Antifungal activity of lichen extracts and lichenic acids

Acetone extracts of the threelichen species Evernia prunastri, Hypogymnia physodes and Cladoniaportentosa were investigated for activityagainst eight plant pathogenic fungi: Pythium ultimum, Phytophthorainfestans, Rhizoctonia solani, Botrytis cinerea, Colletotrichumlindemuthianum, Fusarium solani, Stagonospora nodorum and Ustilagomaydis. Particularly, E. prunastriand H. physodes exhibit total or stronginhibition on P. ultimum, U.maydis and P. infestans growth. Incontrast, Cladonia extracts were lessactive to reduce growth of these fungi.Lichenic acids were also examined forantifungal activity. P. infestans growthwas severely inhibited by evernic acid. P.ultimum and P. infestans growth wereslightly but significantly inhibited by evernicacid and (–) usnic acid, respectively. Growthinhibition of U. maydis was also observedfor the latter lichenic acid. These resultsconfirm the previously observed activities oflichen extracts. This suggests that secondarylichen metabolites might be of potential use asantifungal agents.     

Trichoderma species for biocontrol of soil-borne plant pathogens of pasture species

Soil-borne plant pathogens such as Rhizoctonia solani (Kuhn), Pythium ultimum (Trow) and Sclerotinia trifoliorum (Eriks) can reduce grass and forage legume establishment. The potential for biocontrol of these pathogens by Trichoderma fungi was evaluated. Following dual culture assays, nine Trichoderma isolates (five of Trichoderma atroviride and one each of Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma viride and Trichoderma virens) were chosen for assessment in pot experiments. In the presence of R. solani, perennial ryegrass (Lolium perenne L.) emergence was increased by 60–150% by two isolates of T. atroviride and by 35–212% by the isolate of T. virens, with the increase depending on growing medium and amount of pathogen inoculum. Red clover (Trifolium pratense L.) emergence in the presence of S. trifoliorum was significantly increased by two T. atroviride isolates and the T. hamatum isolate. In the presence of P. ultimum, white clover (Trifolium repens L.) emergence was increased by 25–42% by one isolate of T. atroviride and the T. hamatum isolate. However, for all three pasture species, some Trichoderma isolates reduced seedling emergence. Seedling growth (shoot and root fresh weight/plant) of the three pasture species was significantly increased by one or more T. atroviride isolates. On the basis of these results for both disease reduction and growth promotion, four T. atroviride isolates were selected for field assessment as biocontrol agents of soil-borne pathogens of pasture species.     

Trichoderma spp. tolerance to Brassica carinata seed meal for a combined use in biofumigation

Biofumigation by Brassicaceae green manure or seed meal incorporation into soil is an ecological alternative to chemical fumigation against soil-borne pathogens, based on the release of glucosinolate-derived compounds. This study aimed at investigating the tolerance of the beneficial fungus Trichoderma to these compounds in view to combined utilization with Brassica carinata seed meal (BCSM). Forty isolates of Trichoderma spp. were tested in vitro for tolerance to toxic volatiles released by BCSM and in direct contact with the meal. They were found to be generally less sensitive than the assayed pathogens (Pythium ultimum, Rhizoctonia solani, Fusarium oxysporum), even if a fungistatic effect was observed at the highest dose (10 μmole of sinigrin). Most of them also were able to grow on BCSM and over the pathogens tested. A preliminary experiment of integrating BCSM with Trichoderma in soil was carried out under controlled conditions with the patho-system P. ultimum—sugar beet. BCSM incorporation increased pathogen population, but reduced disease incidence, probably due to indirect mechanisms. The greatest effect was achieved when BCSM was applied in combination with Trichoderma, regardless of meal ability to release isothiocyanate. These findings suggest that disease control can be improved by this integrated approach. This study also highlighted that a reduction of allyl-isothiocyanate concentration in soil could occur due to the activity of some Trichoderma isolates. This effect could protect resident or introduced Trichoderma isolates from depressing effects due to the biocidal compounds, but, on the other hand, could reduce the efficacy of biofumigation against target pathogens.     

