Spore Release Research Articles

Study on the dynamic distribution of spores of powdery mildew pathogen in wheat by rotor airflow of plant protection UAV

Plant protection drones are fast and efficient application machines that are characterised by high application efficiency and no damage to crops. They are particularly suitable for small areas of farmland and mountainous terrain in regions such as Asia and are currently the dominant insecticide application technology in China. The presence of wind is a prerequisite for the spread and dissemination of airborne diseases and it can directly influence the distance and height of ascent of pathogenic spores. This paper investigates the effect of downwash airflow generated by the flight altitude of a plant protection drone on the horizontal distribution, vertical distribution and ground distribution of powdery mildew spores in wheat. Monitoring the changing dynamics of airborne powdery mildew conidia using spore traps. The test results show that: the number of powdery mildew pathogenic spores is related to various factors such as weather, relative humidity and wind speed; the release of spores is greatly influenced by airflow disturbances but has little effect at the early stages of sporulation; the disease is caused by the accumulation process of pathogenic spores and in the control of powdery mildew in wheat, preventive spraying should be carried out within 2–3 days of the germination of pathogenic spores. The study lays the foundation for further in-depth research on the spread of powdery mildew spores and improved pest control, and provides a basis for scientific and rational spraying and control by agricultural drones.

Telescopic peristomes, hygroscopic movement and the spore release model of Regmatodon declinatus (Leskeaceae Bryophyta).

Moss peristome hygroscopic movement plays an important role in protecting and controlling spore release. Recent studies on the peristome's hygroscopic movement and spore release have focussed on mosses with 'perfect' peristomes, such as Brachytheciaceae, whereas the hygroscopic movement type and spore release pattern of 'specialized' peristomes, such as Regmatodon declinatus, are poorly understood. We investigated the relationship between the peristome's hygroscopic movement and spore release in the 'specialized' peristome of R. declinatus by the measurement of peristome hygroscopic movement parameters and the hygroscopic movement spore release test. It was found that: (i) Exostomes (EX) are significantly shorter than endostomes (EN), triggering the hygroscopic movement of telescopic peristomes, in which the EX rapidly elongate while closing in on the EN, and the teeth rapidly converge. (ii) Spore release was minimal when peristome movement was triggered alone. The number of spores released when exposed to wind was 124 times greater than in the absence of wind. Dry capsules released seven times more spores than wet capsules. The study reveals that the hygroscopic movement of 'telescopic' peristomes of R. declinatus did not contribute significantly to spore release. More spores were released when wind and hygroscopic movement acted synergistically. Dry capsules released the maximum number of spores. It was also revealed that structural damage to capsules can facilitate complete spore release. Finally, we modelled the release of R. declinatus spores from initiation to complete release, namely the coupled release model of wind-water-capsule structural damage.

Occurrence of Multispecies Myxozoan Parasite Infestation by Genus <i>Thelohanellus</i> Kudo, 1933 in a Group of <i>Labeo rohita </i>(Hamilton, 1822)

We describe two Thelohanellus Kudo spp. namely, T. bifurcata (34.94x 8.9μm) on gills and T. caudatus (13x8.9μm) on the caudal and dorsal fins, found infecting Labeo rohita (Hamilton, 1822) fingerlings in a wet lab facility of West Bengal India. The L. rohita fingerlings having spores on fins and gills were segregated and gross pathology was observed for one month. It was done to characterise the morphology of mature spores and to examine the histopathology of infected gills. There were 5-6 white cysts (1-2 mm in length) on the gill filament that were mostly persistent during the one-month study period and infected fish (12.5% prevalent) were listless, anorexic, and emaciated before eventually all of them perished. Spores of 4- 7 mm size in 1-4 numbers were found on caudal and dorsal fins which were visible for two weeks before it gets mature and released, whereas infected fish (20%) were active and underwent normal growth. Additionally, it was discovered that 5% of fingerlings had cysts on their gills and fins. The distal tip of the primary gill lamellae is where the gill epithelium ruptures, releasing the spores. The histological inspection of the infected gill clearly demonstrates infective sporoplasm entry, presporogonic stage, plasmodial maturation, and release of spores. Within a month, it was discovered that 100% of the fingerlings with infected gills died. This is because the plasmodial spores on primary gill filaments damage the structural framework of the gill filament and cause epithelial and vascular cell displacement. Our comparative analysis with previous studies support that orders and families of the parasites are strongly correlated with Thellohanellus Kudo sp: a pathogenic strain of T. bifurcata and a non-pathogenic strain of T. caudatus infecting L. rohita. The spore-forming stages of the gill-damaging T. bifurcata (entry, pre-sporogonic, and sporogonic) are extremely pathogenic to fish fingerlings and cause significant damage to gill tissue

Seasonal Periodicity of the Airborne Spores of Fungi Causing Grapevine Trunk Diseases: An Analysis of 247 Studies Published Worldwide.

