Waraporn Sutthisa

This study aimed to identify the causal agent of cassava leaf blight in Thailand and to evaluate the biocontrol potential of Bacillus spp. isolated from the digestive tract of small millipedes. Symptomatic cassava leaves were collected from Maha Sarakham Province, Thailand, and a total of 32 bacterial isolates were obtained and classified into seven morphotypes. Pathogenicity tests revealed that isolates Mnbr-2 and Mnbr-22 induced typical leaf blight symptoms, with Mnbr-2 producing the largest lesion diameter (28.30 ± 3.50 mm) and therefore selected for further study. Based on morphological, biochemical, and 16S rRNA gene analyses, isolate Mnbr-2 was identified as a member of the genus Pantoea and was closely related to Pantoea stewartii, showing 99.75% sequence similarity. These findings provide evidence that Pantoea-associated bacteria are involved in cassava leaf blight in Thailand. The antagonistic activity of 28 Bacillus isolates against P. stewartii Mnbr-2 was evaluated using the agar well diffusion method. Several isolates, including Bacillus velezensis, B. cereus, B. safensis, and B. subtilis, exhibited strong inhibitory activity, with inhibition zones ranging from 25.00 ± 1.50 to 30.00 ± 5.00 mm. Cell-free culture filtrates of selected isolates also suppressed pathogen growth, showing inhibition comparable to that of streptomycin (100 ppm). Detached leaf assays demonstrated that B. cereus N5_TCR and B. velezensis N8_TCR achieved the highest disease suppression under both preventive and curative treatments, with inhibition efficiencies ranging from 91.35%-94.41%. These findings indicate that the selected Bacillus isolates possess strong antagonistic activity and may serve as promising candidates for the development of sustainable and environmentally friendly biocontrol strategies against cassava leaf blight caused by Pantoea.

The present study investigated the phytochemical composition and antibacterial activity of the hexane flower extract of Artabotrys hexapetalus (L.f.) Bhandari. The crude extract obtained from dried flowers was dark yellow, viscous, and possessed a characteristic floral odor, with an extraction yield of 20.78% (w/w). Gas chromatography-mass spectrometry (GC-MS) analysis detected 100 chromatographic peaks corresponding to 94 identified compounds, representing approximately 99.1% of the total peak area. The extract was predominantly composed of fatty acid derivatives (43.67%), followed by sesquiterpenes and oxygenated sesquiterpenes (31.09%), other identified constituents (18.22%), sterols and triterpenoids (5.57%), and monoterpenes (0.55%). The major constituents were 1H-cyclopropa[a]naphthalene derivative (10.08%), cyclohexane (9.50%), 9,12-octadecadienoic acid methyl ester (7.18%), hexadecanoic acid methyl ester (4.91%), and β-sitosterol (4.15%). The antibacterial activity of the extract was evaluated against representative Gram-positive and Gram-negative bacteria using the disc diffusion method. The extract inhibited the growth of all tested bacterial strains, with inhibition zones ranging from 6.33 ± 0.58 to 16.00 ± 1.73 mm. The strongest activity was observed against Salmonella enterica subsp. enterica serovar Typhi, followed by Escherichia coli ATCC 25922 and Bacillus cereus. However, the antibacterial activity was significantly lower than that of streptomycin (P < 0.05). The minimum inhibitory concentration (MIC) values ranged from 0.488 to 500 mg/mL, whereas the minimum bactericidal concentration (MBC) values were either 500 mg/mL or >500 mg/mL, depending on the bacterial strain. These findings demonstrate that the hexane flower extract of A. hexapetalus contains diverse bioactive phytochemicals, particularly fatty acid derivatives and sesquiterpenes, and exhibits broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. The results provide a scientific basis for further studies on the isolation and characterization of antibacterial compounds from A. hexapetalus.

