Bacterial and fungal metabolites of rhizospheric microflora of cotton antagonistic to Rhizoctonia solani Kühn

Summary Growth response ofRhizoctonia solani Kühn, the damping-off fungus, to metabolites of selected antagonistic rhizospheric bacteria and fungi of some Egyptian cotton varieties, namely, two strains ofBacillus subtilis Cohn,Aspergillus terreus Thom, andAspergillus flavus Link produced in culture media containing nitrate- or ammonium-nitrogen sources, proved the potency ofB. subtilis metabolites in inhibitingR. solani mycelial growth whether from nitrate- or ammonium-nitrogen culture media. Metabolite filtrates ofB. subtilis (II) are more potent than those ofB. subtilis (I). Increasing concentration of bacterial metabolite filtrates resulted in a decreased mycelial dry weight ofR. solani. The bacterial inhibitory factor forR. solani mycelial growth is partially affected by heat. Metabolite filtrates ofA. terreus from nitrate-nitrogen are slightly more potent than from ammonium-nitrogen culture media while an opposite relation is evident withA. flavus metabolites. Growth responses ofR. solani to different experimental dilutions of metabolite filtrates ofA. terreus andA. flavus proved the intervention of the nutritive factor in witholding growth of the damping-off fungus.     

Variations in the cultural characteristics of Rhizoctonia solani,and its antagonists: Aspergillus terreus and Aspergillus flavus occurring in the rhizosphere of cotton

Summary Cultural studies onRhizoctonia solani, the causal agent of damping-off of cotton, as well as on two fungi of its antagonistic rhizospheric microflora, namelyAspergillus terreus andAspergillus flavus have shown coincidence of some of their cultural characteristics. However,R. solani produced mycelial growth far ahead both antagonists except at 37° C and at pH 4, at its optimum temperature. It is expected that for a successful biological control ofR. solani in the soil,A. terreus is applied at a soil-temperatured above 35° C in an acid medium.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

Formulation of the Biocontrol FungusCladorrhinum foecundissimumto Reduce Damping-Off Diseases Caused byRhizoctonia solaniandPythium ultimum

Five isolates ofCladorrhinum foecundissimum, added to soilless mix as 10-day-old fresh bran preparations (1.0% w/w), significantly reduced (P≤ 0.05) damping-off of eggplant and pepper caused byRhizoctonia solanistrain R-23. After 4 weeks of growth, plant stands in the biocontrol-amended, pathogen-infested treatments (>80%) were comparable to those in the noninfested controls. Since plant stands were similar at 2 and 4 weeks, most of the disease was preemergence damping-off. The bran preparations also reduced saprophytic growth of the pathogen, and there was an inverse correlation (r²= −0.94) between saprophytic growth and eggplant stand. Added to soilless mix at a rate of 2.0% (w/w), alginate prill containing 20% fermentor-produced biomass of six biocontrol isolates ofC. foecundissimumreduced (P≤ 0.05) damping-off of eggplant caused byR. solani, but only the prill with biomass of isolates Cf-1 or Cf-2 yielded plant stands (>80%) comparable to that in the noninfested control. As with the bran preparations, there was also an inverse correlation (r²= −0.80) between saprophytic growth of R-23 and eggplant stand with the alginate prills. Alginate prill with biomass of Cf-1 or Cf-2 also reduced (P≤ 0.05) damping-off of eggplant and pepper caused by other isolates (195, NG-2, DPR-1) ofR. solani, but only the stands (>80%) of pepper were similar to that in the noninfested control. Alginate prill formulations ofC. foecundissimum(Cf-1, Cf-2, and Cf-3) also reduced (P≤ 0.05) populations of the pathogen and damping-off of eggplant and pepper caused byPythium ultimum(PuZ3). However, although the plant stands in the treatments were not as high as those in the noninfested controls, they were higher than those in the pathogen-infested controls. The treatments also reduced populations ofP. ultimumin the soilless mix so that there were inverse correlations between the pathogen population and eggplant stand (r²= −0.81) and pepper stand (r²= −0.78). Extruded flour/clay granules containing 5.0% biomass of Cf-1 and Cf-2, added toR. solani-infested soilless mix (2.0%), reduced (P≤ 0.05) damping-off of eggplant and pepper. However, only the Cf-2 treatments resulted in stands (>80%) equal to those in the noninfested controls for the crops after 4 weeks of growth. The influence of bran and alginate prill of Cf-1 or Cf-2 on the spatial spread ofR. solaniand its ability to incite damping-off of eggplant showed that prill with Cf-1 or Cf-2 and bran with Cf-2 were equally effective in reducing the spread of the pathogen from the point source of the inoculum to the center of the flats.     

Acetophenone derivatives from Chilean isolate of Trichoderma pseudokoningii Rifai

The novel acetophenone derivative 2′,4′-dihydroxy-3′-methoxymethyl-5′-methylacetophenone and the known 2′,4′-dihydroxy-3′,5′-dimethylacetophenone (clavatol) were isolated from the culture filtrate of a Chilean strain of Trichoderma pseudokoningii. This revised version was published online in July 2006 with corrections to the Cover Date.     

