Fungal Spore Suspension Research Articles

Entrapment of glucose oxidase from Aspergillus niger ISL-09 in poly(acrylamide-co-acrylic acid) hydrogels for improved stability and catalytic efficiency towards industrial applications

The present study highlights the true potential of Aspergillus niger ISL-09 to produce glucose oxidase (GOx) and focuses on improving the catalytic properties of GOx by entrapping it in poly acrylamide-co-acrylic acid (AAm-co-AAc) hydrogels, fabricated by free-radical polymerization method. For this purpose, the optimum conditions for GOx activity were found to be: 15 g soybean meal (SBM) substrate, 20 mL moisture content, 96 h incubation period, and 1.5 mL of fungal spore suspension. The purified extracellular GOx was observed to have 32.4% yield, with 0.02 U/mg specific activity and approximately 80 kDa subunit molecular weight. The GOx was most active at pH 5.5 and 40 °C. The immobilization of GOx in poly (AAm-co-AAc) hydrogels led towards improved stability and catalytic efficiency, resulting in a 21.7% increase in activity compared to free enzyme. The study also examined the potential of GOx in the pharmaceutical and textile industries as Ca-gluconate producer and bleaching agent, respectively. The study concludes that A. niger ISL-09 is a promising source for GOx production under optimal conditions. Furthermore, the immobilization of GOx in poly (AAm-co-AAc) hydrogels can significantly improve its catalytic properties, making it suitable for different industrial applications. However, further scaling up is required for the better implementation in industry.

Potential of Endophytic Fungal Isolates in Improving Productivity and Postharvest Marketable Fruit Qualities of Tomato

In Tanzania, low tomato (Solanum lycopersicum) yield and postharvest marketable fruit qualities remain significant limitations for tomato grower’s profit. Seven endophytic fungal isolates from pyrethrum and Lemongrass leaf and or flowers were evaluated to promote tomato productivity and fruit qualities. The isolates were inoculated by priming the seeds in spore suspension, and the variations in the number of germinated seeds, seedling vigour, yield and fruit qualities were compared across the treatments. It was observed that only three isolates induced more than 50% seed germination. Epf1 and Epl1 isolates showed significant promotion of seed germination (F 0.05 (pdf, 23) = 8.121 and P = 0.001) and seedling vigour. Isolate Epf1 increased fruit number (18.4%) and weight (17%) but reduced fruit size (0.63%); the Epl1 increased fruit size (12%) and reduced fruit number (14.4%). The Epf1 induced delayed fruit ripening, colour development and softening, decaying, and prolonged shelf life by 9 days. These findings indicate that promoting tomato productivity and postharvest marketable fruit qualities is isolate-specific; therefore, it requires diligent screening. Incorporation of the Epf1 isolate through seed priming with fungal spore suspension demonstrated a high potential for increasing tomato productivity and postharvest marketable fruit qualities.

First Report of Verticillium Wilt caused by Verticillium dahliae on Chilli in South Korea.