Grapevine trunk diseases (GTDs) are among the most devastating grapevine diseases globally. GTDs are caused by numerous fungi belonging to different taxa, which release spores into the vineyard and infect wood tissue, mainly through wounds caused by viticultural operations. The timing of operations to avoid infection is critical concerning the periodicity of GTD spores in vineyards, and many studies have been conducted in different grape-growing areas worldwide. However, these studies provide conflicting and fragmented information. To synthesize current knowledge, we conducted a systematic literature review, extracted quantitative data from published papers, and used these data to identify trends and knowledge gaps that need to be addressed in future studies. Our database included 26 papers covering 247 studies and 3,529 spore sampling records concerning a total of 29 fungal taxa responsible for Botryosphaeria dieback (BD), Esca complex (EC), and Eutypa dieback (ED). We found a clear seasonality in the presence and abundance of BD spores, with a peak from fall to spring, more in the northern hemisphere than in the southern hemisphere, but not for EC and ED. Spores of these fungi were present throughout the growing season in both hemispheres, possibly because of higher variability in spore types, sporulation conditions, and spore release mechanisms in EC and ED fungi than in BD. Our analysis has limitations because of knowledge gaps and data availability for some fungi (e.g., basidiomycetes, which cause EC). These limitations are discussed to facilitate further research.

Pengaruh Perendaman Thallus Gracillaria sp. Dalam Media Pupuk Grund Dengan Dosis Berbeda Terhadap Pelepasan dan Penempelan Spora

The objective of this study was to explore the effect of thallus Gracillaria sp. immersion in Ground fertilizer with the addition of an Atonic growth regulator on spore release and attachment and to determine the best dose of the Grund fertilizer with the same addition of growth regulator mentioned previously. From April to June, 2021, this research was conducted at Fisheries Center for Brackish Water Aquaculture/BalaiPerikananBudidaya Air Payau (BPBAP) Takalar. Further, it employed an experimental method with a Completely Randomized Design with four treatments and three replications, i.e., Treatment A SWW (Sterile Seawater), B (1% Ground + 0.2 Atonic), C (2% Ground + 0.2 Atonic), and D (3% Ground + 0.2 Atonic). The results of the ANOVA showed that the use of Ground fertilizer + Atonic growth regulator significantly influenced spore release at the significance level of 5% (sig. value < 0.05). Meanwhile, there was no significant difference in spore attachment at the significance level of 5% (sig. value > 0.05). The best dose from the spore release and attachment was in treatment C (2% Ground + 0.2 Atonic). Keywords: Gracilariasp, Ground Media, Atonic Growth Regulator, Spore Release and Attachment.

Sporulation conditions influence the surface and adhesion properties of Bacillus subtilis spores.

Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca2+ or Mg2+ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca2+ or Mg2+ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines.

Passive fungal spore release from fruit and vegetable solid waste

Food substrates in municipal solid wastes processing facilities and open dumpsites are a source for the release of fungal spores into air and can cause potential health and climate effects. Experiments were conducted in a laboratory scale flux chamber to measure the fungal growth and spore release from representative exposed cut fruit and vegetable substrates. The aerosolised spores were measured using an optical particle sizer. The results were compared to experiments conducted previously with a test species (Penicillium chrysogenum) on a synthetic media (czapek yeast extract agar). Significantly higher surface spore densities were observed for the fungi on the food substrates as compared to that on the synthetic media. The spore flux was high initially and then decreased on continued exposure to air. The spore emission flux normalised to the surface spore densities indicated that the emission from the food substrates was lower than the emissions from the synthetic media. A mathematical model was applied to the experimental data and the observed flux trends were explained in terms of the model parameters. A simple application of the data and the model to release from a municipal solid waste dumpsite was shown.

