Characterization of fungal extracts from Trichoderma isolates: their effects against coffee wilt pathogen (Gibberella xylarioides)

The current research work was designed to evaluate, test, and characterize effective antifungal extracts from Trichoderma isolates against coffee wilt pathogen (Gibberella xylarioides). For extraction of antifungal extracts from fungal mycelium different organic solvents, viz., chloroform, ethanol, methanol, ethyl acetate, n-hexane and butane were used. A direct bioautographic procedure was conducted, involving spraying suspension of Fusarium xylarioides on Thin Layer Chromatography (tlc) plates developed in solvents of varying polarities to detect a number of antifungal substances present in the extracts. Moreover, in vitro antagonistic bioassays were performed to evaluate and determine the potentiality of Trichoderma isolates as biocontrol agents against F. xylarioides. Antifungal extracts were successfully extracted from malt extract agar medium with all organic solvents used except from hexane. Bioautography assay revealed 60 zones of inhibition spots and the highest inhibition zone was observed in aut5 (51 mm) and aut6 (44 mm) with ethanol extract at Rf value of 0.43. In in vitro bioassay, the highest mean inhibitory effect on the growth of the pathogen was achieved by aut2 (77.4%) isolate in dual culture. In general, tlc-directed bioautography assay was found to be useful in isolating active compounds with antifungal activity and all Trichoderma isolates significantly reduced mycelial growth of the test pathogen compared to the control under in vitro condition

Optimization of Culture Conditions and Production of Bio-Fungicides from Trichoderma Species under Solid-State Fermentation Using Mathematical Modeling

Agro-industrial wastes suitable for economical and high mass production of novel Trichoderma species under solid-state fermentation were identified by optimizing the culture conditions using a mathematical model and evaluating the viability of the formulated bio-product. Fourteen inexpensive, locally available, organic substrates and cereals were examined using a one-factor-at-a-time experiment. The fungus colonized nearly all substrates after 21 days of incubation, although the degree of colonization and conidiation varied among the substrates. A mixture of wheat bran and white rice (2:1 w/w) was found to support maximum growth of T. asperellum AU131 (3.2 x 10(7) spores/g dry substrate) and T. longibrachiatum AU158 (3.5 x 10(7) spores/g dry substrate). Using a fractional factorial design, the most significant growth factors influencing biomass production were found to be temperature, moisture content, inoculum concentration, and incubation period (p <= 0.05). Analysis of variance of a Box-Behnken design showed that the regression model was highly significant (p <= 0.05) with F-values of 10.38 (P = 0.0027, T. asperellum AU131) and 12.01 (p < 0.0017, T. longibrachiatum AU158). Under optimal conditions, maximum conidia yield of log(10) (8.6) (T. asperellum AU131) and log(10)(9.18) (T. longibrachiatum) were obtained. For wettable powder Trichoderma species formulations, it was possible to maintain conidial viability at room temperature (25 degrees C) for eight months at concentrations above 10(6) CFU/g

Biological management of coffee wilt disease (Fusarium xylarioides) using antagonistic Trichoderma isolates

Coffee wilt disease (CWD) is a serious threat to the food security of small-scale farmers in Ethiopia, causing significant reductions in coffee yield. Currently, there are no effective control measures available against the causative agent of CWD, Fusarium xylarioides. The main objective of this study was therefore to develop, formulate, and evaluate a range of biofungicides against F. xylarioides, derived from Trichoderma species and tested under in vitro, greenhouse, and field conditions. In total, 175 Trichoderma isolates were screened as microbial biocontrol agents against F. xylarioides. The efficacy of two biofungicide formulations, wettable powder and water dispensable granules, were tested on the susceptible Geisha coffee variety in three different agro-ecological zones in southwestern Ethiopia over three years. The greenhouse experiments were set up using a complete block design, while in the field a randomized complete block design was used, with twice yearly applications of biofungicide. The test pathogen spore suspension was applied to the coffee seedlings by soil drenching, and the subsequent incidence and severity of CWD evaluated annually. The mycelial growth inhibition profiles of the Trichoderma isolates against F. xylarioides ranged from 44.5% to 84.8%. In vitro experiments revealed that T. asperelloides AU71, T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The greenhouse study indicated that wettable powder (WP) of T. asperellum AU131 had the highest biocontrol efficacy (84.3%), followed by T. longibrachiatum AU158 (77.9%) and T. asperelloides AU71 (71.2%); they also had a significant positive impact on plant growth. The pathogen-treated control plants had a disease severity index of 100% across all the field experiments, and of 76.7% in the greenhouse experiments. In comparison to untreated controls, the annual and cumulative disease incidence over the three years of the study period varied from 46.2 to 90%, 51.6 to 84.5%, and 58.2 to 91%, at the Teppi, Gera and Jimma field experimental locations. Overall, the greenhouse and field experiments and in vitro assays support the biocontrol potential of Trichoderma isolates, and T. asperellum AU131 and T. longibrachiatum AU158 in particular are recommended for the management of CWD under field conditions

