Biology subjects

Vetukuri, R. R.

Publications and source records attributed to Vetukuri, R. R..

At least 19 recordsLinked to original sources

bioRxiv2024

Temporal Dynamics of the Tomato Rhizosphere Microbiome in Response to Synthetic Communities of Plant Growth-Promoting Rhizobacteria

In sustainable agriculture, the application of microorganisms to soil is a widely adopted strategy aimed at enhancing soil microbiome functionality, restoring fertility, and recovering biodiversity diminished by intensive farming. While introducing individual beneficial microbes often encounters issues with establishment and persistence, multispecies microbial consortia offer a more robust alternative, providing complementary functions that enhance both their resilience and effectiveness. To test this approach, we designed three synthetic bacterial communities (SynComs), each composed of varying combinations of ten bacterial endophytes previously shown to promote plant growth and exert biocontrol effects. The SynComs were designed with ascending levels of richness (4, 6, and 10 members) and diversity (ranging from 3 to 6 bacterial genera). In growth chamber trials with tomatoes, the SynComs not only promoted plant growth but also induced significant shifts in the rhizosphere bacterial communities, primarily affecting less abundant taxa. The SynComs MIX2 and MIX3, which included Pseudomonas species, exhibited the greatest impact on both plant growth enhancement and shifts within the resident microbial community. Monitoring of the introduced strains over time demonstrated that most bioinoculants successfully established although at very low concentration in the rhizosphere.

microbiology↗

bioRxiv2024

Evaluating the Biocontrol Potential of Pythium oligandrum and Serratia proteamaculans in Controlling Phytophthora plurivora in European beech (Fagus sylvatica)

In recent years, the plant pathogen Phytophthora plurivora has caused severe damage to beech forests in many European countries, including Sweden. In many affected areas, few protective measures are in place due to fears about potential negative impacts on the ecosystem or public health. The research presented in this article assesses the biocontrol potential of the oomycete Pythium oligandrum and the bacterium Serratia proteamaculans against P. plurivora, with experiments performed under both in vitro and greenhouse conditions, which could represent a step forward in developing a safe treatment for European beech forests. The in vitro results revealed that P. oligandrum and S. proteamaculans significantly inhibited pathogen growth and stimulated a shift in the hyphal growth pattern towards shorter, branched hyphae with many hyphal swellings and thickened cell walls. The experiments conducted in greenhouses showed that treating three-month-old beech seedlings with P. oligandrum and S. proteamaculans counteracts the P. plurivora pathogen and reduces disease symptoms on the aerial and underground parts of the plant. GC-MS analysis detected the volatile organic compounds alpha-pinene, 2,5-dimethyl-pyrazine, and 3-methyl-1-butanol from S. proteamaculans; these compounds have the potential to inhibit pathogen growth. The disease suppression demonstrated by these biocontrol agents could thus be related to the synergistic effect of competition for nutrients with the secretion of hydrolytic enzymes and VOCs; moreover, induced systemic resistance may be triggered under greenhouse conditions. In conclusion, using P. oligandrum and S. proteamaculans could represent an environmentally friendly strategy for effectively controlling the disease caused by P. plurivora in beech.

pathology↗

bioRxiv2024

Genetic Diversity of Zymoseptoria tritici Populations in Central and South-eastern Ethiopia

Septoria tritici blotch (STB), caused by the hemibiotrophic fungus Zymoseptoria tritici, is a serious threat to global wheat production, and a major bottleneck to wheat production in Ethiopia. Accurate identification and analysis of the pathogens genetic structure helps inform robust STB management. This study analyzed the molecular identity and genetic structure of 200 Z. tritici isolates retrieved from diseased crops in central and south-eastern regions of Ethiopia. Allelic diversity at 12 simple sequence repeat (SSR) loci was examined for all 200 isolates, and 165 isolates were identified by Sanger sequencing of the internal transcribed spacer (ITS) region of nuclear DNA (rDNA) region. The microsatellites were highly polymorphic, with mean number of alleles (Na) = 6.23, effective alleles (Ne) =2.90, Neis gene diversity (H) =0.57, and polymorphic information content (PIC) =0.59, and an analysis of molecular variance confirmed the presence of low population differentiation (Fixation Index (FST) = 0.02, Gene Flow (Nm) = 14.7), with 95% of the total genetic variation residing within populations, and only 5% residing between populations. The highest genetic diversity (Number of allele (Na) = 9.33, Effective number of allele (Ne) = 3.41 and Neis gene diversity (H) = 0.68) was observed in the Oromia special zone surrounding Finfinnee (OSZ) Z. tritici populations, followed by Arsi and North Shewa populations, indicating that these areas are ideal for multi-location germplasm resistance screening, and also pathogen genetic and genomic analyses. Cluster analyses did not clearly divide the populations into genetically separate clusters according to their geographic sampling areas, probably because of high gene flow. All individual samples showed genetic admixture, and shared genomic backgrounds from two subgroups (K=2). Overall, the SSR markers proved to be highly informative and effective genetic tools for unlocking the pathogens genetic structure. The Z. tritici populations of central and southeast Ethiopia exhibit high genetic diversity, indicating the need to deploy durable and diverse disease management strategies. North Shewa, OSZ, Arsi and West Arsi administrative zones represent hotspots for genetic and genomic analyses of Z. tritici. Moreover, these areas would be excellent locations for host-pathogen interaction studies, and wheat germplasm screening for STB resistance.

