In situ ergot alkaloid detection in three Balansia epichloe-infected grass species.

AIMS: The objectives of this work were to characterize molecularly the morphologically described endophyte Balansia epichloe symbiotic on three grass species, and to determine the in situ production of ergot alkaloids on these three symbiota. METHODS AND RESULTS: Balansia epichloe symbiotic with smut grass (Sporobolus poiretii), love grass (Eragrostis hirsuta) and lace grass (Eragrostis capillaries, a new host) were characterized using DNA barcoding. Laser ablation electro spray ionization (LAESI)-mass spectrometry was used to detect ergot alkaloids in situ for each symbiotum. CONCLUSIONS: The three morphologically described symbionts on the three host grasses were indicated as belonging to the species B. epichloe, DNA barcoding suggested they were related although a cryptic species was suggested. LAESI-mass spectrometry showed that ergot alkaloids were produced in vivo in two hosts but not the third although this same symbiotum was related to one of the ergot alkaloid producing symbiota as revealed by the DNA-barcoding procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: These results established the accumulation of ergot alkaloids in pot culture by a morpho species although there were variations with each species of grass. Barcoding described divergence among species, but considering its limitation, the suggested existence of cryptic species among this morphospecies requires substantiation by studies that are more rigorous.

Screening of Bacillus mojavensis biofilms and biosurfactants using laser ablation electrospray ionization mass spectroscopy.

AIMS: Biofilms are composed of micro-organisms within a matrix of chemically complex polymer compounds and from these structures many unknown competitive factors are suggested that many considered are important consequences for biological control. This research was undertaken to study further the endophyte, Bacillus mojavensis and its relationships to biofilm and two classes of lipopeptides considered relevant for biocontrol of plant pathogens. METHODS AND RESULTS: Laser ablation electrospray ionization mass spectrometry and conventional MS/MS were used to study in situ biofilm production and the production of lipopeptides fengycin and surfactin in different strains of B. mojavensis in plate and test tube culture on two media. All strains were capable of producing biofilm in vitro along with the accumulation of surfactin and fengycin although no concentration-dependent relationship between lipopeptide accumulation and biofilm was observed. CONCLUSION: All strains studied produce biofilms in culture with the accumulated surfactin and fengycin, demonstrating that endophytic bacteria also produced biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that this endophytic species produced biofilms along with two biocontrol compounds of which one, surfactin, considered by others as a quorum sensor, highlighting its ecological role as a signalling mechanism in planta.

Growth-inhibiting effects of concentrations of fusaric acid on the growth of Bacillus mojavensis and other biocontrol Bacillus species.

AIMS: To determine the effects of concentrations of fusaric acid on the growth of several strains of the biocontrol bacterial endophyte Bacillus mojavensis and other species within the Bacillus subtilis group, as well as the genetic relationships within this small group of Gram-positive bacteria, and their antagonisms to Fusarium verticillioides, which produce fusaric acid. METHODS AND RESULTS: The growth of 50 Bacillus strains and species were tested at two concentrations of fusaric acid determined in maize infected by an isolate of F. verticillioides. Molecular characterizations of the strains and species of bacteria were determined with an automated ribotyper. The growth of bacteria measured under both concentrations with an automated turbidometer, Bioscreen, indicated that fusaric acid was toxic to most strains of the bacterial endophyte B. mojavensis. However, the effects of these two concentrations on other Bacillus species varied in that fusaric acid was either bacteriocidal or bacteriostatic to most species. CONCLUSIONS: These data indicate that the concentrations of fusaric acid are inhibitory to the growth of most Bacillus species, some of which are used as biocontrol agents. This suggests that the endophytic and saprophytic states of F. verticillioides and other Fusarium species cannot be controlled by fusaric-acid-sensitive Bacillus species. SIGNIFICANCE AND IMPACT OF STUDY: Mycotoxic Fusarium species, such as F. verticillioides, are competitive because all produce fusaric acid, which is inhibitory to biocontrol bacteria, and mutants tolerant to fusaric acid must be developed in order to be effective on biocontrol bacteria.

Field performance of maize grown from Fusarium verticillioides-inoculated seed.

