Colony Radial Growth Research Articles

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates on Galleria mellonella

Background Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Fungicide-Loaded Liposomes for the Treatment of Fungal Diseases in Agriculture: An Assessment of Botrytis cinerea.

In this work, liposomes loaded with the fungicide, Fludioxonil (FLUD), for the containment of fungal diseases in agriculture were developed. Three types of vesicles with different compositions were compared: (I) plain vesicles, composed of soy phosphatidylcholine and cholesterol; (II) PEG-coated vesicles, with an additional polyethylene glycol coating; and (III) cationic vesicles, containing didodecyldimethylammonium bromide. Nanometric-sized vesicles were obtained both by the micelle-to-vesicle transition method and by the extrusion technique, and encapsulation efficiency, drug loading content, and Zeta potential were determined for all the samples. The extruded and PEGylated liposomes were the most stable over time and together with the cationic ones showed a significant prolonged FLUD release capacity. The liposomes' biological activity was evaluated on conidial germination, germ tube elongation and colony radial growth of the ascomycete Botrytis cinerea, a phytopathogenic fungus affecting worldwide many important agricultural crops in the field as well as in the postharvest phase. The extruded and PEGylated liposomes showed greater effectiveness in inhibiting germ tube elongation and colony radial growth of the fungal pathogen, even at 0.01 µg·mL-1, the lowest concentration assessed.

Phytophthora pseudocryptogea Is Associated with Root and Crown Rot of Nursery-Grown Common Sage in Hungary.

In October 2009, necrotic bark lesions at the root collar and lower stem associated with root rot, reduced growth, and wilting were observed on container-grown 2-year-old common sage (Salvia officinalis L. 'Icterina') in two ornamental nurseries in Somogy and Zala counties in Hungary. The disease occurred at a frequency of 15-20% (100 to 150 symptomatic plants in each nursery). A P. cryptogea-like species was isolated consistently from necrotic root collars of many plants on carrot (CA) PARPB agar. Six isolates from the nursery in Zala county and three isolates from the nursery in Somogy county were deposited in the culture collection of Plant Protection Institute (Budapest, Hungary). All developed slightly petaloid colonies on CA agar. Chlamydospores and gametangia were not present in single and dual culture combinations of isolates. Radial colony growth was the fastest at 25°C (6.8 to 7.4 mm/day) and no growth occurred above 34°C. On mycelial discs floating in nonsterile stream water, persistent, nonpapillate, mostly ovoid to obpyriform sporangia (37.4±3.5 to 47.8±4.6 μm long and 22.3±2.6 to 29.2±3.7 μm wide) and hyphal swellings were produced abundantly. Pathogenicity of one selected isolate from each nursery was tested on 3-month-old seedlings of S. officinalis 'Icterina' in 2010. Isolates were grown for 4 weeks at 20°C on autoclaved millet grains moistened with CA broth. Infested and uninfested grains were mixed with autoclaved soil (30 cm3 grain/liter), and the mixes were used as potting media for transplanting five treated and five control plants per isolate, respectively. Plants were kept in a growth room (20-25°C, 16/8 h dark/light). Pots were flooded for 24 hours on the 1st day and every 2 weeks. All and only treated plants showed symptoms of wilt associated with basal stem and root necrosis within three weeks. The trial was repeated with the same result. The pathogen could be reisolated only from the treated plants. Identity of isolates from nurseries and inoculated plants was confirmed recently by amplification and sequence analysis of the rDNA internal transcribed spacers (ITS) and gene regions of cytochrome c oxidase subunit I (coxI) and β-tubulin (tub) according to Jung et al. (2017). BLASTn searches showed 100% identity and only 97.3-99.0% similarity to the corresponding sequences of authenthic P. pseudocryptogea and P. cryptogea strains, respectively (e.g., GenBank accession nos. KP288336-KP288342, KP288370-KP288372, KP288386-KP288392, MN872725, MN872776). Sequences of the 9 field isolates were deposited in GenBank under accession nos. OR771701-OR771709 (ITS), OR787508-OR787516 (coxI) and OR787517-OR787525 (tub). P. pseudocryptogea was delineated from P. cryptogea sensu lato (Safaiefarahani et al. 2015), which has been reported from S. officinalis in the United States (Koike 1997), and S. leucantha (Cacciola et al. 2002) and S. officinalis (Garibaldi et al. 2015) in Italy. The known natural hosts of P. pseudocryptogea includes plant species in families other than Lamiaceae (cf. Aloi et al. 2023), but it was pathogenic on the lamiaceous Plectranthus scutellarioides in artificial inoculations (Christova 2020). The pathogen is present in European nurseries (Antonelli et al. 2023). This is the first report of P. pseudocryptogea on S. officinalis in Hungary. The causal agent threatens the production of sages and other ornamentals, and its spread in Hungary should be prevented by proper disease management and phytosanitary actions.