plant biology↗

bioRxiv2024

Heterologous Production of Cyprosin B in Nicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization

The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPB{Delta}PSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPB{Delta}PSI) fresh weight. CYPB{Delta}PSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPB{Delta}PSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPB{Delta}PSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPB{Delta}PSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.

molecular biology↗

bioRxiv2024

Genomic loci for sclerotinia stem rot resistance and chlorophyll stability in Brassica napus: integrating GWAS with microbiome insights

Sclerotinia Stem Rot (SSR) disease is one of the most serious diseases affecting the yield and quality of oilseed rape (Brassica napus). Understanding the genetic basis of the resistance trait in oilseed rape to SSR and microbiome composition for enhanced resistance is crucial for developing resistant varieties and sustainably mitigate the impact of the disease. In this study, in a panel of 168 oilseed rape accessions, most resistant (NGB 13503 and NGB 13834) and susceptible (NGB 13497 and NGB 13897) accessions are identified. A Genome-wide association study (GWAS) identified 47 SNPs linked to the SSR lesion length, lesion area, and lesion relative to the leaf area. Among the SNPs significantly linked to lesion length were Bn-A04-p10555408, Bn-A07-p12487549, Bn-A09-p4652268, Bn-A09-p4916858 and to our knowledge, these SNPs have not been previously linked to SSR resistance in oilseed rape. Moreover, the study identified 24 SNPs linked with chlorophyll content before SSR inoculation (SPADH), after the SSR inoculation (SPADI), and chlorophyll index (CI). Maintaining the chlorophyll level is correlated with the SSR resistance. Furthermore, bacterial taxa (e.g. Pseudomonas, Methylobacterium, and Aquabacterium) and fungal taxa (e.g. Mycosphaerellales, Thelebolales, and Akanthomyces) were enriched in the resistant compared to in the susceptible oilseed rape accessions. The SNPs linked to lesion length showed consistent haplotype variation between these selected accessions. Given the absence of complete resistance against SSR, the study provides insights into the significance of maintaining chlorophyll levels and considering microbiome composition for enhancing the level of existing partial resistance to SSR in oilseed rape.

plant biology↗

bioRxiv2024

Genomic Insights and Biocontrol Potential of Ten Bacterial Strains from the Tomato Core Microbiome

Despite their adverse environmental effects, modern agriculture relies heavily on agrochemicals to manage diseases and pests and enhance plant growth and productivity. Some of these functions could instead be fulfilled by endophytes from the plant microbiota, which have diverse activities beneficial for plant growth and health. We therefore used a microbiome-guided top-down approach to select ten bacterial strains from different taxa in the core microbiome of tomato plants in the production chain for evaluation as potential bioinoculants. These taxa included some that are commonly used as biofertilizers and biocontrol agents (Pseudomonas and Bacillus) as well as the less studied genera Leclercia, Chryseobacterium, Glutamicibacter, and Paenarthorbacter. When inoculated in the tomato rhizosphere, these strains promoted plant growth and reduced the severity of Fusarium Crown and Root Rot and Bacterial Spot infections. High-quality genomes for each strain were obtained using Oxford Nanopore long-read and Illumina short-read sequencing, enabling the dissection of their genetic makeup to identify phyto-beneficial traits. This yielded a comprehensive inventory of genes from each strain related to processes including colonization, biofertilization, phytohormones, and plant signaling. Traits directly relevant to fertilization including phosphate solubilization and acquisition of nitrogen and iron were also identified. Moreover, the strains carried several functional genes putatively involved in abiotic stress alleviation and biotic stress management, traits that indirectly foster plant health and growth. The gathered genomic information will be instrumental in planning the use of these bacteria individually or in consortia to enhance plant growth by coupling strains with different traits, effects, and mechanisms of action.