Fusarium verticillioides is an important fungus occupying dual roles in the maize plant. The fungus functions as an endophyte, a fungal/host interaction beneficial to the growth of some plants. At other times, the fungus may function as a mycotoxin producing pathogen. The advantages and/or disadvantages of the endophytic relationship must be established in order to target appropriate sites for controlling diseases and mycotoxins in maize. One possibility could be to ensure seed maize is fungal free prior to planting. Reciprocal inoculations were made with two fungal isolates on seed of two maize genotypes. Yield was measured at harvest by ear and seed characters and vegetative growth at one-month intervals for plant survival, height, weight and stem diameter. Yield and vegetative growth differed among mature plants only once based on seed inoculation status. In 1998, plant weight was reduced and seed weight per ear was increased for the dent maize, GT-MAS: gk, grown from F. verticillioides RRC 374-inoculated seed compared to other seed treatments. Most vegetative characters were reduced at the first collection for Silver Queen plants grown from F. verticillioides-inoculated seed in 1997 and 1999, but not in 1998. However, no significant differences occurred among mature Silver Queen plants during any of the three growing seasons. In conclusion, yield and vegetative growth of mature maize plants grown from F. verticillioides-inoculated seed were equal to or greater than plants grown from non-inoculated seed under south Georgia field conditions during 1997, 1998, and 1999.

Detoxification of corn antimicrobial compounds as the basis for isolating Fusarium verticillioides and some other Fusarium species from corn.

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.

Domain 1.1 of the sigma(70) subunit of Escherichia coli RNA polymerase modulates the formation of stable polymerase/promoter complexes.

The sigma 70 (sigma(70)) subunit of Escherichia coli RNA polymerase specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma(70) is present within polymerase holoenzyme, two of its domains, 2.4 and 4.2, interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma(70), DNA binding is prevented by domain 1.1, the N-terminal domain of the protein. Previous work has demonstrated that the presence of domain 1.1 is required for efficient transcription initiation at the lambda promoter P(R). To investigate whether this is a general property of domain 1.1, we have used five promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays, and in assays assessing the formation and decay of stable, pretranscription complexes. We find that the absence of domain 1.1 does not render the polymerase defective at all of these promoters. Depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerases containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma(70) recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences.

Biological control of Fusarium moniliforme in maize.

Fusarium moniliforme Sheldon, a biological species of the mating populations within the (italic)Gibberella fujikuroi species complex, i.e., population A [= G. moniliformis (Sheld.) Wineland], is an example of a facultative fungal endophyte. During the biotrophic endophytic association with maize, as well as during saprophytic growth, F. moniliforme produces the fumonisins. The fungus is transmitted vertically and horizontally to the next generation of plants via clonal infection of seeds and plant debris. Horizontal infection is the manner by which this fungus is spread contagiously and through which infection occurs from the outside that can be reduced by application of certain fungicides. The endophytic phase is vertically transmitted. This type infection is important because it is not controlled by seed applications of fungicides, and it remains the reservoir from which infection and toxin biosynthesis takes place in each generation of plants. Thus, vertical transmission of this fungus is just as important as horizontal transmission. A biological control system using an endophytic bacterium, Bacillus subtilis, has been developed that shows great promise for reducing mycotoxin accumulation during the endophytic (vertical transmission) growth phase. Because this bacterium occupies the identical ecological niche within the plant, it is considered an ecological homologue to F. moniliforme, and the inhibitory mechanism, regardless of the mode of action, operates on the competitive exclusion principle. In addition to this bacterium, an isolate of a species of the fungus Trichoderma shows promise in the postharvest control of the growth and toxin accumulation from F. moniliforme on corn in storage.

US FDA "Redbook II" immunotoxicity testing guidelines and research in immunotoxicity evaluations of food chemicals and new food proteins.

The rapid advances in the field of immunology and an understanding of the potential adverse effects of xenobiotics on the immune system have resulted in the development of a discipline in toxicology now referred to as immunotoxicology. This discipline has evolved steadily over the last 2 decades as a result of research in the national and international communities. Various US, European, and Japanese regulatory agencies have recognized a need to promulgate testing guidelines for immunotoxicity in support of the approval process involving toxicological testing. The US Food and Drug Administration "Redbook II" guidelines and some of the research conducted in support of the concepts and testing strategies are presented here. Concerns raised with regard to these guidelines are included, as are on-going initiatives in development of experimental approaches for assessing allergic potential and/or hypersensitivity responses to new foods and food constituents.

