​Antagonistic Activity of Panchagavya and Trichoderma spp. against Wilt Complex causing Pathogens of Chickpea (Cicer arietinum L.)

Background: Chickpea wilt complex caused by several soil-borne pathogens is the major yield-reducing malady worldwide. Biological control is one of the best, low-cost and ecologically sustainable method for managing plant diseases caused by soil-borne pathogens. Methods: In this present investigation Panchagavya and Trichoderma spp. were evaluated by following poisoned food technique and dual culture technique against wilt complex causing pathogens i.e. Fusarium oxysporum f. sp. ciceri, Fusarium solani and Macrophomina phaseolina. Result: Among the different isolates of Trichoderma spp. evaluated, Trichoderma viride (AAU isolate) was highly antagonistic to F. oxysporum f. sp. ciceri (52.78%) and F. solani (65.37%) whereas, Trichoderma asperellum (AAU isolate) was highly antagonistic to M. phaseolina (65.93%). Panchagavya at the highest concentration (50%) showed significantly higher efficacy (80.74, 66.62 and 49.67%) in inhibiting the mycelial growth of all three pathogens and at the lowest concentration it was moderately effective.

It Works! Organic-Waste-Assisted Trichoderma spp. Solid-State Fermentation on Agricultural Digestate

This study aimed at valorizing digestate through Trichoderma spp. solid-state fermentation (SSF) to produce a potentially ameliorated fertilizer combined with fungal biomass as a value-added bioproduct. Plant-growth-promoting Trichoderma atroviride Ta13, T. reesei RUT-C30, T. asperellum R, and T. harzianum T-22 were tested on different SSF substrates: whole digestate (WD), digestate dried up with wood sawdust (SSF1), and digestate enriched with food waste and dried up with wood sawdust (SSF2). The fungal biomass was quantified by using a qPCR assay. The growth of the four Trichoderma spp. was only observed on the SSF2 substrate. The highest quantity of mycelium was produced by T. reesei RUT-30 (689.80 ± 80.53 mg/g substrate), followed by T. atroviride Ta13, and T. asperellum R (584.24 ± 13.36 and 444.79 ± 91.02 mg/g substrate). The germination of Lepidium sativum seeds was evaluated in order to assess the phytoxicity of the Trichoderma-enriched substrate. The treatments with 7.5% SSF2-R, 3.75% SSF2-T-22, and 1.8% SSF2-Ta13 equally enhanced the root elongation in comparison to the non-fermented SSF-2. This study demonstrated that digestate, mixed with agro-food waste, was able to support the cultivation of Trichoderma spp., paving the way to the valorization of fermented digestate as a proper biofertilizer.

