
Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus in corn. Non-aflatoxigenic A... more Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus in corn. Non-aflatoxigenic A. flavus isolates are applied to corn fields as a biocontrol to reduce aflatoxin contamination. Direct contact or touch between aflatoxigenic and non-aflatoxigenic isolates dramatically reduces aflatoxin production. To understand the mechanism of touch inhibition, a high-throughput RNA-seq study was conducted to examine gene expression during their interaction. Non-aflatoxigenic strain KD17 and aflatoxigenic strain KD53 were grown separately and in co-culture for 30 and 72 h. Toxin production was high in the aflatoxigenic monoculture and negligible in co-cultures. When grown separately, the toxigenic strain represented 7% and 33% of the combined biomass at 30 and 72 h, respectively. However, only 3% of the sequence reads uniquely aligned to the aflatoxigenic strain during co-culture, indicating growth and/or gene expression of the aflatoxigenic strain was inhibited in response to the non...
Maize genotypes resistant or susceptible to aflatoxin production or contamination were compared f... more Maize genotypes resistant or susceptible to aflatoxin production or contamination were compared for differences in both constitutive and inducible proteins. Five additional constitutive proteins were found to be associated with resistance in over 8 of the 10 genotypes examined. Among these, the 58- and 46-kDa proteins were identified as globulin-1 and globulin-2, respectively. Differences in the ability to induce specific antifungal proteins, such as the higher synthesis of the 22-kDa zeamatin in resistant genotypes, were also observed between resistant and susceptible kernels incubated under germinating conditions (31°C, 100% humidity). Both constitutive and inducible proteins appear to be necessary for kernel resistance. Embryo-killed kernels (unable to synthesize new proteins) supported the highest level of aflatoxins, whereas imbibed kernels (to hasten protein induction) supported the lowest among all treatments. This suggests that the synthesis of new proteins by the embryo pla...
Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flav... more Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 mm membrane. Toxigenic and atoxigenic conidial mixtures (50:50) placed on both sides of these filters restored inhibition. There was ,50% inhibition when a 12 mm pore size filter was used. Conidial and mycelial diameters were in the 3.5-7.0 mm range and could pass through the 12 mm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3-5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition and the major contributor to biological control of aflatoxin contamination.
ABSTRACT A 33kDa protein present in Aspergillus flavus-infected maize embryo was identified as a ... more ABSTRACT A 33kDa protein present in Aspergillus flavus-infected maize embryo was identified as a fungal alkaline protease (ALP). This protein became one of the major extracellular proteins of A. flavus in potato dextrose broth medium culture filtrate after 3 days, but was expressed at low levels or undetectable in A+M medium containing gelatin or starch, respectively. The activity of purified ALP was significantly inhibited in the presence of 0.5mM phenylmethylsulfonyl fluoride (PMSF), or 1.43μμ (200μg/ml) maize 14kDa trypsin inhibitor. Further study demonstrated that reduction of this ALP by PMSF also significantly reduced the level of aflatoxin accumulation in A. flavus culture, suggesting an important role for ALP in the infection of maize kernels and subsequent aflatoxin accumulation.
Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible ... more Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize (Zea mays). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics, and resistance-associated proteins were identified. Here, we report the comparison of maize endosperm proteins from five resistant and five susceptible genotypes, and the identification of additional resistance-associated proteins using the same approach. Ten protein spots were upregulated twofold or higher in resistant lines compared with susceptible ones. Peptide sequencing of these proteins identified them as a globulin-2 protein, late embryogenesis abundant proteins (LEA3 and LEA14), a stress-related peroxiredoxin antioxidant (PER1), heat-shock proteins (HSP17.2), a cold-regulated protein (COR), and an antifungal trypsin-inhibitor protein (TI). The gene encoding one such upregulated protein, PER1, was cloned and overexpressed...
PCR with BOX and ERIC primers was used to analyze DNA of Xanthomonas albilineans and other bacter... more PCR with BOX and ERIC primers was used to analyze DNA of Xanthomonas albilineans and other bacteria associated with sugarcane. Generated fingerprints permitted clear separation of X. albilineans from other bacteria and revealed variation within the species. Good agreement between fingerprint groups and geographic origin and serovars was observed.
