Secondary Metabolites with Herbicidal and Antifungal Activities from Marine-Derived Fungus Alternaria iridiaustralis.

Weed and soil-borne pathogens could synergistically affect vegetable growth and result in serious losses. Investigation of agricultural bioactive metabolites from marine-derived fungus Alternaria iridiaustralis yielded polyketides (1-4), benzopyrones (5-7), meroterpenoid derivatives (8), and alkaloid (9). The structures and absolute configurations of new 1, 3, 5-6, and 8 were elucidated by extensive spectroscopic analyses, as well as comparisons between measured and calculated ECD and 13C NMR data. Compounds 1-4, 6, and 9 showed herbicidal potentials against the radicle growth of Echinochloa crusgalli seedlings. Especially 9 exhibited inhibition rates over 90% at concentrations of 20 and 40 µg/mL, even better than the commonly used chemical herbicide acetochlor. Furthermore, 9 also performed a wide herbicidal spectrum against the malignant weeds Digitaria sanguinalis, Portulaca oleracea, and Descurainia sophia. Compounds 5-8 showed antifungal activities against carbendazim-resistant strains of Botrytis cinerea, with minimum inhibitory concentration (MIC) values ranging from 32 to 128 µg/mL, which were better than those of carbendazim (MIC = 256 µg/mL). Especially 6 exhibited integrated effects against both soil-borne pathogens and weed. Overall, marine-derived fungus A. iridiaustralis, which produces herbicidal and antifungal metabolites 1-9, showed the potential for use as a microbial pesticide to control both weed and soil-borne pathogens.

Streptomyces sp. FX13 inhibits fungicide-resistant Botrytis cinerea in vitro and in vivo by producing oligomycin A.

Botrytis cinerea is one of the most destructive fungal pathogens which can cause gray mold diseases of numerous plant species, while the frequent applications of fungicides also result in the fungicide-resistances of B. cinerea. In this study, a new Streptomyces strain FX13 was obtained to show biocontrol potentials against fungicide-resistant B. cinerea B3-4. Its in vitro and in vivo antifungal mechanisms were further investigated. The results showed that the culture extract of strain FX13 could significantly inhibit the mycelia growth of B. cinerea B3-4 with the EC50 value of 5.40 mg L-1, which was greatly lower than those of pyrisoxazole, boscalid and azoxystrobin. Further bioassay-guided isolation of the extract had yielded the antifungal component SA1, which was elucidated as a 26-membered polyene macrolide of oligomycin A. SA1 could inhibit the mycelia growth, spore germination, germ tube elongation and sporogenesis of B. cinerea B3-4 in vitro, and also showed significant curative and protective effects against gray mold on grapes in vivo. Moreover, SA1 could result in the loss of membrane integrity and the leakage of cytoplasmic contents, which might be related to the accumulation of reactive oxygen species (ROS) and membrane lipid peroxidation. Besides, intracellular adenosine triphosphatase (ATPase) activity and adenosine triphosphate (ATP) content of B. cinerea B3-4 decreased after SA1-treatment. Overall, the oligomycin A-producing strain FX13 could inhibit fungicide-resistant B. cinerea B3-4 in vitro and in vivo, also highlighting its biocontrol potential against gray mold.

Synthesis and antifungal activity of 2-allylphenol derivatives against fungal plant pathogens.

2-Allylphenol (2-AP) is an effective fungicide against a number of plant pathogens, which can be metabolized and bio-transformed to four chemical compounds by Rhizoctonia cerealis. To determine if its degradation affects antifungal activity, two major metabolites derived from 2-AP including 2-(2-hydroxypropyl) phenol and 2-(3-hydroxypropyl) phenol were synthesized. Inhibition of mycelial growth of several plant pathogens by the metabolites was evaluated, and structures of two metabolites were determined by hydrogen nuclear magnetic resonance (1H NMR). Among these metabolites, only 2-(2-hydroxypropyl) phenol inhibited test pathogens effectively. EC50 values of 2-(2-hydroxypropyl) phenol for inhibition of mycelial growth of R. cerealis, Pythium aphanidermatum, Valsa mali and Botrytis cinerea ranged from 1.0 to 23.5µg/ml, which were lower than the parental fungicide 2-AP that ranged from 8.2 to 48.8µg/ml. Hyphae of R. cerealis and P. aphanidermatum treated with 2-(2-hydroxypropyl) phenol were twisted. Newly developed hyphae were slender, twisted and swollen on the tip, while old hyphae were hollow and ruptured. This is the first report indicating the formation of 2-(2-hydroxypropyl) phenol may have contributed to toxicity of 2-allylphenol in control of plant pathogens.

Translocation of Oxathiapiprolin in Bell Pepper Plants and Systemic Protection of Plants Against Phytophthora Blight.