genomics↗

bioRxiv2024

Plant Development Drives Dynamic Shifts in the Root Compartment Microbiomes of Wild and Domesticated Finger Millet Cultivars

BackgroundPlant-microbe interactions in two root compartments - the rhizosphere and endosphere - play vital roles in maintaining plant health and ecosystem dynamics. The microbial communities in these niches are shaped in complex ways by factors including the plants developmental stage and cultivar, and the compartment where the interactions occur. Different plant cultivars provide distinct nutritional and ecological niches and may selectively enrich specific microbial populations through the secretion of root exudates. This gives rise to complex and dynamic plant-microbe interactions; some cultivars promote the recruitment of beneficial symbionts while others may deter pathogens. To clarify these processes, this work investigated the structure of the endosphere and rhizosphere microbial communities of wild type finger millet and five domesticated cultivars across two plant developmental stages. ResultsOur results showed that the plant developmental stage, compartment, and cultivar have varying degrees of impact on root-associated microbiomes. The dominant bacterial phyla in all samples were Proteobacteria, Actinobacteria, and Bacteroidetes, while the dominant fungal phyla were Ascomycota and Basidiomycota. All of these phyla exhibited pronounced variations in abundance. In general, an increased abundance of Actinobacteria in the endosphere was accompanied by a reduced abundance of Proteobacteria. The most pronounced changes in microbial community structure were observed in the rhizosphere during the flowering stage. Changes in the microbiome patterns of the rhizosphere were driven predominantly by the genus Pseudomonas. Moreover, the host plants developmental stage strongly influenced the microbial communities, suggesting that plants can recruit specific taxa based on their need for particular soil consortia. ConclusionsOur results show that both host developmental stage and domestication strongly affect the assembly and structure of the plant microbiome. Moreover, plant root compartments can selectively recruit specific taxa from associated core microbial communities to fulfill their needs in a manner that depends on both the plants developmental stage and the specific root compartment that is involved. These findings show that deterministic selection pressures exerted by plants during their growth and development can significantly affect their microbial communities and have important implications for efforts to create tools for manipulating the microbiome to sustainably improve primary productivity.

microbiology↗

bioRxiv2024

Extracellular biosynthesis of silver nanoparticles using cell suspension of Rhodococcus fascians

Biosynthesis of metallic nanoparticles using biological systems such as bacteria has become an important nanobiotechnology area. In this report, we present the first extracellular biosynthesis of silver nanoparticles (AgNPs) using the gram-positive bacterium Rhodococcus fascians. The AgNPs underwent characterization through various analytical techniques, encompassing UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM). UV-visible spectroscopy revealed the emergence of an absorbance peak at 430 nm due to the synthesis of AgNPs. R. fascians started producing AgNPs after 12 h of incubation, with the highest yield after 48 h. The extent of synthesis was higher when cultures were grown in the dark than in the light. According to TEM and SEM, the AgNPs had a uniform spherical morphology of diameter 10-80 nm. The AgNPs exhibited antifungal efficacy against the virulent filamentous fungi Rhizoctonia solani, Botrytis cinerea, and Fusarium graminearum, which cause root rot, soft rot and head blight on plants, respectively. This research provides evidence on the ability of R. fascians to generate AgNPs from silver nitrate, as well as their subsequent assembly and potential for controlling vascular wilt disease.

microbiology↗

bioRxiv2023

Dicer-mediated RNA silencing is the key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions

The intricate molecular interplay between beneficial fungi and plants is vital to plant growth promotion and induced defense response. This study explored the role of DCL-mediated RNA silencing in the interaction between the biocontrol fungus Clonostachys rosea and wheat roots. We investigated the impact of DCL (Dicer-like) gene deletions in C. rosea on its root colonization ability. Our results revealed that the deletion of dcl2 significantly enhanced C. rosea biomass on wheat roots, indicating a pivotal role of DCL2 in root colonization. Transcriptome sequencing of C. rosea and wheat during their interactions unveiled extensive gene expression changes. In wheat, genes related to stress responses were upregulated during C. rosea interactions, while genes associated with plant cell wall modification and metabolic processes were downregulated, suggesting complex regulatory responses and a trade-off between defense mechanisms and growth promotion. Deletion of C. rosea dcl1 and dcl2 altered the transcriptomic responses of wheat roots during interactions. Wheat genes associated with stress responses were downregulated during interactions with DCL deletion strains. In contrast, genes involved in metabolic processes and growth were upregulated, emphasizing the cross-kingdom regulatory role of C. rosea small RNAs (sRNAs). We identified 18 wheat miRNAs responsive to C. rosea interactions. Furthermore, we identified 24 endogenous and six cross-kingdom potential gene targets for seven and five differentially expressed miRNAs, supported by their inverse gene expression pattern. In C. rosea, we found a large transcriptional reprogramming of genes during interaction with wheat roots. The upregulated genes were associated with carbohydrate and polysaccharide catabolic processes, membrane transporters and effectors. Conversely, downregulated genes were mainly associated with transition metal ion transport and homeostasis processes. The deletion of dcl1 and dcl2 had significant effects on gene expression. A higher number of genes upregulated in WT during the interaction were restored in DCL deletion mutants, suggesting DCL-mediated gene expression regulation. Furthermore, we identified 21 differentially expressed micro-RNA-like RNAs (milRNAs) in C. rosea; nine were DCL-dependent. They had putative gene targets in C. rosea, including transcription factors, effectors, transporters, and enzymes involved in specialized metabolite production. Cross-kingdom RNA silencing was also observed, with seven DCL-dependent C. rosea milRNAs potentially targeting 29 genes in wheat. These findings provide valuable insights into the molecular mechanisms underlying the beneficial interaction between fungi and plant roots. In addition, the study shed light on the role of sRNA-mediated gene regulation in the C. rosea-wheat interaction, with potential implications for sustainable agriculture and biocontrol strategies.

genomics↗

bioRxiv2023

Effects of enriched biochar treatments on CO2 release, soil nitrate and ammonium, and wheat growth parameters in saline soils

The effects of treatment with simple and enriched biochar on microbial respiration, nitrate and ammonium concentrations, and wheat growth parameters in saline soils were investigated using a completely randomized factorial experimental design with three replications, three soil salinity levels (1.5, 4.5 and 9 dS.m-1), and five biochar treatments including control, 2% simple (SB) or enriched biochar (EB) amendment, and 4% SB or EB amendment. The basal respiration rate and the concentrations of ammonium, and nitrate were measured at multiple time points. Additionally, total soil nitrogen, organic carbon, and microbial biomass carbon, microbial biomass nitrogen, and microbial biomass phosphorus were measured together with the height and fresh and dry weight of wheat after a 100-day growth period and at the end of the experiment. Salinity significantly affected basal respiration, nitrate and ammonium concentrations, plant height, and wet and dry weight. Biochar amendment significantly affected pH, basal respiration, nitrate and ammonium concentrations, total soil nitrogen, soil organic carbon, microbial biomass carbon, microbial biomass nitrogen, and microbial biomass phosphorus in both rhizosphere and non-rhizosphere soil, as well as wheat height, and wet and dry weight. The interaction between salinity and biochar significantly affected nitrate and ammonium concentrations and also plant height and fresh and dry weight. Finally, the effects of different biochar amendments and salinity levels on the basal respiration rate and the concentrations of nitrate and ammonium varied significantly over time. Overall, the results obtained show that biochar amendment can significantly moderate the adverse effects of soil salinity, especially if enriched biochar is used.

plant biology↗

bioRxiv2023

Comparative Transcriptome Profiling Provides Insights into the Growth Promotion Activity of Pseudomonas fluorescens strain SLU99 in Tomato and Potato Plants

The use of biocontrol agents with plant growth-promoting activity has emerged as an approach to support sustainable agriculture. During our field evaluation of potato plants treated with biocontrol rhizobacteria, four bacteria were associated with increased plant height. Using two important solanaceous crop plants, tomato and potato, we carried out a comparative analysis of the growth-promoting activity of the four bacterial strains: Pseudomonas fluorescens SLU99, Serratia plymuthica S412, S. rubidaea AV10, and S. rubidaea EV23. Greenhouse and in vitro experiments showed that P. fluorescens SLU99 promoted plant height, biomass accumulation, and yield of potato and tomato plants, while EV23 promoted growth in potato but not in tomato plants. SLU99 induced the expression of plant hormone-related genes in potato and tomato, especially those involved in maintaining homeostasis of auxin, cytokinin, gibberellic acid and ethylene. Our results reveal potential mechanisms underlying the growth promotion and biocontrol effects of these rhizobacteria and suggest which strains may be best deployed for sustainably improving crop yield.