Zerumbone: a potential fungitoxic agent isolated from Zingiber cassumunar Roxb.

The rhizomes of Zingiber cassumunar exhibited strong fungitoxic action against Rhizoctonia solani, the damping-off pathogen. On chemical and spectral investigations, the antifungal compound was found to be zerumbone — a sesquiterpene. Its minimum effective dose against R. solani was 1000 ppm, much lower than some commercial fungicides. Zerumbone had fungistatic activity, a narrow fungitoxic spectrum and was not phytotoxic. Moreover, when used as a seed treatment, zerumbone could control damping-off disease of Phaseolus aureus caused by Rhizoctonia solani by 85.7%.     

The role of rhizosphere bacteria in herbicide-mediated increase in Rhizoctonia solani-induced cotton seedling damping-off

A study was carried out to determine the role of rhizosphere-residing bacteria in the observed pendimethalin- and prometryn-mediated increase in cotton seedling damping-off incidence caused by Rhizoctonia solani. Judging from the results, pendimethalin-mediated increase in disease incidence may be due to a decrease in the populations of indigenous cotton root colonizing bacteria in the presence of pendimethalin. Prometryn-mediated increase in disease may be due to a decrease in the populations of indigenous root colonizing bacteria as well as increased susceptibility of cotton seedlings to R. solani infection in the presence of prometryn. Pendimethalin and prometryn neither affected the growth of R. solani nor caused any visible change in seedling physical characteristics.     

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N′ -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.     

Clavatol and patulin formation as the antagonistic principle of <Emphasis Type="Italic">Aspergillus clavatonanicus</Emphasis>, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2′,4′-dihydroxy-3′,5′-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.     

Flavonoids of Helichrysum chasmolycicum and its antioxidant and antimicrobial activities

From the aerial parts of Helichrysum chasmolycicum P.H Davis, which is an endemic species in Turkey, the flavonoids apigenin, luteolin, kaempferol, 3,5-dihydroxy-6,7,8-trimethoxyflavone, 3,5-dihydroxy-6,7,8,4′-tetramethoxyflavone, apigenin 7-O-glucoside, apigenin 4′-O-glucoside, luteolin 4′-O-glucoside, luteolin 4′,7-O-diglucoside, kaempferol 3-O-glucoside, kaempferol 7-O-glucoside and quercetin 3-O-glucoside were isolated. The methanol extract of the aerial parts of H. chasmolycicum showed antioxidant activity by DPPH method (IC₅₀ 0.92 mg/mL). Antimicrobial activity test was performed on the B, D, E extracts and also 3,5-dihydroxy-6,7,8-trimethoxyflavone and kaempferol 3-O-glucoside which were the major flavonoid compounds obtained from aerial parts of H. chasmolycicum by microbroth dilutions technique. The E (ethanol-ethyl acetate) extract showed moderate antimicrobial activity against Pseudomonas aeruginosa, B (petroleum ether-60% ethanol-chloroform) extract and 3,5-dihydroxy-6,7,8-trimethoxyflavone showed moderate antifungal activity against Candida albicans.     

Isolation of linobiflavonoid, a novel biflavonoid from Linostoma pauciflorum Griff.

A novel biflavonoid, that we have named linobiflavonoid, and the known biscoumarin ether, daphnoretin, were isolated from the root extracts of Linostoma pauciflorum Griff. The structure of linobiflavonoid was determined from interpretation of its NMR spectroscopic data and from a comparison of this data with those of known biflavonoids and biflavones. The known flavones, 5,4′-dihydroxy-7,3′,5′-trimethoxyflavone and 5,4′-dihydroxy-7-methoxyflavone along with stigmasterol were isolated from the vines of the same plant. 4′-Dihydroxy-7,3′,5′-trimethoxyflavone was active against Mycobacterium tuberculosis (MIC 3.13 μM) and KB-oral cavity cancer (IC₅₀ 17.41 μM).     

Exudation of terpenoids by cotton roots

Summary The terpenoid aldehydes, desoxyhemigossypol, desoxy-6-methoxyhemigossypol, hemigossypol, 6-methoxyhemigossypol, gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol, previously reported to be present in cotton roots (Gossypium hivsutum L.) were found on absorbing surfaces adjacent to roots. Infection of hypocotyls byRhizoctonia solani increased the quantity of terpenoids exuded by roots. Because these compounds are antimicrobial, their presence in the rhizosphere should be considered in studies of the microflora of cotton roots.     

Cloning,expression, isolation,and properties of thymidine kinase from herpes simplex virus type 1, strain L2

Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides, particularly 3′-deoxythymidine, 3′-deoxy-2′,3′-didehydrothymidine, 2′,3′-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine, E-5-(2-bromovinyl-2′-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2′,3′-dideoxydehydrothymidine, β-L-2′,3′-dideoxy-3′-thiacytidine, and 3′-fluoro-3′-deoxythymidine. Some properties of the purified enzyme were compared with those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the enzyme.     