Chili pepper (Capsicum spp.) is an important crop in South Korea and is widely used in Korean cuisine, cultivated across a land area of roughly 29.8 thousand hectares, with a total production of 69 thousand tons (Lee et al., 2005; Statista, 2022). In September 2020, two farmer fields in Samcheok (37.444039°N, 129.135875°E; 37.633738°N, 128.911731°E), Gangwon province, South Korea, it is observed that chili pepper leaves showing yellowing and wilting symptoms, with an estimated disease incidence of approximately 10-15%. To identify the causal agents six affected plants were brought to lab. All the plants exhibited vascular discoloration in stem and root. After surface sterilizing small pieces of discolored tissue in 1% NaOCl for 30 s and rinsing twice in sterile distilled water, the tissue pieces were placed on water agar and incubated at 25°C for 10 days. Six pure isolates with consistent morphological characteristics were obtained by single spore isolation. Two representative isolates, NC17601 and NC20845 were selected for identification based morphological and molecular characters. Colonies on potato dextrose agar (PDA) during 10 days of incubation at 25°C in the dark were cottony white initially but progressively became dark as the formation of melanized microsclerotia. Conidiophores were hyaline, vertically branched or not, and had 2 - 4 phialides per node. Phialides were subulate and tapering from base to tip. The colonies produced abundant conidia, which were hyaline, single celled, smooth walled, cylindrical to oval, clustered on phialides, and 3.8 - 7.2 ×2.1 - 3.9 ㎛(mean ± SD: 5.2 ± 0.7 × 2.8 ± 0.5). Microsclerotia were aggregated form, various size and shape, and brown. These are the typical morphology of Verticillium dahliae (Inderbitiz et al. 2011; Yu et al. 2016). The molecular identification was later confirmed through PCR amplification, and sequencing targeting the translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes using the primer sets described by Inderbitiz et al. (2011). The resulting sequences were deposited in GenBank with accession numbers LC761935 to LC761942. The maximum likelihood tree based on combined sequences of ACT, GPD, TEF and TS was inferred using RAxML- HPC2 on XSEDE as implemented on the CIPRES web server. The phylogenetic tree showed that the isolates were sit together with V. dahliae isolates (Ex-type PD322, PD227 and PD502). Pathogenicity tests using two isolates (NC17601 and NC20845) were conducted in the greenhouse, where 10 two weeks old seedlings per isolates (cv. Bigstar) were root-tip cut and then soaked in a fungal spore suspension of 107 conidia ml-1 for 1 h, while 10 seedlings were treated with sterile distilled water as a control. All the treated plants were maintained at 25°C (night)/ 25°C (Day) under natural light. After three weeks, all inoculated plants exhibit growth stunting with vascular discoloration in the stem and roots as compared to asymptomatic control plants. The isolates of V. dahliae were consistently re-isolated from discolored root tissues and identified based on morphological characteristics, thus fulfilling Koch's postulates. In South Korea, V. dahliae has been reported to cause wilt disease in various crops, including Kimchi cabbage and radish (Dumin et al. 2020; Choi et al. 2023). To the best of our knowledge, this is the first report that V. dahliae causing Verticillium wilt of chili pepper in South Korea. Overall, Verticillium dahliae is considered to be a significant threat to agriculture in South Korea, and efforts are being made to develop effective control strategies to mitigate its impact on crops.

Virulence of Local Isolates of The Entomopathogenic Fungus Beauveria bassiana on Egg and Adult Stages of Tomato Leaf Miner, Tuta absoluta

Hasan, A.H., M. Ahmad, O. Hamoudi and M. Moufleh. 2024. Virulence of Local Isolates of The Entomopathogenic Fungus Beauveria bassiana on Egg and Adult Stages of Tomato Leaf Miner, Tuta absoluta. Arab Journal of Plant Protection, 42(3): 340-348. https://doi.org/10.22268/AJPP-001252 The efficacy of four local isolates of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales), (b6 isolated from citrus orchard soil (Harisoon), b7 from greenhouse soil (Alkhrab), b8 from palm weevil pupae (Lattakia) and b10 from olive orchard soil (Mengella), were tested on the egg and adult stages of the tomato leafminer Tuta absoluta by direct spraying for each stage with three different concentrations of fungal spore suspensions 2×104 , 2×106 and 2×108 spores/ml. The results obtained showed that both insect stages were very sensitive to fungal infection to varying degrees and there were significant differences in the hatching rate and adult mortality rate caused by the four isolates in comparison with the control treatment. The b8 and b10 isolates were the most effective on both insect stages at all concentrations tested. Corrected mortality rates at the highest concentration (2×108 spore/ml) were 82.59% for b8 isolate and 86.30% for b10 isolate on eggs and LC50 was 1×106 spores/ml for b8 isolate and 8×105 spores/ml for b10 isolate, whereas the corrected mortality rate for both were 100% on adults, 8 days after treatment, and by using the highest spore concentration. The LT50 was 3.2 days for both isolates and the LC50 was 2×104 spores/ml for b8 isolate and 1×105 spores/ml for b10 isolate under laboratory conditions. Keywords: Biological control, Beauveria bassiana, Tuta absoluta, mortality, LT50, LC50

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

First Report of Verticillium dahliae causing Verticillium Wilt on Radish in South Korea.

