Plant Symbionts Research Articles

A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal.

Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti) synthesizes and responds to N-[(2E, 4E)-2,4-dodecadienoyl] homoserine lactone (2E, 4E-C12:2-HSL). We show that the 2E, 4E-C12:2-HSL QS circuit involves one of four luxR-luxI-type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2E, 4E-C12:2-HSL. The 2E, 4E-C12:2-HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2E, 4E-C12:2-HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S-adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

Current Insights in Fungal Importance-A Comprehensive Review.

Besides plants and animals, the Fungi kingdom describes several species characterized by various forms and applications. They can be found in all habitats and play an essential role in the excellent functioning of the ecosystem, for example, as decomposers of plant material for the cycling of carbon and nutrients or as symbionts of plants. Furthermore, fungi have been used in many sectors for centuries, from producing food, beverages, and medications. Recently, they have gained significant recognition for protecting the environment, agriculture, and several industrial applications. The current article intends to review the beneficial roles of fungi used for a vast range of applications, such as the production of several enzymes and pigments, applications regarding food and pharmaceutical industries, the environment, and research domains, as well as the negative impacts of fungi (secondary metabolites production, etiological agents of diseases in plants, animals, and humans, as well as deteriogenic agents).

Ketols Emerge as Potent Oxylipin Signals Regulating Diverse Physiological Processes in Plants.

Plants produce an array of oxylipins implicated in defense responses against various stresses, with about 600 oxylipins identified in plants to date. Most known oxylipins are the products of lipoxygenase (LOX)-mediated oxygenation of polyunsaturated fatty acids. One of the most well-characterized oxylipins produced by plants is the hormone jasmonic acid (JA); however, the function of the vast majority of oxylipins remains a mystery. One of the lesser-studied groups of oxylipins is comprised of ketols produced by the sequential action of LOX, allene oxide synthase (AOS), followed by non-enzymatic hydrolysis. For decades, ketols were mostly considered mere by-products of JA biosynthesis. Recent accumulating evidence suggests that ketols exhibit hormone-like signaling activities in the regulation of diverse physiological processes, including flowering, germination, plant-symbiont interactions, and defense against biotic and abiotic stresses. To complement multiple reviews on jasmonate and overall oxylipin biology, this review focuses specifically on advancing our understanding of ketol biosynthesis, occurrence, and proposed functions in diverse physiological processes.

Characterization of Arbuscular Mycorrhizal Effector Proteins.

Plants are colonized by various fungi with both pathogenic and beneficial lifestyles. One type of colonization strategy is through the secretion of effector proteins that alter the plant's physiology to accommodate the fungus. The oldest plant symbionts, the arbuscular mycorrhizal fungi (AMF), may exploit effectors to their benefit. Genome analysis coupled with transcriptomic studies in different AMFs has intensified research on the effector function, evolution, and diversification of AMF. However, of the current 338 predicted effector proteins from the AM fungus Rhizophagus irregularis, only five have been characterized, of which merely two have been studied in detail to understand which plant proteins they associate with to affect the host physiology. Here, we review the most recent findings in AMF effector research and discuss the techniques used for the functional characterization of effector proteins, from their in silico prediction to their mode of action, with an emphasis on high-throughput approaches for the identification of plant targets of the effectors through which they manipulate their hosts.

Structurally distinct mitoviruses: are they an ancestral lineage of the Mitoviridae exclusive to arbuscular mycorrhizal fungi (Glomeromycotina)?

Mitoviruses in the family Mitoviridae are the mitochondria-replicating "naked RNA viruses" with genomes encoding only the replicase RNA-dependent RNA polymerase (RdRp) and prevalent across fungi, plants, and invertebrates. Arbuscular mycorrhizal fungi in the subphylum Glomeromycotina are obligate plant symbionts that deliver water and nutrients to the host. We discovered distinct mitoviruses in glomeromycotinian fungi, namely "large duamitovirus," encoding unusually large RdRp with a unique N-terminal motif that is endogenized in some host genomes. More than 400 viral sequences similar to the large duamitoviruses are present in metatranscriptome databases. They are globally distributed in soil ecosystems, consistent with the cosmopolitan distribution of glomeromycotinian fungi, and formed the most basal clade of the Mitoviridae in phylogenetic analysis. Given that glomeromycotinian fungi are the only confirmed hosts of these viruses, we propose the hypothesis that large duamitoviruses are the most ancestral lineage of the Mitoviridae that have been maintained exclusively in glomeromycotinian fungi.