Production of bell pepper is seriously affected by Phytophthora capsici, the causal agent of Phytophthora blight. Limited approaches are available for effective management of the disease. Oxathiapiprolin is a fungicide recently registered in the United States that suppressed P. capsici and reduced Phytophthora blight on bell pepper significantly in our previous studies. It is unknown whether oxathiapiprolin translocates in bell pepper plants systemically after application. Experiments were conducted to determine uptake of oxathiapiprolin by bell pepper plants and its systemic movement in the plant. Quantification of oxathiapiprolin in plant tissues was conducted by high-performance liquid chromatography (HPLC) that detected the compound sensitively and selectively. Percentage of recovery of oxathiapiprolin from plant tissues was calculated by comparing the quantities in plant tissues determined by HPLC with known quantities of the compound added to the plant tissues. Recovery rates of oxathiapiprolin from pepper plant tissues ranged from 87.0 to 119.3%. When oxathiapiprolin was applied to roots of bell pepper plants grown in hydroculture, the compound was detected in the root within 4 h and in the cotyledon, first true leaf, and second true leaf within 8 h. It was detectable in the top new leaf 48 h after application to the root. In greenhouse studies with bell pepper plants grown in pots, oxathiapiprolin was applied as a soil drench at 100 and 400 µg/ml. The compound was detected in the root within 3 days and in the stem and first true leaf within 6 days when applied at 100 µg/ml. It was detected in the root, stem, first true leaf, and top new leaf within 3 days when applied at 400 µg/ml. Phytophthora blight on pepper foliage was significantly reduced when oxathiapiprolin was applied as a soil drench at 100 or 400 µg/ml under greenhouse conditions. This is the first report indicating systemic movement of oxathiapiprolin in pepper plants that provides useful information for designing fungicide application programs for effective management of the disease.

Sensitivity of Phytophthora nicotianae From Tobacco to Fluopicolide, Mandipropamid, and Oxathiapiprolin.

Black shank incited by Phytophthora nicotianae is a devastating disease in the production of tobacco. Fungicides have been commonly used for managing the disease; however, there is only a narrow pool of effective fungicides. A few new fungicides became available in recent years, including fluopicolide, mandipropamid, and oxathiapiprolin, which reduced diseases incited by oomycetes under field conditions. Limited information is available regarding sensitivity of P. nicotianae isolates to these new fungicides. Research was conducted to determine effects of the three new fungicides on P. nicotianae isolates from tobacco in Georgia. Studies with 106 isolates indicated that they did not grow when agar medium was amended with the fungicides at the rate of 1 µg/ml. Twenty isolates were used for in vitro studies to determine sensitivity to the fungicides. Fluopicolide, mandipropamid, and oxathiapiprolin inhibited mycelial growth of the isolates with mean EC50 values (effective concentrations that provide 50% growth reduction) of 0.09, 0.04, and 0.001 µg/ml, respectively. EC50 values of fluopicolide, mandipropamid, and oxathiapiprolin for inhibiting sporangial formation were 0.15, 0.03, and 0.0002 µg/ml, respectively. EC50 values for suppressing zoospore germination averaged 0.16, 0.04, and 0.002 µg/ml for fluopicolide, mandipropamid, and oxathiapiprolin, respectively. Results from the study indicated that P. nicotianae isolates from tobacco in Georgia were sensitive to the fungicides, with lower EC50 for oxathiapiprolin than for fluopicolide and mandipropamid. The information on effectiveness and baseline sensitivity of fungicides on P. nicotianae will facilitate monitoring of resistance development in the pathogen population.

Metabolism of fungicide 2-allylphenol in Rhizoctonia cerealis.

2-Allylphenol is a biomimetic synthetic fungicide that mimics the compound ginkgol found in gingko fruit (Gingko biloba L.). This systemic fungicide can effectively suppress a wide range of plant diseases, including wheat sharp eyespot (Rhizoctonia cerealis). However, its degradation in environment after application is still unknown. To understand this fungicide degradation, major metabolites of 2-allylphenol in R. cerealis were examined. The parent and metabolites of 2-allylphenol were detected and quantified in the mycelia and liquid medium. Results showed that 2-allylphenol was metabolized and bio-transformed by R. cerealis, and four metabolites were found, including 2-(2-hydroxyphenyl) acetic acid (M1), 2-(2, 3-dihydroxypropyl) phenol (M2), 2-(2-hydroxypropyl)-phenol (M3) and 2-(3-hydroxypropyl)-phenol (M4). Based on the results, we propose that the biodegradation pathway is that 2-allylphenol is rapidly oxidized into metabolite M2 and hydrolyzed into M3 and M4, which formed M2, and carboxylation of M2 to 2-hydroxy-3-(2׳-hydroxyphenyl) propionic acid which undergo hydrolyzation and decarboxylation to form M1. 2-Allylphenol can be bio-transformed to new compounds by R. cerealis, suggesting the existence of microbe metabolic pathways for 2-allylphenol.