Radish (Raphanus sativus L.) is an important crop grown in Korea primarily for its use in kimchi, and is cultivated on an area of about 23,000ha. In September 2016, radish leaves were observed with yellowing and wilting symptoms in Gangneung (37.55406°N, 128.84871°E) and Jeongseon (37.42895°N, 128.85882°E), Gangwon province, South Korea. Disease incidence was estimated at approximately 10% in two fields, respectively. About 30% of radish plants (cv. Gwangdongyeoreum) with foliar symptoms exhibited vascular discoloration in the roots. Small pieces of discolored root tissue were surface sterilized in 1% NaOCl for 30s, and then rinsed in sterile water. The tissue pieces were placed on water agar and incubated at 25°C for 10 days. Eight isolates were obtained through single spore isolation, and a representative isolate NC16557 (from Gangneung) was selected for identification. After 14 days, colonies on potato dextrose agar (PDA) were 3.7 cm in diameter and becoming dark due to the formation of microsclerotia. Aerial hyphae were smooth-walled, 3 to 4 μm wide. Conidiophores were erect or slanted, verticillately branched or unbranched, and hyaline. Conidia were hyaline, smooth-walled, non-septate, cylindrical to oval, 4.8 to 6.6 × 2.6 to 3.4 μm (mean 5.7 × 2.9 μm, l/w =2.0, n =80). Microsclerotia were immersed in the agar, composed of rounded, brown-pigmented cells and elongate or irregular in shape, 42 to 60 μm diam. Based on morphology, NC16557 isolate was tentatively identified as Verticillium dahliae (Hawksworth and Talboys, 1970; Isaac, 1967). To confirm taxonomic placement, DNA extracted from mycelia of the same isolate was PCR amplified and sequenced targeting internal transcribed spacer (ITS) regions of rDNA, translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes (Inderbitzin et al., 2011). The sequences were deposited in GenBank with accession numbers MZ723402, and MZ735720-MZ735723, respectively. The sequencing results showed 100% (ITS=479/479, TEF=500/500, ACT=185/185, and TS=295/295) and 99.61% (GPD=257/258) similarity with the sequences of V. dahlia type strain PD322 (LR026889, HQ414624, HQ206921, HQ414909, and HQ414719) by BLAST. Based on the morphology and multigene sequence analysis, the isolate was identified as V. dahliae. Pathogenicity of two isolates (NC16557 and NC16547) was carried out in the greenhouse using ten 10-day-old seedlings (cv. Gwangdongyeoreum) by root-tip cutting and then soaking the roots in a fungal spore suspension of 106 conidia mL-1 for 1 hour. Ten seedlings were treated with sterile distilled water as a control. All plants were placed in the greenhouse at 15°C (night)/25°C (day) with natural light. After 6 weeks, all inoculated plants showed vascular discoloration in the roots while control plants remained asymptomatic. However, the above-ground symptoms of inoculated plants, such as yellowing and wilting, were indistinguishable from control plants. V. dahliae was consistently re-isolated from the symptomatic root tissues and the pathogen identity was confirmed by observing morphological characteristics. Verticillium wilt of radish has been reported from China(Yan et al., 2019). In Korea, this is the first report that V. dahliae causes Verticillium wilt of radish, although Dumin et al. (2020) already reported on Verticillium wilt of Chinese cabbage in Gangwon province, the main production area of Chinese cabbage and radish in summer. With these findings in Chinese cabbage and now radish, it will be critical to identify or develop Verticillium-resistant varieties and other management strategies for the stable production of these crops in Korea.

Effect of Temperature on Efficacy of the Entomopathogenic Fungi Beauveria bassiana and Metarhizum anisopliae Against the Sunn Pest, Eurygaster integriceps Puton

El-Mukhlef, A.A., Sh.A. Kharouf and A. Al-Khalaf. 2023. Effect of Temperature on Efficacy of the Entomopathogenic Fungi Beauveria bassiana and Metarhizum anisopliae Against the Sunn Pest, Eurygaster integriceps Puton. Arab Journal of Plant Protection, 41(2): 134-139. https://doi.org/10.22268/AJPP-41.2.134139 An experiment was conducted in the plant protection laboratory of the Directorate of Agriculture and Agrarian Reform in Raqqa Province during 2013, to assess the effect of temperature and four spores suspension concentrations (1.5×102 , 103 , 104 and 105 spores/insect) on the efficacy of Beauveria bassiana and Metarhizum anisopliae against the Sunn pest (Eurygaster inegriceps Puton). Insect adults dorsal side were inoculated with 1.5×103 spores/insect of B. bassiana and caused insects mortality at a temperature range of 22-31°C, with optimum effect at 25°C, with a LT50 of 5.3 days after inoculation. In contrast, M. anisopliae had a wider temperature range up to 37°C, with optimal effect at 28- 31°C with LT50 of 5 days. No significant differences were observed between the fungal concentrations of 1.5×103 and 1.5×105 . Results obtained for using the four fungal spore suspension concentrations (1.5×102 , 1.5×103 , 1.5×104 , 1.5×105 spores/insect) under optimum conditions for each fungus indicated that the LT50 was close among concentrations, whereas, there was significant difference between the fungal two concentrations 1.5×102 and 1.5×104 , and B. bassiana was most effective at 1.5×104 spores/insect, in contrast to M. anisopliae which was most effective at 1.5×102 spores/insect. Keywords: Beauveria bassiana, Metarhizum anisopliae, Eurygaster inegriceps, Sunn Pest, biological control.