Effects of intermittent exposures of aflatoxin B(1) on hepatic and testicular glutathione S-transferase in rats.

Glutathione S-transferase (GST) plays a major role in the detoxification of the potent hepatocarcinogen aflatoxin B(1) (AFB(1)). This study evaluated the effects of intermittent exposures to AFB(1) on hepatic and testicular GST in rats. Male Fischer 344 rats were fed diets containing AFB(1) (0, 0.01, 0.04, 0.4 and 1.6 ppm) intermittently at 4-week intervals up to 20 weeks. The control animals were fed an AFB(1)-free NIH-31 diet. Rats consuming diets with 0.01 ppm AFB(1) did not show the induction of hepatic or testicular GST activity. Intermittent exposures to AFB(1) at concentrations of 0.04-1.6 ppm significantly increased the GST activities. The increase of the enzyme activity was proportional to the dose and length of AFB(1) exposure.

Efficient inhibition of Escherichia coli RNA polymerase by the bacteriophage T4 AsiA protein requires that AsiA binds first to free sigma70.

The bacteriophage T4 AsiA protein inhibits transcription from host and phage early promoters and is required, along with the T4 MotA protein, for activation of phage middle promoters. During infection, AsiA is found in a tight association with the sigma70 subunit of RNA polymerase. We show that AsiA binds rapidly to free sigma70 at either 4 degrees C or 30 degrees C to form an AsiA-sigma70 complex that with core efficiently reconstitutes the AsiA-inhibited RNA polymerase. In contrast, AsiA does not inhibit transcription after a 15 minute incubation with RNA polymerase holoenzyme at 4 degrees C, and at 30 degrees C an incubation of several minutes is required to inhibit most of the polymerase. We show that the heat step needed for AsiA is not the formation of an active AsiA protein. However, it is consistent with the momentary dissociation of holoenzyme to give free sigma70 and core. Our results indicate that AsiA is either unable to access holoenzyme directly or does so very slowly. Efficient generation of the AsiA-inhibited RNA polymerase requires that AsiA first binds to free sigma70 and then the AsiA-sigma70 complex binds to core to form the Asi-A-inhibited polymerase.

Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts.

During infection, the bacteriophage T4 transcriptional activator MotA, the co-activator AsiA, and host RNA polymerase are needed to transcribe from T4 middle promoters. Middle promoters contain a -10 region recognized by the sigma(70)subunit of RNA polymerase and a MotA box centered at -30 that is bound by MotA. We have investigated how the loss or modification of base determinants within the MotA box sequence 5'TTTGCTTTA3' (positions -34 to -26 of a middle promoter) affects MotA function. Gel retardation assays with mutant MotA boxes are consistent with the idea that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the C.G base-pair at position -30. In particular, the 5-methyl residue on the thymine residue at position -29, a major groove contact, contributes to MotA binding, while converting the T.A at -32 to a C. I base-pair, a change that affects the major but nor the minor groove, yields a MotA box that is similar to wild-type. However, methylation interference analyses indicate that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsXis inhibited by premethylation of guanine and adenine residues, suggesting that binding does not require minor groove contact with any specific T.A base-pair. Using gel retardation analyses, we calculate an apparent dissociation constant of 130 nM for MotA binding to the wild-type MotA box. Previous work has shown that the N-terminal region of MotA is needed for an interaction between MotA and sigma(70). We suggest that this MotA-sigma(70)interaction helps to stabilize the relatively weak interaction of MotA with the -30 region of middle promoter DNA.

Temporal patterns of DNA adduct formation and glutathione S-transferase activity in the testes of rats fed aflatoxin B1: a comparison with patterns in the liver.

