Endophytic Lifestyle Research Articles

Field isolates of Beauveria bassiana exhibit biological heterogeneity in multitrophic interactions of agricultural importance

Beauveria bassiana (Bb) is a widespread entomopathogenic fungus widely used in agriculture for crop protection. Other than pest control, fungi belonging to the B. bassiana complex represent an important microbial resource in agroecosystems, considering their multiple interactions with other microorganisms as antagonists of phytopathogens, or with plants as endophytic colonizers and growth promoters. Here, we characterised field collected or commercial isolates of B. bassiana relative to the environmental factors that affect their growth. We further compared the metabolome, the entomopathogenic potential and biocontrol activity of the tested isolates respectively on the insect pest Spodoptera littoralis or against the fungal plant pathogen Fusarium oxysporum. Our analysis revealed that the B. bassiana complex is characterised by a high level of inter-isolate heterogeneity in terms of nutritional requirements, establishment of intra- or inter-kingdom interactions, and the nature of metabolites produced. Interestingly, certain B. bassiana isolates demonstrated a preference for low nutrient plant-derived media, which hints at their adaptation towards an endophytic lifestyle over a saprophytic one. In addition, there was a noticeable variation among different B. bassiana isolates in their capacity to kill S. littoralis larvae in a contact infection test, but not in an intrahaemocoelic injection experiment, suggesting a unique level of adaptability specific to the host. On the other hand, most B. bassiana isolates exhibited similar biocontrol efficacy against the soil-dwelling ascomycete F. oxysporum f. sp. lycopersici, a pathogen responsible for vascular wilt disease in tomato plants, effectively averting wilting. Overall, we show that the effectiveness of B. bassiana isolates can greatly vary, emphasising the importance of isolate selection and nutritional adaptability consideration for their use in sustainable agriculture.

Comparative genomic analysis of strain Priestia megaterium B1 reveals conserved potential for adaptation to endophytism and plant growth promotion.

In our study, we aimed to explore the genomic and phenotypic traits of Priestia megaterium strain B1, which was isolated from root material of healthy apple plants, to adapt to the endophytic lifestyle and promote plant growth. We identified putative genes encoding proteins involved in chemotaxis, flagella biosynthesis, biofilm formation, secretory systems, detoxification, transporters, and transcription regulation. Furthermore, B1 exhibited both swarming and swimming motilities, along with biofilm formation. Both genomic and physiological analyses revealed the potential of B1 to promote plant growth through the production of indole-3-acetic acid and siderophores, as well as the solubilization of phosphate and zinc. To deduce potential genomic features associated with endophytism across members of P. megaterium strains, we conducted a comparative genomic analysis involving 27 and 31 genomes of strains recovered from plant and soil habitats, respectively, in addition to our strain B1. Our results indicated a closed pan genome and comparable genome size of strains from both habitats, suggesting a facultative host association and adaptive lifestyle to both habitats. Additionally, we performed a sparse Partial Least Squares Discriminant Analysis to infer the most discriminative functional features of the two habitats based on Pfam annotation. Despite the distinctive clustering of both groups, functional enrichment analysis revealed no significant enrichment of any Pfam domain in both habitats. Furthermore, when assessing genetic elements related to adaptation to endophytism in each individual strain, we observed their widespread presence among strains from both habitats. Moreover, all members displayed potential genetic elements for promoting plant growth.IMPORTANCEBoth genomic and phenotypic analyses yielded valuable insights into the capacity of P. megaterium B1 to adapt to the plant niche and enhance its growth. The comparative genomic analysis revealed that P. megaterium members, whether derived from soil or plant sources, possess the essential genetic machinery for interacting with plants and enhancing their growth. The conservation of these traits across various strains of this species extends its potential application as a bio-stimulant in diverse environments. This significance also applies to strain B1, particularly regarding its application to enhance the growth of plants facing apple replant disease conditions.