Enhancement of protease production from Rhizomucor miehei by mutagenesis with ethyl methanesulfonate, ultraviolet, and microwaves- a preliminary study

Rhizomucor miehei protease has been emerging as a potent substitute for the more expensive calf rennet. Enzyme biosynthesis could be improved by using random mutagenesis techniques. In this study, the fungal spore suspension was treated with different concentrations of ethyl methanesulfonate (EMS) (20–80 μg/mL) for different incubation periods (10–60 min). UV-mutagenesis was carried out at different distances (5–25 cm) from the exposure source (253 nm) for different exposure periods (5–25 s). Microwaves (MW) irradiation was done at 2450 MHz for different exposure periods (90–210 s). The best results, in terms of milk-clotting activity (MCA) and milk-clotting activity to proteolytic activity ratio (MCA/PA), were obtained with the protease secreted by the MW-induced mutant (150 s), followed by UV (10 cm*25 s), and then EMS (35 μg/mL*20 min) induced mutants. MW150 mutant presented 3.68 and 2.18 folds improvement in MCA and MCA/PA ratio. Only MW-induced mutant was stable after 8 generations.

Suppression of the insect cuticular microbiomes by a fungal defensin to facilitate parasite infection.

Insects can assemble defensive microbiomes on their body surfaces to defend against fungal parasitic infections. The strategies employed by fungal pathogens to combat host cuticular microbiotas remains unclear. Here, we report the identification and functional characterization of the defensin-like antimicrobial gene BbAMP1 encoded by the entomopathogenic fungus Beauveria bassiana. The mature peptide of BbAMP1 can coat fungal spores and can be secreted by the fungus to target and damage Gram-positive bacterial cells. Significant differences in insect survival were observed between the wild-type and BbAMP1 mutant strains during topical infection but not during injection assays that bypassed insect cuticles. Thus, BbAMP1 deletion considerably reduced fungal virulence while gene overexpression accelerated the fungal colonization of insects compared with the wild-type strain in natural infections. Topical infection of axenic Drosophila adults evidenced no difference in fly survivals between strains. However, the gnotobiotic infections with the addition of Gram-positive but not Gram-negative bacterial cells in fungal spore suspensions substantially increased the survival of the flies treated with ∆BbAMP1 compared to those infected by the wild-type and gene-overexpression strains. Bacterial colony counts and microbiome analysis confirmed that BbAMP1 could assist the fungus to manipulate insect surface bacterial loads. This study reveals that fungal defensin can suppress the host surface defensive microbiomes, which underscores the importance to extend the research scope of fungus-host interactions.

Iron oxide nanoparticles immobilized Aspergillus flavus manganese peroxidase with improved biocatalytic, kinetic, thermodynamic, and dye degradation potentialities

Manganese peroxidase (MnP) has gained significant attention for treating textile waste due to its ability to oxidize and detoxify a wide range of recalcitrant xenobiotics, including synthetic dyes. MnP was isolated from a solid-state culture of Aspergillus flavus using pre-optimized conditions of 5 g wheat bran (66% moisture content), pH 4.5, temperature 30 ℃, carbon source: glucose 2%, nitrogen source: yeast extract 0.02%, 5 ml of homogenous fungal spore suspension and fermentation period of 5 days. The extracted MnP was partially purified using ammonium sulfate precipitation and immobilized on iron oxide nanoparticles. Soluble and immobilized MnP showed the maximum catalytic potential at 35 ℃ and 50 ℃, and optimum pH of 4.0 and 5.0, respectively. Vmax and Km for free MnP were 0.126 U/mg and 0.0886 mM, and 0.140 U/mg and 0.704 mM for the immobilized MnP, respectively. Thermodynamic parameters, including Ea, ΔH*, ΔG*, ΔS* were also investigated for free and immobilized MnP. Finally, the soluble and immobilized MnP were exploited for catalytic degradation of two textile dye pollutants, Direct red 31 and Acid black 234. Free MnP catalyzed 94% decolorization of Direct red 31, while 100% decolorization was achieved with immobilized MnP. On the other hand, Acid black 234 showed 85% and 92% decolorization with free and immobilized MnP, respectively. The magnetically separable immobilized MnP was more thermally stable, can work over a wider pH and temperature range, and be more catalytically vigorous than its free counterpart.