Recognition of Davidsoniella virescens on Fagus sylvatica Wood in Poland and Assessment of Its Pathogenicity.

Davidsoniella virescens is so far only known in North America. However, recently in southern Poland, blackish growth consisting of fungal mycelia and sporulation structures was found on the wood of Fagus sylvatica. As a result of isolation, 17 cultures of this fungus were obtained. All cultures produced an intense sweet odor. This fungus, both in situ and in vitro, abundantly produced perithecia with long necks and asexual stage. Particularly characteristic was the production of variable endoconidia in two types of phialophores differing mainly in the width of the collarette. The nucleotide sequences for five gene fragments of representative cultures were used in phylogenetic analyses: 18S; the internal transcribed spacer regions ITS1 and ITS2, including the 5.8S gene (ITS); 28S region of the ribosomal RNA (rRNA), β-tubulin 2 (TUB2) and translation elongation factor 1-α (TEF1). Based on morphological and phylogenetic analyses, the fungus on European beech in Poland was identified as Davidsoniella virescens. The optimal temperature for radial colony growth was 20 °C. However, the differences between colony diameter at 25 °C compared to that at the optimal temperature were not statistically significant. Six D. virescens isolates were used for pathogenicity assay. They were inoculated into wounds on stems of two-year-old seedlings of Fagus sylvatica and Acer saccharum (36 seedlings of each tree species). Final evaluation was performed 4 months after inoculation. No external symptoms were observed in any A. saccharum seedling, neither in the crown nor on the stem. However, 13.9% of F. sylvatica seedlings showed wilting symptoms throughout the entire crown within 3-6 weeks after inoculation. Moreover, after 4 months on the stems of 30.6% beech seedlings, necrotic lesions with a length of 1.3 to 7.2 cm were formed, without any symptoms of wilting. The most noticeable internal symptom was the discoloration of the wood, which was observed in all inoculated seedlings of both tree species. All D. virescens isolates caused greater wood discoloration in F. sylvatica than in A. saccharum. Most of the differences found in the extent of discoloration between host plants were statistically significant. The discoloration caused by all D. virescens isolates in F. sylvatica was significantly greater than in the control. However, none of the isolates tested on A. saccharum caused significantly greater wood discoloration compared to the control. Pathogenicity tests showed that the D. virescens isolates identified in southern Poland may pose a greater threat to native European beech than to foreign sugar maple.

Breaking down barriers: comprehensive functional analysis of the Aspergillus niger chitin synthase repertoire

BackgroundMembers of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom.ResultsIn this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization.ConclusionThis study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.

Efficiency of bacterial isolates on suppressing brown spot in rice plants

AbstractMany fungicides can control brown spot, caused by Bipolaris oryzae in rice (Oryza sativa L.), although searching for beneficial microorganisms for biocontrol will improve sustainable integrated pest management. The aim of this investigation was the selection of natural suppressors of the brown spot disease in rice. We performed biochemical (E1), molecular antagonism (E2) and severity suppression (E3) studies under laboratory and greenhouse conditions. The E1, E2 and E3 assays were performed with 21 isolates. The experimental design was completely randomized with three replicates. E1 characterized all 21 isolates for the production of extracellular enzymes, siderophores, biofilm, indole‐3‐acetic acid (IAA) and phosphate solubilization. E2 was conducted to test the in vitro antagonism between the 21 isolates and B. oryzae, and E3 was conducted under greenhouse conditions, where seeds of the rice cultivars BRS‐Primavera and Esmeralda were sown in plastic trays containing 3 kg of fertilized soil. After 45 days, rice leaves were sprayed and inoculated with a mixed suspension of bacterial cells (1 × 108 CFU) and B. oryzae. All of the 21 bacterial isolates produced lipase, N‐fixation, siderophore, IAA, biofilm and solubilized phosphorus. Bacillus cereus, Priestia megaterium, Stenotrophomonas nitritireducens, Serratia marcescens and Serratia nematodiphila reduced radial colony growth of B. oryzae and suppressed brown spot severity by up to 90%. This indicates the potential of these isolates for biocontrol of brown spot in rice as a component of rice disease management.

