Dual Culture Method Research Articles

Assessment of Trichoderma Species Isolated From Volcanic Soil of a Durian Field in Sisaket Province, Thailand for Plant Growth Promotion and Biocontrol Potential

Trichoderma species are ubiquitous saprophytic fungi commonly found in soil, with potential as fungal biocontrol agents for plant disease control and growth promotion. This research aimed to assess the attributes of Trichoderma sp. as plant growth promoters, focusing on nitrogen fixation, siderophore production, plant nutrient solubilization and indole-3-acetic acid production. Out of 19 isolates tested, 6 (T05, T19, T25, T27, T28 and T33) showed superior plant growth promotion abilities. These isolates were further evaluated for their capacity to enhance the germination of Khao Dawk Mali 105 rice seeds. Seeds were soaked in a suspension of conidia at a concentration of 1×1012 conidia/mL, and germination rates were measured. The germination rate ranged between 77.67 - 86.67 %, statistically significantly higher than the control rate of 33.33 %. Among the isolates, T05 exhibited the highest promotion of plant growth, evidenced by root length (7.27 cm), shoot length (8.50 cm), fresh weight (0.54 g) and dry weight (0.27 g), all significantly different from the control. Additionally, the potential of isolates to inhibit the fungal pathogen Colletotrichum sp. MN-3 was assessed using a dual culture method, with T35 showing the highest inhibition percentage (60.90 %). Morphological classification and nucleotide sequencing of the ITS1-5.8S-ITS2 region of rDNA gene identified the isolates as T. harzianum, T. reesei, T. asperellum and T. longibrachiatum. These findings underscore the effectiveness of Trichoderma sp. in promoting plant growth and suppressing plant pathogenic fungi. HIGHLIGHTSSix Trichoderma isolates (T05, T19, T25, T27, T28 and T33) exhibited superior plant growth promotion abilities. These isolates significantly enhanced germination rates of Khao Dawk Mali 105 rice seeds compared to the method. Among the isolates, T05 showed the greatest promotion of plant growth, as evidenced by root length, shoot length, fresh weight and dry weight. Trichoderma isolates demonstrated potential for inhibiting the fungal pathogen Colletotrichum sp. MN-3. Morphological classification divided the Trichoderma sp. into 4 groups, and species identification through nucleotide sequencing revealed T. harzianum, T. reesei, T. asperellum and T. longibrachiatum. GRAPHICAL ABSTRACT

Potentiality of Formulated Pseudomonas Fluorescens and Bacillus Subtilis for the Management of Sheath Blight Disease of Rice Under Field Condition

Rice production is severely affected by sheath blight disease in Bangladesh which is caused by a fungus Rhizoctonia solani that is still merely managed by synthetic chemical fungicides. In this study, different isolates of Pseudomonas fluorescens and Bacillus subtilis were evaluated to assess their inhibitory effects against R. solani both in- vitro and field condition following dual culture method in order to formulate technique for the management of sheath blight of rice. Among all the isolates, P. fluorescens (Pf- 7 and Pf- 8) and B. subtilis (Bs-17 and Bs-21) showed the highest in –vitro mycelial growth suppression of the pathogen. Based on the in-vitro mycelial growth suppression performance, fourteen treatments combinations, either single or in consortia of antagonistic bacteria in talc-based formulations were applied by root dipping. All the formulated bacterial isolates showed significant reduction in disease and severity compared to untreated control and chemical fungicide. At 90 DAT, both the disease incidence and severity were found lowest in T11 (31.67 % and 37.33 % respectively) where the seedlings were treated for 24 hours with consortium of Pf-7 + Pf- 8 formulated in talc followed by in T12 (32.00 % and 38.00 %) where the seedlings were treated for 24 hours with consortium of Bs-17 + Bs-21 formulated in talc respectively. Vegetative and yield parameters were also significantly increased in both T11 and T12. The highest yield as well as Benefit Cost Ratio (BCR) were found in T11 (2.50 kg/ m2 and 2.20) followed by T12 (2.49 kg/m2 and 2.18). Thus, the consortia of bacterial isolates (Pseudomonas fluorescens and Bacillus subtilis) with seedling dipping time for 24 hour represents a promising alternative to chemical fungicides for the management of sheath blight of rice in field condition. J Bangladesh Agril Univ 22(3): 342-351, 2024

Molecular Identification and Antifungal Activity of Candida chrysomelidarum and Rhodotorula toruloides Isolated from Indonesian Indigenous Fruit Flacourtia inermis Roxb. (Lobi-lobi) against Pathogenic Mold Aspergillus flavus