First report of Trichoderma afroharzianum causing seed rot on maize in Italy

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 60,602,320 t, covering 588,597 ha (ISTAT 2021). Trichoderma species are widespread filamentous fungi in soil, well known and studied as biological control agents (Vinale et al., 2008). Seeds of a yellow grain hybrid (class FAO 700, 132 days) were collected in September 2020 from an experimental field located in Carmagnola (TO, Italy: GPS: 44°53'11.0"N 7°40'60.0"E) and tested with blotter test (Warham et al., 1996) to assess their phytosanitary condition. Over the 400 seeds tested, more than 50% showed rotting and development of green mycelium typical of the genus Trichoderma. Due to the high and unexpected percentage of decaying kernels, ten colonies were identified by morphological and molecular methods. Single conidia colonies of one Trichoderma (T5.1) strain were cultured on Potato Dextrose Agar (PDA) for pathogenicity tests, and on PDA and Synthetic Nutrient-Poor Agar (SNA) for morphological and molecular identification. The colonies grown on PDA and SNA showed green, abundant, cottony, and radiating aerial mycelium, and yellow pigmentation on the reverse. Colony radius after 72 h at 30°C was of 60-65 mm on PDA and of 50-55 mm on SNA. The isolates produced one cell conidia 2.8 - 3.8 µm long and 2.1 - 3.6 µm wide (n=50) on SNA. Conidiophores and phialides were lageniform to ampulliform and measured 4.5 – 9.7 µm long and 1.6 – 3.6 µm wide (n=50); the base measure 1.5 – 2.9 µm wide and the supporting cell 1.4 – 2.8 µm wide (n=50). The identity of one single-conidia strain was confirmed by sequence comparison of the internal transcribed spacer (ITS), the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (Oskiera et al., 2015). BLASTn searches of GenBank using ITS (OL691534) the partial tef-1α (OL743117) and rpb2 (OL743116) sequences of the representative isolate T5.1, revealed 100% identity for rpb2 to T. afroharzianum TRS835 (KP009149) and 100% identity for tef-1α to T. afroharzianum Z19 (KR911897). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile H2O to 1×106 CFU/ml. Twenty-five seeds were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 23°C under a seasonal day/night light cycle. Twenty primary ears were inoculated, by injection into the silk channel, with 1 ml of a conidial suspension of strain T5.1 seven days after silk channel emergence (BBCH 65) (Pfordt et al., 2020). Ears were removed four weeks after inoculation and disease severity, reaching up to 75% of the kernels of the twenty cobs, was assessed visually according to the EPPO guidelines (EPPO, 2015). Five control cobs, inoculated with 1 ml of sterile distilled water were healthy. T. afroharzianum was reisolated from kernels showing a green mold developing on their surface and identified by resequencing of tef-1α gene. T. afroharzianum has been already reported on maize in Germany and France as causal agent of ear rot of maize (Pfordt et al. 2020). Although several species of Trichoderma are known to be beneficial microorganisms, our results support other findings that report Trichoderma spp. causing ear rot on maize in tropical and subtropical areas of the world (Munkvold and White, 2016). The potential production of mycotoxins and the losses that can be caused by the pathogen during post-harvest need to be explored. To our knowledge this is the first report of T. afroharzianum as a pathogen of maize in Italy.

Trichoderma-Induced Resistance to Botrytis cinerea in Solanum Species: A Meta-Analysis

With the idea of summarizing the outcomes of studies focusing on the resistance induced by Trichoderma spp. against Botrytis cinerea in tomato, the present paper shows, for the first time, results of a meta-analysis performed on studies published from 2010 to 2021 concerning the cross-talk occurring in the tomato–Trichoderma-B. cinerea system. Starting from an initial set of 40 papers, the analysis was performed on 15 works and included nine parameters, as a result of a stringent selection mainly based on the availability of more than one article including the same indicator. The resulting work not only emphasizes the beneficial effects of Trichoderma in the control of grey mold in tomato leaves (reduction in disease intensity, severity and incidence and modulation of resistance genes in the host), but carefully drives the readers to reply to two questions: (i) What are the overall effects of Trichoderma on B. cinerea infection in tomato? (ii) Do the main effects of Trichoderma differ based on the tomato species, Trichoderma species, amount, type and duration of treatment? At the same time, this meta-analysis highlights some weak points of the available literature and should be seen as an invitation to improve future works to better the conceptualization and measure.

Efectos del pretratamiento con Trichoderma y Bacillus en la germinación de semillas de Agave victoriae-reginae T. Moore

La aplicación de tratamientos pregerminativos es fundamental para mejorar las tasas de germinación de las semillas de especies forestales, entre los cuales el uso de microorganismos es uno de ellos. En este estudio se evaluó el porcentaje de germinación de semillas de Agave victoriae-reginae tratadas con Trichoderma spp. y Bacillus spp. Se probaron tres tratamientos: T1 (Trichoderma), T2 (Bacillus) y T3 (Testigo), con tres repeticiones de 100 semillas cada una. Las semillas se sumergieron en una solución de 1 × 106 UFC (tratamientos T1 y T2), y en agua corriente (T3), luego se sembraron sobre Sphagnum peat moss y se registró el porcentaje de germinación diariamente. La germinación comenzó a los 5 días después de la siembra, lo que significa que las semillas no presentaron latencia. Entre el 8° y 12° día se registró un aumento acelerado de la germinación en los tres casos, hasta que la el proceso finalizó a los 26 días, con 85 % de germinación total para semillas tratadas con Trichoderma, 86.7 % con Bacillus y 74 % con el testigo. Se observó un efecto significativo del tratamiento sobre el porcentaje de germinación; incluso el uso de ambos microorganismos aceleró el proceso de germinación con respecto al testigo. Estos resultados sugieren que el uso de Trichoderma spp y Bacillus spp. como tratamientos pregerminativos puede mejorar la germinación de A. victoriae-reginae y su conservación a largo plazo, lo que contribuye a la permanencia de esta especie en peligro de extinción.