The term ‘competitive exclusion’ involving physical blockage of growth or access of the toxigenic... more The term ‘competitive exclusion’ involving physical blockage of growth or access of the toxigenic strain to the seed target has been used to describe the mechanism of biological control of aflatoxin contamination. However, recent evidence suggests that a form of intraspecific aflatoxin inhibition requiring growth of the competing strains together during the infection process in such a way that hyphae physically interact or touch is the trigger for preventing induction of aflatoxin synthesis. This direct touch-based inhibition of aflatoxin synthesis is posited to be the mechanistic basis of biological control in this system. Evidence for this idea comes from the published observations that co-culture of toxigenic and atoxigenic strains in a suspended disc system, in which the hyphae physically interact, prevents aflatoxin production. However, growth of the same strains in the same medium in the two compartments of a filter insert plate well system, separating the atoxigenic and toxig...
Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox)... more Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic aci...
Molecular plant-microbe interactions : MPMI, May 24, 2017
Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcin... more Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcinogenic aflatoxins (AFs). Besides AFs, A. flavus makes many more secondary metabolites (SMs) whose toxicity in insects or vertebrates has been studied. However, the role of SMs in the invasion of plant hosts by A. flavus remains to be investigated. Cyclopiazonic acid (CPA), a neurotoxic SM made by A. flavus, is a nanomolar inhibitor of endoplasmic reticulum calcium ATPases (ECAs) and a potent inducer of cell death in plants. We hypothesized that CPA, by virtue of its cytotoxicity, may serve as a key pathogenicity factor that kills plant cells and supports the saprophytic life style of the fungus while compromising the host defense response. This proposal was tested by two complementary approaches. A comparison of CPA levels among A. flavus isolates indicated that CPA may be a determinant of niche adaptation, i.e., isolates that colonize maize make more CPA than those restricted only to th...
The effect of Avid ® on the induction of synnema-like structure of Aspergillus flavus (61 strains... more The effect of Avid ® on the induction of synnema-like structure of Aspergillus flavus (61 strains) grown on various types of agar media was investigated. Other related species of Aspergillus within the section Flavi, A. parasiticus (14 strains), A. nomius (14 strains), A. psuedotamarii (2 strains), A. tamarii (20 strains) and A. oryzae (5 strains), were also evaluated for comparison. When Avid ® was added into five different solid media (Czapek, V8, Aspergillus flavus and A. parasiticus, corn meal and potato dextrose agar) at 0.004% (v/v), only the A. flavus strains (all 61 tested) were able to produce synnema-like structures, and only in the Czapek agar medium containing Avid ® . The effect of carbon (sucrose, glucose and starch) and nitrogen (NaNO 3 , urea and peptone) sources in Czapek medium containing Avid ® on the induction of a synnema-like structure was also determined, with synnema-like structure induction being found in all three carbon sources but only in NaNO 3 as the ni...
The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammon... more The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammonium salt] was tested at concentrations from 2 to 2,000 g GA per ml for activity against growth and aflatoxin B1 (AFB) production by the mycotoxigenic fungus Aspergillus flavus Link:Fr. The highest concentration (2,000 microg GA per ml) reduced colony diameter of A. flavus strain AF13 by 80%. AFB1 production was inhibited by 90% at this concentration. Reduction in mycelial dry weight and AFB1 production in response to GA application ranged from 17.2 to 97.1% and from 39.1 to 90.1%, respectively. Of four concentrations tested, 2 microg GA per ml was weakly inhibitory. In the kernel screening assay, AFB1 production was inhibited 60 to 91% when kernels were preimmersed or immersed 5 days after incubation in 200 microg GA per ml. Both concentrations (2 and 200 microg GA per ml) reduced seed germination by 25 to 50%. Results indicate that GA has an inhibitory effect on growth and AFB1 producti...