Aspergillus flavus is one of the main contaminating mold species in most feed products, significantly impacting human and animal health. This study aims to isolate the epiphytic yeasts from the fruit Flacourtia inermis Roxb and test their potential ability to act as biocontrol agents for A. flavus. The antagonistic test was performed using a dual culture method, and the potential yeast isolates were then identified using a combination of morphology characterization and molecular sequence analysis. From 42 yeast isolated from F. inermis fruit, 2 yeast isolates (LCA 75 and LCA 102) demonstrated the highest inhibition growth against A. flavus (32.32 and 32.30 %, respectively). The isolates LCA 75 and LCA 102 were identified as Candida chrysomelidarum and Rhodotorula toruloides, respectively. Rhodotorula toruloides, identified as one of the yeast isolates (LCA 102), is an unconventional red yeast demonstrating notable antagonistic effects against A. flavus, inhibiting its growth by 32.30 %. This yeast, also known as Rhodosporidium toruloides, R. rubescens, R. glutinis, or R. gracilis, belongs to the subphylum Pucciniomycotina within the division Basidiomycota. The significant inhibition of mycelium growth and sporulation observed in our study highlights the potential of R. toruloides, along with C. chrysomelidarum (LCA 75), as effective biocontrol agents to reduce mycotoxin contamination in chicken feed by A. flavus. This study represents a significant advancement in the management of A. flavus contamination in animal feed. The findings not only underscore the viability of yeast-based biocontrol strategies but also contribute to a safer, more sustainable approach to preventing mycotoxin proliferation in feedstocks. HIGHLIGHTS Founding Out of 42 yeast isolates, two (LCA 75 and LCA 102) demonstrated the highest inhibition growth against A. flavus, with inhibition rates of 32.32 and 32.30 %, respectively. Identified Yeasts: LCA 75 as Candida chrysomelidarum LCA 102 as Rhodotorula toruloides (alternatively referred to as Rhodosporidium toruloides, R. rubescens, R. glutinis, or R. gracilis). Characteristics of Rhodotorula toruloides Unconventional red yeast belonging to the subphylum Pucciniomycotina within Basidiomycota. Commonly associated with various plant species and found on plant surfaces, including leaves, stems, and fruits. Identified yeast isolates exhibited inhibition activities leading to reduced mycelium growth and sporulation of A. flavus. These yeast isolates have potential as biocontrol agents to prevent mycotoxin contamination in chicken feed caused by A. flavus. GRAPHICAL ABSTRACT

Antifungal metabolites produced by Pseudomonas hunanensis SPT26 effective in biocontrol of fusarium wilt of Lycopersicum esculentum under saline conditions.

In past few years, salinity has become one of the important abiotic stresses in the agricultural fields due to anthropogenic activities. Salinity is leading towards yield losses due to soil infertility and increasing vulnerability of crops to diseases. Fluorescent pseudomonads are a diverse group of soil microorganisms known for promoting plant growth by involving various traits including protecting crops from infection by the phytopathogens. In this investigation, salt tolerant plant growth promoting bacterium Pseudomonas hunanensis SPT26 was selected as an antagonist against Fusarium oxysporum, causal organism of fusarium wilt in tomato. P. hunanensis SPT26 was found capable to produce various antifungal metabolites. Characterization of purified metabolites using Fourier transform infrared spectroscopy (FT-IR) and liquid chromatography-electron spray ionization-mass spectrometry(LC-ESI/MS) showed the production of various antifungal compounds viz., pyrolnitrin, pyochelin and hyroxyphenazine by P. hunanensis SPT26. In the preliminary examination, biocontrol activity of purified antifungal metabolites was checked by dual culture method and results showed 68%, 52% and 65% growth inhibition by pyrolnitrin, 1- hydroxyphenazine and the bacterium (P. hunanensis SPT26) respectively. Images from scanning electron microscopy (SEM) revealed the damage to the mycelia of fungal phytopathogen due to production of antifungal compounds secreted by P. hunanensis SPT26. Application of bioinoculant of P. hunanensis SPT26 and purified metabolites significantly decreased the disease incidence in tomato and increased the plant growth parameters (root and shoot length, antioxidant activity, number of fruits per plant, etc.) under saline conditions. The study reports a novel bioinoculant formulation with the ability to promote plant growth parameters in tomato in presence of phytopathogens even under saline conditions.

Antagonism and mycoparasitism mechanism of T. harzianum against pathogenic fungus species of F. oxysporum and Capnodium sp.

