Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens

Aims: Strains of Trichoderma spp. produce numerous bioactive secondary metabolites. The in vitro production and antibiotic activities of the major compounds synthesized by Trichoderma harzianum strains T22 and T39 against Leptosphaeria maculans, Phytophthora cinnamomi and Botrytis cinerea were evaluated. Moreover, the eliciting effect of viable or nonviable biomasses of Rhizoctonia solani, Pythium ultimum or B. cinerea on the in vitro production of these metabolites was also investigated. Methods and Results: T22azaphilone, 1‐hydroxy‐3‐methyl‐anthraquinone, 1,8‐dihydroxy‐3‐methyl‐anthraquinone, T39butenolide, harzianolide, harzianopyridone were purified, characterized and used as standards. In antifungal assays, T22azaphilone and harzianopyridone inhibited the growth of the pathogens tested even at low doses (1–10 μg per plug), while high concentrations of T39butenolide and harzianolide were needed (>100 μg per plug) for inhibition. The in vitro accumulation of these metabolites was quantified by LC/MS. T22azaphilone production was not enhanced by the presence of the tested pathogens, despite its antibiotic activity. On the other hand, the anthraquinones, which showed no pathogen inhibition, were stimulated by the presence of P. ultimum. The production of T39butenolide was significantly enhanced by co‐cultivation with R. solani or B. cinerea. Similarly, viable and nonviable biomasses of R. solani or B. cinerea increased the accumulation of harzianopyridone. Finally, harzianolide was not detected in any of the interactions examined. Conclusions: The secondary metabolites analysed in this study showed different levels of antibiotic activity. Their production in vitro varied in relation to: (i) the specific compound; (ii) the phytopathogen used for the elicitation; (iii) the viability of the elicitor; and (iv) the balance between elicited biosynthesis and biotransformation rates. Significance and Impact of the Study: The use of cultures of phytopathogens to enhance yields of Trichoderma metabolites could improve the production and application of novel biopesticides and biofertilizers based on the active compounds instead of the living microbe. This could have a significant beneficial impact on the management of diseases in crop plants.     

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis

Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species.Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7–66.4% and 56.5–60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.     

Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461

Rhizoctonia solani is a damping-off pathogen that causes significant crop loss worldwide. In this study, the potential of Muscodor cinnamomi, a new species of endophytic fungus for controlling R. solani AG-2 damping-off disease of plant seedlings by biological fumigation was investigated. In vitro tests showed that M. cinnamomi volatile compounds inhibited mycelial growth of pathogens. Among nine solid media tested, rye grain was the best grain for inoculum production. An in vivo experiment of four seedlings, bird pepper, bush bean, garden pea and tomato were conducted. The results indicated that treatment with 30?g of M. cinnamomi inoculum was the minimum dose that caused complete control of damping-off symptoms of all seedlings after one month of planting. The R. solani-infested soil showed the lowest percentage of seed germination. In addition, M. cinnamomi did not cause any disease symptoms. From the results it is clear that M. cinnamomi is effective in controlling R. solani AG-2 both in vitro and in vivo.     

Antifungal cinnamic acid derivatives from Persian leek (Allium ampeloprasum Subsp. Persicum)

A cinnamic imidate, (1Z,2E)-methyl 3-(-p-hydroxy-m-methoxyphenyl)-N-(-p-hydroxyphenethyl) acrylimidate, named persicoimidate (1), has been isolated and characterized from bulbs and seeds of Persian leek, Allium ampeloprasum Subsp. Persicum. Two cinnamic acid derivatives, N-feruloyl tyramine (2) and N-caffeoyl tyramine (3) were isolated from bulbs and seeds. Compound 2 has been previously reported from garlic and leek, while compound 3 is described in Allium plants for the first time. The chemical structures of the isolated compounds have been elucidated unambiguously by spectroscopic methods, including 2D NMR and MS. The isolated compounds were evaluated for their antifungal activity against four fungal pathogens, the soil-borne pathogen Penicillium italicum, the air-borne pathogens Aspergillus niger and Botrytis cinerea, and the antagonistic fungi Trichoderma harzianum to evaluate the possible involvement of such compounds in resistance to pathogen attack.     

An Interspecies Comparative Analysis of the Predicted Secretomes of the Necrotrophic Plant Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites. Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.     

(10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic Acid Methyl Ester as an Anti-inflammatory Compound from Ehretia dicksonii

The methanol extract of Ehretia dicksonii provided (10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 μg (the inhibitory effect (IE) was 43%). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (5) and (9Z,11E)- 13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE_{500 μg} of 63% and 79%, respectively). Compounds 1, 4 ((9Z,12Z,14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 μg/ml.     