Fisher-344 male rats were fed 1.6 ppm of aflatoxin B1 (AFB1) continuously and intermittently for several weeks. At various time periods, DNA was isolated from the testes and livers and analyzed for AFB1-DNA adducts. The ability of the testis to detoxify AFB1 was also investigated by the glutathione S-transferase (GST) activity assay and compared with that of the liver. The levels of testicular AFB1-DNA adducts were 2.4 to 8.1 times lower than those of the liver after 4 to 16 weeks of continuous treatment and 2.2 to 46.2 times lower after 8 to 20 weeks of intermittent treatment. The testicular DNA adducts markedly decreased over time. By 16 weeks of continuous and 20 weeks of intermittent exposure, they had decreased 37 and 91%, respectively. In contrast, hepatic AFB1-DNA adducts increased four-fold from 4 to 16 weeks of continuous treatment but increased at a much slower rate after intermittent exposure. In both the liver and testis, significant levels of AFB1-DNA adducts persisted for at least 1 month after ending the treatment, suggesting that this type of lesion was poorly repaired. In control rats, the testis showed significantly higher GST activity than the liver. In treated rats, these differences were significant during the first 12 weeks of continuous treatment but not at later times. Tissue-specific differences such as germ-cell depletion and increased testicular detoxification may play an important role in the observed differential pattern of DNA adduct formation between the testis and liver.

The bacteriophage T4 transcriptional activator MotA accepts various base-pair changes within its binding sequence.

During infection, bacteriophage T4 regulates three sets of genes: early, middle, and late. The host RNA polymerase is capable of transcribing early genes, but middle transcription requires the T4-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein, both of which bind to the sigma 70 (sigma70) subunit of RNA polymerase. MotA also binds a DNA sequence (a MotA box), centered at position -30. The identification of more than 20 middle promoters suggested that a strong match to the MotA box consensus sequence (t/a)(t/a)TGCTT(t/c)A was critical for MotA activation. We have investigated how specific base changes within the MotA box sequence affect MotA binding and activation in vitro, and we have identified seven new middle promoters in vivo. We find that an excellent match to the sigma70 -10 consensus sequence, rather than an excellent match to the MotA box consensus sequence, is an invariant feature of MotA-dependent promoters. Many single base changes in the MotA box are tolerated in binding and activation assays, indicating that there is more flexibility in the sequence requirements for MotA than was previously appreciated. We also find that using the natural T4 DNA, which contains glucosylated, 5-hydoxymethylated cytosine residues, affects the ability of particular MotA box sequences to activate transcription. We suggest that MotA and AsiA may function like certain eukaryotic TAFs (TATA binding protein (TBP) associated factors) whose binding to TBP results in transcription from new core promoter sequences.

Effects of Endophytic Infection by Fusarium moniliforme on Corn Growth and Cellular Morphology.

Kernels of corn, Zea mays, were inoculated with Fusarium moniliforme to analyze seedling growth and development during endophytic, symptomless infection. In planta F. moniliforme distribution and seedling growth, expressed as shoot diameter, plant height, leaf length, and dry weight, were examined weekly for 28 days after planting. Even though no visible disease symptoms developed, F. moniliforme was isolated from most segments taken from seedlings grown from inoculated, but not noninoculated, kernels from the earliest to the latest sampling. F. moniliforme did not alter the rate or percentage of kernel germination, but seedlings grown from inoculated kernels had suppressed shoot diameter, plant height, leaf length, and plant weight 7 days after planting. However, seedling growth from inoculated kernels was similar to or greater than that from noninoculated kernels at 28 days. Histological modifications in seedlings grown from inoculated kernels included accelerated lignin deposition in shoots and modified chloroplast orientation in leaves. In summary, gross morphology and histology were altered in corn seedlings during symptomless, endophytic infection by F. moniliforme.

An N-terminal mutation in the bacteriophage T4 motA gene yields a protein that binds DNA but is defective for activation of transcription.

The bacteriophage T4 MotA protein is a transcriptional activator of T4-modified host RNA polymerase and is required for activation of the middle class of T4 promoters. MotA alone binds to the -30 region of T4 middle promoters, a region that contains the MotA box consensus sequence [(t/a)(t/a)TGCTT(t/c)A]. We report the isolation and characterization of a protein designated Mot21, in which the first 8 codons of the wild-type motA sequence have been replaced with 11 different codons. In gel retardation assays, Mot21 and MotA bind DNA containing the T4 middle promoter P(uvsX) similarly, and the proteins yield similar footprints on P(uvsX). However, Mot21 is severely defective in the activation of transcription. On native protein gels, a new protein species is seen after incubation of the sigma70 subunit of RNA polymerase and wild-type MotA protein, suggesting a direct protein-protein contact between MotA and sigma70. Mot21 fails to form this complex, suggesting that this interaction is necessary for transcriptional activation and that the Mot21 defect arises because Mot21 cannot form this contact like the wild-type activator.