Evolutionary history of arbuscular mycorrhizal fungi and genomic signatures of obligate symbiosis

BackgroundThe colonization of land and the diversification of terrestrial plants is intimately linked to the evolutionary history of their symbiotic fungal partners. Extant representatives of these fungal lineages include mutualistic plant symbionts, the arbuscular mycorrhizal (AM) fungi in Glomeromycota and fine root endophytes in Endogonales (Mucoromycota), as well as fungi with saprotrophic, pathogenic and endophytic lifestyles. These fungal groups separate into three monophyletic lineages but their evolutionary relationships remain enigmatic confounding ancestral reconstructions. Their taxonomic ranks are currently fluid.ResultsIn this study, we recognize these three monophyletic linages as phyla, and use a balanced taxon sampling and broad taxonomic representation for phylogenomic analysis that rejects a hard polytomy and resolves Glomeromycota as sister to a clade composed of Mucoromycota and Mortierellomycota. Low copy numbers of genes associated with plant cell wall degradation could not be assigned to the transition to a plant symbiotic lifestyle but appears to be an ancestral phylogenetic signal. Both plant symbiotic lineages, Glomeromycota and Endogonales, lack numerous thiamine metabolism genes but the lack of fatty acid synthesis genes is specific to AM fungi. Many genes previously thought to be missing specifically in Glomeromycota are either missing in all analyzed phyla, or in some cases, are actually present in some of the analyzed AM fungal lineages, e.g. the high affinity phosphorus transporter Pho89.ConclusionBased on a broad taxon sampling of fungal genomes we present a well-supported phylogeny for AM fungi and their sister lineages. We show that among these lineages, two independent evolutionary transitions to mutualistic plant symbiosis happened in a genomic background profoundly different from that known from the emergence of ectomycorrhizal fungi in Dikarya. These results call for further reevaluation of genomic signatures associated with plant symbiosis.

Bacterial Endophytes and Their Contributions to Alleviating Drought and Salinity Stresses in Wheat: A Systematic Review of Physiological Mechanisms

Drought and salinity stresses significantly threaten global wheat productivity, limiting growth and reducing yields, thus endangering food security worldwide. These stresses disrupt physiological processes, impair photosynthesis, and hinder optimal growth and yield by diminishing water uptake, causing osmotic stress, ion toxicity, and oxidative stress. In response, various mitigation strategies have been explored, including breeding for stress-tolerant cultivars, improved irrigation techniques, and the application of exogenous osmoprotectants and soil amendments. Among these strategies, the emergence of rhizospheric and endophytic growth-promoting microorganisms has attracted significant attention. Therefore, a systematic review was undertaken to illustrate the role of endophytic bacteria in enhancing wheat tolerance to drought and salinity stresses. This review analyzes physiological mechanisms and research trends, identifies gaps, and discusses implications for sustainable agriculture. An analysis of the literature related to endophytic bacteria in wheat was conducted using databases of major publishers from 2004 to 2023. The review explores their mechanisms, such as phytohormone production and stress-responsive gene induction, emphasizing their contribution to plant growth and stress resilience. The current research trends indicate a growing interest in utilizing endophytic bacteria to mitigate these stresses in wheat cultivation, with studies focusing on understanding their physiological responses and interactions with wheat plants. Future research should concentrate on elucidating the role of endophytic bacteria in enhancing host plant tolerance to multiple stressors, as well as aspects like endophytic mechanism of action , endophytic lifestyle, and transmission pathways. Overall, endophytic bacteria offer promising avenues for sustainable agricultural practices, aiding in crop resilience and food security amid environmental challenges.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte

BackgroundEndophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications.ResultsThe strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants.ConclusionsStrain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

Trunk Injection Delivery of Biocontrol Strains of Trichoderma spp. Effectively Suppresses Nut Rot by Gnomoniopsis castaneae in Chestnut (Castanea sativa Mill.).

