Non-volatile Antibiotics Research Articles

Selinene Volatiles Are Essential Precursors for Maize Defense Promoting Fungal Pathogen Resistance.

To ensure food security, maize (Zea mays) is a model crop for understanding useful traits underlying stress resistance. In contrast to foliar biochemicals, root defenses limiting the spread of disease remain poorly described. To better understand belowground defenses in the field, we performed root metabolomic profiling and uncovered unexpectedly high levels of the sesquiterpene volatile β-selinene and the corresponding nonvolatile antibiotic derivative β-costic acid. The application of metabolite-based quantitative trait locus mapping using biparental populations, genome-wide association studies, and near-isogenic lines enabled the identification of terpene synthase21 (ZmTps21) on chromosome 9 as a β-costic acid pathway candidate gene. Numerous closely examined β-costic acid-deficient inbred lines were found to harbor Zmtps21 pseudogenes lacking conserved motifs required for farnesyl diphosphate cyclase activity. For biochemical validation, a full-length ZmTps21 was cloned, heterologously expressed in Escherichia coli, and demonstrated to cyclize farnesyl diphosphate, yielding β-selinene as the dominant product. Consistent with microbial defense pathways, ZmTps21 transcripts strongly accumulate following fungal elicitation. Challenged field roots containing functional ZmTps21 alleles displayed β-costic acid levels over 100 μg g-1 fresh weight, greatly exceeding in vitro concentrations required to inhibit the growth of five different fungal pathogens and rootworm larvae (Diabrotica balteata). In vivo disease resistance assays, using ZmTps21 and Zmtps21 near-isogenic lines, further support the endogenous antifungal role of selinene-derived metabolites. Involved in the biosynthesis of nonvolatile antibiotics, ZmTps21 exists as a useful gene for germplasm improvement programs targeting optimized biotic stress resistance.

The role of volatile and non-volatile antibiotics produced by Pseudomonas chlororaphis strain PA23 in its root colonization and control of Sclerotinia sclerotiorum

Pseudomonas chlororaphis strain PA23 has demonstrated excellent biocontrol in the canola phyllosphere. This bacterium produces the non-volatile antibiotics phenazine and pyrrolnitrin as well as the volatile antibiotics nonanal, benzothiazole and 2-ethyl-1-hexanol. In vitro experiments were conducted to study the effects of different mutations on the production of these three organic volatile antibiotics by PA23. In planta experiments in the greenhouse investigated the role of the non-volatile antibiotics on root colonization and biocontrol ability of PA23 against Sclerotinia sclerotiorum on sunflower. Analysis of phenazine- and pyrrolnitrin-deficient Tn mutants of PA23 revealed no differences in production of the three volatile antibiotics. On all sampling dates, PA23 applied alone or in combination with the mutants showed significantly higher (P = 0.05) root bacterial number and Sclerotinia wilt suppression (P = 0.05). Decline of the bacterial population seemed to be inversely proportional to/or negatively correlated with the number of antibiotics produced by PA23 but the relative importance of phenazine or pyrrolnitrin on root colonization and/or wilt suppression was not clear. In several cases, the strains producing at least one antibiotic maintained relatively higher bacterial numbers than non-producing strains. However, by 6 weeks after sowing, there was a rapid and significant (P = 0.05) increase in the proportion of introduced bacteria capable of producing at least one antibiotic over the total bacterial population. Furthermore, combining certain mutants with PA23 reduced the root colonization and biocontrol ability of PA23. Strain PA23-314 (gacS mutant) showed competitive colonization in comparison to the other mutants for most sampling dates.

Evaluation of Fungal Antagonists to Control Damping-Off of Tomato ( Lycopersicon esculentum Mill.) caused by Pythium indicum

Trichoderma viride , T. harzianum , Laetisaria arvalis and other eight isolates of Trichoderma viride , isolated locally. were tested in vitro for their antagonistic activity against Pythium indicum . the causal organism of danlping-off of tomato, by dual agar technique. Among the fungal antagonists tested, Trichoderma viride was found highly inhibitory to P . indicllm. Volatile and non-volatile antibiotics of T . viride inhibited the in vitro growth of P . indicum significantly.