To study the interaction of fluorescently tagged nanoparticles with Aspergillus flavus. Covalentl... more To study the interaction of fluorescently tagged nanoparticles with Aspergillus flavus. Covalently tagged poly(lactic-co-glycolic) acid (PLGA) nanoparticles (PLGA-tetramethylrhodamine [PLGA-TRITC]), and PLGA-TRITC with entrapped coumarin-6 (double-tagged) nanoparticles, were synthesized using an oil-in-water emulsion evaporation method. Nanoparticle interaction with A. flavus was assessed using fluorescent microscopy. PLGA-TRITC nanoparticles associated with the surface of fungal spores and hyphae, with limited fluorescence observed within the interior. With double-tagged nanoparticles, comparatively more red fluorescence (TRITC) was measured on the fungal surface and more green (coumarin-6) on the interior, resulting from uptake of released coumarin-6. The majority of nanoparticles associated with the fungal surface, while smaller nanoparticles were internalized. Surface association between polymeric nanoparticles and A. flavus may facilitate content uptake.
Greenhouse experiments were conducted to determine potential of infectivity titration to evaluate... more Greenhouse experiments were conducted to determine potential of infectivity titration to evaluate resistance of sugarcane to leaf scald disease caused by Xanthomonas albilineans. In two experiments, single-bud cuttings were inoculated with suspensions containing 101, 105, or 108 CFU/ml of X. albilineans. The occurrence of symptoms was recorded every 15 days from 45 to 210 days after inoculation. At the final evaluation date, leaf vascular sap was plated onto selective medium to detect latent infections. ED50 (log10 of the bacterial concentration required to infect 50% of inoculated plants) was estimated for each cultivar based on probit analysis of cumulative infection frequency. Frequency of infected plants varied among inoculum doses and cultivars and resulted in ED50 values ranging from 3.0 to 12.3 and 3.1 to 9.8 in the first and second experiments, respectively. Good agreement between experiments was observed for ED50 values of individual cultivars. Differences in ED50 among cul...
The spread of ratoon stunting disease by the mechanical harvester was followed in disease-free su... more The spread of ratoon stunting disease by the mechanical harvester was followed in disease-free sugarcane plants of cultivars susceptible, intermediate, or resistant to the disease. Incidence in a 12-m row of susceptible cultivar L 62-96 preceded by one, two, or four diseased plants was 30, 60, and 75%, respectively, after one harvest and 84, 98, and 99%, respectively, after two harvests. Incidence in intermediate cultivar CP 65-357 was 10, 11, and 9% in the first ratoon and 26, 24, and 44% in the second ratoon. No spread was detected in resistant cultivar L 60-25. Gradients were apparent in consecutive 3-m segments of rows (.)
The spread and increase of ratoon stunting disease (RSD) resulting from two mechanical harvests w... more The spread and increase of ratoon stunting disease (RSD) resulting from two mechanical harvests were compared in eight sugarcane cultivars at two locations. RSD spread and increase were detected in the ratoon crops grown after each harvest and varied among cultivars and locations. Disease spread and increase were greater in plants grown from stalks collected at the first harvest than in the first ratoon growth from the harvested field. RSD infection was determined using five disease detection methods: alkaline-induced metaxylem autofluorescence; microscopic examination of xylem sap; and dot blot, evaporative-binding, and tissue blot enzyme immunoassays. The tissue blot enzyme immunoassay was the most accurate RSD detection method. The dot blot and evaporative-binding enzyme immunoassays were the least sensitive for detection of RSD-infected stalks, and alkaline-induced metaxylem autofluorescence was least accurate for correct identification of noninfected stalks. The results indicat...
ABSTRACT Eleven soybean cultivars with different levels of susceptibility to virulent isolate SG9... more ABSTRACT Eleven soybean cultivars with different levels of susceptibility to virulent isolate SG915 ofCalonectria ilicicola were examined for reaction to metabolites produced by the isolate. When the culture filtrate from isolate SG915 was applied to trifoliates from 11 cultivars, cvs. ‘Cajun’ and ‘Asgrow 7986’ exhibited reduced wilting severity. However, there was no correlation between sensitivity to culture filtrate and susceptibility to the fungal isolate. Wilting severity on cv. ‘Riverside 699’ was greatest when trifoliates were treated with culture filtrates from isolates SG915 (highly virulent) and C31 (less virulent). The dilution end-point for culture filtrates of virulent isolate SG915 was determined to be 1:8. Nonautoclaved culture filtrates caused complete wilt of soybean trifoliates after 36 h, but autoclaved culture filtrates demonstrated a reduced ability to wilt leaves. Electrolyte leakage from treated leaf tissues increased over time regardless of the concentrations of culture filtrate tested. The greatest electrolyte losses were observed during the initial 30 min incubation of leaf tissues. The highest concentration of culture filtrate (50%, v/v) induced more electrolyte loss than the low concentration (10%, v/v) or control. These results suggest that toxic metabolites ofC. ilicicola may be involved in disease development with leaf symptom expression.