Characterization of pre-transcription complexes made at a bacteriophage T4 middle promoter: involvement of the T4 MotA activator and the T4 AsiA protein, a sigma 70 binding protein, in the formation of the open complex.

Bacteriophage T4 middle promoters have excellent matches to the -10 consensus sequence for the sigma 70 subunit of Escherichia coli RNA polymerase, but a binding site for the T4 transcriptional activator MotA replaces the sigma 70 -35 consensus. E. Coli RNA polymerase transcribes from middle promoters with or without the activator. In contrast, transcription by T4-modified E. coli RNA polymerase, which is present during T4 infection, requires NotA. We show that transcription by unmodified polymerase from the T4 middle promoter P uvsx is independent of the specific sequences within the -35 region, and the Dnase I footprint obtained with unmodified polymerase and P uvsx resembles those seen previously with E. coli extended -10" promoters. In contrast, although T4-modified polymerase alond binds P uvsx, promoter unwinding and detection of a Dnase I footprint requires MotA. This footprint is significantly different from that obtained with unmodified polymerase, starting upstream of around position -20. Previous work has indicated that the T4 AsiA protein, which binds tightly to sigma 70, is the phage modification required for MotA activation. We show that in the presence of AsiA, MotA, and otherwise unmodified polymerase, Dnase I protection of P uvsx is now similar to that obtained with the fully modified polymerase and MotA up to around position -40. However, protection upstream of -40 is still similar to that seen with unmodified polymerase. Our results support the idea that MotA-dependent activation requires AsiA binding to sigma 70 to achieve specific protein-DNA contacts within the -20 to -40 region of a middle promoter.

Fusaric acid and pathogenic interactions of corn and non-corn isolates of Fusarium moniliforme, a nonobligate pathogen of corn.

Fusarium moniliform is a nonobligate parasite of corn, which exists as a complex of closely related fungi from different mating population or biological species. Strains of this fungus isolated from corn, have been determined to belong to mating populations A, although other populations have been isolated from corn. The ultrastructural association of the fungus with corn during growth, and the effects of the host on suppression of disease suppression are reviewed. This fungus enters a relationship with corn cultivars that is not always pathogenic. Pathogenesis is delayed, if it ever occurs. F. moniliforme can exist entirely as an endophyte, systemically colonizing kernels, remaining there until germination upon which the fungus infects the emerging seedlings. The symptomless association persists during the growth cycle of corn, and the resulting endophytic hyphae may be the source of mycotoxin production. The host's ability to suppress the fungus appears to be related to one class of compounds, the cyclic hydroxamic acids and their decomposition products, which can be catabolized by the fungi of mating population A but not C.

Identification and characterization of bacterial endophytes of rice.

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.

The bacteriophage T4 middle promoter PuvsX: analysis of regions important for binding of the T4 transcriptional activator MotA and for activation of transcription.

Bacteriophage T4 middle promoters, which are transcribed using phage-modified host RNA polymerase and the T4 transcriptional activator, MotA, match the host sigma 70 consensus sequence at -10, but they have a different consensus ((t/a)(t/a)TGCTT(t/c)A) (a MotA box) at -30. While the T4 middle promoter PuvsX has these -10 and -30 motifs, it also has matches to the MotA box at -35, -51, -70, and -87. We show that MotA binds to PuvsX DNA, footprinting a region that includes the MotA boxes at -30, -35, and -51. Very high levels of MotA are required for footprinting and gel-shift experiments, and protein-DNA complexes formed in the presence of both phage-modified polymerase and MotA are more resistant to HindIII cleavage than those formed with either protein alone. These results suggest that MotA-DNA interactions may be stabilized by phage-modified polymerase. Sequences between -18 and -38 are absolutely required for MotA activation of transcription, but sequences upstream of -38 are stimulatory, particularly when chloride instead of glutamate is the major anion. Our results dissect PuvsX into a core promoter, downstream of -38, which is required for MotA activation, and an upstream region that enhances transcription especially under conditions less favourable for protein-DNA interactions.