Gnomoniopsis castaneae is responsible for brown or chalky nut rot in sweet chestnut (Castanea sativa), causing heavy reductions in nut production. Controlling it is challenging, due to its inconspicuous infections, erratic colonization of host tissues and endophytic lifestyle. Fungicides are not applicable because they are prohibited in chestnut forests and strongly discouraged in fruit chestnut groves. Trichoderma species are safe and wide-spectrum biocontrol agents (BCAs), with a variety of beneficial effects in plant protection. This study tested selected strains of T. viride, T. harzianum and T. atroviride for their ability to suppress G. castaneae. Field experiments were conducted in four chestnut groves (two test plots plus two controls) at two sites with a different microclimate. As the size of the trees were a major drawback for uniform and effective treatments, the Trichoderma strains were delivered directly by trunk injection, using the BITE® (Blade for Infusion in TrEes) endotherapic tool. The BCA application, repeated twice in two subsequent years, significantly reduced nut rot incidence, with a more marked, presumably cumulative, effect in the second year. Our data showed the tested Trichoderma strains retain great potential for the biological control of G. castaneae in chestnut groves. The exploitation of Trichoderma spp. as biopesticides is a novelty in the forestry sector and proves the benefits of these microbes in plant disease protection.

Needle pathogens of Rhytismataceae: Current knowledge and research opportunities for conifer foliar diseases

AbstractNeedle diseases caused by pathogens within Rhytismataceae are increasing in frequency globally where pines are either a predominate species or are grown commercially, and yet remain poorly understood. Here, we provide background information on the current knowledge and management strategies of needle pathogens in the Rhytismataceae and discuss research opportunities using molecular tools to fill knowledge gaps. Many of the aggressive needle pathogens within this group lack morphological features to delimit species and are difficult to grow in axenic cultures, which complicates scientific investigations. Molecular research has greatly advanced knowledge of this unique group of pathogens. Nucleotide sequences from multiple gene loci identified greater diversity within this group of pathogens than previously thought, as new and cryptic species are being discovered in broader geographic regions. High throughput sequencing and analyses revealed that an endophytic lifestyle is a common trait among fastidious if not obligate needle pathogens within Rhytismataceae. Further, molecular analyses showed the impact these pathogens have on the diversity of endophytic microbiota is underestimated. Early detection in healthy‐looking needles using PCR‐based markers greatly improves capabilities for disease diagnosis and monitoring. Despite growing number of molecular studies on Rhytismataceae needle pathogens, fundamental questions on the taxonomy, phylogeny, ecology and pathology of many Rhytismataceae needle pathogens remain unanswered. Nonetheless, with the projected changes in the occurrence and severity of Rhytismataceae pathogens, increased accessibility to molecular tools vastly expands the potential to gain more insights into understudied needle pathosystems to help define and address its impact under changing environments and consequently inform forest disease management more effectively.

Endophytic fungi: Tracing the evolutionary roots and exploring the diversity of plant-fungal symbioses

The endophytic lifestyle, characterized by a symbiotic relationship between fungi and their host plants, is pervasive throughout the fungal kingdom. However, the question of whether all fungi have ancestors with endophytic lifestyles remains a topic of ongoing debate. This review paper aims to explore this question by examining the evolutionary history of endophytism in fungi, the diversity of endophytic fungi, and the ecological and host-specific factors that have influenced the evolution and diversification of endophytic lifestyles. While it is clear that not all fungi descend from endophytic ancestors, the prevalence and diversity of endophytism across the fungal phylogeny suggest that this lifestyle has evolved multiple times in response to various ecological and host-specific pressures. Future research, integrating modern genomic tools and experimental approaches, will likely shed more light on the complex evolutionary trajectories of endophytic fungi, and potentially contribute to applications in agriculture, industry, and medicine.

Zinc oxide nanoparticles enhance plasmid transfer among growth-promoting endophytes in Arabidopsis thaliana