Control of fusarium wilt of Solanum melongena by Trichoderma spp.

Biological control of wilt of egg plant (Solanum melongena L.) caused by Fusarium solani was made with the application of five Trichoderma species, T. harzianum, T. viride, T. lignorum, T. hamatum and T. reesei. The effect of volatile and non-volatile antibiotics of Trichoderma origin on growth inhibition of the wilt pathogen was studied. T. harzianum showed maximum growth inhibition (86.44 %) of the pathogen through mycoparasitism. The non-volatiles produced by the Trichoderma species exhibited 100 % growth inhibition of the pathogen under in vitro condition. Production of siderophores and fungal cell wall degrading enzymes, chitinase and β-1,3-glucanase were found. Treatments with two most efficient Trichoderma species, T. harzianum and T. viride resulted in the decreasing population of Fusarium solani in soil thereby deterring disease incidence in field condition.

Characterization of Antagonistic Potential of Trichoderma Spp. against some Soil Borne Plant Pathogens

Ten isolates of Trichoderma spp. isolated from rhizosphere of different crops were evaluated for their antagonistic potential against five soil borne plant pathogens, viz., Rliizoctonia solani, Fusarium oxysporum f sp radicis-lycopersici, Macrophomina phaseolina and Sclerotium rolfsii using dual culture technique and production of volatile and non-volatile antibiotics. Sclerotial antagonism by the biocontrol strains was tested with R. solani and S. rolfsii . The isolate T 2 , T 4 and T 7 against Pythium sp., T 1 , T 7 and T 10 against F. oxysporum f. sp radicislycopersici, T 3 and T 9 against R. solani , T 7 against S. rolfsii and T 6 against M. phaseolina were the most efficient. The highest percentage inhibition of respective pathogen through the production of certain metabolites of Trichoderma isolates was recorded with T 7 against Pythium sp, and Fusarium oxysporum sp. radicis-lycopersici , T 1 against R. solani , T 5 and T 6 against S. rolfsii and T 9 isolate against M. phaseolina . Trichoderma isolate T 9 and T 2 were most effective in inhibition of sclerotia formation, production and germination of sclerotia of R. solani and S. rolfsii , respectively.

A GacS deficiency does not affectPseudomonas chlororaphisPA23 fitness when growing on canola, in aged batch culture or as a biofilm

Pseudomonas chlororaphis PA23 is a biocontrol agent that protects against the fungal pathogen Sclerotinia sclerotiorum. Employing transposon mutagenesis, we isolated a gacS mutant that no longer exhibited antifungal activity. Pseudomonas chlororaphis PA23 was previously reported to produce the nonvolatile antibiotics phenazine 1-carboxylic acid and 2-hydroxyphenazine. We report here that PA23 produces additional compounds, including protease, lipase, hydrogen cyanide, and siderophores, that may contribute to its biocontrol ability. In the gacS mutant background, generation of these products was markedly reduced or delayed with the exception of siderophores, which were elevated. Not surprisingly, this mutant was unable to protect canola from disease incited by S. sclerotiorum. The gacS mutant was able to sustain itself in the canola phyllosphere, therefore, the loss of biocontrol activity can be attributed to a reduced production of antifungal compounds and not a declining population size. Competition assays between the mutant and wild type revealed equivalent fitness in aged batch culture; consequently, the gacS mutation did not impart a growth advantage in the stationary phase phenotype. Under minimal nutrient conditions, the gacS-deficient strain produced a tenfold less biofilm than the wild type. However, no difference was observed in the ability of the mutant biofilm to protect cells from lethal antibiotic challenge.