Tomato (Lycopersicon esculentum Mill.) production is severely affected by bacterial wilt, a destructive disease caused by Ralstonia solanacearum, which poses a significant challenge to growers worldwide. This study aimed to develop powdered formulations of Bacillus velezensis MSU01 and evaluate their efficacy in controlling the disease and enhancing plant growth. Laboratory evaluation using the agar well diffusion assay demonstrated strong antagonistic activity of B. velezensis MSU01 against R. solanacearum, with an inhibition zone of 13.8 mm. Three powder formulations-talcum-based, corn starch-based, and kaolin-based were prepared and evaluated for physicochemical properties, bacterial viability during storage, seed germination, and disease control efficacy. All formulations showed excellent solubility and stable pH (7.03-8.67), with initial moisture contents ranging from 0.20%-2.17%. After 60 days of storage at room temperature, the kaolin-based formulation maintained the highest viable cell count (6.30 ± 0.30 log CFU/g), while the corn starch-based formulation showed the highest moisture content (5.24 ± 0.30%). The corn starch-based formulation most effectively reduced bacterial wilt, lowering disease incidence to 10.00 ± 0.00% and severity to 5.33 ± 4.16%, outperforming the chemical control (streptomycin). Seed germination rates were highest with the talcum-based formulation (96.67 ± 5.77%), comparable to the positive control and significantly greater than the negative control. Although shoot and root lengths did not differ significantly among treatments, all B. velezensis MSU01 formulations enhanced plant growth compared to the pathogen-only treatment. Overall, the corn starch-based formulation showed the greatest potential as a sustainable and efficient biocontrol agent for managing bacterial wilt in tomato.

Tomato bacterial wilt, caused by Ralstonia solanacearum, poses a significant challenge to crop production. This study screened 101 bacterial isolates from soil samples collected in Maha Sarakham Province, Thailand, for their antagonistic activity against R. solanacearum. The selection process involved initial screening based on visual inhibition zones using the paper disc diffusion method, followed by quantitative assessment of bacterial growth inhibition. Among the isolates, SK3-24 exhibited the highest inhibitory effect, forming an inhibition zone of 40.00 ± 17.32 mm, as measured using the standard zone of inhibition method, followed by SK3-20 (18.17 ± 1.50 mm). Morphological, biochemical, and molecular analyses (16S rRNA gene sequencing) identified SK3-24 as Bacillus subtilis. To enhance its application, SK3-24 was formulated into alginate-based biopellets, which maintained high bacterial viability (1.20 × 10⁸ CFU/g) after nine months of storage at 4 °C. Greenhouse trials demonstrated that these biopellets effectively suppressed disease incidence and severity in tomato seedlings and seeds. At 21 days post-inoculation, treated plants showed no disease symptoms and exhibited enhanced growth, with plant height (16.14 ± 1.32 cm) and leaf number (19.40 ± 1.34) comparable to healthy controls. These findings underscore the potential of SK3-24 biopellets as a sustainable and effective biocontrol strategy for managing tomato bacterial wilt while promoting plant health. HIGHLIGHTS This study explores the identification of effective antagonistic bacterial strains against R. solanacearum through 16S rDNA sequencing and aims to develop a bioformulation for sustainable management of tomato bacterial wilt. By integrating strain characterization, screening, and bioformulation development, this research seeks to deliver an environmentally friendly alternative to chemical pesticides, ensuring enhanced tomato production while safeguarding environmental and human health. GRAPHICAL ABSTRACT

Mango anthracnose, caused by Colletotrichum gloeosporioides, is a significant disease affecting mango production. This study aimed to develop and evaluate a bio-formulation of Serratia nematodiphila GCSR38 for controlling mango anthracnose. The antagonistic activity of S. nematodiphila GCSR38 was tested using the dual culture method, achieving a 40.21% inhibition rate against C. gloeosporioides. Three powder formulations were developed using a low-heat drying process, differing in their composition of corn starch, talcum, calcium carbonate, and carboxymethyl cellulose (CMC). The formulations exhibited varying physical characteristics, water solubility, and moisture stability, with pH values ranging from 7.90 to 8.00. The survival of S. nematodiphila GCSR38 in the powder formulations remained substantial over 120 days, with final bacterial counts between 2.50 × 107 and 5.10 × 107 CFU/g. Efficacy testing on mango leaves showed that Bio-Formulation 3 provided the highest inhibition of C. gloeosporioides, achieving 51.00% inhibition in preventive application and 48.30% in curative application. These results were comparable to the chemical fungicide Carbendazim (56.11% and 49.31%, respectively), with no significant differences observed. Preventive application proved more effective than curative treatment. Overall, S. nematodiphila GCSR38 shows promise as a biocontrol agent for mango anthracnose, offering an eco-friendly alternative to chemical fungicides.