Nanoparticles (NPs) hold great promise for agricultural applications, yet their potential impact on exogenous gene transfer within plant remains poorly understood. In this study, we utilized the non-conjugative plasmid pCAMBIA1300, harboring the bialaphos resistance (bar) gene expressed in plant and the kanamycin resistance (kanR) gene as selectable marker in bacteria. Our results revealed a significant increase in the transfer of plasmid (via carrier Escherichia coli DH5α), both intra- and inter-species within the endophyte, when Arabidopsis thaliana was exposed to environmentally relevant level of zinc oxide (ZnO) NPs at a concentration of 0.7 μg/mL throughout its lifespan. Intriguingly, the plasmid exhibited selective transfer to growth-promoting endophytes, such as Enterobacter, Serratia, and Achromobacter, with the presence of ZnO NPs expanding the pool of potential recipients. This result is due to the facilitation of an endophytic and mutualistic lifestyle of invasive E. coli DH5α and the enrichment of beneficial bacteria aided by ZnO NPs. The plant's descendant generations did not express the bar gene, and the endophytes carrying the exogenous plasmid did not transmit it to sub sequent generation. This research provides crucial insights for assessing the potential risks associated with gene contamination and ensuring the safe and sustainable use of NPs in agriculture.

Endophyte genomes support greater metabolic gene cluster diversity compared with non-endophytes in Trichoderma.

Trichoderma is a cosmopolitan genus with diverse lifestyles and nutritional modes, including mycotrophy, saprophytism, and endophytism. Previous research has reported greater metabolic gene repertoires in endophytic fungal species compared to closely-related non-endophytes. However, the extent of this ecological trend and its underlying mechanisms are unclear. Some endophytic fungi may also be mycotrophs and have one or more mycoparasitism mechanisms. Mycotrophic endophytes are prominent in certain genera like Trichoderma, therefore, the mechanisms that enable these fungi to colonize both living plants and fungi may be the result of expanded metabolic gene repertoires. Our objective was to determine what, if any, genomic features are overrepresented in endophytic fungi genomes in order to undercover the genomic underpinning of the fungal endophytic lifestyle. Here we compared metabolic gene cluster and mycoparasitism gene diversity across a dataset of thirty-eight Trichoderma genomes representing the full breadth of environmental Trichoderma's diverse lifestyles and nutritional modes. We generated four new Trichoderma endophyticum genomes to improve the sampling of endophytic isolates from this genus. As predicted, endophytic Trichoderma genomes contained, on average, more total biosynthetic and degradative gene clusters than non-endophytic isolates, suggesting that the ability to create/modify a diversity of metabolites potential is beneficial or necessary to the endophytic fungi. Still, once the phylogenetic signal was taken in consideration, no particular class of metabolic gene cluster was independently associated with the Trichoderma endophytic lifestyle. Several mycoparasitism genes, but no chitinase genes, were associated with endophytic Trichoderma genomes. Most genomic differences between Trichoderma lifestyles and nutritional modes are difficult to disentangle from phylogenetic divergences among species, suggesting that Trichoderma genomes maybe particularly well-equipped for lifestyle plasticity. We also consider the role of endophytism in diversifying secondary metabolism after identifying the horizontal transfer of the ergot alkaloid gene cluster to Trichoderma.

Genome Sequence and Characterisation of Peribacillus sp. Strain AS_2, a Bacterial Endophyte Isolated from Alectra sessiliflora

Peribacillus sp. AS_2, a leaf endophytic bacterium isolated from the medicinal plant Alectra sessiliflora, was previously identified using the 16S rRNA gene sequence. The draft genome of AS_2 had a 5,482,853 bp draft circular chromosome, 43 contigs, N50 of 360,633 bp and an average G + C% content of 40.5%. Whole genome sequencing and phenotypic analysis showed that AS_2 was Gram-positive, endospore-forming, motile, and rod-shaped and it showed a high sequence similarity with P. frigoritolerans DSM 8801T. Genomic digital DNA–DNA hybridisation (dDDH) between strain AS_2 and Peribacillus frigoritolerans DSM 8801T and P. castrilensis N3T was 84.8% and 79.2%, respectively, and the average nucleotide identity (ANI) of strain AS_2 with P. frigoritolerans DSM 8801T and P. castrilensis N3T was 97.0% and 96.7%, respectively. The antiSMASH software predicted a total of eight secondary metabolite gene clusters comprising non-ribosomal peptide synthetase (NRPS) type koranimine, terpenes, and siderophore clusters. Strain AS_2 also displayed genes involved in endophytic lifestyle and antibiotic resistance gene clusters such as small multidrug resistance antibiotic efflux pumps (qacJs). Using the multilocus sequence analysis (MLSA), together with the phenotypic data and genomic analysis, we demonstrated that strain AS_2 is a subspecies of P. frigoritolerans DSM 8801T. Genome sequencing of Peribacillus sp. AS_2 from medicinal plants provides valuable genomic information and allows us to further explore its biotechnological applications.