Responses of soil bacterial and fungal communities to the long-term monoculture of grapevine.

Soil microorganisms are essential for the long-term sustainability of agricultural ecosystems. However, continuous grapevine replanting can disrupt the stability of soil microbial communities. We investigated the bacterial and fungal abundance, diversity, and community composition in rhizosphere soils with continuous grapevine replanting for 5, 6, 7 (Y5, Y6, and Y7; short-term), and 20 (Y20; long-term) years with high-throughput sequencing. Results showed that diversities and abundances of bacterial and fungal communities in Y20 were significantly lower than in other samples. The bacterial and fungal community compositions were markedly affected by the replanting time and planting year. After short-term grapevine replanting, relative abundances of potential beneficial bacteria and harmful fungi in rhizosphere soils were higher compared to long-term planting. Bacterial and fungal communities were significantly correlated with available nitrogen (AN), available phosphorus, available potassium (AK), and pH. AK and AN were the primary soil factors related to the shift of bacterial and fungal communities. Bacterial and fungal co-occurrence patterns were remarkably affected by replanting time, showing that fallow land harbored co-occurrence networks more complex than those in other groups, with the Y20 group showing the lowest complexity. Then, we isolated the dominant fungi in grapevine rhizosphere soil after continuous replanting and verified the harmful effects of three candidate strains through pot experiments. The results showed that 12days post-treating the soil with fungal spore suspensions significantly inhibited grapevine seedlings' growth, whereas Fusarium solani inhibited plant growth. Overall, we showed that F. solani might be a potentially harmful fungus related to grapevine replant diseases. KEY POINTS: • Continuous grapevine planting reduced soil microbe diversities/abundances. • Beneficial bacteria and harmful fungi increased after short-term replanting. • F. solani may be a harmful fungus related to grapevine replant diseases.

First Report of Blackleg DiseaseCaused by Epicoccum sorghinumon on Lavender (Lavandula stoechas) in China.

Lavender (Lavandula angustifolia Mill) is an ornamental plant and worldwidely grown for its aromatic and pharmacological qualities. In June 2020, the symptoms of blackleg disease on lavender plants were observed, with more than 50% incidence in Chaohu city (117°38'19.12″N, 31°47'18.94″W) of Anhui Province, China. The disease symptoms progressed from stem wilt and necrosis to prolonged necrosis and bending of leaves, and all infected lavender plants died eventually. Ten necrotic stem lesions werecollectedfrom ten independent plants for the isolation of pathogen. All samples were washed in 70% ethanol for 1 minute, rinsed twice in sterile distilled water and placed on water agar (WA) plates containing 30 mg/liter of kanamycin. All 16 fungal isolates were transferred onto potato dextrose agar (PDA) and incubated at 26°C for 5 days, and all fungal colonies were isolated consistently, which produced redish-gray mycelium at 26°C with a 12-h photoperiod on PDA media. They developed black pycnidia with abundant hyaline, unicellular, oval shaped conidia (4.5 to 5.9 × 2.1 to 2.5 μm) after 14 days. DNA was extracted (10-day-old culture) using the Fungal DNA Mini Kit (Omega Bio-tek, China), according to the manufacturer's protocol. The internal transcribed spacer (ITS), beta-tubulin (β-tub) and translation elongation factor 1-alpha (tef1-α) genes of three isolates were amplified using the primers: ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995) and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The ITS(MT883331), β-tub(MT896891) andtef1-α (MT874165) genes were sequenced and analyzed through BLASTn. The ITS sequence showed 99.81% with Epicoccum sorghinum (GenBank Accession No. MK020690.1). The β-tub and tef1-α showed 100% homology with Epicoccum sorghinum (GenBank AccessionMN554062.1 and MN512426.1), respectively. To complete Koch's postulates, pathogenicity tests were performed by spraying the fungal spore suspension (1×105 CFU/ml) prepared from 14-day-old cultures onto needling wounded stems of 1-year-old potted healthy L. angustifolia plants. The healthy plants were sprayed with sterilized water onto needling wounded stems served as negative control. Wilting and stem necrosis were observed 5 days afterinoculation and incubation in a growth chamber at 26°C, with a 12-h photoperiod. All fungal infected plants died after 10 days, while, the control plants remained healthy. The fungus was re-isolated from the lesions of the inoculated plants and verified. Based on morphological characteristics, sequence analysis and pathogenicity test, the pathogen was identified as E. sorghinum. The pathogen has been observed previously on many plants such as tea (Bao et al. 2019) and taro (Liu et al. 2018), in China. To our knowledge, this is the first report of E. sorghinum causing blackleg disease of lavender in China and worldwide.

