Investigation of some endophytic fungi from five medicinal plants with growth promoting ability on maize (Zea mays L.).

AIMS: This study aimed to identify endophytic fungi from Anthemis altissima, Matricaria parthenium, Cichorium intybus, Achillea millefolium, and A. filipendulina with plant-promoting ability on the ZP684 maize hybrid-cultivar. METHODS AND RESULTS: Plants were collected from northeast-Iran and endophytic fungi were isolated and identified using partial large subunit nrDNA, internal transcribed spacer, translation elongation factor, and ß-tubulin genetic markers. Endophytic fungi that improved seed germination were studied under greenhouse conditions. Ninety-seven endophytic fungi were identified. Preussia africana, Bjerkandera adusta, Schizophyllum commune, Alternaria embellisia, Trichaptum biforme, Septoria malagutii, A. consortiale, Verticillium dahliae, Fusarium avenacearum, and Trametes versicolor significantly improved seed-germination. Alternaria consortiale produced the highest level of indole-3-acetic acid-like compounds and maize growth-promoting. CONCLUSIONS: Plant fungal colonization frequency increased with orthometric height. Sampling location Chahar Bagh at 2230 m contained the most endophytic fungi. Fusarium and Alternaria were the most frequently isolated endophytic genera. Therefore, medicinal plants are potential hosts for endophytic fungi that may be suitable biofertilizer agents in agriculture. SIGNIFICANCE AND IMPACT OF THE STUDY: This study helps to better understand the ecosystem functions by investigating of endophytic fungi distribution under different ecological conditions. Finding effective isolates among these microorganisms with a suitable plant-promoting ability on crops may help to reduce the use of chemical fertilizers in an agroecosystem.

Evaluation of colonization and mutualistic endophytic symbiosis of Escherichia coli with tomato and Bermuda grass seedlings.

Escherichia coli is generally considered a bacterium associated with animal microbiomes. However, we present evidence that E. coli may also mutualistically colonize roots of plant species, even to the extent that it may become endophytic in plants. In this study we used GFP tagged E. coli to observe its colonization and effects on tomato (Solanum lycopersicum) and Bermuda grass (Cynodon dactylon) seedling development and growth. Inoculation with the bacterium significantly improved root development of both seedlings tested. Treatment also increased the photosynthetic pigments in Bermuda grass seedlings. However, effects on shoot length in both seedlings were not significant. This bacterium was found to produce indole acetic acid (IAA) up to 8.68 ± 0.43 µg ml-1 in the broth medium amended with tryptophan. Effects on seedling root growth could, in part, be explained by IAA production. Bacteria successfully colonized the root surfaces and interiors of both seedlings. Tagged bacteria expressing the GFP were observed in the vascular tissues of Bermuda grass seedling roots. Seedlings with bacteria showed greater survival and were healthier than seedlings without bacteria, indicating that E. coli set up a successful mutualistic symbiosis with seedlings. E. coli is not commonly considered to be a plant endophyte but is more generally considered to be a crop contaminant. In this study we show that E. coli may also be an endophyte in plant tissues.

Endophytic Microbes Are Tools to Increase Tolerance in Jasione Plants Against Arsenic Stress.

Seed microbiota is becoming an emergent area of research. Host plant microbial diversity is increasingly well described, yet relatively little is known about the stressors driving plant endomicrobiota at the metaorganism level. The present work examines the role of horizontal and vertical transmission of bacterial microbiota in response to abiotic stress generated by arsenic. Horizontal transmission is achieved by bioaugmentation with the endophyte Rhodococcus rhodochrous, while vertical transmission comes via maternal inheritance from seeds. To achieve this goal, all experiments were conducted with two Jasione species. J. montana is tolerant to arsenic (As), whereas J. sessiliflora, being phylogenetically close to J. montana, was not previously described as As tolerant. The Jasione core bacterial endophytes are composed of genera Pseudomonas, Ralstonia, Undibacterium, Cutibacterium, and Kocuria and family Comamanadaceae across different environmental conditions. All these operational taxonomic units (OTUs) coexisted from seeds to the development of the seedling, independently of As stress, or bioaugmentation treatment and Jasione species. R. rhodochrous colonized efficiently both species, driving the endomicrobiota structure of Jasione with a stronger effect than As stress. Despite the fact that most of the OTUs identified inside Jasione seeds and seedlings belonged to rare microbiota, they represent a large bacterial reservoir offering important physiological and ecological traits to the host. Jasione traits co-regulated with R. rhodochrous, and the associated microbiota improved the host response to As stress. NGS-Illumina tools provided further knowledge about the ecological and functional roles of plant endophytes.