Estimation of the infestation rate and parasitism rate of the citrus leafminer Phyllocnistis citrella in a Thomson orchard near a greenhouse where beneficials are reared in Boufarik-Blida (Algeria)

The citrus leafminer Phyllocnistis citrella causes severe damage to young citrus shoots in Algeria. It therefore indirectly delays fruit set and consequently production and crop profitability. The endophytic lifestyle of the insect's larvae has made chemical control difficult, making the use of other control agents essential.
 To this end, a study on the population dynamics of Phyllocnistis citrella and the incidence of the parasite was carried out for 08 months in 2021 (from January to August 2021) in the Boufarik-Blida region (Algeria) on the Thomson variety.
 The main results gathered from the sampling show that the parasitism rate reached 68% and the infection rate 39%, due to the intervention of auxiliaries (Semielacher petiolatus, Cirostichus phyllocnistoides) from the greenhouse where these species are reared near the Thomson orchard. The citrus leafminer population is declining. Parasite brokering has a positive effect on the defense against Phyllocnistis citrella and represents an effective method of reinforcing biological control of this pest. In addition, the installation of mini greenhouses for the rearing of beneficials in orchards infested by citrus leafminer can be proposed as a means of reinforcing biological control. Moreover, regional containment is a healthy alternative in the context of integrated pest management, helping the ecosystem to reduce pollution. For this reason, it is worthwhile scheduling containment periods in agricultural regions.

Lifestyle changes in Botryosphaeriaceae as evidenced by ancestral genome expansion and horizontal gene transfer

AbstractBotryosphaeriaceae (Botryosphaeriales, Dothideomycetes, Ascomycota) encompasses commonly encountered opportunistic pathogens that cause stem cankers on woody plants. Lifestyles of Botryosphaeriaceae species could vary as endophytes, pathogens and saprobes and one species can have one or more lifestyles. Therefore, this family is an excellent candidate to study the relationships among lifestyles and lifestyle changes. It is postulated that this family has saprobic ancestors, and the mechanisms by which they evolved from nonpathogenic ancestors to woody pathogens remain unclear. Here, we present an analysis of 18 Botryosphaeriaceae genomes, including four newly generated high-quality genomes of Botryosphaeriaceae strains. We compared Botryosphaeriaceae genomes with phylogenetically closely related Dothideomycetes taxa including plant pathogens and saprobes which revealed significant net gene family expansion in Botryosphaeriaceae. This gene expansion is prominent in the early ancestors before the divergence of genera of Botryosphaeriaceae. This expansion affected the pathogenicity-related genes and detoxification genes. Furthermore, we analysed horizontal gene transfer, which is a mechanism of transfer to genetic material between organisms that are not in a parent–offspring relationship and identified widespread putative intra-kingdom horizontal gene transfer events in this family. Most were transferred during the evolution of ancient ancestors of Botryosphaeriaceae, before the divergence of the modern genera and were enriched in pathogenicity-related genes and detoxification genes. Furthermore, The RNA sequencing analysis of the Botryosphaeriaceae species Lasiodiplodia theobromae revealed that pathogenicity-related genes and detoxification genes, including those obtained through gene family expansion and horizontal gene transfers, were significantly induced after the infection of plant hosts rather than before infection. These insights reveal critical roles for gene family expansion and horizontal gene transfers in the evolutionary adaptation of Botryosphaeriaceae in the infection of woody plants. We postulate that the pathogenic lifestyle of Botryosphaeriaceae species evolved from saprobic or endophytic lifestyles in the early divergence of this family. However, there are few endophytic genomes available for closely related species of Botryosphaeriaceae, thus further studies are necessary to clarify the evolutionary relationships of the endophytes.