The purpose of this study was to test the ability of T. harzianum to inhibit the growth of pathogenic mold species and to determine the effectiveness of T. harzianum mold species in inhibiting the growth of pathogenic molds, as well as investigate how T. harzianum and pathogenic molds interact with each other. The Microbiology Laboratory of the Department of Biology, Faculty of Mathematics and Natural Sciences, State University of Malang, is where this research was conducted. The dual culture method was used for this test by using Czapek Agar (CA) medium. The isolated molds were then incubated for 4x24 hours at 25O–27OC. After that, the antagonism power was calculated. The results of macroscopic and microscopic observations were used to assess the mechanism of T. harzianum mold antagonism against pathogenic molds. The results showed that Trichoderma mold species were more resistant to the pathogenic mold Fusarium oxysporum than the pathogenic mold Capnodium. The antagonistic power of T. harzianum was 80%, with the antagonistic power of Capnodium sp. at 66.7%. The mechanism of mycoparasitism occurs when the hyphae of T. harzianum attach or entangle the hyphae of pathogenic molds, causing damage to the hyphal structure and inhibiting the growth of pathogenic molds.

Antagonistic potential of Trichoderma strains isolated from Musa paradisiaca cv. Malnad Rasbale grown farmyards against Foc race 4 pathogen

Foc race 4 is a causative pathogen for Panama wilt disease of Musa Paradisiaca cv. Malnad Rasbale. The cost-effective measure to control rather than the usage of agrochemicals is still not available for this cultivar. Trichoderma strains act as an antagonistic agent against different phytopathogenic fungi including many pathogenic races of Panama wilt-causing pathogens. An attempt has been made to recognize the mode of action of this antagonistic agent in in vitro conditions and interaction between six Trichoderma strains. Foc race 4 was first investigated by dual plate culture method on PDA medium. This study revealed the potential of native strain KUVKU-TH02 for the biological control of Foc race 4 pathogen affecting Malnad Rasbale cultivar in in vivo conditions rather than native isolates KUVKU-TH01, KUVKU-TV01 and KUVKU-TV02. Observations revealed the lysis of hyphal ending in inhibited colonies of the fungal pathogen. Pure culture of isolated fungal strains incubated on potato dextrose broth made a path to isolate DNA for identification and molecular characterization studies. Upon DNA sequencing, native isolates sequences were deposited to NCBI genebank to gain accession IDs. The phylogenetic tree built showed the evolutionary relationship between the isolates and also the potency of native biocontrol isolates against procured isolates.

Antagonistic activity of Trichoderma sp. against pathogens in the leaves of Allium ascalonicum L.

Pathogenic fungi pose constraints and reduce shallot production. Trichoderma sp. is an antagonistic fungus capable of controlling pathogen growth in shallots. The aim of this study was to determine the characteristics of Trichoderma sp. and pathogens in shallots and to assess the antagonistic ability of Trichoderma sp. against shallot pathogens (Allium ascalonicum L.). Trichoderma sp. and shallot pathogens were isolated using a serial dilution agar plate method using potato dextrose agar medium with 7-day incubation. The antagonistic activity of Trichoderma sp. against shallot pathogens was evaluated based on the dual culture method. In this study, we successfully isolated Trichoderma harzianum from the shallot leaf and its root systems. Moreover, four morphologically distinctive pathogens from shallot roots and leaves were successfully isolated (l Aspergillus sp., Colletotrichum sp., Phytium sp. and Penicillium sp. T. harzianum was found to have the ability to inhibit 23.45% growth of Aspergillus sp, 26.19% growth of Colletotrichum sp., 75.40% growth of Phytium sp., and 40.38% growth of Penicillium sp. In conclusion, the isolated T. harzianum had a strong antagonistic activity against some pathogens in the shallot, but the activity was weak against some others.

Characterization and evaluation of native Trichoderma harzianum P39 for biocontrol potentials against five seed-borne fungal pathogens of rice

Plant seeds can harbour pathogens that limits crop growth and productivity. This study isolated, characterised and evaluated native Trichoderma harzianum for antagonism against five (Alternaria alternata, Aspergillus oryzae, Bipolaris oryzae, Curvularia lunata and Fusarium verticillioides) fungal pathogens isolated from rice seeds. Evaluation was through simultaneous inoculation, a pathogen against the biocontrol agent, using the dual culture method in a PDA growth medium. Results confirmed the T. harzianum isolate had 98.27% pairwise identity with T. harzianum P39 (KX381171.1. accession number). The isolate showed increasing percentage inhibition, against all the pathogens, with increasing days after inoculation. The highest values ranged from 53.02%, A. oryzae to 70.06%, F. verticillioides (the least and most susceptible respectively). The overall inhibition percentages of A. alterata and B. oryzae (12th day after inoculation) were not significantly different from that of F. verticillioides. Trichoderma harzianum P39 can be formulated as biological seed dressing agent.