Screening of sunflower associated bacteria as biocontrol agents for plant growth promotion.

Deleterious effects of artificially applied chemicals have highlighted the significance of biocontrol agents as suitable substitute for sustainable agriculture. In present study, three endophytic bacterial strains SV7, SV10 and LV19 showed extensive range of antifungal as well as plant growth promoting activities signifying potential to accomplish the requirement. Phylogenetic analysis revealed 100% similarity of three strains with taxon Firmicute. However, there was division among these isolates on basis of subgroups as SV7 belonged to Exiguobacterium auranticum, SV10 belonged to Paenibacillus sp. and LV19 was best fit in subgroup Priestia koreensis. All strains showed antifungal activity against Fusarium oxysporum on three different media (PDA, NA, LA) with maximum activity (53%) of LV19 strain on NA and least activity (13%) on PDA medium as recorded by zones of inhibition. In growth promotion experiments, combination of LV19 with Fusarium significantly suppressed chances of Fusarium wilt which is commonly caused by Fusarium oxysporum in sunflower plants. Diverse growth parameters (seed germination percentage, lengths and fresh weights of root and shoot) were significantly increased from 34 to 909% over pathogen infected plants only which was further proved by their root colonization analysis. Based on most efficient growth promotion by LV19 strain, expression of five plant defense related genes (SOD, PAL, NPR1, PR5, Chitinase) was evaluated revealing enhanced expression by 1.7-270-folds in consortium of LV19 and Fusarium. Thus, current study provided a scientific justification that bacterial strains in specific LV19 (Priestia koreensis) could be further developed as biocontrol agent with potential of plant growth promotion.

First Report of Leaf Spot Disease Caused by Stemphylium vesicarium on Fagopyrum esculentum in China.

Common buckwheat (Fagopyrum esculentum) is a widely cultivated non-grass cereal. It has a considerable market value with nutritional qualities and ability to treat or prevent cancers, hypertension and diabetes (Cawoy et al., 2006). In July-August 2018 and 2019, leaves exhibiting unfamiliar symptoms were observed in Huan County and Huachi County, Gansu, China, with 15% incidence and moderate to severe severity across the field. Initial symptoms consisted of small chlorotic, circular to oval leaf spots. As disease progressed, the spots enlarged and turned light brown to brown with chlorotic margins. When the disease was severe, the leaf spots coalesced and the leaves became prematurely chlorotic and senescent. Spots occurred mostly but not exclusively on older foliage. Diseased tissues were surface sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, placed on PDA medium and incubated at 20 °C for 48-72 h. A total 24 isolates were obtained and purified through single-spore cultures, 19 of which were characterized. Colonies on PDA of all 19 isolates were identical, exhibiting a light gray color, with whitish aerial mycelium that later turned light brown on the reverse of the culture plates, and sporulated sparsely. Conidia were brown colored, cylindrical, and borne singly, often had three main transverse septa, at which points there were conspicuous constrictions, and measured (17-37) µm long × (13-21) µm wide (n=30) in V8 juice agar, (22-38) µm long × (11-19) µm wide (n=30) in PCA, (21-41) µm long × (13-20) µm wide (n=30) in SNA, the mean length/width ratio was 1.6 to 2.0 in V8 juice agar, 1.6 to 2.5 in PCA, 1.3 to 2.2 in SNA. Conidiophores were unbranched, 5.1 to 7.9 µm wide. Dictyospores were produced on well-differentiated conidiophores, the apical cell of conidiophore was slightly to distinctly swollen. Based on morphological features, the isolates were tentatively identified as a member of the Stemphylium vesicarium species complex (Koike et al., 2013; Simmons, 1967). Genomic DNA of representative isolate B1 was extracted, and the internal transcribed spacer (ITS) region and calmodulin gene (cmdA) were amplified using ITS1/4, V9G/ITS4, and CALDF1/CALDR1, respectively. The resulting sequences were deposited in GenBank (acc. nos. MT629829, MW406903, MW417122). Nucleotide BLAST similarity analysis of the sequence fragment of ITS and cmdA from isolate B1 resulted in higher than 99% (99.32% for ITS1/4, 100% for V9G/ITS4, and 100% for CALDF1/CALDR1) identity with S. vesicarium strains (GenBank acc. nos. LC512757, MH863402 and MH206181). Based on morphological features and molecular data, the buckwheat isolates were identified as S. vesicarium. To verify pathogenicity, the back side of leaves from six asymptomic plants were inoculated by spraying the spore suspension (104 spores/ml) harvested from isolate B1 grown on V8 juice agar for 20 days. Control plants were sprayed with sterile water. Each plant was covered with a black plastic bag for 48 h and then was kept in a greenhouse. Stemphylium spot symptoms were observed on all inoculated leaves after 14 days, whereas control leaves were symptomless. The pathogen was reisolated from symptomatic leaf spots, micromorphological features and colony characters of the reisolated fungi were identical to the original isolate. To our knowledge, this is the first documentation of leaf spot of buckwheat caused by S. vesicarium in China, and the first characterization of a Stemphylium foliar pathogen on this crop.