Use of Biostimulants: Towards Sustainable Approach to Enhance Durum Wheat Performances

The use of biostimulant (BS) holds a promising and environmental-friendly innovation to address current needs of sustainable agriculture. The aim of the present study is twofold: (i) assess the potential of durum wheat seed coating with microbial BS (‘Panoramix’, Koppert), a mix of Bacillus spp., Trichoderma spp., and endomycorrhiza, compared to two chemical products (‘Spectro’ and ‘Mycoseeds’) through germination bioassay, pot and field trials under semi-arid conditions, and (ii) identify the most effective method of BS supply (‘seed coating’, ‘foliar spray’, and ‘seed coating + foliar spray’) under field conditions. For this purpose, three modern durum wheat cultivars were tested. ‘Panoramix’ was the most efficient treatment and enhanced all germination (germination rate, and coleoptile and radicle length), physiological (relative water content, chlorophyll content, and leaf area), and agro-morphological (plant height, biomass, seed number per spike, thousand kernel weight, and grain yield) attributes. Unexpectedly, the individual application of ‘Panoramix’ showed better performance than the combined treatment ‘Panoramix + Spectro’. Considering the physiological and agro-morphological traits, the combined method ‘seed coating + foliar spray’ displayed the best results. Principal component analysis confirmed the superiority of ‘Panoramix’ treatment or ‘seed coating + foliar spray’ method. Among tested durum wheat cultivars, ‘Salim’ performed better especially under ‘Panoramix’ treatment, but in some case ‘Karim’ valorized better this BS showing the highest increase rates. Based on these study outcomes, ‘Panoramix’ might be used as promising sustainable approach to stimulate durum wheat performance.

Antagonistic screening of Trichoderma spp. isolated from patchouli rhizosphere

The objective of this research was to determine the antagonistic activity of Trichoderma spp. isolated from patchouli rhizosphere (Pogostemon cablin Benth.). Another objective was to perform antagonistic screening of these fungi to inhibit the growth of the wilted pathogen Fusarium spp. In vitro research was conducted in the Laboratory of Plant Pathology, Universitas Syiah Kuala, from January to June 2020. The study used a completely randomised design with five treatments and three replications. The antagonistic screening was carried out by using the dual culture method of Trichoderma spp. against Fusarium spp. with the medium of Potato Dextrose Agar (PDA). The result showed that five isolates of Trichoderma have different antagonistic percentages in inhibiting the Fusarium. The highest antagonistic activity was found from isolate 2 and the lowest value was shown by isolate 3.

OPTIMIZING THE PRODUCTION OF A FUNCTIONAL TYPE A RECOMBINANT ENDOCHITINASE FROM Trichoderma asperellum IN Escherichia coli

