Biocontrol Potential and Catabolic Profile of Endophytic Diaporthe eres Strain 1420S from Prunus domestica L. in Poland—A Preliminary Study

Recently, Diaporthe has been considered the most frequently isolated genera of endophytic fungi, having a broad spectrum of host plants and a worldwide distribution. The endophytic Diaporthe strain used in the present work came from the Fungal Collection of Phytopathology and Mycology Subdepartment, University of Life Sciences in Lublin (Poland), and was isolated from healthy Prunus domestica shoots during previous studies. Due to the possibility of using the Diaporthe endophytes as a promising option for plant disease management, the main goal of the research was to study the antagonistic effect of endophytic Diaporthe strain against six phytopathogens: Verticillium dahliae, Botrytis cinerea, Fusarium avenaceum, F. sprotrichioides, Alternaria alternata, and Trichothecium roseum based on the dual culture assay and to determine the catabolic profile of the endophyte by using Biolog FF Plates. The dual-culture test assay revealed the ability of the endophytic Diaporthe to limit the growth of all tested pathogens. The growth inhibition percentage ranged from 20% (V. dahliae) to 40% (T. roseum). A distinct zone of inhibition occurred between the endophytic Diaporthe and the pathogens T. roseum, V. dahliae, and B. cinerea in the co-growth combinations. As for the catabolic profile results, the most intensive utilization of carbon substrates was observed after 168 h of incubation. The growth of the analyzed strain was observed on 79 media containing carbohydrates, carboxylic acids, amino acids, amines and amides, polymers, and others. The most effective decomposition was observed in the polymers group, the least in amines and amides. Molecular identification indicated that this strain was closely related to the Diaporthe eres species complex.

Fungi Detected in the Previous Year’s Leaf Petioles of Fraxinus excelsior and Their Antagonistic Potential against Hymenoscyphus fraxineus

Studies on fungal communities in the previous year’s leaf petioles of Fraxinus excelsior found in litter in five ash stands in southern Poland were made in 2017. Fungi were identified on the basis of isolation from 300 surface sterilized leaf petioles and by in situ inventory of fruit bodies (on 600 petioles, in spring and autumn). Identification was based on morphology of colonies and fruit bodies, and sequencing of ITS region of the rRNA gene cluster. In total, 2832 isolates from 117 taxa (Ascomycota—100; Basidiomycota—15; Mucoromycota—2 taxa) were obtained with the isolation method. The most frequent taxa (with frequency >10%) were: Nemania serpens, Hymenoscyphus fraxineus, Alternaria sp. 1, Boeremia sp., Helotiales sp. 1, Epicoccum nigrum, Venturia fraxini, Fusarium sp., Fusarium lateritium, Nemania diffusa, Typhula sp. 2 (in descending order). In total, 45 taxa were detected with the in situ inventory method. Eleven taxa were classified as dominant: Hymenoscyphus fraxineus, Venturia fraxini, Leptosphaeria sp. 2, Cyathicula fraxinophila, Typhula sp. 2, Hypoderma rubi, Pyrenopeziza petiolaris, Cyathicula coronata, Hymenoscyphus scutula, Leptosphaeria sclerotioides and Hymenoscyphus caudatus. Among 202 leaf petioles colonized by H. fraxineus, 177 petioles also showed fructification of 26 other fungi. All the isolated saprotrophs were tested in dual-culture assay for antagonism to two strains of H. fraxineus. Three interaction types were observed: type A, mutual direct contact, when the two fungi meet along the contact line (occurred with 43.3% of test fungi); type B, with inhibition zone between colonies (with 46.9% of test fungi); type C, when the test fungus overgrows the colony of H. fraxineus (with 9.8% of test fungi). The possible contribution of the fungal saprotrophs in limiting of the expansion of H. fraxineus in ash leaf petioles, which may result in reduction in the inoculum of ash dieback causal agent, is discussed.