Aims: Itraconazole and coumarin-6 loaded polylactic-co-glycolic acid-nanoparticles (PLGA-ITZ- and... more Aims: Itraconazole and coumarin-6 loaded polylactic-co-glycolic acid-nanoparticles (PLGA-ITZ- and PLGA-C6-NPs) were synthesized and tested for fungal cell uptake and antifungal ability based on particle size. Materials & Methods: PLGA-ITZ- and PLGA-C6-NPs were synthesized using an oil-in-water emulsion evaporation method. Fungal cell uptake and antifungal activity of the polymeric NPs was tested on Aspergillus flavus. Results: PLGA-C6-NPs of 203 nm associated with fungal cell surfaces and internalized efficiently, while 1206 nm NPs associated with cell surfaces were internalized less efficiently. Antifungal studies of PLGA-ITZ-NPs of 232, 630 and 1060 nm showed differences in inhibitory activity with 232 nm NPs showing superior activity at the lowest ITZ concentration of 0.003 mg/ml, followed by 630 and 1060 nm NPs. No differences in antifungal activity were observed at higher ITZ concentrations. Conclusion: The PLGA-ITZ-NP system can increase bioavailability of ITZ by improving its...
Proceedings of 1st International Electronic Conference on Toxins, 2021
Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus in corn. Non-aflatoxigenic A... more Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus in corn. Non-aflatoxigenic A. flavus isolates are applied to corn fields as a biocontrol to reduce aflatoxin contamination. Direct contact or touch between aflatoxigenic and non-aflatoxigenic isolates dramatically reduces aflatoxin production. To understand the mechanism of touch inhibition, a high-throughput RNA-seq study was conducted to examine gene expression during their interaction. Non-aflatoxigenic strain KD17 and aflatoxigenic strain KD53 were grown separately and in co-culture for 30 and 72 h. Toxin production was high in the aflatoxigenic monoculture and negligible in co-cultures. When grown separately, the toxigenic strain represented 7% and 33% of the combined biomass at 30 and 72 h, respectively. However, only 3% of the sequence reads uniquely aligned to the aflatoxigenic strain during co-culture, indicating growth and/or gene expression of the aflatoxigenic strain was inhibited in response to the non...
Maize genotypes resistant or susceptible to aflatoxin production or contamination were compared f... more Maize genotypes resistant or susceptible to aflatoxin production or contamination were compared for differences in both constitutive and inducible proteins. Five additional constitutive proteins were found to be associated with resistance in over 8 of the 10 genotypes examined. Among these, the 58- and 46-kDa proteins were identified as globulin-1 and globulin-2, respectively. Differences in the ability to induce specific antifungal proteins, such as the higher synthesis of the 22-kDa zeamatin in resistant genotypes, were also observed between resistant and susceptible kernels incubated under germinating conditions (31°C, 100% humidity). Both constitutive and inducible proteins appear to be necessary for kernel resistance. Embryo-killed kernels (unable to synthesize new proteins) supported the highest level of aflatoxins, whereas imbibed kernels (to hasten protein induction) supported the lowest among all treatments. This suggests that the synthesis of new proteins by the embryo pla...
Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flav... more Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 mm membrane. Toxigenic and atoxigenic conidial mixtures (50:50) placed on both sides of these filters restored inhibition. There was ,50% inhibition when a 12 mm pore size filter was used. Conidial and mycelial diameters were in the 3.5-7.0 mm range and could pass through the 12 mm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3-5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition and the major contributor to biological control of aflatoxin contamination.