Antifungal Potential of Bacillus subtilis subsp. inaquosorum RLS76 for Management of Fusarium Wilt Disease of Bt- Cotton

Fusarium wilt is a destructive disease affecting Bt cotton that is caused by Fusarium oxysporum f. sp. vasinfectum. This seed and soilborne disease pose a significant risk to Bt cotton production worldwide, including India. Therefore, to cope with this problem in an eco-friendly manner, we explored the potentiality of the rhizobacterial isolate possessing antifungal activity as a natural arsenal against the fungal pathogen fusarium wilt of Bt cotton. The rhizobacterial isolate RLS76 inhibited 85.39% of the fungal phytopathogen in the dual culture method. The RLS76 isolate's 16S rRNA partial gene sequence was determined to be Bacillus subtilis subsp. inaquosorum RLS76, with a closest phylogenetic affiliation of 99.53%. The crude extract of Bacillus subtilis subsp. inaquosorum RLS76 demonstrated 34.38% inhibition of the growth of the pathogen after a week of incubation by using an agar well diffusion method. Bacillus subtilis subsp. inaquosorum RLS76 produced volatile organic compounds that inhibited the fungal growth of phytopathogens by 54.84% in a closed environment. Also, GCMS analysis of the crude extract of Bacillus subtilis subsp. inaquosorum RLS76 identified four compounds, namely Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-; Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); 2,5-cyclohexadiene-1,4-dione, 2,5-Dihydroxy-3-methoxy-6-methyl-; 9,12-Octadecadienoic acid (Z,Z)-,phenylmethyl ester. Out of four compounds, three exhibit antimicrobial activity except 2,5-Dihydroxy-3-methoxy-6-methyl. The RLS76 strain of Bacillus subtilis subsp. inaquosorum displayed a transition in hue, shifting from blue to orange, surrounding the colony on CAS medium by producing siderophore qualitatively. The quantitative siderophore produced in the crude extract was 86.06 SU after 60-72 h of incubation. The RLS76 strain of Bacillus subtilis subsp. inaquosorum also showed the ability to produce a proteolytic enzyme outside the cell. This enzyme caused a noticeable clear zone around the colony when the bacteria were grown on a skim-milk agar plate. The Bacillus subtilis subsp. inaquosorum RLS76 also exhibited phosphate solubilization activity with the clear halo around the colonies on NBRIP medium. All these results suggest that Bacillus subtilis subsp. inaquosorum RLS76 exhibits good biocontrol as well as a plant growth promoting candidate against the phytopathogen of Fusarium wilt of Bt cotton.

Screening Plant Growth-Promoting Bacteria with Antimicrobial Properties for Upland Rice.

This study explores beneficial bacteria isolated from the roots and rhizosphere soil of Khao Rai Leum Pua Phetchabun rice plants. A total of 315 bacterial isolates (KK001 to KK315) were obtained. Plant growth-promoting traits (phosphate solubilization and indole-3-acetic acid (IAA) production), and antimicrobial activity against three rice pathogens (Curvularia lunata NUF001, Bipolaris oryzae 2464, and Xanthomonas oryzae pv. oryzae) were assessed. KK074 was the most prolific in IAA production, generating 362.6 ± 28.0 μg/ml, and KK007 excelled in tricalcium phosphate solubilization, achieving 714.2 ± 12.1 μg/ml. In antimicrobial assays using the dual culture method, KK024 and KK281 exhibited strong inhibitory activity against C. lunata, and KK269 was particularly effective against B. oryzae. In the evaluation of antimicrobial metabolite production, KK281 and KK288 exhibited strong antifungal activities in cell-free supernatants. Given the superior performance of KK281, taxonomically identified as Bacillus sp. KK281, it was investigated further. Lipopeptide extracts from KK281 had significant antimicrobial activity against C. lunata and a minimum inhibitory concentration (MIC) of 3.1 mg/ml against X. oryzae pv. oryzae. LC-ESI-MS/MS analysis revealed the presence of surfactin in the lipopeptide extract. The crude extract was non-cytotoxic to the L-929 cell line at tested concentrations. In conclusion, the in vitro plant growth-promoting and disease-controlling attributes of Bacillus sp. KK281 make it a strong candidate for field evaluation to boost plant growth and manage disease in upland rice.