Rhizospheric Fungal spp. of Tomato (Solanum lycopersicum L.) and Their Effect on Tomato Plant Growth and on Alternaria solani, the Causative Agent of Early Blight of Tomato

Tomato (Solanum lycopersicum L.) is a widely consumed crop throughout the world including Sri Lanka. Use of agrochemicals for enhanced yields and managing diseases associated with tomato results in deleterious impacts on soil microflora, fauna and leads to human health problems. A possible alternative is the use of rhizospheric microorganisms, as they are reported to improve productivity and protect various crops. Hence this study aimed to isolate rhizospheric fungal assemblages of tomato plants and evaluate their ability to solubilize phosphate while, controlling Alternaria solani, the causative agent of Early Blight of tomatoes. Rhizospheric fungi were isolated from commercial tomato cultivations in two agro-ecological zones (WM3b and IM2b), using soil dilution plate technique. Composite samples of soil attached to the root systems of five healthy plants, uprooted from each field were prepared and 10 g of each sample was used for the isolations. Twenty-seven fungal genera were isolated and Trichoderma and Penicillium were the frequently isolated genera. Phosphate solubilization ability of each fungal species was evaluated using Pikovskaya's agar and liquid medium, and four isolates (Eupenicillium sp., Talaromyces purpureogenus, Paecilomyces sp., and Purpureocillium lilacinum) with the highest phosphate solubilizing abilities were evaluated on tomato plant growth under greenhouse condition. The inocula of each fungal sp. were prepared by adding fifteen, 6mm diameter mycelial discs from each 5-day-old culture into polypropylene bags containing 90 g of sterile rice husk medium and incubated for 02 weeks at room temperature. Each pot contained a 4.6 kg layer of non- sieved sterilized potting medium (Sand: topsoil: Compost at 2:1:1 ratio) and a 2.5 kg layer of sieved medium on top, which was mixed with 15 g of inoculum. Three-week old, healthy tomato seedlings (variety-Bathiya) were planted (with 3 replicates) and allowed to grow with regular watering. Control pots contained potting medium with uninoculated sterile rice husk. Growth parameters such as Root and shoot lengths, fresh and dry weights of roots, shoots, and leaves were evaluated at 02-week intervals for 02 months and the results were analysed using ANOVA with Tukey‘s pairwise comparison. Plants treated with T. purpureogenus and P. lilacinum showed significant increases (p≤0.05) in all growth parameters in every evaluation. Dual culture plate assay on the Potato Dextrose Agar was used to test the effect of rhizospheric fungal spp. on the radial colony growth of A. solani, and Eupenicillium sp. showed the highest percentage inhibition (85.09±5.68%). Microscopic observations of inhibition zones between two colonies showed the presence of coils, loops, knobs, and haustoria, formed by inhibitory rhizospheric fungal species. Assays for Chitinase and Glucanase enzyme production showed that Mortierella sp.-1, P. lilacinum, Trichoderma sp.-1, and Acremonium sp. were the highest chitinase producers, whereas P. lilacinum was the highest glucanase producer. These results indicate the ability of some rhizospheric fungal species of tomato to increase the growth of tomato plants significantly by phosphate solubilization and ability to inhibit the growth of foliar pathogen A. solani under in vitro conditions. 
 Keywords: Rhizospheric fungi, Phosphate solubilization, Dual culture plate, Plant growth parameters

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates in Galleria mellonella

Background Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

In vitro application of Eruca vesicaria subsp. sativa leaf extracts and associated metabolites reduces the growth of Oomycota species involved in Kiwifruit Vine Decline Syndrome.