Improved formulation and lyophilization cycle for rBCG vaccine.

To improve the conventional BCG vaccine in cake appearance and integrity, a new formulation with corresponding freeze drying cycle was developed for a recombinant BCG vaccine. The new formulation contains mannitol as a bulking agent, and trehalose, sucrose and sodium glutamate as stabilizers. The formulation and freeze drying cycle were tested with different super cooling rates and secondary drying temperatures, with or without an annealing process. Thermodynamic behavior was characterized using differential scanning calorimetry (DSC). Varying the secondary drying temperature and presence/absence of an annealing step caused marked differences in cake thermodynamic profiles irrespective of different cooling rates. The annealing process allowed efficient crystallization of the mannitol. Failure to crystallize the bulking agent had the potential to depress the Tg' and compromise storage stability in the final lyophile by crystallizing from the solid during storage, even when the secondary drying temperature was as high as 40°C. The improved formulation and freeze drying cycle resulted in good recovery of 53.2% during lyophilization and a higher survival rate of 61.7% in an accelerated stability study than the conventional BCG formulation and cycle. In summary, full crystallization was necessary for the mannitol bulking formulation. The freeze dried rBCG vials obtained using the formulation and drying cycle developed here met the requirements of BCG vaccine in good cake appearance, high viability post freeze drying and heat stability during storage.

Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes.

OBJECTIVE: It is well documented that natural killer (NK) cells provide host defense against tumors and viruses. We previously showed that lifestyle affects human NK and LAK activities. In order to explore the underlying mechanism, we investigated the effect of lifestyle on intracellular perforin, granulysin, and granzymes A/B in peripheral blood lymphocytes (PBL). METHODS: 114 healthy male subjects, aged 20-59 years, from a large company in Osaka, Japan were selected with informed consent. The subjects were divided into groups reporting good, moderate, and poor lifestyles according to their responses on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress). Peripheral blood was taken, and numbers of NK, T, perforin, granulysin, and granzymes A/B-expressing cells in PBL were measured by flow cytometry. RESULTS: Subjects with good or moderate lifestyle showed significantly higher numbers of NK, and perforin, granulysin, and granzymes A/B-expressing cells and a significantly lower number of T cells in PBL than subjects with poor lifestyle. Among the eight health practices, cigarette smoking, physical exercise, eating breakfast, and balanced nutrition significantly affect the numbers of NK, T cells, perforin, granulysin, and/or granzymes A/B-expressing cells, and alcohol consumption significantly affects the number of granzyme A-expressing cells. On the other hand, mental stress, sleeping, and working hours had no effect on those parameters. CONCLUSIONS: Taken together, these findings indicate that poor lifestyle significantly decreases the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells in PBL.

X-ray repair cross-complementing group 1 polymorphisms and cancer risks in Asian populations: a mini review.

X-ray repair cross-complementing group 1 (XRCC1) is an important DNA repair protein. Arg194Trp, Arg280His, and Arg399Gln are three polymorphisms of XRCC1 that commonly exist in human. In this context, we obtained the relevant articles through a PubMed search and examined the association of XRCC1 polymorphisms and the risk of cancer in Asian populations. Generally, a single XRCC1 polymorphism is weakly associated with cancer in Asian populations. However, when combined with other genetic polymorphisms or such lifestyle factors as smoking, XRCC1 polymorphisms show a stronger association with the risk of cancer. The interaction of the 399Gln/Gln genotype and smoking might be associated with a three-fold increase in the risk of cancer. In this paper we provide some important information for practical future cancer prevention programs. To further clarify the association of XRCC1 polymorphisms and cancer risks, additional studies are required from the perspectives of epidemiology and in vitro.

Micronuclei in EM9 cells expressing polymorphic forms of human XRCC1.

X-ray repair cross-complementing gene 1 (XRCC1) is involved in base excision repair (BER) through interaction with other BER enzymes, and polymorphisms in XRCC1 appear to increase the risk of various cancers. We evaluated how three XRCC1 polymorphisms, Arg194Trp, Arg280His and Arg399Gln, affect the extent of DNA damage and repair using the micronucleus assay. XRCC1 cDNAs containing the wild-type sequence and the three polymorphisms were overexpressed in EM9 cells, which lack the full sequence needed to perform XRCC1 functions. Normal human XRCC1 cDNA corrected the defect in EM9 cells. Only XRCC1 cDNA containing the Arg399Gln polymorphism did not fully correct the DNA repair defect in EM9 cells. These results indicate that the Arg399Gln polymorphism, but not the Arg194Trp or Arg280His polymorphism, influences the ability of XRCC1 to repair DNA. This study may provide a model that can be used to evaluate the functional significance of polymorphisms in DNA repair genes.