Biodiversity of Costus speciosus Phylloplane Fungi in Baturaden Botanical Gardens and Antagonist Testing against Fusarium oxysporum

Indonesia has an abundant biodiversity of natural resources, one of which is phylloplane fungi. This abundant phylloplane mold needs further investigation to utilize its potential as an antagonistic agent. This study aims to prove the existence and to determine the types of phylloplane fungi species found on the leaf surface of pacing tawar (sweet ginger or crepe-ginger) plants, to analyze and determine the level of diversity and dominance of phylloplane, and to determine the types of phylloplane fungi species that have the antagonistic ability of the phytopathogenic mold Fusarium oxysporum. This research method includes isolating crepe-ginger phylloplane fungi using a contact plate, isolates purification, macromorphological and micromorphological identification, diversity and dominance index calculation, and antagonist test using a dual culture method. This study used a descriptive analysis research design. The isolation results obtained 57 species of crepe-ginger plants leaf phylloplane molds consisting of eight genera (Alternaria, Aspergillus, Cladosporium, Curvularia, Fusarium, Mucor, Penicillium, and Trichoderma). The results of the diversity index calculation with a value of 2.02 showed that the diversity of phylloplane fungi was moderate. The result of the dominance index calculation, with a value of 0.1521, shows no tendency for phylloplane fungi species to dominate. The results of the antagonist test showed that 15 isolates could antagonize the F. oxysporum with an inhibitory proportion of up to 82% obtained from T. viride isolates with code FD5B3.2

Biological control of the native endophytic fungus Pochonia chlamydosporia from the root nodule of Dolichos lablab on Fusarium wilt of banana TR4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense, Tropical Race 4 (TR4) is a soil-borne disease, and it is devastating. At present, the biological control using antagonistic microorganisms to mitigate TR4 is one of the best strategies as a safe and green way. Yunnan has abundant and diverse microbial resources. Using the dual-culture method, the antagonistic endophytic fungi against TR4 were isolated and screened from the root nodule of Dolichos lablab. The effect of the highest antagonistic activity strain on the morphology of the TR4 mycelium was observed using the scanning electron microscope. According to morphological characteristics and sequence analysis, the strain was identified. The biocontrol effect and plant growth promotion were investigated by greenhouse pot experiment. Using the confocal laser scanning microscope and the real-time fluorescence quantitative PCR, the dynamics of TR4 infestation and the TR4 content in banana plant roots and corms would also be detected. In this study, 18 native endophytic fungi were isolated from a root nodule sample of Dolichos lablab in the mulch for banana fields in Yuxi, Yunnan Province, China. The YNF2217 strain showed a high antagonistic activity against TR4 in plate confrontation experiments, and the inhibition rate of YNF2217 is 77.63%. After TR4 culture with YNF2217 for 7 days in plate confrontation experiments, the morphology of the TR4 mycelium appeared deformed and swollen when observed under a scanning electron microscope. According to morphological characteristics and sequence analysis, the strain YNF2217 was identified as Pochonia chlamydosporia. In the greenhouse pot experiment, the biocontrol effect of YNF2217 fermentation solution on TR4 was 70.97% and 96.87% on banana plant leaves and corms, respectively. Furthermore, YNF2217 significantly promoted the growth of banana plants, such as plant height, leaf length, leaf width, leaf number, pseudostem girth, and both the aboveground and underground fresh weight. Observations of TR4 infestation dynamics in banana roots and corms, along with real-time fluorescence quantitative PCR, verified that YNF2217 inoculation could significantly reduce the TR4 content. Therefore, YNF2217 as P. chlamydosporia, which was found first time in China and reported here, is expected to be an important new fungal resource for the green control of Fusarium wilt of banana in the future.