In vitro antagonistic activity of Trichoderma harzianum and T. viride strains compared to carbendazim fungicide against the fungal phytopathogens of Sorghum bicolor (L.) Moench

Background High losses of sorghum crops due to fungal diseases, such as grain mold and stalk rot, are economically harmful and cause increased use of environmentally damaging chemical fungicides, which also are detrimental to human health. Hence, finding safe and effective ways to manage fungal diseases of sorghum is urgently needed. Results In the present study, the antagonistic activities of Trichoderma viride and T. harzianum against different pathogenic fungal strains were evaluated in vitro using a dual culture assay. Furthermore, the anti-mycotic activity of Trichoderma spp. culture filtrates was evaluated against different fungal strains using a food poisoning technique. Additionally, the antifungal activities of ethyl acetate extracts of T. viride and T. harzianum against different pathogens were evaluated using a disk diffusion method. As indicated by the dual culture assay, T. harzianum suppressed 66.8, 69.5, 68.7, 54.6, 84.12, and 71.39% of the mycelial growth of Curvularia lunata, Exserohilum rostratum, Fusarium chlamydosporum, F. incarnatum, F. proliferatum, and Macrophomina phaseolina, respectively. T. viride was more effective for controlling the growth of these pathogens, inhibiting 81.0, 89.0, 63.0, 70.7, 84.4, and 71.8% of mycelial growth, respectively. Both E. rostratum and M. phaseolina showed resistance to carbendazim fungicide at all tested concentrations, whereas the fungicidal concentrations of carbendazim against C. lunata, F. chlamydosporum, and F. incarnatum strains were 2.50, 1.50, and 2.00 ppm, respectively. Furthermore, F. proliferatum was sensitive to carbendazim fungicide at all tested concentrations. Antifungal assays of the ethyl acetate extracts of T. viride and T. harzianum indicated the potent activity of these extracts against fungal phytopathogens with different susceptibility patterns. F. chlamydosporum was the most sensitive to the extracts of T. viride and T. harzianum with minimum inhibitory concentrations of 0.5 and 1.0 mg/disk, respectively. Conclusion The potent suppression of sorghum phytopathogens by T. viride and T. harzianum makes them potential sources of safe and effective natural fungicides compared to carbendazim fungicide.

Post-Harvest Non-Conventional and Traditional Methods to Control Cadophora luteo-olivacea: Skin Pitting Agent of Actinidia chinensis var. deliciosa (A. Chev.)

Cadophora luteo-olivacea represents a critical problem for kiwifruit in the post-harvest phase, mainly for its little note epidemiology. The study presented some results about the possibility of preserving kiwifruit from skin pitting symptoms using alternative methods to fungicides. By in vitro assays, antagonist mechanisms of action against pathogen isolates were tested. Trichoderma harzianum (Th1) showed the highest inhibitory activity against C. luteo-olivacea isolates by volatile, non-volatile, and by dual culture assay, displaying an inhibition respectively by 90%, 70.6%, and 78.8%, and with respect to Aureobasidium pullulans (L1 and L8) by 23.3% and 25.8%, 50% and 34.7%, and 22.5% and 23.6%, respectively. Further, the sensitivity on CFU and mycelial growth of C. luteo-olivacea isolates to fludioxonil, and CaCl2 was tested, displaying interesting EC50 values (0.36 and 0.92 g L−1, 22.5 g L−1, respectively). The effect of Brassica nigra defatted meal was tested as biofumigation assays and through FT-IR (Fourier-Transform Infrared) spectroscopy. The above-mentioned treatments were applied in vivo to evaluate their efficacy on kiwifruits. Our data demonstrated that alternative solutions could be considered to control postharvest pathogens such as C. luteo-olivacea.

Screening of Fungal Endophytes for their Biocontrol Potential against Rhizopus sp. Isolated from Diseased Cassava (Manihot esculenta Crantz)