Enhanced production of extracellular lipase by ethyl methane sulfonate from soil fungal strain in submerged and solid-state fermentation condition

Enhancement in the production of enzyme by utilizing different strains of microbe is one of the main prospects in biotechnology. In the present work, ethyl methane sulfonate (EMF) was selected as the chemical mutagen for inducing mutagenesis in fungi. It is a cheap method to induce random mutation as compared to other methods of recombinant technologies. Strain improvement was done by incubating the fungal spore suspension at variable concentrations of EMS i.e. 4% (v/v) and 10% (v/v) for the time period of 60, 90, and 120 min respectively. The set of control was treated with distilled water only. The fungal colonies were found to be maximum in control plate as compared to the EMF exposed plates. The number of fungal colonies was reduced as we raised the exposure time of EMF. Specific activity and the lipase activity of wild strain and hyperproducer were evaluated under the submerged (SmF) and solid-state fermentation (SSF). The wild strain denoted the 3.2 U/ml/min of enzymatic activity under SmF and 15.87 U/g/min of activity under SSF. In contrast, the best enzymatic activity was represented by S2St1 at 10% of EMS after the time period of 60 min i.e. 11.7 U/ml/min under SmF and 99.35 U/g/min under SSF after the time period of 72 hrs. Statistical analysis by using one-way ANOVA determined that the value of F calculated was lower than the F tabulated. So, there was a significant relation between the EMS percentage and time of exposure among the mutated strains. In conclusion, this soil fungal strain can be utilized to produce lipase enzyme for numerous industrial applications.

Asymbiotic seed germination, in vitro seedling development, and symbiotic acclimatization of the Mexican threatened orchid Stanhopea tigrina

Stanhopea tigrina commonly known in Mexico as “cabeza de vibora” or “calaverita”, is an endemic orchid from the mountain cloud forest of this country and it is currently an endangered species. The objective of this study was to stablish an in vitro propagation system for S. tigrina in order to contribute to the development of alternatives in the preservation of this species. Therefore, seeds from a mature capsule were cultivated in a Murashige and Skoog basal medium. Then, 16-week-old seedlings, counting weeks from the moment of germination, were transplanted into several treatments fortified with organic extracts (OE) or 6-benzylaminopurine (BAP) to induce the proliferation of the seedlings. These treatments fortified with OE and 5.0 mg of L− 1 BAP developed 1.25 ± 0.35 shoots with no significant differences. For the rooting process, the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid were tested either alone or in combination with salicylic acid or coconut water. The addition of 100 mL L− 1 of coconut water, alone or in combination with 2.5 or 5.0 mg L− 1 of IAA auxin were the best treatments for root production. For the establishment of symbiotic acclimatization, the vitroplants were inoculated with fungal spore suspensions of Trichoderma hamatum and Penicillium sp. The vitroplants to which T. hamatum was added as a symbiote achieved 100% survival and developed an average height of 2.54 ± 0.33 cm. In conclusion, the in vitro propagation protocol proposed in this study was effective for the symbiotic obtaining and acclimatization of S. tigrina plants. Stanhopea tigrina is an endemic orchid to Mexico and currently threatened. This research was designed in order to contribute to its conservation through the use of plant biotechnology. In this study it has been shown that the use of organic extracts and symbiotic acclimatization with fungi can be a useful method to regenerate and massively propagate this orchid.

Biological Control of the Raspberry Eriophyoid Mite Phyllocoptes gracilis Using Entomopathogenic Fungi

There is an urgent need to develop biological control methods against the eriophyoid mite, Phyllocoptes gracilis, which causes significant losses in organic raspberry production in Europe. The use of entomopathogenic fungi (EF) is a sustainable alternative to conventional chemical pesticides, reducing the risks of pesticide resistance and other negative environmental impacts of agriculture. The objective of this study was to assess the pathogenicity of three strains of EF, two of Beauveria bassiana and one of Metarhizium anisopliae, on P. gracilis under laboratory conditions. Fungal spore suspensions (1 × 107 spores per mL) were sprayed on detached leaves infested with P. gracilis. Treated mites were kept under controlled conditions (25 ± 3 °C, 72 ± 10% relative humidity and photoperiod of 16:8 (light/dark)) and mite mortality was assessed three, five and seven days after inoculation. At all three measurement points (days after inoculation), the mortality of P. gracilis was highest for B. bassiana strain BB 1.1 and M. anisopliae strain MA 10.1. Our data demonstrate that EFs are promising candidates for the development of biological control agents against P. gracilis in raspberry crops.