Biocontrol potential of rhizospheric fungi against Rigidoporus microporus, the causal agent of white root rot disease in tea plants

White root rot disease (WRD), caused by Rigidoporus microporus, poses a serious threat to various tropical trees, resulting in substantial crop damage and even plant death. However, there is still a lack of information regarding WRD in tea plants. Due to soil-borne in nature, synthetic fungicide is not recommended to control WRD. Therefore, biocontrol using natural microorganisms found in the tea's rhizosphere has been explored as a potential solution. This study aimed to identify rhizospheric fungi of tea plants with antagonistic properties that can be used as a biocontrol agent against R. microporus. The study involved a field survey and laboratory works had been conducted in a tea plantation at Rancabali, West Java and the Phytopathology Laboratory, Faculty of Agriculture, Universitas Padjadjaran from March to October 2021. The disease incidence and severity were assessed in three blocks of land representing different topographic conditions of the plantation while antagonism tests were conducted using the completely randomized design and dual culture method. The results showed that the highest occurrence of WRD was in the central part of the hill with the disease incidence and severity of 70 and 50%, respectively. Thirteen fungal isolates were obtained from the rhizosphere of tea plants, all of which were found to inhibit the growth of R. microporus. These isolates belonged to the genera of Trichoderma, Gliocladium, Paecilomyces, Penicillium, Aureobasidium, Acremonium and Rhizopus. The considerable inhibition of R. microporus colony growth was performed by the isolates of Aureobasidium sp. (51.23%), Rhizopus sp. (50%), and Trichoderma sp. (43.96%). The results suggested that rhizospheric fungi of tea plants have the potential as biocontrol agents against R. microporus.

Cultural and Morphological Characterization of Streptomyces and Interaction Study with Sclerotium rolfsii by SEM

Stem rot caused by Sclerotium rolfsii Sacc. is a major constraint in the production and productivity of the groundnut. Management of stem rot disease is very difficult due to soil borne nature, wide host range of S. rolfsii and lack of disease resistance in existing commercial cultivars. Biological control with potential Streptomyces is receiving greater attention all over the world. Studies were conducted on collection and isolation of Streptomyces, their mechanism of action, cultural and morphological characteristics, its efficacy against S. rolfsii with Scanning electron microscope(SEM). Roving survey was conducted in major groundnut growing regions of Andhra Pradesh. During survey 180 soil samples were collected from groundnut rhizosphere and 50 Streptomyces isolates were isolated from different soil samples. Twenty Streptomyces isolates were tested for their growth rate and antifungal activity against groundnut stem rot pathogen, S.rolfsii under in vitro conditions using dual culture method. Among twenty isolates of Streptomyces, five isolates showed maximum inhibition of S. rolfsii. These five isolates were characterized based on cultural and morphological characters. The study involved in differentiating various characters viz., type of spore chain, colony margins, shape of spore chains, colour of spore mass and spore surface. The morphology of the spore chains varied across the isolates showing the filamentous (Ggd), spiral (Kyd and Lrp) and Rectus flexibilis (Kdr and Mkc). SEM studies revealed that inhibition of the growth of S. rolfsii by Streptomyces was mainly due to exhibition of irregular distortions at contact site of the pathogen and collapse of the fungal hyphae and this might be due to the production of secondary metabolites and extracellular enzymes.

Prospecting the Effectiveness of Fungicides and Bioagents against Colletotrichum gloeosporioides (Penz.) Penz & Sacc.

Aims: The leaf spot caused by Colletotrichum gloeosporioides (Penz.) Penz & Sacc. is one of the most serious disease in all mango growing regions of the world. In vitro bioassay of fungicides and bioagents were performed to evaluate the effectiveness of different fungicides and bioagents against C. gloeosporioides.
 Study Design: Poison food techique for fungicides bioassay and dual culture technique for bioagents bioassay.
 Place and Duration of Study: The laboratory studies were conducted in the Department of Plant Pathology, N. M. College of Agriculture, Navsari Agricultural University, Navsari, Gujarat, India during 2020–2021.
 Methodology: The poison food technique assessed the efficacy of systemic, contact and combination fungicides against C. gloeosporioides. While the dual culture method was employed to gauge the potency of biocontrol agents, against C. gloeosporioides. 
 Results: In laboratory screening, systemic fungicides such as carbendazim and difenconazole were shown to have 100% growth suppression at 500 ppm. Mancozeb and copper oxychloride, two contact fungicides, demonstrated 100% growth inhibition of C. gloeosporioides at 2500 ppm . The combi-product fungicides tricyclazole 18% + mancozeb 62%, hexaconazole 4% + zineb 68%, and carbendazim 12% + mancozeb 63% were shown to exhibit 100% growth inhibition at 2000 ppm. Using the dual culture method, five distinct bioagents were tested in vitro for their ability to prevent the development of C. gleosporiodes. Trichoderma harzianum showed up as one of them to be a powerful and effective antagonist of C. gleosporiodes.
 Conclusion: In vitro fungicide and bioagent testing provides preliminary data on the effectiveness of fungicides against pathogens, guiding field testing. The study evaluated the effectiveness of fungicides and bioagents in suppressing the leaf spot pathogen of mango.