The aim of this study was to screen for the bio-control potential of fungal endophytes isolated from cassava against a test pathogen of cassava. Fungal endophytes and pathogen were isolated and identified from healthy and diseased cassava respectively. The isolated fungal endophytes were screened for their biocontrol potential against a test pathogen using the dual culture and culture filtrate assay. Fusarium sp., Botryosphaeria sp., Colletotrichum sp., yeast isolate 1 and 2 were the isolated fungal endophytes while the pathogen was Rhizopus sp. The effect of endophytes on the mycelia growth of Rhizopus sp. using the dual culture assay indicated that yeast isolate 1 & 2 and Colletotrichum sp. were effective in inhibiting the mycelia growth of the test pathogen while Fusarium sp. and Botryosphaeria sp. were not effective. The co-culture of yeast isolate 2 with the test pathogen gave the lowest mycelia growth (1.66a±0.09) at day 2. The effect of endophytic culture filtrate on the mycelia growth of Rhizopus sp. showed that Fusarium sp. gave the lowest mycelia growth in the three days observed. The findings from this study suggested that the test endophytes have biocontrol potential against Rhizopus sp. The biocontrol abilities of the test endophytes vary using the dual culture and culture filtrate assay.

Weed-Associated Fungal Endophytes as Biocontrol Agents of Fusariumoxysporum f. sp. cubense TR4 in Cavendish Banana

The antagonistic activity of fungal endophytes isolated from weeds growing in Cavendish banana farms was determined against Fusarium oxysporum f. sp. cubense TR4 (Foc TR4) causing Fusarium wilt of Cavendish banana. Forty-nine out of the total 357 fungal endophytes from the roots of weeds exhibited antagonistic activity against Foc TR4. High inhibitory activity at 79.61–99.31% based on dual culture assay was recorded in endophytes Lasiodiplodia theobromae TDC029, Trichoderma asperellum TDC075, Ceratobasidium sp. TDC037, Ceratobasidium sp. TDC241, and Ceratobasidium sp. TDC474. All five endophytes were identified through DNA sequencing with 86–100% identity. Endophyte-treated Grand Naine and GCTCV 218 plantlets showed significantly lower disease incidence (p = 0.014), significantly lower degree of leaf yellowing (p = 0.037) and rhizome discoloration (p = 0.003). In addition, the cultivar Grand Naine was consistently highly susceptible compared with the tolerant cultivar GCTCV 218.

Isolation, Identification and In Vitro Test for the Biocontrol Potential of Trichoderma viride on Fusarium oxysporum f. sp. Lycopersici

Introduction: Tomato production in Ethiopia is challenged by many pests and diseases. Fusarium wilt is one of the most important diseases of tomato affecting its productivity. Methods: Tomato tissue and soil samples were collected from tomato farmlands around Aksum town to isolate and identify pathogenic Fusarium species and Trichoderma species with biocontrol efficacy. Samples were processed in the Aksum University Biotechnology laboratory following standard procedures. Results and Discussion: Eight Fusarium and five Trichoderma isolates were obtained. Six of the Fusarium isolates were identified as Fusarium oxysporum, whereas the remaining two were Fusarium equiseti and Fusarium circinatum. Detached leaf bioassay of the F. oxysporum on tomato leaves showed leaf lesion on the tomato variety, Melka oda. The isolated Trichoderma strains were screened for biocontrol potential against virulent F. oxysporum in vitro. The Trichoderma isolate showing the highest biocontrol efficacy against the virulent Fusarium was morphologically identified as Trichoderma viride. in vitro F. oxysporum-T. viride dual culture assay demonstrated that T. viride inhibits the growth of F. oxysporum f.sp. lycopersici with 76.94% growth inhibition. Conclusion: Fusarium oxysporum is prevalent in tomato growing farmlands covered in this study. T. viride identified in this study is an effective biocontrol agent for the identified F. oxysporum fsp. lycopersici in vitro.

Antagonistic Potential of Novel Endophytic Bacillus Strains and Mediation of Plant Defense against Verticillium Wilt in Upland Cotton

Verticillium wilt caused by Verticillium dahliae is a threatening disease of cotton, causing economic loss worldwide. In this study, nine endophytic Bacillus strains isolated from cotton roots exhibited inhibitory activity against V. dahliae strain VD-080 in a dual culture assay. B. altitudinis HNH7 and B. velezensis HNH9 were chosen for further experiments based on their high antagonistic activity. The secondary metabolites of HNH7 and HNH9 also inhibited the growth of VD-080. Genetic marker-assisted detection revealed the presence of bacillibactin, surfactin, bacillomycin and fengycin encoding genes in the genome of HNH7 and HNH9 and their corresponding gene products were validated through LC-MS. Scanning electron microscopy revealed mycelial disintegration, curling and shrinkage of VD-080 hyphae after treatment with methanolic extracts of the isolated endophytes. Furthermore, a significant reduction in verticillium wilt severity was noticed in cotton plants treated with HNH7 and HNH9 as compared to control treatments. Moreover, the expression of defense-linked genes, viz., MPK3, GST, SOD, PAL, PPO and HMGR, was considerably higher in plants treated with endophytic Bacillus strains and inoculated with VD-080 as compared to control.

