Volatile Antibiotics Research Articles

Fighting plant pathogens with cold-active microorganisms: biopesticide development and agriculture intensification in cold climates.

Cold-adapted (CA) microorganisms (= psychrophiles or psychrotolerants) are key players of many ecological interactions in natural ecosystems. Some of them can colonize the rhizosphere of plants and cause damage to their hosts; others, on the contrary, protect plants from their pathogens through direct and indirect mechanisms, thus promoting plant growth and development. These "protective" microbes are known as biocontrol agents (BCA). BCA either limit or inhibit the growth of plant pathogens, owing to the excretion of a panoply of secondary metabolites (including soluble and volatile antibiotics, siderophores, quorum sensing interfering agents). BCA can also control plant pathogens through indirect mechanisms, including competence for nutrients and space, or else by interfering with their chemical communication. That explains why some of these BCA have been included in the formulation of commercial biopesticides, which are environmentally friendly products containing live cells used to control plant diseases and pests. At present, the development of biopesticides from mesophilic microorganisms is an established technology. Unfortunately, these biopesticides are not active at low temperatures. On the other hand, the information concerning the potential use of CA-BCA for the same goal is at its infancy. Here, we review the current knowledge concerning the isolation, identification, and characterization of CA microbes which act as antagonists of plant pathogens, including the mechanisms they deploy to antagonize plant pathogens. We also illustrate their biotechnological potential to develop CA biopesticides and discuss their utility in the context of mountainous agriculture. KEY POINTS: • Many naturally occurring cold-active microbes antagonize plant pathogens. • The mechanisms of biocontrol exerted by these microbes are either direct or indirect. • Cold-active biocontrol agents can be used to develop biopesticides. • Cold-active biopesticides are crucial for sustainably intensifying agriculture in cold climates.

Diallylthiosulfinate (Allicin), a Volatile Antimicrobial from Garlic (Allium sativum), Kills Human Lung Pathogenic Bacteria, Including MDR Strains, as a Vapor

Garlic (Allium sativum) has potent antimicrobial activity due to allicin (diallylthiosulfinate) synthesized by enzyme catalysis in damaged garlic tissues. Allicin gives crushed garlic its characteristic odor and its volatility makes it potentially useful for combating lung infections. Allicin was synthesized (>98% pure) by oxidation of diallyl disulfide by H2O2 using formic acid as a catalyst and the growth inhibitory effect of allicin vapor and allicin in solution to clinical isolates of lung pathogenic bacteria from the genera Pseudomonas, Streptococcus, and Staphylococcus, including multi-drug resistant (MDR) strains, was demonstrated. Minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were determined and compared to clinical antibiotics using standard European Committee on Antimicrobial Susceptibility Testing (EUCAST) procedures. The cytotoxicity of allicin to human lung and colon epithelial and murine fibroblast cells was tested in vitro and shown to be ameliorated by glutathione (GSH). Similarly, the sensitivity of rat precision-cut lung slices (PCLS) to allicin was decreased by raising the [GSH] to the approximate blood plasma level of 1 mM. Because allicin inhibited bacterial growth as a vapor, it could be used to combat bacterial lung infections via direct inhalation. Since there are no volatile antibiotics available to treat pulmonary infections, allicin, particularly at sublethal doses in combination with oral antibiotics, could make a valuable addition to currently available treatments.

EFFICACY OF SECONDARY METABOLITES AND EXTRACELLULAR LYTIC ENZYMES OF PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR) IN CONTROLLING FUSARIUM WILT OF CHICKPEA