Biocontrol of Fruit Discoloring and Fruit Rot Disease in Longan (Dimocarpus longan) by Using Antagonistic Bacillus spp.

Fruit discoloration and fruit rot disease in longan is a serious disease caused by the pathogenic fungi Pestalotiopsis oxyanthi and Lasiodiplodia pseudotheobromae. This study investigated the potential of antagonistic bacteria as an eco-friendly alternative to traditional fungicides for managing longan diseases caused by these fungal pathogens. Two bacterial isolates, UP-JLS050 and UP-JLS067, demonstrated significant inhibitory activity against the pathogenic fungi P. oxyanthi and L. pseudotheobromae, with high inhibitory activity. The bacterial strains were isolated from leaves and soil in a longan garden, and dual culture methods showed that the isolates UP-JLS050 and UP-JLS067 exhibited the highest inhibitory activity on the fungal pathogens, with percentage inhibitions of 72.67 ± 2.31 and 70.37 ± 1.28 %, respectively. Biochemical methods and molecular techniques, including sequencing of the 16S rDNA gene, were then used to identify the isolates. This confirmed their similarity to Bacillus subtilis and Bacillus amyloliquefaciens with 100 and 99 % identity, respectively. In vitro tests on longan fruit indicated that these antagonistic bacteria, administered at a concentration of 108 CFU/mL, significantly reduced disease incidence by up to 45 % after 9 days of incubation. The incubation of B. subtilis (UP-JLS050) and B. amyloliquefaciens (UP-JLS050) reduced disease incidence by 45 ± 5.77 and 32.5 ± 5.00 %, respectively. HIGHLIGHTS Fruit Discoloration and Rot in Longan: Caused by pathogenic fungi, namely Pestalotiopsis oxyanthi and Lasiodiplodia pseudotheobromae Antagonistic Bacteria for Disease Control: This study explores the potential of antagonistic bacteria as an eco-friendly alternative to traditional fungicides for managing longan diseases caused by these fungal pathogens Effective Inhibition: Two bacterial isolates, UP-JLS050 and UP-JLS067, demonstrated significant inhibitory activity against oxyanthi and L. pseudotheobromae, with inhibitions exceeding 70 % on PDA medium Identification of Bacterial Strains: Molecular techniques, including 16S rDNA gene sequencing, confirmed the identity of these isolates as subtilis and B. amyloliquefaciens Reduced Disease Incidence: In vitro tests on longan fruit indicated that these antagonistic bacteria, administered at a concentration of 108 CFU/ml, significantly reduced disease incidence by up to 45 % after 9 days of incubation GRAPHICAL ABSTRACT

Categorization of mycotoxin producing fungi in leguminous crops and its eco-friendly management under in vitro condition

Four legume crops, viz., groundnut (Arachis hypogea L.), soybean (Glycine max (L.) Merr.) of Kharif season, chickpea (Cicer arietinum L.), and lentil (Lens esculentum L.) of Rabi season belonging to family Fabaceae were taken for investigation of seed-borne mycoflora. Since these are severely affected by many plant-pathogenic fungi at various stages of the whole life cycle, they were chosen for the study. Many post-harvest fungal pathogens cause severe loss by contaminating the grains with various mycotoxins and also deteriorate the nutritional quality of grains. The goal of any agricultural system is to increase the yield of crops as per the population demand. Various eco-friendly management practices were performed on highly mycotoxin-producing isolates of Aspergillus flavus and A. niger isolated from these grains. Four Trichoderma isolates (T2, T3, T4 and T5) were used as a biocontrol agent in the dual culture method to check antagonism between various Aspergillus isolates and Trichoderma isolates. It gave a satisfactory result in controlling mycelial growth. Achyranthes aspera leaf and stem phytoextract (10%, 20%, 30%, and 40%) was used as a botanical treatment in the well diffusion method, which successfully restricts the Aspergillus spp. mycelial growth.