In vitro efficacy of Beauveria bassiana (Balsamo) Vuillemin against Tetranychus urticae Koch on tuberose

Bio efficacy of virulent isolate of entomopathogenic fungi, Beauveria bassiana (MK918495) was evaluated against two spotted spider mite, Tetranychus urticae on tuberose under pot culture condition at Insectary, Department of Agricultural Entomology, TNAU, Coimbatore. For effective working of any insect pathogen, it is mandatory to develop an effective formulation. Keeping this point in view, in the present study, compatibility of the fungal isolate with two different oils, viz., ground nut and sunflower were tested at different concentrations, viz., 1, 3, 5 and 10%. The results revealed that the groundnut oil was compatible with B. bassiana at all concentrations with respect to colony growth and spore yield compared to sunflower oil which was compatible only at one percent. Based on the results, oil in water and oil based formulations were prepared. At higher concentration of 10%, groundnut oil recorded the radial colony growth of 36.90 mm diameter and spore yield of 1.2 x 10 8 spores mL -1 . Among the seven formulations (A-G) tested, formulation D which contained surfactant mixture having HLB value of 10.00 (53.33% Tween 80 and 46.67% Span 80) formed a better suspension with less flocculation layer and dispersion of conidia in spray fluid and was found to be better as it formed a good emulsion with water by mere shaking. Results of pot culture studies revealed that groundnut oil based fungal formulation was effective with a cumulative mean mite population reduction of 64.98% followed by crude fungal spore suspension (62.16%) and both were statistically on par with each other. Standard chemical check, Fenazaquin 10% EC @ 1.5 mL L -1 recorded significantly highest population reduction of mites (77.20%).Â

Novel inactivation of the causative fungal pathogenof white-nose syndrome with methoxsalen plusultraviolet A orB radiation.

White-nose syndrome is a fungal disease responsible for the rapid decline of North American bat populations. This study addressed a novel method for inactivating Pseudogymnoascus destructans, the causative agent of WNS, using ultraviolet A (UVA) or B (UVB) radiation in combination with methoxsalen, a photosensitizer from the furanocoumarin family of compounds. Fungal spore suspensions were diluted in micromolar concentrations of methoxsalen (50–500 μM), then exposed to fixed doses of UVA radiation (500–5000 mJ/cm2), followed by plating on germination media. These plates were examined for two to four weeks for evidence of spore germination or inactivation, along with resultant growth or inhibition of P. destructans colonies. Pretreatment of fungal spores with low doses of methoxsalen resulted in a UVA dose-dependent inactivation of the P. destructans spores. All doses of methoxsalen paired with 500 mJ/cm2 of UVA led to an approximate two-log10 (~99%) reduction in spore viability, and when paired with 1000 mJ/cm2, a four-log10 or greater (>99.99%) reduction in spore viability was observed. Additionally, actively growing P. destructans colonies treated directly with methoxsalen and either UVA or UVB radiation demonstrated UV dose-dependent inhibition and termination of colony growth. This novel approach of using a photosensitizer in combination with UV radiation to control fungal growth may have broad, practical application in the future.

Toxicological Studies and Histopathological Changes on Black Bean Aphid, Aphis craccivora Induced by Entomopathogenic Fungi, Metarhizium anisopliae and Purpureocillium lilacinum

Toxicological, histopathological and ultrastructure studies of Metarhizium anisopliae and Purpureocillium lilacinum infected Aphis craccivora nymph were investigated in this study. comparison between fungal spore suspensions and fungal culture filterates virulence effects of the two tested entomopathogenic fungi against A. craccivora nymph were done under laboratory condition and the toxicological bioassay results revealed that P.lilacinum has a high toxic effect than M. anisopliae in which the LC50 of fungal spore suspensions and culture filterates after 7 days post-treatment was (7×102, 1×105 spore/ml and 3.2, 4.7 % ) for P. lilacinum and M.anisopliae, respectively. Treated nymph with spore suspensions was examined using both light and transmission electron microscope to study histopathological and ultrastructure changes compared with the control one. This comparison suggested that there is a deformation of the nymph body, complete destruction of all internal organs with an extensive invasion of the fungal spores throughout all internal body regions.