Characteristics of root endophytic fungi communities associated with genetically modified plants

Root endophytic fungi (EF) spend at least parts of their life cycle inside plant tissues without apparent harm to the host. There is a hypothesis that the endophytic lifestyle is a common strategy for most fungi and they have endophytic ancestors [1]. By receiving habitat and nutrients EF can increase the solubility of nutrients in the plant rhizosphere, stimulate plant growth, and activate the plant’s systemic resistance to stress. One of the alternatives to the use of pesticides is the use of resistant transgenic plants, but the potential effects of crop modifications on their associated microorganisms are poorly studied.
 The EF communities of transgenic lines of cotton, sugar cane, and maize containing the expressed Cry1 protein from Bacillus thuringiensis were compared with communities of non-transgenic plants. There were no significant differences in the composition of the EF community [2, 3]. The introduction of phosphinothricin-N-acetyltransferase and imazapyr herbicide resistance genes for corn and sugar cane also did not affect on EF communities but did affect the bacterial community [3, 4]. The similar effect was observed for transgenic maple plants [5]. The stage of plant development had a more significant effect on EF community than the fact of transformation itself [1].
 We believe that the fungal community is more conservative and the introduction of herbicide resistance or toxin synthesis genes into the plant genome has a significantly lesser effect on EF community than on the bacterial one.

Bacteria Associated with Acute Oak Decline: Where Did They Come From? We Know Where They Go.

Acute oak decline is a high-impact disease causing necrotic lesions on the trunk, crown thinning and the eventual death of oak. Four bacterial species are associated with the lesions-Brenneria goodwinii, Gibbsiella quercinecans, Rahnella victoriana and Lonsdalea Britannica-although an epi-/endophytic lifestyle has also been suggested for these bacteria. However, little is known about their environmental reservoirs or their pathway to endophytic colonisation. This work aimed to investigate the ability of the four AOD-associated bacterial species to survive for prolonged periods within rhizosphere soil, leaves and acorns in vitro, and to design an appropriate method for their recovery. This method was trialled on field samples related to healthy and symptomatic oaks. The in vitro study showed that the majority of these species could survive for at least six weeks within each sample type. Results from the field samples demonstrated that R. victoriana and G. quercinecans appear environmentally widespread, indicating multiple routes of endophytic colonisation might be plausible. B. goodwinii and L. britannica were only identified from acorns from healthy and symptomatic trees, indicating they may be inherited members of the endophytic seed microbiome and, despite their ability to survive outside of the host, their environmental occurrence is limited. Future research should focus on preventative measures targeting the abiotic factors of AOD, how endophytic bacteria shift to a pathogenic cycle and the identification of resilient seed stock that is less susceptible to AOD.

First report of Neofusicoccum parvum causing stem blight and dieback of highbush blueberry in the Czech Republic.

Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips is a cosmopolitan pathogen causing dieback of multiple diverse woody hosts including highbush blueberry (Vaccinium corymbosum L.). This fungus can survive inside colonized plants without causing any symptoms for several years. Once the endophytic lifestyle is switched to the parasitic one, the symptoms of dieback can rapidly occur (bronze leaves, necroses under the bark, apoplexy) and the plant usually declines within a few weeks (Slipper and Wingfield 2007). In August 2022, blueberry plants displaying symptoms described above were observed in a production orchard located in Hovorany, the Czech Republic. Around 3 % of 1000 observed plants were symptomatic. In order to identify the pathogen, leaves, stems and roots of three diseased plants were collected, sectioned into small pieces (5 × 5 mm), surface sterilized (60 s in 75% ethanol, followed by 60 s in 1% sodium hypochlorite and rinsed three times using sterile distilled water), plated on potato dextrose agar (PDA) supplemented with 0.5 g/liter of streptomycin sulfate (PDAS) (Biosynth, Staad, Switzerland) and incubated at 25°C for 2 weeks at dark. Newly developed mycelia were immediately transferred to fresh PDA plates and purified by single-spore or hyphal-tip isolation. In total 33 fungal isolates were obtained. All the 33 isolates shared similar morphology and resembled Botryosphaeriaceae spp. Colonies on PDA (7 d at 25°C) were felty, white to iron grey in the centre. Conidiomata were observed on sterile pine needles on 2 % water agar (WA) at 25°C under near-UV light after 2 wks (110-220 × 60-175 μm). Conidia (n=30) were cylindrical to ellipsoidal, hyaline, 0(-1)-septate, (3.8-8.1 × 2-3 μm). Two representative isolates were deposited at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 149846 and CBS 149847). The partial internal transcribed spacer (ITS) regions, beta-tubulin gene (tub2) and translation elongation factor 1-alpha (tef) gene were amplified from genomic DNA of both isolates following primers and protocols previously described (Eichmeier et al. 2020). Newly generated sequences were deposited in NCBI GenBank (acc. nos. ITS: OQ376566, OQ376567; tub2: OQ401701, OQ401702 and tef: OQ401699, OQ401700), being >99% identical (ITS 483/484 nt, tub2 426/430 nt and tef 230/232 nt) with the ex-type ITS (AY236943), tub2 (AY236888) and tef (AY236917) sequences of N. parvum strain CMW9081. Phylogenetically, newly obtained isolates grouped with ex-type and another three cultures of N. parvum in the three gene-based phylogenetic tree with strong 98/1.0 (BP/PP) support. To confirm pathogenicity, one-year-old canes of ten two-year-old V. corymbosum plants grown in pots were wounded by a 5 mm diam cork borer, and a 5-mm mycelial plug of a 7-day-old culture of both (CBS 149846 and CBS 149847) strains (five plants per strain) was inserted into the wound. Ten plants were inoculated with sterile PDA plugs and served as controls. Wounds were covered by sterile wet cotton, sealed with Parafilm® and inoculated plants were maintained in a growth chamber at 20 °C with 12/12 h light/dark period. Within two weeks, inoculated shoots changed colour from green to dark brown and exhibited dark necroses under the bark; after one month inoculated plants declined, while controls remained symptomless. The pathogen was reisolated from the inoculated plants with 100 % re-isolation rate, and its identity confirmed by sequencing ITS region. The experiment was repeated. Neofusicoccum parvum causing dieback of highbush blueberry was already reported from Australia, California, Chile, China, Italy, Mexico, Portugal and Uruguay (Rossman et al. 2023). Pecenka et al. (2021) reported a presence of another pathogen - Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the same plantation. This suggests that stem blight and dieback of highbush blueberry is caused by more than one Botryosphaeriaceae spp. as it was previously proved by Xu et al. (2015). To the best of our knowledge, this is the first report of stem blight and dieback of highbush blueberry caused by N. parvum in the Czech Republic.

Transcriptional plasticity of schizotrophic Sclerotinia sclerotiorum responds to symptomatic rapeseed and endophytic wheat hosts.

Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, is also a reciprocal endophytic fungus in monocot plants. However, it is unclear how it responds to two distinct plants. Comparative transcriptomic analyses uncovered a network rewiring, with 14.1% up-differentially expressed genes overlapping upon colonizing the endophytic host wheat and a symptomatic host rapeseed. The divergent transcript of genes originating from host-specific induction is central to infection and colonization, including genes related to appressorium, oxalic acid, carbohydrate-active enzymes, and effector-like proteins. We confirmed that appressorium is required for S. sclerotiorum during colonization in symptomatic hosts but not in endophytic wheat via the observation of the infection process of ΔSs-caf1, a compound appressorium formation-defective mutant with hypovirulence to symptomatic hosts. SS1G_10617 and SS1G_13809 are components of the starch degradation pathways. ΔSS1G_10617 displayed lower virulence on symptomatic host plants and abnormal endophytic growth on wheat, and ΔSS1G_13809 only showed an abnormal symbiotic relationship with wheat, implying that starch may be the major carbon source for S. sclerotiorum in the symbiosis with wheat. Our results herein suggest that S. sclerotiorum modulates biphasic colonization via a divergent transcriptional landscape. This study contributes to a better understanding of schizotrophic fungi and provides new clues for cultivating disease-resistant varieties and adjusting reasonable farming strategies. IMPORTANCE The broad host range of fungi with differential fungal responses leads to either a pathogenic or an endophytic lifestyle in various host plants. Yet, the molecular basis of schizotrophic fungal responses to different plant hosts remains unexplored. Here, we observed a general increase in the gene expression of S. sclerotiorum associated with pathogenicity in symptomatic rapeseed, including small protein secretion, appressorial formation, and oxalic acid toxin production. Conversely, in wheat, many carbohydrate metabolism and transport-associated genes were induced, indicating a general increase in processes associated with carbohydrate acquisition. Appressorium is required for S. sclerotiorum during colonization in symptomatic hosts but not in endophytic wheat. These findings provide new clues for understanding schizotrophic fungi, fungal evolution, and the emergence pathways of new plant diseases.