Plant growth promoting rhizobacteria (PGPR) and its metabolic products may play a pivotal role in controlling wilt disease in chickpea plants caused by Fusarium oxysporum f. sp. ciceris and promote plant growth under greenhouse and field conditions. The obtained data indicated that both PGPR strains viz. Bacillus subtilis and B. megaterium were able to produce indole acetic acid (IAA), siderophore, hydrogen cyanide (HCN), extracellular compound and volatile antibiotics in vitro. In addition, both PGPR strains produced mycolytic enzymes viz. chitinase, β-1, 3-glucanase and protease in growth media. Bacillus megaterium produced greater amounts of secondary metabolites than B. subtilis. Under laboratory condition, cell cultures, culture filtrates, metabolic precipitates with acetone, ethanol and ammonium sulfate inhibited mycelial growth of the target pathogen and the percentage of inhibition varied from 26.07 % to 48.82 %. Bacillus megaterium inhibited mycelial growth more than B. subtilis. Also, the metabolic precipitate significantly suppressed mycelial growth of pathogen more than cell cultures or culture filtrates and the ethanol precipitate was the most inhibitive. Under greenhouse and field conditions, both PGPR strains used as seed soaking in cell cultures, culture filtrates and/or metabolic precipitates significantly reduced area under wilt progress curve (AUWPC) compared with untreated seeds (control). B. megaterium was more efficient for controlling wilt disease in chickpea than B. subtilis and the metabolic precipitates reduced AUWPC more than cell cultures or its filtrates. Ethanol precipitate recorded the highest reduction of AUWPC either under greenhouse or field conditions. Using cell cultures, cultures filtrates and metabolic precipitates of both PGPR strains significantly increased growth parameters (plant height and number of branches per plant), yield components (numbers of pod and seed /plant, weight of 100 seed, total yield / feddan and protein content in seeds) compared with untreated seeds (control) during growing seasons 2013-14 and 2014-15 under field conditions. Ethanol precipitate of B. megaterium or B. subtilis recorded the highest growth parameters and yield components in both growing seasons. Generally, metabolic precipitation recorded the best results for controlling wilt disease under greenhouse and field conditions and improved plant growth and increased yield components more than cell cultures or culture filtrates of both tested PGPR strains.

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86 % as Trichoderma asperellum, 33.33 % as Trichoderma harzianum, 14.29 % as Trichoderma tomentosum, 4.76 % as Trichoderma koningiopsis, and 4.76 % as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.

Biocontrol of Cytospora canker of poplar in north-east China with Trichoderma longibrachiatum

Summary Trichoderma isolate T05, collected and isolated from a poplar (Populus simonii × Populus nigra) stand in the province of Heilongjiang, China, was tested for its efficacy in controlling Cytospora chrysosperma in vitro and Cytospora canker of poplar in the field. Trichoderma isolate T05 was identified as Trichoderma longibrachiatum in sect. Longibrachiatum based on both morphological characteristics and phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) oligonucleotide sequences and the short intron of tef1. During dual culturing, the inhibitory effect of T. longibrachiatum T05 on C. chrysosperma increased over time, and inhibition up to 81% was observed 120 h after inoculation. Hyphae of T. longibrachiatum T05 grew alongside, coiled and sporulated around the pathogen hyphae, which were generally observed to be wrinkled, collapsed, disrupted and degraded. Culture filtrates of T. longibrachiatum T05 that were sterilized by either bacterial filtration or autoclaving suppressed the growth of the pathogen colony. Filtrate sterilized by filtration showed greater inhibitory activity than the autoclaved filtrate, which still had some inhibitory activity, demonstrating that some inhibitory substances were heat unstable and some heat stable. Cytospora chrysosperma C01 colony growth was also reduced by volatile antibiotics produced by T. longibrachiatum T05, with inhibition of 54% observed after 96 h. In a field trial, Cytospora canker on poplar trunks was controlled with efficacy similar to that of routine chemical control by applying a T. longibrachiatum preparation.

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.

Biocontrol of a root rot of kale by Muscodor albus strain MFC2

Pythium ultimum is an oomycetous root rot pathogen that causes significant crop production losses on many crops including kale (Brassica oleracea), an economically important vegetable in Thailand. An endophytic fungus from Thailand designated Muscodor albus MFC2 controlled P. ultimum both in vitro and on kale seedlings grown under outdoor conditions via the production of volatile antibiotics. Ten-day old M. albus MFC2 PDA cultures killed P. ultimum in vitro. Thoroughly mixing three PDA plates of 10-day old M. albus MFC2 into a 500 g mixture of commercial soil and field soil did not adversely affect kale seed germination. The same amount of M. albus MFC2 could restore seedling emergence in P. ultimum inoculated soil to a level close to that of a non-infested control. In addition, M. albus MFC2 did not cause any disease symptoms, but rather seemed to promote the growth of kale in the presence or absence of P. ultimum for up to eight weeks after planting.