Draft Genome Sequence of the Volatile Organic Compound-Producing Antarctic Bacterium Arthrobacter sp. Strain TB23, Able To Inhibit Cystic Fibrosis Pathogens Belonging to the Burkholderia cepacia Complex

Arthrobacter sp. strain TB23 was isolated from the Antarctic sponge Lissodendoryx nobilis. This bacterium is able to produce antimicrobial compounds and volatile organic compounds (VOCs) that inhibit the growth of other Antarctic bacteria and of cystic fibrosis opportunistic pathogens, respectively. Here we report the draft genome sequence of Arthrobacter sp. TB23.     

Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1

Bacillus atrophaeus CAB-1 displays a high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. We extracted and identified lipopeptides and secreted proteins and volatile compounds produced by strain CAB-1 to investigate the mechanisms involved in its biocontrol performance. In vitro assays indicated all three types of products contributed to the antagonistic activity against the fungal pathogen Botrytis cinerea. Each of these components also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry revealed that the major bioactive lipopeptide was fengycin A (C15–C17). We isolated the crude-secreted proteins of CAB-1 and purified a fraction with antifungal activity. This protein sequence shared a high identity with a putative phage-related pre-neck appendage protein, which has not been reported as an antifungal factor. The volatile compounds produced by CAB-1 were complex, including a range of alcohols, phenols, amines, and alkane amides. O-anisaldehyde represented one of the most abundant volatiles with the highest inhibition on the mycelial growth of B. cinerea. To our knowledge, this is the first report on profiling three types of antifungal substances in Bacilli and demonstrating their contributions to plant disease control.     

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.     

Viral cross-class transmission results in disease of a phytopathogenic fungus

Interspecies transmission of viruses is a well-known phenomenon in animals and plants whether via contacts or vectors. In fungi, interspecies transmission between distantly related fungi is often suspected but rarely experimentally documented and may have practical implications. A newly described double-strand RNA (dsRNA) virus found asymptomatic in the phytopathogenic fungus Leptosphaeria biglobosa of cruciferous crops was successfully transmitted to an evolutionarily distant, broad-host range pathogen Botrytis cinerea. Leptosphaeria biglobosa botybirnavirus 1 (LbBV1) was characterized in L. biglobosa strain GZJS-19. Its infection in L. biglobosa was asymptomatic, as no significant differences in radial mycelial growth and pathogenicity were observed between LbBV1-infected and LbBV1-free strains. However, cross-species transmission of LbBV1 from L. biglobosa to infection in B. cinerea resulted in the hypovirulence of the recipient B. cinerea strain t-459-V. The cross-species transmission was succeeded only by inoculation of mixed spores of L. biglobosa and B. cinerea on PDA or on stems of oilseed rape with the efficiency of 4.6% and 18.8%, respectively. To investigate viral cross-species transmission between L. biglobosa and B. cinerea in nature, RNA sequencing was carried out on L. biglobosa and B. cinerea isolates obtained from Brassica samples co-infected by these two pathogens and showed that at least two mycoviruses were detected in both fungal groups. These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi. This study also provides new insights for using asymptomatic mycoviruses as biocontrol agent.Subject terms: Virology, Fungi     

Evaluation of human neutrophil elastase inhibitory effect of iridoid glycosides from Hedyotis diffusa

Five iridoid glycosides were isolated from the MeOH extract of Hedyotis diffusa, and their structures were elucidated as E-6-O-p-methoxycinnamoyl scandoside methyl ester (1), Z-6-O-p-methoxycinnamoyl scandoside methyl ester (2), E-6-O-p-feruloyl scandoside methyl ester (3), E-6-O-p-coumaroyl scandoside methyl ester (4), and Z-6-O-p-coumaroyl scandoside methyl ester (5) by interpretation of their spectroscopic data. All the isolated compounds were evaluated for human neutrophil elastase inhibitory effect, and compound 1 showed potent activity with an IC₅₀ value of 18.0 μM. The molecular docking simulation suggested a structural model for the inhibition of human neutrophil elastase by compound 1.     

Control Effect and Possible Mechanism of the Natural Compound Phenazine-1-Carboxamide against Botrytis cinerea