LC-MS based identification of stylosin and tschimgine from fungal endophytes associated with Ferula ovina.

OBJECTIVES: Ferula ovina is an Iranian medicinal plant. Tschimgine and stylosin are two of its major monoterpene derivatives. In this study, we proceeded to investigate some fungal endophytes from F. ovina that can produce plant secondary metabolites. MATERIALS AND METHODS: The isolated endophytic fungi were fermented in potato dextrose broth (PDB) medium and their extracts were screened for the presence of the plant compounds by liquid chromatography-tandem mass spectrometry (LC-MS). Endophytes identification was performed by morphological and molecular methods. Three markers (ITS, LSU, and TEF1) were used for accurate molecular identification. RESULTS: Forty isolates from 9 different genera of endophytic fungi were identified, of which two recently reported species of O. ferulica and Pithoascus persicus were able to produce tschimgine and stylosin. CONCLUSION: These fungi can be used as a substitute for the production of plant's medicinal compounds independent of wild populations of the source plant.

Mercury resistance and bioremediation mediated by endophytic fungi.

The present study proposes the use of endophytic fungi for mercury bioremediation in in vitro and host-associated systems. We examined mercury resistance in 32 strains of endophytic fungi grown in culture medium supplemented with toxic metal concentrations. The residual mercury concentrations were quantified after mycelial growth. Aspergillus sp. A31, Curvularia geniculata P1, Lindgomycetaceae P87, and Westerdykella sp. P71 were selected and further tested for mercury bioremediation and bioaccumulation in vitro, as well as for growth promotion of Aeschynomene fluminensis and Zea mays in the presence or absence of the metal. Aspergillus sp. A31, C. geniculata P1, Lindgomycetaceae P87 and Westerdykella sp. P71 removed up to 100% of mercury from the culture medium in a species-dependent manner and they promoted A. fluminensis and Z. mays growth in substrates containing mercury or not (Dunnett's test, p < 0.05). Lindgomycetaceae P87 and C. geniculata P1 are dark septate endophytic fungi that endophytically colonize root cells of their host plants. The increase of host biomass correlated with the reduction of soil mercury concentration due to the metal bioaccumulation in host tissues and its possible volatilization. The soil mercury concentration was decreased by 7.69% and 57.14% in A. fluminensis plants inoculated with Lindgomycetaceae P87 + Aspergillus sp. A31 and Lindgomycetaceae P87, respectively (Dunnet's test, p < 0.05). The resistance mechanisms of mercury volatilization and bioaccumulation in plant tissues mediated by these endophytic fungi can contribute to bioremediation programs. The biochemical and genetic mechanisms involved in bioaccumulation and volatilization need to be elucidated in the future.

Endophytic fungal communities of Polygonum acuminatum and Aeschynomene fluminensis are influenced by soil mercury contamination.

The endophytic fungal communities of Polygonum acuminatum and Aeschynomene fluminensis were examined with respect to soil mercury (Hg) contamination. Plants were collected in places with and without Hg+2 for isolation and identification of their endophytic root fungi. We evaluated frequency of colonization, number of isolates and richness, indices of diversity and similarity, functional traits (hydrolytic enzymes, siderophores, indoleacetic acid, antibiosis and metal tolerance) and growth promotion of Aeschynomene fluminensis inoculated with endophytic fungi on soil with mercury. The frequency of colonization, structure and community function, as well as the abundant distribution of taxa of endophytic fungi were influenced by mercury contamination, with higher endophytic fungi in hosts in soil with mercury. The presence or absence of mercury in the soil changes the profile of the functional characteristics of the endophytic fungal community. On the other hand, tolerance of lineages to multiple metals is not associated with contamination. A. fluminensis depends on its endophytic fungi, since plants free of endophytic fungi grew less than expected due to mercury toxicity. In contrast plants containing certain endophytic fungi showed good growth in soil containing mercury, even exceeding growth of plants cultivated in soil without mercury. The data obtained confirm the hypothesis that soil contamination by mercury alters community structure of root endophytic fungi in terms of composition, abundance and species richness. The inoculation of A. fluminensis with certain strains of stress tolerant endophytic fungi contribute to colonization and establishment of the host and may be used in processes that aim to improve phytoremediation of soils with toxic concentrations of mercury.