Efficacy ofTrichoderma chitinases againstRhizoctonia solani, the rice sheath blight pathogen

Thirty-five strains ofTrichoderma viride andT. harzianum were screened for their antagonistic ability against the rice sheath blight pathogen,Rhizoctonia solani. The strains that inhibited/overgrew the phytopathogenic fungus were considered effective. Light microscopic studies showed the antagonism of the hyphae of effectiveTrichoderma strains towards their host hyphae. Chitinase activity ofTrichoderma culture filtrates was enhanced, when colloidal chitin was used as the sole carbon source, instead of glucose. Chitinase pattern differed among the four select strains. The chitinase isoforms are induced differentially by carbon sources. The chitin affinity column fraction ofTrichoderma culture filtrate inhibited,in vitro, the growth ofR. solani.     

Control of fusarium wilt of <Emphasis Type="Italic">Solanum melongena</Emphasis> by <Emphasis Type="Italic">Trichoderma</Emphasis> 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.     

Trichoderma aureoviride URM 5158 and Trichoderma hamatum URM 6656 are Biocontrol Agents that act against Cassava Root rot through different Mechanisms

Trichoderma has been used to manage a large number of pathogens, but there is a gap in the mechanisms used by these biocontrol agents regarding the physiological response of cassava plants (Manihot esculenta) when it is subjected to cassava root rot. The aims of this study were to investigate the antagonist activity of ten Trichoderma isolates against Fusarium solani on potato dextrose Agar (PDA), to quantify the chitinase production, to select and test in vivo the best isolate from each experiment and to assess the physiological response of cassava to the production of oxidative enzyme complex production (ascorbate peroxidase, catalase, peroxidase and polyphenol oxidase). All Trichoderma isolates have shown competitive capability against F. solani, and Trichoderma hamatum URM 6656 showed the highest inhibition of pathogen growth (88.91%). All isolates have shown chitinase activity, but Trichoderma aureoviride URM 5158 produced the highest amount of chitinase. T. hamatum URM 6656 and T. aureoviride URM 5158 were selected to be applied in vivo. The two Trichoderma strains reduced 64 and 60% of the disease severity in the shoot and 82 and 84% in the root. Cassava plants infected with Trichoderma have shown the highest peroxidase and ascorbate peroxidase production. Our results have indicated that T. aureoviride URM 5158 is an effective biocontrol agent against cassava root rot caused by F. solani, because it presented competitive antagonist capability in vitro, the highest chitinase production, and reduced the cassava root rot severity. The application of T. aureoviride has led to the maximum enzyme activity of reactive oxygen species group in cassava plants.     

In vitro and greenhouse evaluations of Trichoderma isolates for biological control of potato wilt disease (Fusarium solani)

The biological efficacy of Trichoderma species may differ due to variations in ecosystems. This study was conducted to assess the biocontrol efficacy of some native Trichoderma isolates against Fusarium solani, an important causal agent of potato wilt disease under laboratory and greenhouse conditions at Shahrood Agricultural Research Centre, Shahrood, Iran, during 2006–2007. Fourteen isolates were collected among which eight showed promising ability in inhibiting the growth of the pathogen through dual culture and production of volatile and non-volatile inhibitors but T. brevicompactum (T1), T. longibrachiatum (T5) and T. asperellum (T2) were almost better than other isolates in inhibiting the mycelial growth of the pathogen in comparison to control in the above three tests (p ≤ 0.01). Isolates performing mycoparasitism under in vitro condition were evaluated against the disease in pot culture under greenhouse condition. In all treatments in which Trichoderma isolates + F. solani were involved lower disease incidence was noticed in comparison to Fusarium-infested control (p ≤ 0.05). Best disease control was observed in potted plants treated with F. solani + T. longibrachiatum (T5) with 6.25% disease incidence in comparison to Fusarium-infested control, in which disease incidence was observed to be 75%. Interaction of T. brevicompactum (T1) and F. solani also indicated good control of the disease by 12.50% of disease incidence.     

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.     