Antifungal compounds, GC-MS analysis and toxicity assessment of methanolic extracts of Trichoderma species in an animal model

Fungi of the genus Trichoderma have been marketed for the management of diseases of crops. However, some Trichoderma species may produce toxic secondary metabolites and it should receive due attention to ensure human safety. In this study, we investigated the in vitro antagonistic potential of T. asperellum AU131 and T. longibrachiatum AU158 as microbial biocontrol agents (MBCAs) against Fusarium xylarioides and the associated antagonistic mechanism with bioactive substances. Swiss albino mice were used to evaluate the in vivo toxicity and pathogenicity of T. asperellum AU131 and T. longibrachiatum AU158 methanolic extracts and spore suspensions, respectively, in a preliminary safety assessment for use as biofungicides. Gas Chromatography-Mass Spectrometry (GC-MS) was used to profile volatile organic metabolites (VOCs) present in the methanolic extracts. The agar diffusion assay of the methanolic extracts from both T. asperellum AU131 and T. longibrachiatum AU158 were effective at a concentration of 200 mu g/mL (1x10(7) spores/mL), causing 62.5%, and 74.3% inhibition, respectively. A GC-MS analysis of methanolic extracts from both bioagents identified 23 VOCs which classified as alcohols, acids, sesquiterpenes, ketones and aromatic compounds. The oral administration of methanolic extracts and spore suspensions of each Trichoderma species to female Swiss albino mice over 14 days did not show any significant signs of toxicity, mortality or changes to body weight. It can be concluded that the tested spore suspensions and methanolic extracts were not pathogenic or toxic, respectively, when administered to Swiss albino mice at various doses

In vitro evaluation of Trichoderma isolates for the control of sorghum anthracnose (Colletotrichum species)

Sorghum (Sorghum bicolor L.) is one of the most important grain crops grown in Ethiopia for food security. However, the production of sorghum is highly affected by anthracnose diseases. The present study aimed to test, evaluate, and characterize potential biocontrol agents of Trichoderma species against two pathogenic isolates of Colletotrichum species. Sorghum infected leaf, sheath, stalk, and soil samples were collected from Wolkait districts for the isolation of Colletotrichum isolates. In this study, seven Trichoderma isolates were evaluated against two pathogenic Colletotrichum isolates in dual culture techniques and through the production of volatile and non-volatile inhibitors. The study examined the effect of pH and temperature on the mycelia growth and spore yield of Trichoderma isolates. In vitro screening results showed that the proportion of isolates with antagonistic activities was highest for the AUC-1 isolate followed by AUC-2 isolate. The analysis revealed that all Trichoderma isolates were highly antagonistic against AUC-1 whereas AU-97, AU-131, AU-11, and AU-12 isolates displayed over 75% inhibition of mycelial growth on AUC-2 isolate. Under dual culture test, the highest mean inhibitory effect on the growth of the test pathogens was achieved by AU-11 isolate (90.29%) against AUC-1 and AU-97 isolate (81.1%) against AUC-2 while AU-32 isolate showed the lowest mean inhibitory effect in plates as compared to the controls growing alone