Exploring fungal endophytes as biocontrol agents against rice blast disease

The rice blast disease caused by <i>Pyricularia oryzae</i> Cavara is among the most severe plant diseases in the world. The utilization of natural antagonists, such as fungal endophytes, can be a good method to control the spread of this disease. In the present study, 68 fungal isolates were obtained from 160 rice samples collected in Guilan province fields, Iran. Morphological and molecular characteristics were used for the identification of these fungi. According to the results, the fungal isolates were <i>Alternaria citri</i> (Penz.) Mussat, <i>Alternaria infectoria</i> Simmons, <i>Aspergillus fumigatus</i> Fresen, <i>Fusarium verticillioides</i> (Sacc.) Nirenberg, <i>Trichoderma harzianum</i> Rifai, <i>Trichoderma virens</i> (Mill., Giddens & Foster) Arx, <i>Trichoderma viride</i> Pers., <i>Ulocladium consortiale</i> (Thum) Simmons, and <i>Pyricularia oryzae</i> . Eight isolates that did not cause any plant disease were selected for biocontrol studies <i>in vitro</i> . The dual culture, culture filtrate, slide culture, and volatile metabolites methods were used in search of natural endophytic antagonists of <i>Pyricularia oryzae</i>. It was found that <i>T. harzianum, T. virens, T. viride, A. fumigatus</i>, and <i>A. citri</i> isolates caused the highest percentage of mycelial growth inhibition of <i>P. oryzae in vitro</i>. In the greenhouse experiments, all of the studied isolates reduced the <i>P. oryzae</i> disease rating, with <i>T. harzianum</i> isolate being the most effective antagonist (27.36% reduction in the disease rating), followed by <i>T. virens, T. viride</i>, and <i>A. fumigatus</i> (20.75%, 17.92%, and 16.98%, respectively). Furthermore, in greenhouse conditions, these antagonistic fungi increased the height, fresh weight, and dry weight of plants. The results of this research showed that there are various fungi in the natural flora of rice plants that possess potential biological properties against rice blast disease that can be implemented into practice.

In vitro screening of fungal endophytes from sandalwood (Santalum album) as antagonists to phytopathogens

Abstract. Simamora AV, Hahuly MV, Nenotek PS, Kana YR, Kasim M, Pollo R, Ola AR, Pramatana F. 2024. In vitro screening of fungal endophytes from sandalwood (Santalum album) as antagonists to phytopathogens. Biodiversitas 25: 361-371. Endophytic fungi possess bioactive compounds and generate secondary metabolites like their host plants. The phytochemical compounds found in sandalwood plants (Santalum album L.) are recognized for their anti-pathogenic properties, suggesting the potential of sandalwood endophytic fungi as effective anti-phytopathogens. Alternaria solani and Fusarium oxysporum threaten tomato plants, whereas Phytophthora palmivora is particularly detrimental to cocoa plants. The aims of this study were: (i) to obtain isolates of endophytic fungi from roots, stems, and leaves of sandalwood plants and (ii) to assess the ability of endophytic fungi as anti-phytopathogens in vitro. Endophytic fungi were isolated through a direct plating method and next by purification and identification. All obtained endophytic fungi underwent testing for their antagonistic potential against A. solani, F. oxysporum, and P. palmivora in vitro through the dual culture method. The inhibition percentage was analyzed using variance analysis and further examined with the 5% HSD test. The study successfully isolated 104 endophytic fungal isolates from the sandalwood; 29 isolates were from the roots, 33 from the stems, and 42 from the leaves. All endophytic isolates demonstrated the ability to inhibit the growth of A. solani, F. oxysporum, and P. palmivora in vitro, with various inhibition percentages ranging from 26.7 to 83.3%. Further research will concentrate on secondary metabolites from selected isolates showing inhibition levels exceeding 70%.

Test antagonism of several Trichoderma species in suppressing the growth of Alternaria sp. in vitro

Alternaria sp. is a pathogenic fungus that causes purple blotch disease in onion plants. Losses from this disease lead to a decrease in yield by more than 70% and seed damage to 100%. For this reason, a study was conducted to test the antagonistic ability of several Trichoderma species in suppressing the growth of Alternaria sp. in-vitro as an alternative to plant disease control that is safer and more environmentally friendly. Alternaria sp. isolate. derived from onion plants located in Bale Atu, Bukit District, Bener Meriah Regency, Aceh Province. The Trichoderma species used are T. asperellum, T. harzianum, T. virens, and Trichoderma sp. Antagonism testing was carried out using a dual culture method in a 9 cm diameter petri dish. The results showed that, T. asperellum, T. harzianum, T. virens, and Trichoderma sp. able to suppress the growth of Alternaria sp. on observation of 5 HSI. The antagonistic mechanism that occurs is competition against space, nutrition, antibiosis and mycoparasites. The best percentage of resistance occurred in the treatment of T. virens, which was 77.78%. next followed by the inhibitory power of Trichoderma sp. by 72.39%, T. harzianum by 64.80%, and T. asperellum by 60.77%.