Mycoparasitism studies of Trichoderma species against three phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production

Trichoderma spp. are used for biocontrol of several plant pathogens. However, their efficient interaction with the host needs to be accompanied by production of secondary metabolites and cell wall-degrading enzymes. Three parameters were evaluated after interaction between four Trichoderma species and plant-pathogenic fungi: Fusarium solani, Rhizoctonia solani and Sclerotinia sclerotiorum. Trichoderma harzianum and T. asperellum were the most effective antagonists against the pathogens. Most of the Trichoderma species produced toxic volatile metabolites, having significant effects on growth and development of the plant pathogens. When these species were grown in liquid cultures with cell walls from these plant pathogens, they produced and secreted β-1,3-glucanase, NAGAse, chitinase, acid phosphatase, acid proteases and alginate lyase.     

A quantitative bioassay for extracellular metabolites that antagonize growth of filamentous fungi,and its use with biocontrol fungi

A bioassay and an empirically derived formula were developed to quantify fungitoxic effects. This bioassay can be easily performed and objectively read, and it is suitable for low-volume samples originating from aqueous or organic solvents. The formula defines the Inhibition Index (I), a single value that incorporates both the response to concentrations of the inhibitory compound and the persistence of inhibition. Antagonistic efficacy of metabolites produced by biocontrol strains of Trichoderma spp. were measured based on inhibition of growth of Rhizoctonia solani. Although the bioassay itself was not influenced by pH or light conditions, these factors affected metabolite production or activity. Aqueous extracts from light-grown germlings of Trichoderma virens inhibited R. solani more than extracts from germlings grown in the dark. Low pH increased the inhibitory activity of extracts from T. virens. Tests of fungal strains developed for biocontrol demonstrated that the bioassay reflected their activity both in the field and in other in vitro tests. The bioassay and formula are readily adapted for use with other fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Do Pathogenic Fungi Have the Potential to Inhibit Biocontrol Fungi?

Metabolites produced by the soilborne plant pathogen Rhizoctonia solani (R-23) and Pythium ultimum (PuZ3) grown on cellophane disks placed on potato-dextrose agar (PDA), molasses brewer's yeast agar (MYA) and wheat bran extract agar (BEA) did not significantly affect the rate of growth of isolates of the antagonists Trichoderma viride (TS-I-R3. Tv-101), T. harzianum (Th-57, Th-87), T. hamatum (Tm-23, TRI-4), or Gliocladiun virens (GI-3, GI-21) when these antagonists were grown on the three agars containing pathogen metabolites. However. in some instances. density of antagonist mycelium growing on the agar media as well as the observable production of antagonist conidia on the agar media were reduced. Using four antagonists in liquid cultures of potato-dextrose broth (PDB) containing metabolites of the pathogens grown on bran extract broth, metabolites from R-23 significantly reduced mycelial dry weight of Th-87 and GI-21 but not that of TRI-4 and GI-3. On the contrary. metabolites of PuZ3 increased the mycelial dry weight of all four antagonists, Metabolites of R-23 reduced production of conidia of only TRI-4; metabolites of PuZ3 significantly reduced production of conidia of all four antagonists. Pathogen metabolites did not affect germination of conidia in the system used.     

The toolbox of Trichoderma spp. in the biocontrol of Botrytis cinerea disease

Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma–plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR‐prime phase). Finally, we discuss the ISR‐boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.     

Decay of Sclerotia of Botrytis cinerea by Trichoderma spp. at Low Temperatures

In vitro, tests were conducted at 10°C and 5°C against sclerotia of Botrytis cinerea with 58 isolates of Trichoderma spp., highly antagonistic at 24°C but differing in their cold tolerance. Some isolates macerated and colonized sclerotia even at 5 °C. With 19 isolates of Trichoderma spp. less than 10 % of the sclerotia remained viable after 42 d at 5 °C. Conidia ol some Trichoderma spp. germinated at 5 °C within a few days and reached germination rates higher than 80 %. It seems to be feasible to use selected isolates of Trichoderma spp. for biological control of sclerotia of ß. cinerea also during the colder season.     