Veterinary drug albendazole inhibits root colonization and symbiotic function of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi (AMF) are plant symbionts that have a pivotal role in maintaining soil fertility and nutrient cycling. However, these microsymbionts may be exposed to organic pollutants like pesticides or veterinary drugs known to occur in agricultural soils. Anthelminthics are veterinary drugs that reach soils through the application of contaminated manures in agricultural settings. Their presence might threaten the function of AMF, considered as sensitive indicators of the toxicity of agrochemicals to the soil microbiota. We determined the impact of the anthelminthic compounds albendazole and ivermectin on the establishment and functionality of the symbiosis between the model-legume Lotus japonicus and the AMF Rhizophagus irregularis. Our analyses revealed negative effects of albendazole on the development and functionality of arbuscules, the symbiotic organelle of AMF, at a concentration of 0.75μg g-1. The impairment of the symbiotic function was verified by the reduced expression of genes SbtM1, PT4 and AMT2;2 involved in arbuscules formation, P and N uptake, and the lower phosphorus shoot content detected in the albendazole-treated plants. Our results provide first evidence for the toxicity of albendazole on the colonization capacity and function of R. irregularis at concentrations that may occur in agricultural soils systematically amended with drug-containing manures.

Salmonella Type III Secretion Effector SrfJ: A Glucosylceramidase Affecting the Lipidome and the Transcriptome of Mammalian Host Cells.

Type III secretion systems are found in many Gram-negative pathogens and symbionts of animals and plants. Salmonella enterica has two type III secretion systems associated with virulence, one involved in the invasion of host cells and another involved in maintaining an appropriate intracellular niche. SrfJ is an effector of the second type III secretion system. In this study, we explored the biochemical function of SrfJ and the consequences for mammalian host cells of the expression of this S. enterica effector. Our experiments suggest that SrfJ is a glucosylceramidase that alters the lipidome and the transcriptome of host cells, both when expressed alone in epithelial cells and when translocated into macrophages in the context of Salmonella infection. We were able to identify seventeen lipids with higher levels and six lipids with lower levels in the presence of SrfJ. Analysis of the forty-five genes, the expression of which is significantly altered by SrfJ with a fold-change threshold of two, suggests that this effector may be involved in protecting Salmonella from host immune defenses.

The structure of root-associated fungal communities is related to the long-term effects of plant diversity on productivity.

Root-associated fungi could play a role in determining both the positive relationship between plant diversity and productivity in experimental grasslands, and its strengthening over time. This hypothesis assumes that specialized pathogenic and mutualistic fungal communities gradually assemble over time, enhancing plant growth more in species-rich than in species-poor plots. To test this hypothesis, we used high-throughput amplicon sequencing to characterize root-associated fungal communities in experimental grasslands of 1 and 15 years of age with varying levels of plant species richness. Specifically, we tested whether the relationship between fungal communities and plant richness and productivity becomes stronger with the age of the experimental plots. Our results showed that fungal diversity increased with plant diversity, but this relationship weakened rather than strengthened over the two time points. Contrastingly, fungal community composition showed increasing associations with plant diversity over time, suggesting a gradual build-up of specific fungal assemblages. Analyses of different fungal guilds showed that these changes were particularly marked in pathogenic fungi, whose shifts in relative abundance are consistent with the pathogen dilution hypothesis in diverse plant communities. Our results suggest that root-associated fungal pathogens play more specific roles in determining the diversity-productivity relationship than other root-associated plant symbionts.

Urban land uses shape soil microbial abundance and diversity

Soil microbial biodiversity provides many useful services in cities. However, the ecology of microbial communities in urban soils remains poorly documented, and studies are required to better predict the impact of urban land use. We characterized microbial communities (archea/bacteria and fungi) in urban soils in Dijon (Burgundy, France). Three main land uses were considered – public leisure, traffic, and urban agriculture – sub-categorized in sub-land uses according to urban indexes and management practices. Microbial biomass and diversity were determined by quantifying and high-throughput sequencing of soil DNA. Variation partitioning analysis was used to rank soil physicochemical characteristics and land uses according to their relative contribution to the variation of soil microbial communities. Urban soils in Dijon harbored high levels of microbial biomass and diversity that varied according to land uses. Microbial biomass was 1.8 times higher in public leisure and traffic sites than in urban agriculture sites. Fungal richness increased by 25 % in urban agriculture soils, and bacterial richness was lower (by 20 %) in public leisure soils. Partitioning models explained 25.7 %, 46.2 % and 75.6 % of the variance of fungal richness, bacterial richness and microbial biomass, respectively. The organic carbon content and the C/N ratio were the best predictors of microbial biomass, whereas soil bacterial diversity was mainly explained by soil texture and land use. Neither metal trace elements nor polycyclic aromatic hydrocarbons contents explained variations of microbial communities, probably due to their very low concentration in the soils. The microbial composition results highlighted that leisure sites represented a stabilized habitat favoring specialized microbial groups and microbial plant symbionts, as opposed to urban agriculture sites that stimulated opportunistic populations able to face the impact of agricultural practices. Altogether, our results provide evidence that there is scope for urban planners to drive soil microbial diversity through sustainable urban land use and associated management practices.