Antagonistic potential of biocontrol agents against Botryodiplodia theobrome causing die-back of bottle brush (Callistemon citrinus)

Biological control of die-back of bottle brush (Callistemon citrinus) caused by Botryodiplodia theobrome was made with the application of antagonistic agents like Trichoderma viride, T. lignorum, T. harzianum, Aspergillus niger and Penicillium citrinum. The effect of volatile and non-volatile antibiotics of Trichoderma origin on growth inhibition of the die-back pathogen was studied. T. harzianum showed maximum growth inhibition (75.33%) of the pathogen through mycoparasitism and the non-volatiles produced by the same agent exhibited its excellent antagonism to the growth of the pathogen (91.11%) under in vitro condition and that the effect was also proved to be durable.

IN VITROINTERACTIONS BETWEENTRICHODERMASPP. ANDOPHIOSTOMA ULMIAND THEIR IMPLICATIONS FOR THE BIOLOGICAL CONTROL OF DUTCH ELM DISEASE AND OTHER FUNGAL DISEASES OF TREES

Summary The in vitro antagonism between T. harzianum and T. polysporum and O. ulmi in relation to biological control of Dutch elm disease was studied. The results of isolation from trees inoculated with pellets of Trichoderma spp. suggest that elms are not colonised. Direct interactions between the antagonist and pathogen were not detected by light or scanning electron microscopy of dual cultures. T. harzianum was shown to produce volatile and non-volatile antibiotics which were inhibitory to the growth of O. ulmi. The results are discussed in relation to the biological control of fungal tree diseases.

Control of white rot of garlic by antagonistic fungi under controlled environmental conditions

Several fungi were isolated from soil samples collected in areas with a high incidence of white rot disease of garlic at Amarantina county, Minas Gerais, Brazil. After the screening in vitro for antagonists to Sclerotium cepivorum, three fungi were found to be highly inhibitory to the pathogen. These fungi were identified as Trichoderma harzianum Rifai, Paecilomyces lilacinus (Thom.) Samson, and a Penicillium sp. The three antagonists produced in vitro nonvolatile antibiotics towards Sc. cepivorum. These substances were thermolabile (120 °C, 15 min) and significantly inhibited the growth of Sc. cepivorum. Interactions between the pathogen and each antagonist, studied by a dual slide-mount technique, showed that Pa. lilacinus and Penicillium sp. caused an inhibition halo to Sc. cepivorum although hyphal contact never occurred. Trichoderma harzianum showed inhibitory activity at a distance, and after contact with Sc. cepivorum, caused hyphal cytoplasm disintegration and cell wall collapse. This detrimental effect occurred either after direct penetration of the cell wall or after the formation of "coilings." The efficacy of the three antagonists in the biocontrol of white rot was tested under controlled environmental conditions in a nonsterilized, artificially infested soil (1 sclerotia per gram of soil). Trichoderma harzianum VL1 applied as a conidial suspension (106 conidia/mL) during the transplanting period significantly reduced the severity of white rot and increased the number of healthy plants when compared with the untreated control. Trichoderma harzianum gave significantly better protection to the plants than Coniothyrium minitans, a known biocontrol agent of sclerotia-forming fungi. Paecilomyces lilacinus, Penicillium sp., and the mixture of the three antagonists gave no protection against the disease.

Antagonistic properties of species-groups of Trichoderma: I. Production of non-volatile antibiotics

Isolates from different species-groups of Trichoderma were tested for production of non-volatile antibiotics, by an agar layer technique. Preliminary studies on the chemical nature of these antibiotics were made. Many isolates produced non-volatile antibiotics active against a range of fungi. The ability to produce such antibiotics varied between isolates of the same species-group as well as between isolates of different species groups. The susceptibility of fungi to these antibiotics varied widely; Fomes annosus (Fr.) Cooke was the most susceptible and Fusarium oxysporum Schlecht, ex Fr. the most resistant of the test fungi used. Gliotoxin and viridin were not produced, but other chloroform-soluble antibiotics, including trichodermin and peptide antibiotics were detected.