Plants as the Extended Phenotype of Endophytes-The Actual Source of Bioactive Compounds.

For thousands of years, plants have been used for their medicinal properties. The industrial production of plant-beneficial compounds is facing many drawbacks, such as seasonal dependence and troublesome extraction and purification processes, which have led to many species being on the edge of extinction. As the demand for compounds applicable to, e.g., cancer treatment, is still growing, there is a need to develop sustainable production processes. The industrial potential of the endophytic microorganisms residing within plant tissues is undeniable, as they are often able to produce, in vitro, similar to or even the same compounds as their hosts. The peculiar conditions of the endophytic lifestyle raise questions about the molecular background of the biosynthesis of these bioactive compounds in planta, and the actual producer, whether it is the plant itself or its residents. Extending this knowledge is crucial to overcoming the current limitations in the implementation of endophytes for larger-scale production. In this review, we focus on the possible routes of the synthesis of host-specific compounds in planta by their endophytes.

Unraveling Azospirillum's colonization ability through microbiological and molecular evidence.

It is known that members of the bacterial genus Azospirillum can promote the growth of a great variety of plants, an ability harnessed by the industry to create bioproducts aimed to enhance the yield of economically relevant crops. Its versatile metabolism allows this bacterium to adapt to numerous environments, from optimal to extreme or highly polluted. The fact of having been isolated from soil and rhizosphere samples collected worldwide and many other habitats proves its remarkable ubiquity. Azospirillum rhizospheric and endophytic lifestyles are governed by several mechanisms, leading to efficient niche colonization. These mechanisms include cell aggregation and biofilm formation, motility, chemotaxis, phytohormone and other signaling molecules production, and cell-to-cell communication, in turn, involved in regulating Azospirillum interactions with the surrounding microbial community. Despite being infrequently mentioned in metagenomics studies after its introduction as an inoculant, an increasing number of studies detected Azospirillum through molecular tools (mostly 16S rRNA sequencing) as part of diverse, even unexpected, microbiomes. This review focuses on Azospirillum traceability and the performance of the available methods, both classical and molecular. An overview of Azospirillum occurrence in diverse microbiomes and the less-known features explaining its notorious ability to colonize niches and prevail in multiple environments is provided.

Red clover root-associated microbiota is shaped by geographic location and choice of farming system.

This study evaluated the red clover (Trifolium pratense) root-associated microbiota to clarify the presence of pathogenic and beneficial microorganisms in 89 Swedish field sites. 16S rRNA and ITS amplicon sequencing analysis were performed on DNA extracted from the red clover root samples collected to determine the composition of the prokaryotic and eukaryotic root-associated microbe communities. Alpha and beta diversities were calculated and relative abundance of various microbial taxa and their co-occurrence were analyzed. Rhizobium was the most prevalent bacterial genus, followed by Sphingomonas, Mucilaginibacter, Flavobacterium, and the unclassified Chloroflexi group KD4-96. The Leptodontidium, Cladosporium, Clonostachys, and Tetracladium fungal genera known for endophytic, saprotrophic, and mycoparasitic lifestyles were also frequently observed in all samples. Sixty-two potential pathogenic fungi were identified with a bias toward grass pathogens and a higher abundance in samples from conventional farms. We showed that the microbial community was mainly shaped by geographic location and management procedures. Co-occurrence networks revealed that the Rhizobiumleguminosarum bv. trifolii was negatively associated with all fungal pathogenic taxa recognized in this study.