Extracellular Vesicles in the Arbuscular Mycorrhizal Symbiosis: Current Understanding and Future Perspectives

The arbuscular mycorrhizal (AM) symbiosis is an ancient and highly conserved mutualism between plant and fungal symbionts, in which a highly specialized membrane-delimited fungal arbuscule acts as the symbiotic interface for nutrient exchange and signaling. As a ubiquitous means of biomolecule transport and intercellular communication, extracellular vesicles (EVs) are likely to play a role in this intimate cross-kingdom symbiosis, yet, there is a lack of research investigating the importance of EVs in AM symbiosis despite known roles in microbial interactions in both animal and plant pathosystems. Clarifying the current understanding of EVs in this symbiosis in light of recent ultrastructural observations is paramount to guiding future investigations in the field, and, to this end, this review summarizes recent research investigating these areas. Namely, this review discusses the available knowledge regarding biogenesis pathways and marker proteins associated with the various plant EV subclasses, EV trafficking pathways during symbiosis, and the endocytic mechanisms implicated in the uptake of these EVs. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effects of pavements on diversity and activity of mycorrhizal symbionts associated with urban trees

This study investigated the impact of different types of soil sealing on the communities of a group of beneficial plant symbionts, arbuscular mycorrhizal fungi (AMF), colonizing the roots of two shade trees, Celtis australis and Fraxinus ornus, frequently grown in urbanized sites. Such plants were grown in an experimental site, in northern Italy, established in November 2011 and subjected to four different pavement treatments: impermeable monolithic asphalt, permeable pavers, permeable concrete and unpaved soil. The diversity and composition of root AMF communities were assessed by PCR denaturating gradient gel electrophoresis of partial 18S rRNA gene, AMF taxa were identified by amplicon sequencing and mycorrhizal colonization was evaluated after root clearing and staining. For the first time, our molecular work revealed that impermeable pavements induced shifts in the composition of AMF communities associated to the roots of C. australis and F. ornus and impacted on the percentage of mycorrhizal root length. When the root-zone was covered with permeable pavements, a similar AMF community as that observed in the unpaved soil was detected, providing novel information to be utilised for reducing the disturbance caused by specific types of soil sealing on AMF symbionts, which play a key role in plant nutrition and health. A total of 45 AMF sequence types were detected, with Sclerocystis and Septoglomus as the most abundant phylotypes, accounting for 84% of the sequences. The predominance of Sclerocystis species in the roots of both tree species under impermeable pavements indicated their high and unforeseen tolerance towards harsh environmental conditions. Such species could be utilized as AMF inocula specifically selected for their proven resilience in paved sites, in order to exploit their ability to boost biogeochemical processes fundamental for energy fluxes and plant nutrition and health.

Disentangling arbuscular mycorrhizal fungi and bacteria at the soil-root interface.

Arbuscular mycorrhizal fungi (AMF) are essential components of the plant root mycobiome and are found in approximately 80% of land plants. As obligate plant symbionts, AMF harbor their own microbiota, both inside and outside the plant root system. AMF-associated bacteria (AAB) possess various functional traits, including nitrogen fixation, organic and inorganic phosphate mobilization, growth hormone production, biofilm production, enzymatic capabilities, and biocontrol against pathogen attacks, which not only contribute to the health of the arbuscular mycorrhizal symbiosis but also promote plant growth. Because of this, there is increasing interest in the diversity, functioning, and mechanisms that underlie the complex interactions between AMF, AAB, and plant hosts. This review critically examines AMF-associated bacteria, focusing on AAB diversity, the factors driving richness and community composition of these bacteria across various ecosystems, along with the physical, chemical, and biological connections that enable AMF to select and recruit beneficial bacterial symbionts on and within their structures and hyphospheres. Additionally, potential applications of these bacteria in agriculture are discussed, emphasizing the potential importance of AMF fungal highways in engineering plant rhizosphere and endophyte bacteria communities, and the importance of a functional core of AAB taxa as a promising tool to improve plant and soil productivity. Thus, AMF and their highly diverse bacterial taxa represent important tools that could be efficiently explored in sustainable agriculture, carbon sequestration, and reduction of greenhouse gas emissions related to nitrogen fertilizer applications. Nevertheless, future studies adopting integrated multidisciplinary approaches are crucial to better understand AAB functional diversity and the mechanisms that govern these tripartite relationships.