To develop new agents against strawberry grey mould and to aid in the development of biological pesticides, we investigated the inhibitory effect of a natural compound, phenazine-1-carboxamide (PCN), against Botrytis cinerea using a growth rate assay. Additionally, indoor toxicity and the in vitro control effect of PCN were further studied to determine its potential mechanisms of action on B. cinerea. PCN was inhibitory against B. cinerea with a 50% effective concentration (EC₅₀) of 108.12 μg/mL; the toxicity of PCN was equivalent to that of carbendazim (CBM). The best in vitro control effect of PCN against grey mould in strawberry (fruit) reached 75.32%, which was slightly higher than that of CBM. The field control effect of PCN against grey mould reached a maximum of 72.31% at a PCN concentration of 700 μg/mL, which was 1.02 times higher than that of CBM. Fungistatic activity was observed at low concentrations of PCN, while high concentrations of PCN resulted in fungicidal activity against B. cinerea. This natural compound strongly inhibited both spore and sclerotium germination of B. cinerea, with the best relative inhibition rates of 77.03% and 82.11%, respectively. The inhibitory effect of PCN on mycelial growth of B. cinerea was significant and reached levels of 87.32%. Scanning electron microscopy observations revealed that after 48 h of PCN treatment, the mycelia appeared loose, locally twisted, and folded, with exudation of contents; the mycelia was withered and twisted, with edge burrs, deformations, ruptures and a sheet-like structure. Transmission electron microscopy observations revealed that after 48 h of PCN treatment, the structure of the cell nucleus was unclear and the vacuoles had ruptured; additionally, various organelles exhibited disordered structures, there were substantial non-membrane transparent inclusions, the cells were plasmolysed, the cell walls were collapsed in some cases, and the hyphal tissue was essentially necrotic. A PCN dosage of 35–140 μg/mL had no effect on the cell membrane permeability of the mycelia, while a PCN dosage of 700 μg/mL resulted in significant permeability. PCN inhibited B. cinerea toxin; the mycotoxin level was approximately 0.41 of the value recorded for the control at a PCN dosage of 700 μg/mL. PCN affected the activity of pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β-glucosidase (BG); the lowest activities of PMG, PG, BG and Cx reached 0.3 U/mg, 0.62 U/mg, 0.64 U/mg, and 0.79 U/mg, respectively, after treatment with 700 μg/mL PCN.     

Volatile organic compounds of some Trichoderma spp. increase growth and induce salt tolerance in Arabidopsis thaliana

Many beneficial effects of Trichoderma spp. on plant growth and/or resistance to biotic/abiotic stresses can result from the production of bioactive compounds including volatile organic compounds (VOCs). We evaluated the effects of the volatile mixtures from 13 strains of different Trichoderma species on induction of tolerance to salt stress (100 mM NaCl) as well as growth promotion of Arabidopsis thaliana. Plants responded differently due to the presence of VOCs from various Trichoderma species ranging from both growth promotion and induction of salt tolerance to no significant changes under any of the conditions tested. In plants exposed for 2 weeks to VOCs of the selected strain, i.e. Trichoderma koningii, there was less H₂O₂ accumulation under salt stress compared to that in control plants. This result may reflect the possible role of VOCs of this strain in plant protection against oxidative damage under salt stress. Together, induction of salt tolerance using VOCs should be added to the known mechanisms of plant vigor enhancement by Trichoderma spp.     

Biocontrol potential of Trichoderma species against mango malformation pathogens

Malformation disease of Mango (Mangifera indica L.) caused by Fusarium moniliforme var. subglutinans is one of the most destructive diseases, which is a major production constraint in the mango-growing regions of India. In this study, The bioagents Trichoderma viride (Tr1), Trichoderma virens (Tr2) and Trichoderma harzianum (Tr3) were evaluated in culture with the pathogens to monitor the antagonistic effect and their volatile compound and culture filtrates (non-volatile compound). It was found that all the three isolates of bioagents significantly checked the growth of F. moniliforme var. subglutinans. In dual culture, the best result was obtained with T. harzianum followed by T. virens and T. viride. A similar result was also observed in the case of culture filtrates ofTrichoderma spp. The results clearly showed that inhibition of the growth of the fusaria isolates by T. harzianum was significantly superior to T. viride andT.virens. In case of antifungal activity of volatile compounds released by Trichoderma isolates, it was also observed that T. virens was more superior to T.harzianum and T. viride.     

New C9-polyacetylenes from the essential oil of the highly endangered species Baccharis palustris Heering (Asteraceae)