The insecticidal activity of the endophytic fungus Isaria fumosorosea Wize isolated from the leaves of the Ricinus communis L. against Locusta migratoria L. and Acanthoscelides obtectus Say

Abstract This study was conducted to evaluate the insecticidal activity of the endophytic fungus Isaria fumosorosea Wize isolated from Ricinus communis L. against Locusta migratoria L. and Acanthoscelides obtectus Say. To evaluate the insecticidal activity, a mixture of fungal extract and spore suspensions (1x107, 1x106 and 1x105 spores/ml of dissolved extract) were applied against the insects. It was found that I. fumosorosea had a good insecticidal activity, positively proportional to concentration and time after exposure (24, 48, 72 hours). A maximum mortality rate of 80% for A. obtectus and 70% for L. migratoria at 1x107 spores/ml of dissolved extract was recorded. Preliminary phytochemical analyses of the fungal extract revealed the presence of alkaloids, flavonoids and phenols. Additionally, a fungal development on the insects bodies has also been noticed; that is undoubtedly explained the recorded insecticidal activity.

First Report of Aspergillus parasiticus on Barley Grain in Serbia

Barley (Hordeum vulgare L.) is a secondary grain crop in Serbia used commercially for animal feed, seed, and human food applications. The production of barley in the 2016 to 2017 growing season reached a record yield of almost 400,000 metric tons (USDA 2017). Aspergillus contamination has been rare in the agroecological conditions of cereal-growing areas in Serbia. Changes in climatic factors, such as occurrence of high temperatures and prolonged droughts, increased frequency of Aspergillus spp. Species Aspergillus parasiticus was isolated from maize grain for the first time in Serbia in 2012 and from wheat grains in 2017 (Nikolic et al. 2018). We hypothesized that these pathogens can also be present in barley fields in Serbia. Barley spikes exhibiting bleaching were sampled at the beginning of June 2017 grown in northern Serbia. In severe infections, barley spikes get a dry look with awns that stand upright and firm. The incidence of the disease of the bleached spiked in the field was 15 to 20%. From each representative sample, 100 shriveled grains were collected. After surface sterilization with bleach/distilled water 1:3, 100 grains per sample (10 per Petri dish) were placed on potato dextrose agar and incubated at 25°C for 7 days. After isolation, 50% of isolates were identified as Alternaria spp., 20% as Fusarium spp., approximately 15% as Aspergillus spp. In order to reliably identify individual species of fungi, the fragments of colonies were transferred to malt extract agar (MEA) and Czapek yeast agar (CYA) and incubated in the dark for 7 days. The fungal colonies were dark green. The reverse side was pale yellow. The average colony diameter was 65 mm. Conidia were spherical and rough with thick walls. The average size of conidia was 5.1 µm. Colonies were floccose and thin on MEA and CYA. Based on growth and morphological characteristics, isolates were determined as A. parasiticus (Pitt and Hocking 2009). Molecular detection of Aspergillus species was done by using PCR-restriction fragment length polymorphism analysis of the aflR-aflJ (genes for aflatoxin biosynthesis) intergenic spacer. The restriction enzyme BglII was able to cut the PCR product of A. parasiticus at one restriction site, resulting in two fragments of 363 and 311 bp (El Khoury et al. 2011). A. parasiticus CBS 100926 was used as a reference isolate. The pathogenicity of 20 isolates was verified on a group of 20 randomly selected spikes in four replicates (Mesterhazy et al. 1999). A 7-day-old culture of each isolate was used for the preparation of the spore suspension (1 × 10⁶ spores/ml). Inoculation was carried out after 50% of plants reached the anthesis stage. Groups of 20 selected spikes were sprayed from all sides with 20 ml of fungal spore suspension. Control spikes were inoculated by applying an equal amount of sterile distilled water. The infection rate was estimated after 3 weeks on a 1 to 7 scale, with 1 = 0 to 5%, 2 = 5 to 15%, 3 = 15 to 30%, 4 = 30 to 50%, 5 = 50 to 75%, 6 = 75 to 90%, and 7 = 90 to 100% infected spike area. The average infection rate was 3.2. The pathogen was reisolated from the inoculated spikes and identified as A. parasiticus, with the aim to confirm Koch’s postulates. Developed symptoms were similar to those observed on spikes collected from the field. Control spikes did not show any symptoms of the disease. These results confirmed the pathogenicity of A. parasiticus on H. vulgare. To the best of our knowledge, this is the first report of the occurrence of A. parasiticus on barley grain in Serbia. Because A. parasiticus is known to be a severe aflatoxin producer and climatic changes can increase the frequency of this fungus, further studies are necessary to improve strategies for food safety and quality.