Production of Inter- and Intra-strain Hybrids of Trichoderma spp. by Protoplast Fusion and Evaluation of Their Biocontrol Activity Against Soil-borne and Foliar Pathogens

Protoplast fusion techniques were used for the production of new antagonistic strains of Trichoderma spp. Two British and two Italian strains of Trichoderma with different biocontrol potential against Botrytis cinerea, Sclerotinia sclerotiorum and Pythium ultimum, were treated with u.v. radiation and mutants resistant to hygromycin B or propiconzole were selected. Protoplasts were obtained from these mutants after digestion of young hyphae with Novozym 234 and used in inter- and intra-strain protoplast fusion experiments. Hybrids were obtained in 13 crosses isolated on fungicide-amended media by the inheritance of resistance markers from both parental strains. Selected, fast-growing, stable mutants were tested in biocontrol trials against P. ultimum on lettuce seedlings and B. cinera on grape bunches in comparison with their parental strains. Intra-strain hybrids derived from cross XIII were tested in vitro for their mycoparasitic ability on sclerotia of S. sclerotiorum. A high degree of variability in the biocontrol and the mycoparasitic ability of the fusants was observed but no significant increase in the activity was accomplished after fusion, the hybrids being generally less activewas accomplished after fusion, the hybrids being generally less active than their parental strains.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

Use of Trichoderma harzianum and Gliocladium vitens for the Biological Control of Post-emergence Damping-off and Root Rot of Cucumbers Caused by Pythium ultimum

The antagonist strains Gliocladium virens G2 and Trichoderma harzianum T3 originally isolated from Pythium suppressive peat, and two benomyl-resistant strains of T. harzianum, T12B and T95, were evaluated as biological control agents of damping-off and root rot of cucumbers in sphagnum peat caused by Pythium ultimum. All strains were equally effective when applied as 1 % peat-bran preparations, whereas the effectiveness of disease control was reduced at higher concentrations of the antagonists. The two wild-type strains were also found to be effective when applied as conidial suspensions, and in this case no reduction in disease control was seen at higher concentrations. G. virens G2 and T. harzianum T12B showed antibiotic activity against P. ultimum in in vitro tests; however there were no signs of mycoparasitism of P. ultimum by any of the antagonist strains.     

Antifungal Activities of N-Arylbenzenesulfonamides against Phytopathogens and Control Efficacy on Wheat Leaf Rust and Cabbage Club Root Diseases

A set of N-arylbenzenesulfonamides with various substituents at the arylamine and benzenesulfonyl positions were prepared, and their antifungal properties were measured in vitro against such plant pathogenic fungi as Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, and Botrytis cinerea. Compounds 3, 4, 8, 9, 10, 14, 16, 18, 20, 21, 24 and 27 had antifungal activity over a broad spectrum of the phytopathogenic fungi tested, where 50% of inhibition (ED₅₀) was in the range of 3-15 μg/ml. Based on the in vitro activity, six derivatives (3, 4, 10, 18, 21 and 27) were selected and tested further for their fungicidal efficacy in vivo. The fungicidal efficacy of 10, 21 and 27 had a disease control value of over 85% at 50 μg/ml against wheat leaf rust, while that of 4 was selective against cabbage club root disease.     

Diversity Profile and Dynamics of Peptaibols Produced by Green Mould Trichoderma Species in Interactions with Their Hosts Agaricus bisporus and Pleurotus ostreatus

Certain Trichoderma species are causing serious losses in mushroom production worldwide. Trichoderma aggressivum and Trichoderma pleuroti are among the major causal agents of the green mould diseases affecting Agaricus bisporus and Pleurotus ostreatus, respectively. The genus Trichoderma is well‐known for the production of bioactive secondary metabolites, including peptaibols, which are short, linear peptides containing unusual amino acid residues and being synthesised via non‐ribosomal peptide synthetases (NRPSs). The aim of this study was to get more insight into the peptaibol production of T. aggressivum and T. pleuroti. HPLC/MS‐based methods revealed the production of peptaibols closely related to hypomurocins B by T. aggressivum, while tripleurins representing a new group of 18‐residue peptaibols were identified in T. pleuroti. Putative NRPS genes enabling the biosynthesis of the detected peptaibols could be found in the genomes of both Trichoderma species. In vitro experiments revealed that peptaibols are potential growth inhibitors of mushroom mycelia, and that the host mushrooms may have an influence on the peptaibol profiles of green mould agents.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

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.