Bacterial cellulose: A highly versatile nanomaterial.

Bacterial cellulose: A highly versatile nanomaterial.

Fungi associated with ornamental plants in some Nurseries in Al-Qurayyat, Jouf region, Saudi Arabia.

Fungi communities are important soil components as decomposers and plant symbionts, and they play an important part in natural ecological and biogeochemical processes. In this study, isolation and identification of terrestrial and zoosporic fungi were detected. Sixty-seven fungal species from thirty-four genera were isolated from 45 soil samples obtained randomly from nurseries in Al-Qurayyat, Jouf reagon, Saudi Arabia using the soil dilution technique on glucose-Czapek's agar medium, cellulose-Czapek's agar, and Potato dextrose agar medium.Authentic fungus manuals were then used to identify and characterise the mycoflora. A total of 46 fungal species belonging to 22 terrestrial fungal genera were recovered on glucose-Czapek's agar, 38 species belonging to 20 terrestrial fungal genera were recovered on cellulos-Czapek's agar and 27 fungal species belonging to 15 terrestrial fungal genera were recovered on PDA medium while 12 species belonging to 7 genera zoosporic fungal genera were discovered. The most common terrestrial fungal genera were Aspergillus, Penicillium, Fusarium, Trichoderma, Acremonium, and Cladosopium while in zoosporic fungus. Allomyces was the most prevalent, followed by Achlya and Pythium.

Missing symbionts – emerging pathogens? Microbiome management for sustainable agriculture

Plant diversification and co-evolution shaped the plant microbiome and vice versa. This resulted in a specific composition of the plant microbiome and a strong connection with the host in terms of functional interplay. Symbionts are part of the microbiota, and important for the plant’s germination and growth, nutrition, as well as stress protection. However, human activities in the Anthropocene are linked to a significant shift of diversity, evenness and specificity of the plant microbiota. In addition, and very importantly, many plant symbionts are missing or no longer functional. It will require targeted microbiome management to support and reintroduce them. In future agriculture, we should aim at replacing harmful chemicals in the field, as well as post-harvest, by using precision microbiome engineering. This is because the plant microbiome is connected across systems and crucial for human and planetary health. This commentary aims to inspire holistic studies for the development of solutions for sustainable agriculture in framework of the One Health and the Planetary Health concepts.

Using stable isotopes in hummingbird breath to estimate reliance on supplemental feeders.

Understanding the ecological consequences of supplemental feeding to both hummingbirds and the plants they pollinate is complicated by logistical challenges associated with assessing relative dietary resource use with commonly applied observational methods. Here, we describe the results of research conducted to assess the relative use of feeder and flower nectar by Broad-tailed (Selasphorus platycercus) and Rufous hummingbirds (Selasphorus rufus) using two distinct methodological variations to measure the δ13C values of exhaled CO2. Because of the relatively quick time in which both species switch from exogenous to endogenous resources to fuel metabolism, our experiment allowed us to assess resource use at two timescales. Our results suggest variability in the relative contributions of the two dietary sources within and among species and timescales, with most birds employing a mixture of feeder and flower sugars as fuel sources. This diversity in relative resource use may mitigate potential negative effects of supplemental feeding on hummingbirds and their plant symbionts.

Stochastic nuclear organization and host-dependent allele contribution in Rhizophagus irregularis

BackgroundArbuscular mycorrhizal (AM) fungi are arguably the most important symbionts of plants, offering a range of benefits to their hosts. However, the provisioning of these benefits does not appear to be uniform among AM fungal individuals, with genetic variation between fungal symbionts having a substantial impact on plant performance. Interestingly, genetic variation has also been reported within fungal individuals, which contain millions of haploid nuclei sharing a common cytoplasm. In the model AM fungus, Rhizophagus irregularis, several isolates have been reported to be dikaryotes, containing two genetically distinct types of nuclei recognized based on their mating-type (MAT) locus identity. However, their extremely coenocytic nature and lack of a known single nucleus stage has raised questions on the origin, distribution and dynamics of this genetic variation.ResultsHere we performed DNA and RNA sequencing at the mycelial individual, single spore and single nucleus levels to gain insight into the dynamic genetic make-up of the dikaryote-like R. irregularis C3 isolate and the effect of different host plants on its genetic variation. Our analyses reveal that parallel spore and root culture batches can have widely variable ratios of two main genotypes in C3. Additionally, numerous polymorphisms were found with frequencies that deviated significantly from the general genotype ratio, indicating a diverse population of slightly different nucleotypes. Changing host plants did not show consistent host effects on nucleotype ratio’s after multiple rounds of subculturing. Instead, we found a major effect of host plant-identity on allele-specific expression in C3.ConclusionOur analyses indicate a highly dynamic/variable genetic organization in different isolates of R. irregularis. Seemingly random fluctuations in nucleotype ratio’s upon spore formation, recombination events, high variability of non-tandemly repeated rDNA sequences and host-dependent allele expression all add levels of variation that may contribute to the evolutionary success of these widespread symbionts.