Antagonistic activity of selected strains of Bacillus thuringiensis against Rhizoctonia solani of chili pepper

The aim of this work was to determine, in vitro, the antagonistic effectiveness of 60 strains of Bacillus thuringiensis against damping-off and root and stem rot caused by Rhizoctonia solani. The strains were obtained from the International Collection of Entomopathogenic Bacillus at the FCB-UANL. During the in vitro dual culture assay only 16 of the strains displayed an inhibitory effect. Six strains were chosen to be screened simultaneously by volatile antibiotics, thermostability and seedling assay. In the volatile antibiotics assay, the strains GM-11 and GM-121 showed the best inhibitory effect over R. solani growth. None of the strains showed an efficient antagonistic effect during the thermoestability assay. In seedling assay, majority of the antagonistic isolates, GM-23, GM-11and GM-121, were effective in the reduction of R. solani infection. In addition, GM-23 increased the length of pepper seedlings. These results suggest that the B. thuringiensis strains studied have an excellent potential to be used as bio-control agents of R. solani in chili pepper.

Muscodor albus E-6, an endophyte of Guazuma ulmifolia making volatile antibiotics: isolation, characterization and experimental establishment in the host plant

Muscodor albus is an endophytic fungus, represented by a number of isolates from tropical tree and vine species in several of the world's rainforests, that produces volatile organic compounds (VOCs) with antibiotic activity. A new isolate, E-6, of this organism, with unusual biochemical and biological properties, has been obtained from the branches of a mature Guazuma ulmifolia (Sterculiaceae) tree growing in a dry tropical forest in SW Ecuador. This unique organism produces many VOCs not previously observed in other M. albus isolates, including butanoic acid, 2-methyl-; butanoic acid, 3-methyl-; 2-butenal, 2-methyl-; butanoic acid, 3-methylbutyl ester; 3-buten-1-ol, 3-methyl; guaiol; 1-octene, 3-ethyl-; formamide, N-(1-methylpropyl); and certain azulene and naphthalene derivatives. Some compounds usually seen in other M. albus isolates also appeared in the VOCs of isolate E-6, including caryophyllene; phenylethyl alcohol; acetic acid, 2-phenylethyl ester; bulnesene; and various propanoic acid, 2-methyl- derivatives. The biological activity of the VOCs of E-6 appears different from the original isolate of this fungus, CZ-620, since a Gram-positive bacterium was killed, and Sclerotinia sclerotiorum and Rhizoctonia solani were not. Scanning electron micrographs of the mycelium of isolate E-6 showed substantial intertwining of the hyphal strands. These strands seemed to be held together by an extracellular matrix accounting for the strong mat-like nature of the mycelium, which easily lifts off the agar surface upon transfer, unlike any other isolate of this fungus. The ITS-5.8S rDNA partial sequence data showed 99 % similarity to the original M. albus strain CZ-620. For the first time, successful establishment of M. albus into its natural host, followed by recovery of the fungus, was accomplished in seedlings of G. ulmifolia. Overall, isolates of M. albus, including E-6, have chemical, biological and structural characteristics that make them potentially useful in medicine, agricultural and industrial applications.

An endophytic Gliocladium sp. of Eucryphia cordifolia producing selective volatile antimicrobial compounds

An endophytic isolate of Gliocladium sp. was obtained from the Patagonian Eucryphiacean tree— Eucryphia cordifolia, known locally as “ulmo”. The fungus was identified on the basis of its morphology and aspects of its molecular biology. This fungus produces a mixture of volatile organic compounds (VOC's) lethal to such plant pathogenic fungi as Pythium ultimum and Verticillum dahliae, while other pathogens were only inhibited by its volatiles. Some of the same volatile bioactive compounds exuded by Gliocladium sp. such as 1-butanol, 3-methyl-, phenylethyl alcohol and acetic acid, 2-phenylethyl ester, as well as various propanoic acid esters, are also produced by Muscodor albus, a well known volatile antimicrobial producer. In fact, M. albus was used as a selection tool to effectively isolate Gliocladium sp. since it is resistant to VOC's produced by M. albus. However, the primary volatile compound produced by Gliocladium sp. is 1,3,5,7-cyclooctatetraene or [8]annulene, which by itself, was an effective inhibitor of fungal growth. The authenticated VOC's of Gliocladium sp. were inhibitory to all, and lethal to some test fungi in a manner that nearly mimicked the gases of Gliocladium sp. itself. This report shows that the production of selective volatile antibiotics by endophytic fungi is not exclusively confined to the Muscodor spp.