The essential oil composition of the aerial parts from Baccharis palustris Heering (Asteraceae), a highly endangered species, was analyzed by GC/qMS, GC/HRMS-TOF, FT-IR, UV–vis and NMR spectroscopy using 1D and 2D techniques. To our knowledge, this is the first report on the essential oil composition of B. palustris, as well as the first phytochemical study on this plant species. The volatile extracts were obtained from different phenologicals stages of plant, at flowering stage (FS) and vegetative stage (VS), where 51 components were identified accounting for 96.3 % and 99.0 % of the oil, respectively. The oil was rich in polyacetylene compounds (> 75 %), being the main components identified the new natural C₉-polyacetylenes 1-nonene-3,5-diyne (1, here named as baccharisdiyne) (52.7–65.0 %), 1,7(Z)-nonadiene-3,5-diyne [2, 7(Z)-dehydro-baccharisdiyne] (14.4–17.8 %), and 1,7(E)-nonadiene-3,5-diyne [3, 7(E)-dehydro-baccharisdiyne] (1.5–2.4 %). In addition, the known polyacetylenic compounds (Z)-lachnophyllum acid methyl ester (4) (4.3–5.3 %) and (E)-lachnophyllum acid methyl ester (5) (0.2 %) were also identified. Moreover, GC/qMS analysis allowed the identification of other 46 components in the essential oil samples, mainly mono- and sesquiterpenoids. In B. palustris, C₉-polyacetylenes probably derive biogenetically from a C₁₀ precursor: baccharisdiyne (1) would be derived from either or both lachnophyllum methyl ester geometric isomers (4 or 5), by enzymatic hydrolysis followed by decarboxylation. Dehydro-baccharisdiynes (2 and 3) could be produced by a similar pathway starting from the corresponding matricaria acid methyl ester isomers, one of which was tentatively identified at trace-level in B. palustris essential oil.     

Chemical constituents from Alnus mandshurica (Callier) Hand.-Mazz. and their chemotaxonomic significance

A new phenol compound, (9S)-9-hydroxy-9-[(2-hydroxyphenyl)methoxy]-nonanoic acid methyl ester (1) was isolated from the stem bark of Alnus mandshurica (Callier) Hand.-Mazz., along with eight known compounds (2–9). The structure of compound 1 was determined by spectral analyses, including HR-ESI-MS, 1D and 2D NMR (COSY, HMQC and HMBC) experiments. All the isolated compounds were reported for first time from A. mandshurica. Furthermore, compounds 3–9 were found in the family Betulaceae for the first time.     

Avocado roots treated with salicylic acid produce phenol-2,4-bis (1,1-dimethylethyl), a compound with antifungal activity

We demonstrated the ability of salicylic acid (SA) to induce a compound in avocado roots that strengthens their defense against Phytophthora cinnamomi. The SA content of avocado roots, before and after the application of exogenous SA, was determined by High-Performance Liquid Chromatography (HPLC). After 4 h of SA feeding, the endogenous level in the roots increased to 223 μg g⁻¹ FW, which was 15 times the amount found in control roots. The methanolic extract obtained from SA-treated avocado roots inhibited the radial growth of P. cinnamomi. A thin layer chromatographic bioassay with the methanolic extract and spores of Aspergillus showed a distinct inhibition zone. The compound responsible for the inhibition was identified as phenol-2,4-bis (1,1-dimethylethyl) by gas chromatography and mass spectrometry. At a concentration of 100 μg/mL, the substance reduced germinative tube length in Aspergillus and radial growth of P. cinnamomi. A commercial preparation of phenol-2,4-bis (1,1-dimethylethyl) caused the same effects on mycelium morphology and radial growth as our isolate, confirming the presence of this compound in the root extracts. This is the first report of the induction of this compound in plants by SA, and the results suggest that it plays an important role in the defense response of avocado.     

Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC₅₀ value of 36.2 μM, compared with EC₅₀ values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.     

The synthesis of folic acid-like compounds by Lactobacillus arabinosus

In the presence of p-aminobenzoic acid (PABA), Lactobacillus arabinosus synthesizes one or more compounds with folic acid (FA)-like activity during growth. The total FA activity formed is proportional to the amount of PABA added, up to 200 mμg./tube. Most of the FA activity is in a bound form which is present chiefly in the cells, and the remainder is present in free form which is mainly in the culture medium. Increasing levels of sulfanilamide in the medium competitively inhibit the utilization of PABA for growth of L. arabinosus and decrease the amount of free and combined FA compounds formed.Equimolecular amounts of p-aminobenzoylglutamic acid and PABA have approximately the same growth-promoting and antisulfanilamide activities for L. arabinosus under the conditions employed, and lead to the synthesis of similar amounts of the FA-like compound by L. arabinosus.Thymidine can replace PABA for growth of L. arabinosus and its activity is inhibited very little by sulfanilamide. Thymine and thymidylic acid are much less effective than thymidine. Crystalline vitamin B_c conjugate and vitamin B₁₂ cannot replace PABA for growth of L. arabinosus and do not augment the antisulfonamide activity of PABA.The growth-promoting and antisulfanilamide activities of synthetic folid acid (pteroylglutamic acid), citrovorum factor, and formylfolic acid preparations for L. arabinosus are much poorer than those of equimolecular amounts of PABA. The possibility that the compound(s) with FA-like activity produced by L. arabinosus may differ from pteroylglutamic acid, citrovorum factor, or formylfolic acid is discussed.