Biocontrol of Pythium Damping-Off on Pepper (Capsicum Annuum) with Selected Fungal and Rhizobacterial Agents

Pythium ultimum is a common soilborne pathogen causing serious losses of pepper seedlings in nurseries and few weeks post-planting. Two pepper associated-P. ultimum isolates (P1 and P2) were shown pathogenic to pepper cv. Altar causing post-emergence damping-off with P2 isolate being the most aggressive. Fungal and bacterial antagonists have been evaluated in vitro and in vivo for their ability to suppress P. ultimum. In dual culture assay, Trichoderma harzianum, T. viride and Gliocladium virens inhibited pathogen radial growth by 18.54, 17.52 and 15.24%, respectively, relative to control, while none of the tested bacteria was shown able to significantly inhibit pathogen growth. However, drastic changes in pathogen hyphae expressed as strong lysis, the formation of mycelial cords and mycoparasitism have been observed. Pepper seeds treated with fungal antagonists’ conidial suspensions showed 60, 50 and 60% less pre-emergence damping-off infections, respectively, compared to the positive control. When tested as root dipping, only G. virens resulted in 40% reduced post-emergence damping-off. An improved seedlings fresh weight, by 79.31 and 76%, was respectively induced by G. virens-, and T. viride-based treatments while an increment of 27.58, 25.33 and 22.22 % was recorded following treatments with G. virens, T. viride and T. harzianum, relative to the positive control. The majority of tested bacterial isolates, applied as a seed treatment, had significantly improved the emergence percentage of inoculated seedlings as compared to control with Burkholderia glathei isolate 35 being the most efficient. When applied as root dipping, reduction of post-emergence damping-off ranged between 40 and 100% with Pseudomonas aureofaciens isolate 314 being the most effective agent. Seedlings treated with P. aureofaciens (314) and Bacillus pumilus (420) showed 35.38 and 28.51% higher heights, respectively. Plant weight was enhanced by 73.06, 61.18, 77.39, 61.8 and 67.93% over control following treatments with P. aureofaciens isolates 314 and 31, Bacillus pumilus 420, P. fluorescens and P. putida 227.

Beneficial Bacteria Identified for the Control of Botrytis cinerea in Petunia Greenhouse Production

Botrytis cinerea infects most major greenhouse crops worldwide. With its increasing resistance to conventional fungicides and the movement of the greenhouse industry toward more sustainable production practices, alternative methods of control are needed. The objective of this study was to evaluate a collection of 60 bacterial strains through both a dual-culture assay and greenhouse trials to identify strains with biocontrol activity against B. cinerea. For the dual-culture assay, each bacterial strain was streaked on potato dextrose agar medium with B. cinerea. The B. cinerea growth reduction and the zone of inhibition were measured. Thirty-five strains reduced the growth of B. cinerea. All strains were also tested in an initial greenhouse trial in which Petunia × hybrida ‘Carpet Red Bright’ was sprayed and drenched with the bacteria biweekly for 6 weeks. All open flowers were tagged, and plants were inoculated with B. cinerea (1 × 104 conidia per 1 ml). Disease severity indices calculated from the daily flower gray mold severity ratings of all tagged flowers were used to identify the seven top-performing strains. These seven strains were then evaluated in a greenhouse validation trial. The methods were similar to those of the initial greenhouse trials except that replicate numbers were increased. Three strains (Pseudomonas protegens AP54, Pseudomonas chlororaphis 14B11, and Pseudomonas fluorescens 89F1) were selected for the ability to reduce B. cinerea infection in a greenhouse production setting. These strains can be used in future studies to develop additional biocontrol products for the management of B. cinerea in floriculture crops.