Jackfruit (Artocarpus heterophyllus Lam.), a tropical plant widely cultivated for its edible fruit, has been traditionally used in folk medicine for its potential therapeutic properties. Despite its extensive use, limited studies have comprehensively evaluated the antimicrobial efficacy and chemical composition of its various plant parts. This study investigates the antimicrobial properties and chemical composition of 95% ethanol extracts from the bark, leaves, peel, and seeds of jackfruit, aiming to explore their potential as natural antimicrobial agents. The antimicrobial activity was assessed using the paper disc diffusion method at 250 mg/ml. The bark extract exhibited significant inhibition against Salmonella Typhi ATCC16122, Staphylococcus aureus, S. aureus DMST20645, Candida albicans, and C. albicans ATCC10231, with inhibition zones ranging from 6.89±0.70 to 9.67±0.58 mm. Leaf extracts were active against S. Typhi, S. aureus, and C. albicans (7.00±0.58 to 7.66±0.30 mm). Peel extracts inhibited Serratia marcescens and C. albicans (8.33±1.15 to 9.33±0.58 mm), while seed extracts inhibited S. Typhi, S. marcescens, and C. albicans (8.33±1.15 to 10.33±0.58 mm). Minimum inhibitory concentrations (MIC) ranged from 0.97 mg/ml to 125 mg/ml, with minimum bactericidal concentrations (MBC) between 62.5 and >500 mg/ml. Gas Chromatography-Mass Spectrometry (GC-MS) identified bioactive compounds such as linoleic acid, β-sitosterol, stigmasterol, tocopherols, and cycloartenol, known for antimicrobial and antioxidant properties. These findings underscore the potential of jackfruit extracts as natural agents in medicinal and industrial applications.

This study aimed to develop and evaluate a bioformulation combining Trichoderma asperellum MSU007 and the plant growth-promoting rhizobacterium (PGPR) Bacillus velezensis MSU01 to enhance rice growth. Coexistence assays demonstrated that T. asperellum MSU007 and the PGPR B. velezensis MSU01 could grow concurrently without mutual inhibition. A powdered bio-formulation was created that showed effective water solubility and moisture content stability across the three formulations. Viability assessments indicated that T. asperellum MSU007 and the PGPR maintained high survival rates over a 60 day storage period, with formulation 3 (T. asperellum MSU007 and B. velezensis MSU01 at a 3:1 ratio) consistently exhibiting the highest viability. The effect of the bioformulation on rice growth was significant. Formulation 3 was the most effective in enhancing rice seed germination and seedling growth. It achieved 100% germination by the fourth day and consistently promoted improvements in shoot and root lengths, as well as in the fresh and dry weights of shoots and roots. Formulations 1 and 2 also showed positive effects but were less effective than formulation 3. In summary, the Trichoderma-PGPR bioformulations, particularly formulation 3, significantly enhanced rice growth, offering a promising solution for improving crop yield.