Molecular detection and identification of Diatrypaceous airborne spores in Australian vineyards revealed high species diversity between regions.

The grapevine trunk disease, Eutypa dieback (ED), causes significant vine decline and yield reduction. For many years, the fungus Eutypa lata was considered the main pathogen causing ED of grapevines in Australia. Recent studies showed other Diatrypaceous fungi were also associated with vines exhibiting dieback symptoms but there is limited information on how these fungal pathogens spread in vineyards. Thus, information on the spore dispersal patterns of Diatrypaceous fungi in different wine regions will assist in identifying high-risk infection periods in vineyards. Using more than 6800 DNA samples from airborne spores collected from eight wine regions in south-eastern Australia over 8 years using a Burkard spore trap, this study investigated the diversity and abundance of Diatrypaceous species, using multi-faceted molecular tools. A multi-target quantitative PCR (qPCR) assay successfully detected and quantified Diatrypaceous spores from 30% of the total samples with spore numbers and frequency of detection varying between regions and years. The high-resolution melting analysis (HRMA) coupled with DNA sequencing identified seven species, with E. lata being present in seven regions and the most prevalent species in the Adelaide Hills, Barossa Valley and McLaren Vale. Cryptovalsa ampelina and Diatrype stigma were the predominant species in the Clare Valley and Coonawarra, respectively while Eutypella citricola and Eu. microtheca dominated in the Hunter Valley and the Riverina regions. This study represents the first report of D. stigma and Cryptosphaeria multicontinentalis in Australian vineyards. This study further showed rainfall as a primary factor that triggers spore release, however, other weather factors that may influence the spore release in different climatic regions of Australia still requires further investigation.

Spore dispersal patterns of the ascomycete fungus Ramularia collo-cygni and their influence on disease epidemics

Ramularia leaf spot is a major economic disease of barley caused by the dothidiomycete fungus Ramularia collo-cygni. The fungus has a complex life cycle which includes extensive late season spore release events and a seed-borne phase. Predicting disease epidemics during the growing season remains a difficult challenge. To better understand the interaction between spore movement and disease epidemics, spore samplers were set up in Germany (2 sites over 4 years), Poland (7 sites over 2 years) and the UK (2 sites over 9 years), where the disease has been observed. Spore concentrations were determined using a real-time PCR assay, and meteorological data were obtained from co-located automatic stations. Spore release events were seen to peak in June on mainland Europe and July in the UK. The pattern of spore release was broadly similar across countries with earlier peaks in mainland Europe. A relationship was observed in the UK between July spore levels and disease in following winter barley crops. Rainfall and temperature were proposed as significant drivers of spore release in these months. The major environmental parameter associated with spore release across the two UK sites was crop surface wetness, although some site-specific interactions were noted for rainfall and wind movement. Regression analysis of spore patterns and disease epidemics indicates a relationship between spore levels 75 and 105 days pre-harvest and final disease levels in UK winter barley crops. This relationship was not observed in spring barley. The implications on risk forecasts are discussed.

Venturioscypha nigropila (Hyphodiscaceae, Helotiales) – a new genus and species from xeric Pinus bark

A new genus and species, <em>Venturioscypha nigropila</em>, is proposed for a minute inoperculate discomycete with long, cylindrical, partly flexuous, dark blackish-brown, smooth, finally thick-walled hairs. It has been collected repeatedly in Europe on dead, corticated branches of <em>Pinus</em> spp. attached to living or recently dead trees. At first glance the species resembles members of <em>Pirottaea</em> (<em>Pyrenopezizaceae</em>), but the relationship is shown by molecular phylogenetics to be close to <em>Hyphodiscus</em>, <em>Hyphopeziza</em>, <em>Fuscolachnum</em>, and <em>Venturiocistella</em> (<em>Hyphodiscaceae</em>). These genera differ in having hairs with more or less conspicuous warts, in <em>Hyphopeziza</em> also with glassy solidifications, and <em>Venturiocistella</em> in having in addition long, stiff, thick-walled, apically acute, dark brown hairs, which are warted in their lower part. The hair wall of <em>Venturioscypha</em> appears superficially smooth, but the surface is inconspicuously pitted as viewed under light microscopy. <em>Venturioscypha</em> is unique in <em>Hyphodiscaceae</em> by its peculiar hairs, inamyloid asci with a thin apical wall that ruptures irregularly by a terminal split at spore discharge, spores with a delicate sheath, and apothecial proliferation.