Extruded Granular Formulation with Biomass of Biocontrol Gliocladium virens and Trichoderma spp. to Reduce Damping-off of Eggplant Caused by Rhizoctonia solani and Saprophytic Growth of the Pathogen in Soil-less Mix

Extruded granular formulations containing rice flour, gluten, Pyrax, vermiculite, canola oil, and fermentor-produced biomass of isolates of Gliocladium virens (Gl-3, Gl-21 and Gl-32), Trichoderma hamatum (TRI-4 and 31-3), T. harzianum (Th-32 and Th-87) and T. viride (Tv-101) were evaluated for their effect on the reduction of eggplant damping-off caused by Rhizoctonia solani, reduction of pathogen inoculum and proliferation of the isolates in a soil-less mix. Granules with all isolates except 31-3 significantly (P < 0.01) reduced damping-off, and granules with Gl-3, Gl-21, Gl-32, TRI-4 and Th-87 yielded stands comparable to that (90%) of the non-infested control. Granules with isolates Gl-21 and TRI-4 were the most effective in the reduction of saprophytic growth of R. solani, and there was a significant inverse correlation (r ² = - 0.82) between eggplant stand and saprophytic growth of the pathogen over all treatments. Isolate propagules proliferated to about 10⁷ colony-forming units (CFU) g^?1 of soil-less mix after a 6-week incubation, but there was no correlation between the number of CFU and eggplant stand or saprophytic growth reduction of the pathogen. Granules with Gl-21 and TRI-4 amended to pathogen-infested soil-less mix at a rate as low as 0.06% significantly (P < 0.05) reduced damping-off and pathogen saprophytic growth, and a rate of 0.25% of Gl-21 granules resulted in an eggplant stand comparable to that of the non-infested control. There was no significant correlation between the rate of granule amendment and the proliferation of Gl-21 and TRI-4. Granules of Gl-21 and TRI-4 also significantly prevented the spread of R. solani in flats of eggplant seedlings when the biocontrol granules were applied to the soil-less mix 1 day before the pathogen inoculum.     

5,8-Dihydroxy-3,6,7-trimethoxyflavone from Gnaphalium gaudichaudianum

5,8-Dihydroxy-3,6,7-trimethoxyflavone and 5,8-dihydroxy-6,7-dimethoxyflavone were isolated from aerial parts of Gnaphalium gaudichaudianum and identified by spectral data.     

Quinoline alkaloids as intercalative topoisomerase inhibitors

Quinoline alkaloids are abundant in the Rutaceae, and many have exhibited cytotoxic activity. Because structurally related antitumor alkaloids such as camptothecin and fagaronine are known to function as intercalative topoisomerase poisons, it is hypothesized that cytotoxic Stauranthus alkaloids may also serve as intercalative topoisomerase inhibitors. To test this hypothesis theoretically, ten Stauranthus quinoline alkaloids were examined for potential intercalation into DNA using a molecular docking approach. Four of the alkaloids (stauranthine, skimmianine, 3′,6′-dihydroxy-3′,6′-dihydrostauranthine, and trans-3′,4′-dihydroxy-3′,4′-dihydrostauranthine) were able to intercalatively dock consistently into DNA. In order to probe the intermolecular interactions that may be responsible for intercalation of these quinoline alkaloids, density functional calculations have been carried out using both the B3LYP and M06 functionals. M06 calculations indicated favorable π–π interactions between either skimmianine or stauranthine and the guanine–cytosine base pair. Furthermore, the lowest-energy face-to-face orientation of stauranthine with guanine is consistent with favorable dipole–dipole orientations, favorable electrostatic interactions, and favorable frontier molecular orbital interactions. Likewise, the lowest-energy face-to-face orientation of stauranthine with the guanine–cytosine base pair reveals favorable electrostatic interactions as well as frontier molecular orbital interactions. Thus, not only can quinoline alkaloids dock intercalatively into DNA, but the docked orientations are also electronically favorable.      

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Splitting of the cyclic 3′, 5′-adenosine monophosphate in cell-free system ofEscherichia coli

Broken cells ofEscherichia coli contain an enzyme system breaking down cyclic 3′,5′-adenosine monophosphate (Ado-3′,5′-P). The enzyme splitting this nucleotide is located in the supernatant fraction at 20,000 ×g. Some characteristics of the enzyme were studied. In contrast with the animal enzyme theEscherichia coli enzyme is not inhibited by caffeine.     

Biological Control of Phoma and Pythium Damping-Off of Sugar-Beet with Pythium oligandrum

Flooding freshly harvested oospores in sterile distilled water (SDW) for several days enhanced germination in 3 out of 4 isolates of Phythium oligandrum. Treatment of SDW-flooded oospores with myo-inositol increased germinability during the first 20 days of storage at 15°C. Seed dressing with oospores of P. oligandrum controlled pre- and post-emergence damping-off of sugar-beet caused by soil-borne P. ultimum and seed-borne Phoma betae. For some isolates, flooded oospores in SDW and treatment with myo-inositol increased efficacy of the seed dressing. However, no significant control of damping-off caused by Rhizoctonia solani was observed. On corn-meal agar, P. oligandrum coiled around and penetrated hyphae of P. ultimum and R. solani, but did not interfere with Ph. betae.