ABSTRACT A 33kDa protein present in Aspergillus flavus-infected maize embryo was identified as a ... more ABSTRACT A 33kDa protein present in Aspergillus flavus-infected maize embryo was identified as a fungal alkaline protease (ALP). This protein became one of the major extracellular proteins of A. flavus in potato dextrose broth medium culture filtrate after 3 days, but was expressed at low levels or undetectable in A+M medium containing gelatin or starch, respectively. The activity of purified ALP was significantly inhibited in the presence of 0.5mM phenylmethylsulfonyl fluoride (PMSF), or 1.43μμ (200μg/ml) maize 14kDa trypsin inhibitor. Further study demonstrated that reduction of this ALP by PMSF also significantly reduced the level of aflatoxin accumulation in A. flavus culture, suggesting an important role for ALP in the infection of maize kernels and subsequent aflatoxin accumulation.
Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible ... more Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops such as maize (Zea mays). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics, and resistance-associated proteins were identified. Here, we report the comparison of maize endosperm proteins from five resistant and five susceptible genotypes, and the identification of additional resistance-associated proteins using the same approach. Ten protein spots were upregulated twofold or higher in resistant lines compared with susceptible ones. Peptide sequencing of these proteins identified them as a globulin-2 protein, late embryogenesis abundant proteins (LEA3 and LEA14), a stress-related peroxiredoxin antioxidant (PER1), heat-shock proteins (HSP17.2), a cold-regulated protein (COR), and an antifungal trypsin-inhibitor protein (TI). The gene encoding one such upregulated protein, PER1, was cloned and overexpressed...
PCR with BOX and ERIC primers was used to analyze DNA of Xanthomonas albilineans and other bacter... more PCR with BOX and ERIC primers was used to analyze DNA of Xanthomonas albilineans and other bacteria associated with sugarcane. Generated fingerprints permitted clear separation of X. albilineans from other bacteria and revealed variation within the species. Good agreement between fingerprint groups and geographic origin and serovars was observed.
The term ‘competitive exclusion’ involving physical blockage of growth or access of the toxigenic... more The term ‘competitive exclusion’ involving physical blockage of growth or access of the toxigenic strain to the seed target has been used to describe the mechanism of biological control of aflatoxin contamination. However, recent evidence suggests that a form of intraspecific aflatoxin inhibition requiring growth of the competing strains together during the infection process in such a way that hyphae physically interact or touch is the trigger for preventing induction of aflatoxin synthesis. This direct touch-based inhibition of aflatoxin synthesis is posited to be the mechanistic basis of biological control in this system. Evidence for this idea comes from the published observations that co-culture of toxigenic and atoxigenic strains in a suspended disc system, in which the hyphae physically interact, prevents aflatoxin production. However, growth of the same strains in the same medium in the two compartments of a filter insert plate well system, separating the atoxigenic and toxig...
Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox)... more Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic aci...
Molecular plant-microbe interactions : MPMI, May 24, 2017
Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcin... more Aspergillus flavus, an opportunistic pathogen, contaminates maize and other key crops with carcinogenic aflatoxins (AFs). Besides AFs, A. flavus makes many more secondary metabolites (SMs) whose toxicity in insects or vertebrates has been studied. However, the role of SMs in the invasion of plant hosts by A. flavus remains to be investigated. Cyclopiazonic acid (CPA), a neurotoxic SM made by A. flavus, is a nanomolar inhibitor of endoplasmic reticulum calcium ATPases (ECAs) and a potent inducer of cell death in plants. We hypothesized that CPA, by virtue of its cytotoxicity, may serve as a key pathogenicity factor that kills plant cells and supports the saprophytic life style of the fungus while compromising the host defense response. This proposal was tested by two complementary approaches. A comparison of CPA levels among A. flavus isolates indicated that CPA may be a determinant of niche adaptation, i.e., isolates that colonize maize make more CPA than those restricted only to th...