Identification, molecular characterization, and plant growth promoting activities of endophytic fungi of Jasminum sambac, Camellia sinensis, and Ocimum basilicum

ObjectivesEndophytic fungi are widely known to reside in plant tissues causing no harm to host plants or even no noticeable change. They may colonize host plants for a part of their life cycle or may complete the entire life cycle since host provide a variety of novel metabolites. Despite being in a close relationship with medicinal host plants, the diversity and metabolic spectrum of endophytic fungi have not been researched well. Their diversity and ecological niche as plant symbionts make them attractive targets in the search for novel biochemicals. MethodsWe aimed to isolate, purify, and characterize the endophytic fungal population of medicinal host plants cultivated in Riyadh, Saudi Arabia. The goal was achieved through morphological identification and internal transcribed spacer (ITS) regions in rRNA analyses to determine phylogeny and diversity. Three aromatic and medicinal plants Jasminum sambac, Camellia sinensis, and Ocimum basilicum collected from the Riyadh region, Saudi Arabia were investigated. In total, 84 isolates were purified and they were grouped into 20 operational taxonomic units (OTUs) as per their sequences of ITS regions in rRNA. ResultsTwenty species of endophytic fungi were grouped in 12 genera i.e. Neopestalotiopsis, Trichoderma, Fusarium, Colletotrichum, Myrothecium, Chaetomium, Alternaria, Phoma, Curvularia, Cladosporium, Neodidymelliopsis, and Aspergillus and all isolates belonged to Ascomycota phylum. J. sambac was found dominant among other and had a relative frequency of 27%. C. sinensis was next with 18.7% relative frequency. The diversity was prominently recorded in leaf organs over stem and roots while roots exhibited the lowest diversity. Isolates also produced indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme, and siderophores with variable magnitudes that could be assigned to their wide range of metabolic variations from species to species. ConclusionConclusively, J. sambac, C. sinensis, and O. basilicum are a good source of endophytic fungi with certain plant growth-promoting traits. Overall, Alternaria was found as the most predominant genus in terms of colonization rate. Further deternminations are required to screen the beneficial compounds released by these endophytic fungi.

Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics.

The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search database (iPtgxDB) types. Searches against standard and 20-fold smaller Ribo-seq data-informed custom iPtgxDBs confirmed 47 annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by combining MS and Ribo-seq approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are part of predicted operons and/or are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.

Trichoderma harzianum as fungicide and symbiont: is it safe for human and animals?

Trichoderma species are considered as biological control agents against numerous phytopathogenic fungi. They are also helpful for plants as plant symbiont. This study aimed to identify harmful effects of Trichoderma in laboratory animals. In the first step, inhalation toxicity was studied. Six rats as control received a spray of bio-formulation without spores. Ten rats as treatment A received 1.00× 106 colony-forming unit (CFU) of Trichoderma spores and ten rats as treatment B received 1.00 × 107 CFU per test of Trichoderma spores. The harmful effects of Trichoderma were obvious especially in the lungs, liver and kidney, and some blood parameters were abnormal. In the second step, we studied acute oral toxicity by gavage. Four rats as control received bio-formulation without spores. Six rats as treatment A received 1.00 × 106 CFU per test of Trichoderma spores. Six rats as treatment B received 1.00 × 107 CFU per test of Trichoderma spores. The harmful effects of Trichoderma were noticeable more in the liver and kidney tissues. For dermal toxicity study, two rabbits as control received bio-formulation without spores by rubbing on the surface of the skin. Treatment groups A and B received 1.00 × 106 and 1.00 × 107 CFU per test of Trichoderma spores, respectively (four rabbits for each group). The liver and kidney and some blood parameters were abnormal. Trichoderma has some harmful effects on tissues and organs and although it is a natural product, it should be used under cautions.