This study aimed to determine whether leaf extracts from seven Eruca vesicaria subsp. sativa cultivars and their biochemically active compounds (glucosinolates and downstream-derived products) inhibit mycelia growth of three well-known pathogenic oomycetes, Phytopythium chamaehyphon, Phytopythium vexans and Phytophthora citrophthora; being the most significant in the development of Kiwifruit Vine Decline Syndrome (KVDS). Leaf extract quantity of 10, 20 and 30 mg were inoculated in Petri dish (90 mm Ø, each 22 mL of liquid medium - Potato Dextrose Agar), for in vitro bioassays. A pathogen plug was placed in the centre of each plate and the Oomycota colony perimeter was marked 5 days after inoculation. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days after inoculation, further elaborated with Image J software image analysis. Growth rates for all strains were inhibited by around 67% after 15 days. This was most pronounced when applying the highest concentration of leaf extract. By using Liquid Chromatography Mass Spectrometry (LC-MS) and Gas Chromatography Mass Spectrometry (GC-MS), fifteen glucosinolate compounds, of which glucosativin was found in the highest quantity, were identified. Concentrations of hydrolysis products produced by leaves (erucin and sativin) were also investigated, and were significantly associated with colony radial growth, especially towards Pp. chamaehyphon and Pp. vexans. Three downstream products of glucosinolates (two pure isothiocyanates, AITC and PEITC; and one indole I3C; all commonly present in Brassicaceae) were also tested, and a statistically significant inhibition of growth was observed at the highest concentration (0.6 µL).

Eugenia caryophyllata, Cinnamomum sp., Mentha piperita ve Thymus vulgaris esansiyel yağların Aspergillus niger suşuna karşı antifungal aktivitesi

Aspergillus species are pollutants found in both food and air. The increase in the metabolic activity of Aspergillus leads to spoilage of foodstuffs and large economic losses. In addition, some Aspergillus species have the ability to produce aflatoxins and ochratoxins, secondary metabolites called, namely, mycotoxins. Especially mycotoxins are very important in terms of food safety and human health. Since the protection of human and animal health and the prevention of economic losses is a very important issue, our study aimed to determine the antifungal activity of Eugenia caryophyllata Thunb., Cinnamomum sp., Mentha piperita L. and Thymus vulgaris L. essential oils (EO) against Aspergillus niger Tiegh. NRRL 321 strain. In the second step, MIC and MFC values of EOs were determined. It was determined that the MIC value of Cinnamomum sp, M. piperita and T. vulgaris EOs was 0.01 µL/mL, and E. caryophyllata EO was 0.5 µL/mL. It was determined that E. caryophyllata, Mentha piperita and Thymus vulgaris EOs completely inhibited radial colony growth at MIC, 2xMIC and 4xMIC values. It was determined that the inhibition of radial growth of Cinnamomum sp. EO varies depending on the concentration, and the inhibition rate increases as the concentration increases. As a result, evaluations should be made considering the in vivo conditions that the tested EOs showed strong antifungal activity against the A. niger strain.

Synergistic antifungal properties of lauraldehyde and geraniol against Aspergillus flavus in pistachio

Natural plant-derived compounds have been recognized as promising fungicides for reducing Aspergillus flavus growth in agricultural commodities. In this study, the synergistic antifungal activity of plant-derived compounds against A. flavus growth in stored pistachios was investigated. The minimum inhibitory concentration (MIC) of lauraldehyde, geraniol, dihydrocoumarin, and p-anisaldehyde against A. flavus in potato dextrose broth (PDB) were 0.40, 0.20, 0.40, and 0.80 mg/mL, respectively. Whereas, after screening 6 groups of agents using the checkerboard method, only one combination, i.e., that of lauraldehyde and geraniol (SLG), showed synergistic effect (fractional inhibitory concentration index = 0.5) on A. flavus. In addition, at 2 MIC, SLG completely inhibited the spore germination of A. flavus, colony radial growth, and aflatoxin biosynthesis. At the same time, the mycelial growth was inhibited by more than 98%. Further research found that fumigation with SLG significantly inhibited the growth of A. flavus in stored pistachios and reduced aflatoxin biosynthesis. Moreover, the pistachios with SLG fumigation exhibited higher soluble protein content, lower malonaldehyde (MDA) content, and lower carbonyl values, indicating that fumigation with SLG could effectively delay the trend of quality deterioration in stored pistachios. These results supported SLG as a potential green antifungal agent for the shelf-life extension of stored pistachios.