plant biology↗

bioRxiv2022

Characterization of environmental effects on flowering and plant architecture in an everbearing strawberry F1-hybrid by meristem dissection and gene expression analysis

Floral transition in the cultivated everbearing strawberry is a hot topic because these genotypes flower perpetually and are difficult to maintain in a non-flowering state. However, it has rarely been studied using morphogenetic and molecular analyses simultaneously. We therefore examined morphogenetic effects and the activation of genes involved in floral induction and initiation in seedlings of an everbearing F1-hybrid. Seedlings were grown at 12, 19, and 26{degrees}C under 10-h SD and 20-h LD conditions. We observed a strong environmental influence on meristem development and a FLOWERING LOCUS T1 (FaFT1)-SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (FaSOC1) pathway similar to that in the everbearing woodland strawberry. The everbearing cultivar showed typical features of a quantitative LD plant, flowering earlier under LD than SD conditions at all temperatures. We also found that floral induction is facilitated by FaFT1 upregulation under LD conditions, while FaSOC1 upregulation in the apex leads to photoperiod-independent floral initiation. Moreover, we confirmed the strawberry meristem identity gene FRUITFULL (FaFUL) can also be used as an early indicator of floral initiation in EB cultivars. This study also highlights the advantages of using seed-propagated F1-hybrids for genetic studies because are genetically identical, and not biased by a previous flowering history.

plant biology↗

bioRxiv2022

Bioassay evaluation, GC-MS Analysis and Acute Toxicity Assessment of Methanolic Extracts of Trichoderma species in an Animal Model

Fungi of the genus Trichoderma have been marketed for the control of crop pests, weeds, and diseases. However, some Trichoderma species may produce toxic secondary metabolites; the existence of these compounds in bioformulated products, along with their relative risk, 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 a biocontrol agent against F. 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 crude extracts and spore suspensions, respectively, in a preliminary safety assessment for use as biofungicides. Metabolite profiling of the crude extracts were performed by Gas Chromatography-Mass Spectrometry (GC-MS). The agar diffusion assay of the crude extracts from both T. asperellum AU131 and T. longibrachiatum AU158 were the most effective at a concentration of 200g/mL, causing 62.5%, and 74.3% inhibition, respectively. A GC-MS analysis of crude extracts from both bioagents identified 23 volatile secondary metabolites classified as alcohols, acids, sesquiterpenes, ketones and aromatic compounds. The oral administration of crude extracts (doses of 600, 1200, 2000 and 5000 mg/kg body weight) and spore suspensions (doses of 1 x 106, 1 x 107, 1 x 108 and 1 x 109 spores/mL) to female Swiss albino mice over 14 days did not show any significant signs of toxicity, mortality or changes to body weight. The highest dose, 5000 mg/kg, can be used to determine the no observed effect level of crude extracts and spore suspensions of T. asperellum AU131 and T. longibrachiatum AU158 when calculating safety margins. It can be concluded that these two Trichoderma species can serve as a biocontrol agents against F. xylarioides and the mechanism for this function was due to the secondary metabolites with effective bioactive substance. Moreover, the tested spore suspensions and crude extracts were not pathogenic or toxic, respectively, when administered to Swiss albino mice at various doses.

microbiology↗

bioRxiv2022

Comparative small RNA and degradome sequencing provide new insights into antagonistic interactions in the biocontrol fungus Clonostachys rosea

Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small-RNAs (sRNAs) are vital components of post-transcriptional gene regulation, although their role in gene expression regulation during mycoparasitism remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tags sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were down-regulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing de-suppression (up-regulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated gene targets for 282 differentially expressed sRNAs. Analysis of sRNA gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. While the regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum highlighting common and mycohosts-specific gene regulation of C. rosea. By combining these results with transcriptome data collected in similar experimental conditions during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism. ImportanceSmall-RNAs (sRNAs) are emerging as key players in pathogenic and symbiotic fungus-plant interactions, however, their role in fungal-fungal interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and plant pathogenic mycohots Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel miRNAs and their potential gene targets at endogenous level, and at a cross-species level in B. cinerea and F. graminearum indicating a role of cross-species RNAi in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.

microbiology↗

bioRxiv2022

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. Shannons biodiversity index (H), the evenness (E), Simpsons 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 Ethiopias ongoing coffee wilt disease crisis.

microbiology↗

bioRxiv2021

A family of cell wall transglutaminases is essential for appressorium development and pathogenicity in Phytophthora infestans