Effects of polyamines on the early development of cystocarps, spore release, survival, and germination rate of Gracilariopsis lemaneiformis

Effects of polyamines on the early development of cystocarps, spore release, survival, and germination rate of Gracilariopsis lemaneiformis

Seasonal dynamics of inoculum of Botryosphaeriaceae in macadamia orchards in Australia

AbstractMacadamia (Macadamia integrifolia and M. tetraphylla and their hybrids) is a tree nut crop affected by Botryosphaeria branch dieback. The disease results in branch and tree death in commercial orchards, so reducing the productivity of the Australian macadamia industry. There is a dearth of knowledge of the disease cycle in macadamia, which confounds disease management. This study investigated the dynamics of pathogen inoculum associated with Botryosphaeria branch dieback and the influence of environmental factors (temperature, rainfall and relative humidity) on the seasonal spore release within the orchards. Diseased and asymptomatic plant parts from macadamia trees were collected each season for 3 years in commercial orchards. Fungal structures (pycnidia and pseudothecia) mainly were (71%) observed on symptomatic wood samples. A comparison of the number of spores released from the fungal structures between summer and autumn revealed high concentrations of Lasiodiplodia pseudotheobromae from diseased wood (4.8 × 104 conidia/cm2) and Neofusicoccum luteum from blighted leaves (2.1 × 103 conidia/cm2). The peak airborne conidia dispersal period, quantified using microscopy and quantitative PCR assays, was consistently detected after rainfall and strongly associated with relative humidity over 70% and temperatures between 20 and 27°C during summer and autumn. Botryosphaeriaceae isolates obtained from the pycnidia on the macadamia trees caused severe branch dieback symptoms when inoculated on detached macadamia twigs, which suggests both airborne and rain‐splashed spores from fungal structures on the trees contribute to the disease in macadamia orchards.

Cork harvest planning and climate: High air humidity favors availability of airborne inoculum of Diplodia corticola

The canker disease caused by Diplodia corticola is one of the most important emerging pathologies of cork oak (Quercus suber) in western Europe. The fungus is dispersed by borer insects, although it is also thought that the spores can be dispersed by wind and rain. The aim of this study was to evaluate the presence of airborne inoculum of D. corticola in managed cork oak stands during cork harvesting season. Semi-passive spore traps were set in eight sampling plots in Catalonia (north-eastern Spain) in summer of 2020. Traps were replaced every week and the number of D. corticolas spores per sampling event was estimated based on a specific nested-qPCR protocol. Spatial-temporal distribution of airborne inoculum accumulation along sampling areas was analyzed using generalized additive models (GAMs). The availability of airborne inoculum resulted rather low with noticeable accumulation peaks in some of the sampled areas. The fitted GAM revealed a positive effect of high air humidity during the sampling period on the availability of spores. This study represents the first attempt to model the spore release of this emerging pathogen, and it provides insights for developing D. corticola canker control strategies based on the precise timing of cork harvesting operations.

Nanofibers with genotyped Bacillus strains exhibiting antibacterial and immunomodulatory activity.

Biofilm-associated diseases such as periodontitis are widespread and challenging to treat which calls for new strategies for their effective management. Probiotics represent a promising approach for targeted treatment of dysbiosis in biofilm and modulation of host immune response. In this interdisciplinary study, nanofibers with two autochthonous Bacillus strains 27.3.Z and 25.2.M were developed. The strains were isolated from the oral microbiota of healthy individuals, and their genomes were sequenced and screened for genes associated with antimicrobial and immunomodulatory activities, virulence factors, and transferability of resistance to antibiotics. Spores of two Bacillus strains were incorporated individually or in combination into hydrophilic poly(ethylene oxide) (PEO) and composite PEO/alginate nanofibers. The nanofiber mats were characterised by a high loading of viable spores (> 7 log CFU/mg) and they maintained viability during electrospinning and 6months of storage at room temperature. Spores were rapidly released from PEO nanofibers, while presence of alginate in the nanofibers prolonged their release. All formulations exhibited swelling, followed by transformation of the nanofiber mat into a hydrogel and polymer erosion mediating spore release kinetics. The investigated Bacillus strains released metabolites, which were not cytotoxic to peripheral blood mononuclear cells (PBMCs) in vitro. Moreover, their metabolites exhibited antibacterial activity against two periodontopathogens, an antiproliferative effect on PBMCs, and inhibition of PBMC expression of proinflammatory cytokines. In summary, the developed nanofiber-based delivery system represents a promising therapeutic approach to combat biofilm-associated disease on two fronts, namely via modulation of the local microbiota with probiotic bacteria and host immune response with their metabolites.