The effect of Avid ® on the induction of synnema-like structure of Aspergillus flavus (61 strains... more The effect of Avid ® on the induction of synnema-like structure of Aspergillus flavus (61 strains) grown on various types of agar media was investigated. Other related species of Aspergillus within the section Flavi, A. parasiticus (14 strains), A. nomius (14 strains), A. psuedotamarii (2 strains), A. tamarii (20 strains) and A. oryzae (5 strains), were also evaluated for comparison. When Avid ® was added into five different solid media (Czapek, V8, Aspergillus flavus and A. parasiticus, corn meal and potato dextrose agar) at 0.004% (v/v), only the A. flavus strains (all 61 tested) were able to produce synnema-like structures, and only in the Czapek agar medium containing Avid ® . The effect of carbon (sucrose, glucose and starch) and nitrogen (NaNO 3 , urea and peptone) sources in Czapek medium containing Avid ® on the induction of a synnema-like structure was also determined, with synnema-like structure induction being found in all three carbon sources but only in NaNO 3 as the ni...
The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammon... more The herbicide glufosinate-ammonium (GA) [butanoic acid, 2-amino-4-(hydroxymethylphosphinyl)-ammonium salt] was tested at concentrations from 2 to 2,000 g GA per ml for activity against growth and aflatoxin B1 (AFB) production by the mycotoxigenic fungus Aspergillus flavus Link:Fr. The highest concentration (2,000 microg GA per ml) reduced colony diameter of A. flavus strain AF13 by 80%. AFB1 production was inhibited by 90% at this concentration. Reduction in mycelial dry weight and AFB1 production in response to GA application ranged from 17.2 to 97.1% and from 39.1 to 90.1%, respectively. Of four concentrations tested, 2 microg GA per ml was weakly inhibitory. In the kernel screening assay, AFB1 production was inhibited 60 to 91% when kernels were preimmersed or immersed 5 days after incubation in 200 microg GA per ml. Both concentrations (2 and 200 microg GA per ml) reduced seed germination by 25 to 50%. Results indicate that GA has an inhibitory effect on growth and AFB1 producti...
To study the interaction of fluorescently tagged nanoparticles with Aspergillus flavus. Covalentl... more To study the interaction of fluorescently tagged nanoparticles with Aspergillus flavus. Covalently tagged poly(lactic-co-glycolic) acid (PLGA) nanoparticles (PLGA-tetramethylrhodamine [PLGA-TRITC]), and PLGA-TRITC with entrapped coumarin-6 (double-tagged) nanoparticles, were synthesized using an oil-in-water emulsion evaporation method. Nanoparticle interaction with A. flavus was assessed using fluorescent microscopy. PLGA-TRITC nanoparticles associated with the surface of fungal spores and hyphae, with limited fluorescence observed within the interior. With double-tagged nanoparticles, comparatively more red fluorescence (TRITC) was measured on the fungal surface and more green (coumarin-6) on the interior, resulting from uptake of released coumarin-6. The majority of nanoparticles associated with the fungal surface, while smaller nanoparticles were internalized. Surface association between polymeric nanoparticles and A. flavus may facilitate content uptake.
Greenhouse experiments were conducted to determine potential of infectivity titration to evaluate... more Greenhouse experiments were conducted to determine potential of infectivity titration to evaluate resistance of sugarcane to leaf scald disease caused by Xanthomonas albilineans. In two experiments, single-bud cuttings were inoculated with suspensions containing 101, 105, or 108 CFU/ml of X. albilineans. The occurrence of symptoms was recorded every 15 days from 45 to 210 days after inoculation. At the final evaluation date, leaf vascular sap was plated onto selective medium to detect latent infections. ED50 (log10 of the bacterial concentration required to infect 50% of inoculated plants) was estimated for each cultivar based on probit analysis of cumulative infection frequency. Frequency of infected plants varied among inoculum doses and cultivars and resulted in ED50 values ranging from 3.0 to 12.3 and 3.1 to 9.8 in the first and second experiments, respectively. Good agreement between experiments was observed for ED50 values of individual cultivars. Differences in ED50 among cul...