Isolation, morphological characterization, and screening a virulence of Beauveria bassiana and Metarhizium robertsii as bioagents

Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.

Isolation, morphological characterization, and screening virulence of Beauveria bassiana and Metarhizium robertsii fungal isolates on Galleria mellonella.

Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 7 spores ml -1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day -1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 8 conidial ml -1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.

Morphological and Cultural Variability among Exserohilum turcicum Isolates Causing Turcicum Leaf Blight Disease of Maize in Manipur

This present experiment was conducted May–October, 2021 and April–October, 2022) in the Laboratory, Department of Plant Pathology, College of Agriculture, Central Agricultural University, Imphal, Manipur, India to study the different morphological and cultural characterizations of the pathogen, Exserohilum turcicum responsible for causing the Northern corn leaf blight (NCLB)/ Turcicum leaf blight (TLB) disease of maize. A total of 10 accessions were used for this experiment which were collected from different districts of Manipur. Based on the colony colour, they were classified as black, greyish-black and light greyish. Pigmentation was the same, i.e., black for all. Based on sporulation they were categorised as excellent, good and moderate. Different types of growth patterns were also observed from different types of accessions, i.e., flattened, slightly raised fluffy and raised cottony. The margins were recorded as regular and irregular. The range of fresh and dry weight, varied from 12.12 g–5.15 g and 1.61 g–0.82 g, respectively. Among six different types of media used to measure radial colony growth, maize leaf extract was found to be the highest and corn meal agar was the least. The growth rate for up to 7 days was observed and it was found that OQ689065 was considered as fastest-growing among all while, OQ689062 was considered as slowest. As per the length of the conidia, the maximum and minimum length was found to be 91.70 µm and 57.35 µm. Similarly, the maximum and minimum width was found to be 18.01 µm and 11.09 µm. The average septation of 10 accessions varied from 6.4 to 4.3. The statistical analyses performed during this study were found significant.

Isolation, morphological characterization, and screening a virulence of Beauveria bassiana and Metarhizium robertsii as bioagents

Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.

Screening bacterial isolates for biocontrol of sheath blight in rice plants

Sheath blight (Rhizoctonia solani) causes significant yield losses in rice (Oryza sativa L.). Its sustainable management needs an efficient biocontrol agent. The objective was to screen bacterial isolates as an antagonist to R. solani and identify the most efficient ones as sheath blight suppressors under greenhouse conditions. Two assays (E1 and E2) were performed in a completely randomized design with three replications. E1 tested 21 bacterial isolates antagonists to R. solani in vitro. E2 was conducted under greenhouse conditions, with rice cultivar BRS Pampeira sown in plastic pots (7 kg) containing fertilized soil. Sixty old plants were inoculated with a segment of a toothpick containing fragments of R. solani, followed by spray inoculation of a bacterial suspension (108 CFU/mL). The severity of the disease was determined by calculating the relative lesion size formed on the colm. Isolates BRM32112 (Pseudomonas nitroreducens), BRM65929 (Priestia megaterium), and BRM65919 (Bacillus cereus) reduced R. solani colony radial growth by 92.8, 77.56, and 75.56%, respectively while BRM63523 (Serratia marcescens), BRM65923 and BRM65916 (P. megaterium) and BRM65919 (B. cereus) with 23.45, 23.37, 23.62, and 20.17 cm, respectively were effective at suppressing sheath blight in greenhouse, indicating their potential as a biofungicide for sheath blight suppression.

Functional Characterization of the GNAT Family Histone Acetyltransferase Elp3 and GcnE in Aspergillus fumigatus.

Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a pivotal role in the regulation of gene expression and diverse biological processes. However, the function of GNAT family HATs, especially Elp3, in the opportunistic human pathogenic fungus Aspergillus fumigatus is largely unknown. To investigate the roles of the GNAT family HATs Elp3 and GcnE in the A. fumigatus, we have generated and characterized individual null Δelp3 and ΔgcnE mutants. The radial growth of fungal colonies was significantly decreased by the loss of elp3 or gcnE, and the number of asexual spores (conidia) in the ΔgcnE mutant was significantly reduced. Moreover, the mRNA levels of the key asexual development regulators were also significantly low in the ΔgcnE mutant compared to wild type (WT). Whereas both the Δelp3 and ΔgcnE mutants were markedly impaired in the formation of adherent biofilms, the ΔgcnE mutant showed a complete loss of surface structure and of intercellular matrix. The ΔgcnE mutant responded differently to oxidative stressors and showed significant susceptibility to triazole antifungal agents. Furthermore, Elp3 and GcnE function oppositely in the production of secondary metabolites, and the ΔgcnE mutant showed attenuated virulence. In conclusion, Elp3 and GcnE are associated with diverse biological processes and can be potential targets for controlling the pathogenic fungus.

First Report of Dieback of Salix acmophylla Caused by Paecilomyces formosus in Iran.

Salix acmophylla Boss. (Brook willow), is native to Iran and mainly grows in natural riparian landscapes. It has excellent potential to use in erosion and phytoremediation projects. Since 2018, dieback has been observed on S. acmophylla at the Gilan-e Gharb forest (34°05'59.9"N, 46°00'52.6"E) in Kermanshah province, Iran. Affected trees showed wilting and yellowing of leaves, cankers, branch dieback, and decline (Fig. 1A, B). Small pieces (5 mm2) from the canker margins of 10 affected tree branches were surface sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, samples were rinsed three times with sterile H2O, plated on potato dextrose agar (PDA) supplemented with 100 mg/l streptomycin sulfate and 25 μg/mL chloramphenicole, and incubated at 25°C for 5 days. The same fungus was isolated from all samples. Pure cultures were obtained by monosporic isolation. A representative isolate, RU-PaMa-S1, was used for morphological and molecular characterization and deposited in the Fungal Reference Collection of the Ministry of Jihad-e Agriculture at the Iranian Research Institute of Plant Protection, Tehran, Iran. Radial growth of colonies on PDA and Czapek yeast extract agar (CYA) were 30 and 25 mm after seven days at 25 °C in the dark, respectively. Fungal colonies grew more rapidly on malt extract agar (MEA), and reaches a diameter of about 70 mm within 7 days at 25 °C in the dark. Color of colony on MEA, PDA, and CYA was light yellow to yellow-brown, which gets darker upon aging. In biochemical assay, our isolate grow and produced acid in culture on creatine sucrose agar medium. The conidiophore was irregularly branched out with one to seven phialides. The phialides were in whorls or solitary, flask shaped with cylindrical bases, and narrow apices. Conidia were one-celled, hyaline to yellow brown in mass, smooth, and ellipsoidal to cylindrical with truncate ends. Conidia were 3.0-9.6 (4.5) × 1.7-4.0 (2.6) µm (n = 50). Chlamydospores were present in two week old cultures. The morphology matched the description of Paecilomyces formosus (Sakag., May., Inoue and Tada) Houbraken and Samson (Samson et al., 2009). For an accurate identification, genomic DNA of RU-PaMa-S1 was extracted to amplify ITS regions, and β-tub gene with ITS1/ITS4 (White et al. 1990), and βt2a/βt2b (Glass and Donaldson 1995) primers, respectively. The sequences were deposited in GenBank (ITS: OL891502, β-tub OL944475). A BLAST search of GenBank showed that the ITS, and β-tub sequences of RU-PaMa-S1 were similar to those of Paecilomyces formosus. Baed on the multigene phylogeny and morphology, RU-PaMa-S1 isolate was identified as Paecilomyces formosus (Fig. 2). Molecular analyses based on sequencing of the ITS region, and parts of the β-tubulin and calmodulin genes, showed that P. formosus may be composed of three species, including P. maximus, P. lecythidis and P. formosus. However, the three species could be distinguished by only molecular data and not by microscopical examination (Samson et al. 2009). To fulfill Koch's postulates, 10 detached and 10 attached healthy branches from two 20-year-old S. acmophylla plants at the Gilan-e Gharb forest in Kermanshah province, Iran, were inoculated with 5-mm mycelial plugs of 5-day-old culture of RU-PaMa-S1 isolate (Fig. 1 C). Control branches were treated with agar plugs. The detached inoculated branches were stored in sterilized glass jar containing moist sponge and incubated at 25-30°C for 35 days until the appearance of the canker symptoms. After 35 days, inoculated branches were cut from the trees and transferred to the laboratory to check disease development and re-isolation of the pathogen. All inoculated points on detached and intact branches showed lesions similar to those observed on naturally infected trees, whereas controls were symptomless (Fig. 1 D, E). The same fungus was re-isolated from these lesions. P. formosus has been reported to infect a broad range of hosts, including Amygdalus scoparia, Malus domestica, Pistatia vera, and Querqus brantii (Sabbagh and Khosravi Moghaddam 2016; Sabernasab et al. 2019; Azizi et al. 2020). This is the first report of P. formosus on S. acmophylla in Iran and in the world. This finding provides crucial information on this high-risk disease to S. acmophylla and other forest trees and basis for identifying management strategies.