Spore dispersal patterns of the ascomycete fungus Ramulariacollo-cygni and their influence on disease epidemics

Ramularia leaf spot is a major economic disease of barley caused by the dothidiomycete fungus Ramulariacollo-cygni. The fungus has a complex life cycle which includes extensive late season spore release events and a seed-borne phase. Predicting disease epidemics during the growing season remains a difficult challenge. To better understand the interaction between spore movement and disease epidemics, spore samplers were set up in Germany (two sites over 4 years), Poland (seven sites over 2 years) and the UK (two sites over 9 years), where the disease has been observed. Spore concentrations were determined using a real time PCR assay, and meteorological data were obtained from co-located automatic stations. Spore release events were seen to peak in June on mainland Europe and July in the UK. The pattern of spore release was broadly similar across countries with earlier peaks in mainland Europe. A relationship was observed in the UK between July spore levels and disease in following winter barley crops. Rainfall and temperature were proposed as significant drivers of spore release in these months. The major environmental parameter associated with spore release across the two UK sites was crop surface wetness, although some site-specific interactions were noted for rainfall and wind movement. Regression analysis of spore patterns and disease epidemics indicates a relationship between spore levels 75–105 days pre harvest and final disease levels in UK winter barley crops. This relationship was not observed in spring barley. The implications on risk forecasts are discussed.

Spore production monitoring reveals contrasting seasonal strategies and a trade‐off between spore size and number in wood‐inhabiting fungi

Abstract Traits related to reproduction and dispersal drive the assembly and dynamics of species communities and can explain and predict how species respond to habitat loss and fragmentation and to the changing climate. For fungi, such links remain poorly known. We examine how spore production rate, a key demographic trait, is influenced by the interaction between environmental conditions and species traits. We monitored the spore production of 97 wood‐inhabiting fungal species on 107 decaying logs for 2 years and analysed the data with a hierarchical community model. Our analysis demonstrates clear species differences in seasonal patterns, with spring and summer release dominating in perennial species, contrary to the commonly held view of autumn as the primary “mushroom season”. Many species follow a diurnal pattern with a higher spore release rate during the night. Such patterns in release timing have important implications for dispersal, as shown by recent model simulations. The overall level of spore release was negatively correlated with spore size, providing new evidence that fungi face the classic trade‐off of investing either in the number or size of offspring. We found that different species within the functional group of wood‐inhabiting fungi display alternative strategies in spore release timing and along the trade‐off between offspring size and number. Linking our findings to previously reported correlations between spore size and other traits, we propose a new conceptualization of life history strategies in wood‐inhabiting fungi, with implications for species' ability to survive the ongoing biodiversity crisis. Read the free Plain Language Summary for this article on the Journal blog.

Reproductive phenology of the kelp Ecklonia radiata at its Australian warm-range edge and the influence of environmental factors

Context Range-edge populations persist closer to their physiological thresholds, often limiting reproductive output. Kelps are foundation species on temperate reefs, but their reproductive phenology at range edges remains poorly understood. Aims We assess the reproductive phenology of Ecklonia radiata at its eastern Australian warm range edge in relation to local environmental conditions and make comparisons with populations throughout Australia. Methods E. radiata fertility was monitored over multiple years and environmental predictors of fertility were assessed using generalised additive models. Fertility responses were compared with data from centre and cold range-edge populations. Key results The proportion of fertile E. radiata plants increased as daylength declined and the length of fertile tissue was positively correlated with temperature. The magnitude of spore release was variable and responded to episodic swell and rainfall events. This is contrary to spore-release data from centre and cold range-edge populations, which demonstrated seasonal patterns correlated with temperature. Conclusion Spore release was lower at the warm range edge, yet year-round spore release demonstrated flexibility in response to local environmental conditions. Implications Flexible reproductive phenology of E. radiata at its warm range edge may be advantageous for sustaining populations at low latitudes under climate change.