The spread of ratoon stunting disease by the mechanical harvester was followed in disease-free su... more The spread of ratoon stunting disease by the mechanical harvester was followed in disease-free sugarcane plants of cultivars susceptible, intermediate, or resistant to the disease. Incidence in a 12-m row of susceptible cultivar L 62-96 preceded by one, two, or four diseased plants was 30, 60, and 75%, respectively, after one harvest and 84, 98, and 99%, respectively, after two harvests. Incidence in intermediate cultivar CP 65-357 was 10, 11, and 9% in the first ratoon and 26, 24, and 44% in the second ratoon. No spread was detected in resistant cultivar L 60-25. Gradients were apparent in consecutive 3-m segments of rows (.)
The spread and increase of ratoon stunting disease (RSD) resulting from two mechanical harvests w... more The spread and increase of ratoon stunting disease (RSD) resulting from two mechanical harvests were compared in eight sugarcane cultivars at two locations. RSD spread and increase were detected in the ratoon crops grown after each harvest and varied among cultivars and locations. Disease spread and increase were greater in plants grown from stalks collected at the first harvest than in the first ratoon growth from the harvested field. RSD infection was determined using five disease detection methods: alkaline-induced metaxylem autofluorescence; microscopic examination of xylem sap; and dot blot, evaporative-binding, and tissue blot enzyme immunoassays. The tissue blot enzyme immunoassay was the most accurate RSD detection method. The dot blot and evaporative-binding enzyme immunoassays were the least sensitive for detection of RSD-infected stalks, and alkaline-induced metaxylem autofluorescence was least accurate for correct identification of noninfected stalks. The results indicat...
ABSTRACT Eleven soybean cultivars with different levels of susceptibility to virulent isolate SG9... more ABSTRACT Eleven soybean cultivars with different levels of susceptibility to virulent isolate SG915 ofCalonectria ilicicola were examined for reaction to metabolites produced by the isolate. When the culture filtrate from isolate SG915 was applied to trifoliates from 11 cultivars, cvs. ‘Cajun’ and ‘Asgrow 7986’ exhibited reduced wilting severity. However, there was no correlation between sensitivity to culture filtrate and susceptibility to the fungal isolate. Wilting severity on cv. ‘Riverside 699’ was greatest when trifoliates were treated with culture filtrates from isolates SG915 (highly virulent) and C31 (less virulent). The dilution end-point for culture filtrates of virulent isolate SG915 was determined to be 1:8. Nonautoclaved culture filtrates caused complete wilt of soybean trifoliates after 36 h, but autoclaved culture filtrates demonstrated a reduced ability to wilt leaves. Electrolyte leakage from treated leaf tissues increased over time regardless of the concentrations of culture filtrate tested. The greatest electrolyte losses were observed during the initial 30 min incubation of leaf tissues. The highest concentration of culture filtrate (50%, v/v) induced more electrolyte loss than the low concentration (10%, v/v) or control. These results suggest that toxic metabolites ofC. ilicicola may be involved in disease development with leaf symptom expression.
Aims: Itraconazole and coumarin-6 loaded polylactic-co-glycolic acid-nanoparticles (PLGA-ITZ- and... more Aims: Itraconazole and coumarin-6 loaded polylactic-co-glycolic acid-nanoparticles (PLGA-ITZ- and PLGA-C6-NPs) were synthesized and tested for fungal cell uptake and antifungal ability based on particle size. Materials & Methods: PLGA-ITZ- and PLGA-C6-NPs were synthesized using an oil-in-water emulsion evaporation method. Fungal cell uptake and antifungal activity of the polymeric NPs was tested on Aspergillus flavus. Results: PLGA-C6-NPs of 203 nm associated with fungal cell surfaces and internalized efficiently, while 1206 nm NPs associated with cell surfaces were internalized less efficiently. Antifungal studies of PLGA-ITZ-NPs of 232, 630 and 1060 nm showed differences in inhibitory activity with 232 nm NPs showing superior activity at the lowest ITZ concentration of 0.003 mg/ml, followed by 630 and 1060 nm NPs. No differences in antifungal activity were observed at higher ITZ concentrations. Conclusion: The PLGA-ITZ-NP system can increase bioavailability of ITZ by improving its...
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Papers by Ken Dasmann