ISOLATION AND SCREENING OF FUNGI ASSOCIATED WITH DISEASED RIVER CATFISH, Pangasius hypophthalmus

The production of river catfish Pangasius hypophthalmus trade has increased significantly over the past decade. Concerning the high demand for river catfish, P. hypophthalmus, these freshwater fish are susceptible to fungi infection that can cause mortality to individual and fish eggs. This study has been conducted to identify the morphology of isolated fungi and their extracellular- enzymes for virulent screening. Three fishes with apparent signs of infection such as ulcerative, haemorrhages and dermal lesions were collected from the fish cage culture at Kampung Serada, Kuala Terengganu. Fungi were isolated from the fish externally (fin, skin, mouth, gill) and internal organs (kidney and liver). The fungal morphology was characterised macroscopically by observing the colony features such as margin, pigmentation (colour), elevation, texture and shape of fungi on an agar plate. Meanwhile, for micromorphology observation were characterised the fungi structure includes the spore, hyphae, and conidiaphore. Based on the identification using a general key to characteristics moulds, two fungi have been classified into two genera: Rhizopus-like sp and Geomyces-like sp. These fungi proceeded with extracellular enzyme tests such as the proteolysis assay, amylases test and lipases test. The reaction enzymes of fungi from the amylases test showed the clearing zone around the radial colony growth of fungi. The test strain of fungi screened during the present investigation proved to be an efficient producer of protein and polysaccharide degrading enzymes. Rhizopus-like sp and Geomyces-like sp to produce extracellular amylase enzyme indicated the virulent characteristic of the isolate and the ability of those fungi to initiate infection or resist harsh environments and treatments.

ANTIFUNGAL PROPERTIES OF CORIANDRUM SATIVUM EXTRACTS ON FUSARIUM SPP. IN VITRO

Fusarium spp. cause significant plant diseases and are responsible for food contamination with mycotoxins. Sustainable crop protection relies on a combination of different measures; among others, plants contain bioactive compounds providing antifungal and antimicrobial properties. Coriander seed extracts have a composition rich in fatty acids, sterols, essential oils, polyphenols etc. Therefore, this study aimed to evaluate the antifungal properties of coriander essential oil and coriander extract on F. culmorum and F. oxysporum f. sp. lycopersici in vitro. The essential oil was obtained by Clevenger apparatus and the extract with the CO2 extraction method. A potato dextrose agar medium enriched with extracts was inoculated with Fusarium spp. discs, incubated and the radial growth of colonies was measured 3 and 7 days after inoculation. Results revealed that the coriander essential oil had higher antifungal properties on Fusarium spp. than the coriander extract. No radial colony growth of F. culmorum was observed at 1000 µL/L concentration of the essential oil. In contrast, the coriander extract had moderate antifungal properties, which were higher for F. oxysporum than for F. culmorum. Both plant extracts showed potential to be used as antifungal compounds and thus could help to make plant protection more sustainable.