Factors influencing the airborne sporangia concentration of Phytophthora infestans and its relationship with potato disease severity

Forecasting systems are widely used to predict the application of fungicides for managing late blight. However, airborne inoculum has rarely been included in these forecasting systems. Monitoring the sporangia in crop environments may offer an opportunity to improve late blight forecast systems by integrating pathogen pressure. Hence, this experiment aiming to analyze relationships between weather based risk systems and sporangia levels in the atmosphere of potato crops. The experiments were conducted during two growing seasons in a potato field. During the study, the concentration of Phytophthora infestans in the air, the weather conditions, the phenology of cultivars and r-AUPDC during the crop cycle were recorded. The weather-based risk of late blight was estimated using infection pressure (IP) and the daily risk value (DRV) based on hourly relative humidity (RH) and temperature (T). The effect of weather parameters on sporangia levels was analyzed. IP and DRV showed a strong positive correlation with sporangia concentration, standing out the pronounced effect of RH on the sporangia levels. Analysis of the hourly sporangia concentration within a day showed an increase in the sporangia concentration from 9 h to 18 h. This increase in sporangia was linked to an increase in T, spore release, and a decrease in RH. Our results identified a T of 10 °C and RH of 80% as the minimum threshold for significant sporangia concentration in the air. However, maximum sporangia level was found in the air at 88% (average relative humidity) and 17 °C (average temperature). Finally, the effect of weekly P. infestans sporangia was observed on cultivars with different susceptibility to late blight.

Improvement of Alternaria Leaf Blotch and Fruit Spot of Apple Control through the Management of Primary Inoculum

Alternaria spp. is the causal agent of apple leaf blotch and fruit spot, diseases of recent appearance in Spain. The overwinter inoculum of Alternaria spp. is the source of primary infections in apple, thus the aim of this work was to optimize the control of infection through two environmentally friendly inoculum-management strategies, the removal of winter fallen leaves and the treatment of leaves with the biological agent Trichoderma asperellum to inhibit or prevent inoculum development in commercial orchards. The results of commercial orchard trials showed that leaf aspiration and application of T. asperellum on the ground have efficacy to reduce fruit spot between 50 and 80% and leaf blotch of between 30 and 40% depending on the year. The efficacies on the reduction of leaf blotch were slightly lower than of fruit spot. Disease reduction has been related to a reduction of total spores released during the season. Results of dynamics of spore release indicate that factors influencing spore release were rainfall and temperature. In conclusion, the use of environmentally friendly strategies combined with standard fungicides, and with monitoring environmental conditions, might allow a reduction in the number of phytosanitary applications, thus achieving the goal of reducing their use.

Enhanced encapsulation efficiency and controlled release of co-encapsulated Bacillus coagulans spores and vitamin B9 in gellan/κ-carrageenan/chitosan tri-composite hydrogel

The current study, for the first time, attempts to co-encapsulate Bacillus coagulans spores as probiotics and vitamin B9 in the polysaccharide-based matrix for their targeted delivery. Instead of vegetative cells, probiotic spores were chosen owing to their higher stability. The matrix, tri-composite hydrogel, was synthesized from gellan, κ-carrageenan, and chitosan through self-assembly devoid of chemical cross-linkers. Hence, it was found suitable for application in the co-encapsulation of bioactive compounds. The synthesized hydrogel showed remarkable encapsulation efficiency for folic acid and probiotic spores, both individually and in combination. At acidic pH, loaded hydrogel exhibited 28.42 % and 45.14 % release of spores and folic acid, respectively, which was comparatively lower than the trends observed under neutral and alkaline pH. These results were correlated with the release pattern observed during in vitro digestibility studies. Moreover, spore conversion to vegetative cells and its high colonization were observed in the simulated intestinal phase. Therefore, the matrix maintained viability and stability of co-encapsulated folic acid and bacterial spores in gastric pH while they were slowly released in the intestinal phase. These promising findings pave the way to develop a natural matrix for co-encapsulating various bioactive compounds and probiotics.