Optimization of Culture Conditions and Production of Bio-Fungicides from Trichoderma Species under Solid-State Fermentation Using Mathematical Modeling

Agro-industrial wastes suitable for economical and high mass production of novel Trichoderma species under solid-state fermentation were identified by optimizing the culture conditions using a mathematical model and evaluating the viability of the formulated bio-product. Fourteen inexpensive, locally available, organic substrates and cereals were examined using a one-factor-at-a-time experiment. The fungus colonized nearly all substrates after 21 days of incubation, although the degree of colonization and conidiation varied among the substrates. A mixture of wheat bran and white rice (2:1 w/w) was found to support maximum growth of T. asperellum AU131 (3.2 × 107 spores/g dry substrate) and T. longibrachiatum AU158 (3.5 × 107 spores/g dry substrate). Using a fractional factorial design, the most significant growth factors influencing biomass production were found to be temperature, moisture content, inoculum concentration, and incubation period (p ≤ 0.05). Analysis of variance of a Box–Behnken design showed that the regression model was highly significant (p ≤ 0.05) with F-values of 10.38 (P = 0.0027, T. asperellum AU131) and 12.01 (p &lt; 0.0017, T. longibrachiatum AU158). Under optimal conditions, maximum conidia yield of log10 (8.6) (T. asperellum AU131) and log10(9.18) (T. longibrachiatum) were obtained. For wettable powder Trichoderma species formulations, it was possible to maintain conidial viability at room temperature (25 °C) for eight months at concentrations above 106 CFU/g

Fig 3 -

Chromatograms of the volatile organic compounds identified from T. asperellum AU131 (A) and T. longibrachiatum AU158 (B) crude extracts.</p

Biodiversity of the Genus Trichoderma in the Rhizosphere of Coffee (Coffea arabica) Plants in Ethiopia and Their Potential Use in Biocontrol of Coffee Wilt Disease

The present study investigated the distribution status and biodiversity of Trichoderma species surveyed from coffee rhizosphere soil samples from Ethiopia and their potential for biocontrol of coffee wilt disease (CWD) caused by Fusarium xylarioides. Trichoderma isolates were identified based on molecular approaches and morphological characteristics followed by biodiversity analysis using different biodiversity indices. The antagonistic potential of Trichoderma isolates was evaluated against F. xylarioides using the dual confrontation technique and agar diffusion bioassays. A relatively high diversity of species was observed, including 16 taxa and 11 undescribed isolates. Trichoderma asperellum, T. asperelloides and T. longibrachiatum were classified as abundant species, with dominance (Y) values of 0.062, 0.056 and 0.034, respectively. Trichoderma asperellum was the most abundant species (comprising 39.6% of all isolates) in all investigated coffee ecosystems. Shannon&rsquo;s biodiversity index (H), the evenness (E), Simpson&rsquo;s biodiversity index (D) and the abundance index (J) were calculated for each coffee ecosystem, revealing that species diversity and evenness were highest in the Jimma zone (H = 1.97, E = 0.76, D = 0.91, J = 2.73). The average diversity values for Trichoderma species originating from the coffee ecosystem were H = 1.77, D = 0.7, E = 0.75 and J = 2.4. In vitro confrontation experiments revealed that T. asperellum AU131 and T. longibrachiatum AU158 reduced the mycelial growth of F. xylarioides by over 80%. The potential use of these Trichoderma species for disease management of F. xylarioides and to reduce its impact on coffee cultivation is discussed in relation to Ethiopia&rsquo;s ongoing coffee wilt disease crisis

Effect of temperature and pH on <i>Trichoderma</i> species growth.

Different alphabets depicted in superscript indicate mean treatments that are significantly different according to Tukey’s HSD posthoc test at p ≤ 0.05 each value is an average of 3 replicate samples ± standard error.</p

Volatile organic compounds of <i>T</i>. <i>asperellum</i> AU131 and <i>T</i>. <i>longibrachiatum</i> AU158 identified by GC-MS.

Volatile organic compounds of T. asperellum AU131 and T. longibrachiatum AU158 identified by GC-MS.</p