Transglutaminases (TGases) are enzymes highly conserved among prokaryotic and eukaryotic organisms, where their role is to catalyse protein cross-linking. One of the putative TGases of Phytophthora infestans has previously been shown to be localised to the cell wall. Based on sequence similarity we were able to identify six more genes annotated as putative TGases and show that these seven genes group together in phylogenetic analysis. All of the seven proteins are predicted to contain transmembrane helices and both a TGase domain and a MANSC domain, the latter of which was previously shown to play a role in protein stability. Chemical inhibition of transglutaminase activity and silencing of the entire family of the putative cell wall TGases are both lethal to P. infestans indicating the importance of these proteins in cell wall formation and stability. The intermediate phenotype obtained with lower drug concentrations and less efficient silencing displays a number of deformations to germ tubes and appressoria. Both chemically treated and silenced lines show lower pathogenicity than the wild type in leaf infection assays. Finally, we show that appressoria of P. infestans possess the ability to build up turgor pressure and that this ability is decreased by chemical inhibition of TGases.

molecular biology↗

bioRxiv2021

Mycorrhizal inoculation and treatment with biochar and compost from pruning waste improve the qualitative properties of a calcareous soil under wheat cultivation

Most calcareous soils have relatively low levels of organic matter. To address this issue and improve the qualitative properties of calcareous soils, soils can be treated with mycorrhizal fungi and/or exogenous organic material such as biochar or compost derived from tree pruning waste. To evaluate the effect of pruning waste biochar (PWB) and pruning waste compost (PWC) derived from apple and grape trees combined with arbuscular mycorrhizal fungi (AMF) on the biological indices of calcareous soils, a rhizobox study on wheat plants using a completely randomized design was conducted under greenhouse conditions. The studied factors included the source of the type of organic material applied (PWB, PWC, and control), the nature of the microbial inoculation (inoculation with AMF or no inoculation), and the zone to which the treatments were applied (rhizosphere and non-rhizosphere soil). At the end of the plant growth period, organic carbon (OC), microbial biomass carbon (MBC), microbial biomass phosphorous (MBP), microbial respiration (BR), substrate-induced respiration (SIR), alkaline (ALP), acid (ACP) phosphatase enzyme activities in the rhizosphere and non-rhizosphere soils, and root mycorrhizal colonization were determined. Simultaneous application of a source of organic matter and AMF inoculation significantly increased the OC and biological indices of soil relative to those observed when applying organic matter without AMF inoculation. Additionally, MBC, MBP, ACP and ALP - enzymes activities in the rhizosphere zone were significantly higher than in the non-rhizosphere. AMF increased BR and SIR levels in the rhizosphere by 13.06% and 7.95% compared to non-rhizosphere, respectively. It can be concluded that in calcareous soils with low organic carbon contents, organic amendments such as PWC and PWB can improve soil biological properties by increasing microbial activity and changing the properties of the rhizosphere.

microbiology↗

bioRxiv2021

The role of Dicer-dependent RNA interference in regulating cross-species communication during fungus-fungus interactions

Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs. Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated. In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea. Moreover, the {Delta}dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum, and reduced production of 62 secondary metabolites (SM) including yellow-coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea {Delta}dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea, and 11 was upregulated in the {Delta}dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B. cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the {Delta}dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology. Author summaryRNA interference (RNAi) is a conserved cellular mechanism mediated by small RNAs (sRNAs) regulating biological processes through the targeted destruction or modulation of RNA filaments necessary for protein synthesis. Dicer-like endoribonucleases (DCL) play a vital role in the RNAi pathway by generating sRNAs. In this study, we identified two DCL-encoding genes in the mycoparasitic fungus Clonostachys rosea and investigated a role of DCL-mediated RNAi in interference interactions between Clonostachys rosea and the two important fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohost). Using transcriptome (sRNA and mRNA) sequencing and secondary metabolome analysis approach, we found that the dcl mutants were not able to produce 11 sRNAs predicted to finetune the regulatory network of genes known to be involved in production of hydrolytic enzymes, antifungal compounds, and membrane transporters needed for antagonistic action of C. rosea. We also found C. rosea sRNAs putatively targeting known virulence factors in the mycohost, indicating RNAi-mediated cross-species communication. Our study expanded the understanding of underlying mechanisms of cross-species communication during interference interactions and showed that DCL-mediated RNAi is an important regulator of parasitic fungus-fungus interactions. The results pose the base for future works studying the role of DCL-based cross-species RNAi in fungal interactions.

genomics↗