Chitinases from the genus Trichoderma fungi are mainly responsible for their anti-fungal activities, which allow them to become the most widely used fungal biocontrol. Therefore, several Trichoderma chitinases have been cloned and expressed to facilitate their production and applications. A previous study of the same authors has characterized an endochitinase from a relatively novel Trichoderma spp., Trichoderma asperellum. To produce this enzyme more economically and efficiently, we reported the synthesis and expression of its synthetic encoding gene in the Escherichia coli M15 strain and established the optimal conditions for preparative scale production of the enzyme in its functional form. By lowering the induction temperatures, we observed substantial improvement in the expression levels of the active enzyme. At 30 oC and 0.5 mM IPTG induction, 1 L of cells yielded approximately 80 - 100 mg of soluble protein, accounting for about 9-11 % of total soluble protein. This figure may be an underestimation of the actual yield, as deduced from the SDS-PAGE data. The recombinant enzyme can be retrieved by simple repeated freezing and thawing cycles and purified to near homogeneity using Ni-NTA chromatography. The purified enzyme showed in vitro colloidal chitin hydrolysis activity. These results could be scaled up to produce soluble 42 kDa chitinase in E. coli. The study demonstrated an economical method to produce chitinases for various agricultural and environmental applications.

Screening and Identification of Trichoderma Strains isolated from Natural Habitats in China with Potential Agricultural Applications

Trichoderma spp. are widely distributed in natural habitats and have been evaluated as a potential biocontrol agent (BCA) for disease control and plant growth promotion. In this study, 1308 Trichoderma strains were obtained from the plant rhizosphere soil, above-ground plants, and decaying wood from natural habitats in China. Among them, 49 Trichoderma strains showed a good inhibitory effect, especially against Botrytis cinerea, Fusarium oxysporum, and Colletotrichum gloeosporioides with inhibition rate above 85% in the dual culture test. Among these 49 strains, the 13 strains with broad-spectrum inhibitory effects also significantly promoted the seed germination of five crops (rice, cucumber, tomato, melon, and pakchoi) and root growth of four crop seedlings (watermelon, tomato, eggplant, and chili). Furthermore, these strains showed effective colonization in the rhizosphere and root of cucumber. Trichoderma strains SC012 and NX043 showed the highest chitinase and β-1,3-glucanase activity among all strains. Based on the morphological characterization and phylogenetic analysis of the nuclear ribosomal internal transcribed spacer (ITS) and translation elongation factor 1 (tef1), twelve Trichoderma strains were identified as Trichoderma asperellum and one as Trichoderma afroharzianum. This study suggests that the 13 Trichoderma strains are promising BCAs and could be developed as biofertilizers and biological pesticides for agricultural applications.

Study of Trichoderma Spp. Application on the Incidence of Moler Disease and Shallot's Growth and Yield

Fusarium wilt disease/moler is a concern in onion cultivation. Many farmers apply chemical pesticides such as azoxystrobin and difenoconazole to control the diseases. Both of these chemical pesticides are currently exhibiting a decline in effectiveness, prompting farmers to increase the dosage and frequency of application. Recommendations for biological control, including Trichoderma spp.. This study aimed to determine the influence of application time of of Trichoderma spp. in various media types on the incidence of moler disease and shallot growth and yield. This study was conducted in Tabalong district, South Kalimantan, from February to June 2019 using a completely randomized design (CRD) factorial comprising two nested designs. The first factor was the type of media in which Trichoderma spp.. was applied, namely solid and liquid. The second factor was the application time of Trichoderma spp.. Level 1 was the application of Fusarium sp. seven days before planting and Trichoderma spp.. seven days after planting. Level 2 included the application of Trichoderma spp.. seven days before planting and Fusarium sp. simultaneously during planting. Level 3 involved the administration of Trichoderma spp.. concurrently with planting and Fusarium sp. seven days later. This investigation used one positive control (without treatment) and one negative control (shallots inoculated with Fusarium). Each experimental unit was consisted of ten plants and replicated three times. The parameters observed were the incubation period of Fusarium sp., disease incidence, number of leaves, plant height, number of cloves, and average clove weight. The study found that the application time of Trichoderma on various types of media affected the incidence of Fusarium disease, the number of leaves, and the number of shallot cloves.When compared to liquid media, the application of solid media resulted in a reduced incidence of Fusarium disease and a greater frequency of flares. The application time on solid media gave a low response to moles. As for liquid media, the lowest incidence of moler disease occurred at the seven days before planting.