Shiitake mushrooms ( Lentinus edodes (Berk.) Sing.) have been widely recognized for their bioactive properties, including antimicrobial activity. This study aimed to investigate the antibacterial potential and chemical composition of shiitake mushroom extracts prepared using different solvents (95% ethanol, ethyl acetate, and chloroform). The highest extraction yield (31.16%) was obtained with 95% ethanol. The antimicrobial activity of the extracts was evaluated using the paper disc diffusion method against nine pathogenic bacterial isolates, including Gram‐positive ( Bacillus cereus , Staphylococcus aureus , and S. aureus DMST20654) and Gram‐negative ( Escherichia coli , E. coli ATCC25922, Enterobacter cloacae , Pseudomonas aeruginosa , Serratia marcescens , and Salmonella enterica serovar Typhi ATCC16122) bacteria. Plates were incubated at 37°C for 24 h. All experiments were performed in triplicate. The ethyl acetate extract exhibited the strongest antibacterial activity, with the largest inhibition zone observed for E. coli ATCC25922 (30.00 ± 0.00 mm), followed by S. enterica serovar Typhi ATCC16122 (28.33 ± 2.00 mm). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays confirmed the superior antibacterial potential of the ethyl acetate extract, particularly against S. aureus DMST20654 (MIC = 1.95 mg/mL, MBC = 31.25 mg/mL). Gas chromatography–mass spectrometry (GC–MS) analysis identified key bioactive compounds, including ergosterol (62.38%, %Prob 62.6 in the chloroform extract) and linoleic acid (28.65%, %Prob 56.5 in the ethyl acetate extract), which are known for their antimicrobial properties. The findings highlight the potential of ethyl acetate‐extracted shiitake mushroom compounds as natural antibacterial agents, with applications for food preservation, nutraceuticals, and pharmaceuticals.

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

Damping-off disease represents a considerable hazard to the cultivation of Chinese kale (Brassica oleracea var. alboglabra), leading to seedling death and notable economic repercussions. This study explores the efficacy of guava leaf extracts, obtained through different extraction solvents, against Pythium aphanidermatum, a significant pathogen causing damping-off disease in Chinese kale (Pak Kha-naa) cultivation in Thailand. Guava leaf extract was obtained using different solvents: hexane, ethyl acetate and ethanol. Subsequently, the extract was diluted to concentrations of 1,000, 1,500 and 3,000 ppm to assessment the inhibitory effects on P. aphanidermatum utilizing the poisoned plate technique. Results revealed that only ethanol-extracted guava leaf extract at 3,000 ppm exhibited significant inhibition of P. aphanidermatum, achieving complete inhibition. At 1,500 ppm, ethanol-extracted guava leaf extract demonstrated inhibition comparable to carbendazim. Greenhouse trials further confirmed the efficacy of pre-planting seeds coating with ethanolic guava leaf extract at concentrations of 1,500 and 3,000 ppm in reducing damping-off disease incidence in Chinese kale. Chemical analysis of guava extract with GC-MS revealed the presence of 58 compounds in the hexane extract, with copaene, β-caryophyllene, calamenene and caryophyllene oxide being major constituents. The ethyl acetate extract contained 53 compounds, including β-caryophyllene and squalene, while caryophyllene was a predominant component among the 40 chemicals identified in the ethanol extract. Methylparaben was exclusively present in the ethanol extract, showcasing inhibitory effects on the fungus. This research sheds light on the potential of guava leaf extracts as a sustainable solution for disease management in Chinese kale cultivation. HIGHLIGHTSEthanolic extract of guava leaf at 3,000 ppm completely inhibits the pathogen Pythium aphanidermatum. Seed coated with ethanol-extracted guava leaf extract at concentrations of 1,500 and 3,000 ppm reduced damping-off disease incidence in Chinese kale in greenhouse trials. Chemical analysis via GC-MS revealed diverse compounds in guava leaf extracts, with notable constituents such as copaene, β-caryophyllene, calamenene and caryophyllene oxide in the hexane extract, β-caryophyllene and squalene in the ethyl acetate extract and caryophyllene predominance in the ethanol extract. Identification of methylparaben exclusively in the ethanol extract suggests its potential inhibitory effects on the fungus. This study underscores the promising potential of guava leaf extracts as a sustainable solution for disease management in Chinese kale cultivation, offering insights into alternative methods for controlling damping-off disease. GRAPHICAL ABSTRACT