The role of saprophytic microflora in the development of Fusarium ear blight of winter wheat caused by Fusarium culmorum

Saprophytic microflora may interact with ear blight pathogens of wheat and contribute to the poor performance of fungicides against this disease in vivo. A glasshouse experiment and in vitro experiments were conducted to determine the relationship between saprophytic microflora and Fusarium culmorum and to determine fungicide effects on Alternaria alternata, Botrytis cinerea, Cladosporium herbarum and Fusarium culmorum. Inoculation of winter wheat ears (cultivar Avalon) in the glass-house with A. alternata, B. cinerea or C. herbarum at GS 69 before inoculation with F. culmorum at GS 69 led to a decrease in the severity of Fusarium ear blight (FEB). The mean percentage of spikelets infected was reduced from 19% for ears which had been inoculated with the pathogen alone to 4%, 6% and 5% for ears which had been inoculated with A. alternata, B. cinerea and C. herbarum, respectively. In vitro, the mycelial growth of F. culmorum was reduced when inoculated opposite A. alternata, B. cinerea and C. herbarum. The antagonism was shown to be due to production of non-volatile and volatile antibiotics by the saprophytes. In vitro experiments showed that F. culmorum and the three saprophytes showed differential sensitivity to a range of fungicides including benomyl, chlorothalonil, fluquinconazole, flusilazole, flutriafol, prochloraz, pyrimethanil and tebuconazole. These experiments showed that A. alternata, B. cinerea and C. herbarum suppressed the mycelial growth of F. culmorum in vitro and reduced the severity of FEB when inoculated onto plants before the pathogen. The four species showed differential sensitivity to fungicides in vitro. It is possible then that fungicides disrupt the normal suppression of FEB by saprophytic microflora on the ears of wheat.

Biological control of damping-off caused byPythium ultimum andRhizoctonia solani usingTrichoderma spp. applied as industrial film coatings on seeds

Conidia of sevenTrichoderma strains were applied on cucumber or radish seeds as a simple methyl cellulose coating or through an industrial film coating process. The seeds were sown in a peat-based soil artificially infested byR. solani orP. ultimum. Four strains controlled damping-off caused byR. solani when applied as a simple coating or as an industrial film-coating. Also, four strains significantly reduced damping-off caused byP. ultimum in cucumber. A correlation was found between production of volatile antibiotics in vitro and control ofP. ultimum. Survival during storage varied according to the strain. Better survival was observed for two strains, with a decrease in conidial viability of one order of magnitude after storage for three and five months at 15 ° C and 4 ° C, respectively. The results show the feasibility of biocontrol of seedling diseases by some antagonists applied onto seeds through an industrial film-coating process.

STIMULATION OF SEX ORGAN FORMATION IN PHYTOPHTHORA BY ANTAGONISTIC SPECIES OF TRICHODERMA

SUMMARYTwo methods are described for the induction of sex organ formation in heterothallic Phytophthora species in response to Trichoderma, a volatile method and a direct inoculation (‘contact’) method.Tested for their ability to induce sexual reproduction in Phytophthora, isolates of Trichoderma koningii, T. piluliferum, T. polysporum and T. viride were found to be active, and isolates of T. hamatum, T. harzianum, T. longibrachiatum and T. pseudokoningii inactive. Response in Phytophthora was, without exception, confined to the A2 compatibility type. Response of different Phytophthora species, however, was more variable. All forty‐one isolates of P. cam‐bivora and P. cinnamomi tested responded to Trichoderma viride. Isolates of Phytophthora cryptogea, P. drechsleri, and P. nicotianae were apparently polymorphic for response. The black pepper form of P. palmivora responded, whereas P. palmivora forms 1 and 2 did not. A minimum induction period of c. 24 h exposure to volatiles of active Trichoderma was apparently necessary for consistent oogonial formation to occur. Oogonial formation occurred on a range of sterol‐containing media but not in their absence and over a wide range of conditions of temperature and light.Antagonism of Phytophthora cultures by active Trichoderma isolates was readily apparent both in the volatile and contact methods. The ability of Trichoderma isolates to induce a sexual response in Phytophthora was correlated with their ability to produce volatile antibiotics. Possible implications of the ‘Trichoderma effect’ for the taxonomy, reproductive physiology and genetics of Phytophthora spp. are considered.