Mycelial Diameter Research Articles

Inhibitory Activity of Propolis Extracts From Stingless Bees (Meliponini) on the Development of Phytopathogenic Fungi: in Vitro Evaluation

Objective: The present study aimed to assess the alcoholic extracts of propolis from Tetragona clavipes, Scaptotrigona bipunctata, Tetragonisca angustula, and Melipona quadrifasciata. Theoretical Framework: There is growing interest in the bioactive metabolites of propolis and honey produced by different species of the group of native stingless bees (NSB) belonging to the Meliponini tribe. Method: The extracts were prepared in a proportion of 30 g of propolis to 70 mL of 96 % v/v ethanol (1:2). Afterwards, its chemical constituents were characterized by HPLC. A PDA medium was used to evaluate the antifungal activity, with the extracts added to the melting medium (55 °C) in different concentrations. The effect of increasing concentrations (1.6 %, 3.2 %, 6.4 %, and 12.0 % v/v) of extracts on the mycelial growth of the fungi Sclerotinia sclerotiorum, Fusarium sp., Colletotrichum gloeosporioides, Botrytis cinerea, and Botryosphaeria sp. was evaluated. Fungal development was determined by measuring mycelial diameter on the 14th day of inoculation. Results and Discussion: The antioxidant activity and the contents of flavonoids and phenolic compounds were identified in all samples of propolis extracts from ANSF. The T. clavipes propolis extract had the highest content of phenolic compounds (5,593 mgEAG·100 mL-1). Among the chemical compounds, naringin, gallic acid, hesperidin, and ferulic acid were identified in greater quantities. The fungi evaluated in this work were partially inhibited by most SNB alcoholic extracts, mainly at concentrations of 1.6 % and 3.2 % v/v compared to the control treatment (ethanol 96 % v/v). Changes in the hyphal network and the reduction or absence of conidia were also observed when combined with different alcoholic propolis extracts and the control with 96 % v/v ethanol. Research Implications: The results demonstrate that the chemical components of the alcoholic extracts of NSB propolis have the potential for controlling phytopathogenic fungi of agricultural interest. Originality/Value: This study contributes by assessing and highlighting the potential applications of NSB as natural products in the alternative control of phytopathogenic fungi of agricultural interest.

VdP5CDH is involved in melanin formation, stress resistance and play a regulatory role in virulence of Verticillium dahliae.

Verticillium dahliae, a soil-borne fungal pathogen, can cause cotton Verticillium wilt. In this study, VdP5CDH, the member of the ALDH_F4-17 family of carboxylate dehydrogenases, was identified in the genome of V. dahliae and investigated function in regulating virulence by generating gene deletion mutants and complementary mutants. Homologous recombination method was used to construct mutants, transcriptome sequencing revealed gene-related metabolic pathways, and disease degree of cotton was observed through pathogen infection experiments. The conidial surface of VdP5CDH deletion strains was dented and shriveled, and the number of conidial spores increased. Compared with the wild-type (WT), the mycelial diameter of deletion mutants increased by 10.59%-11.16%, the mycelial growth showed irregular branching patterns, and misaligned arrangement. Although capable of penetrating cellophane, deletion mutants were unable to produce melanin. VdP5CDH was mainly associated with glucose metabolism, nitrogen metabolism, ABC transporter activity as well as various amino acid metabolic processes. After gene knockout, raffinose and pectin were used as the main carbon sources to promote the growth of strains and the growth rate of deletion strains in the medium containing raffinose was higher than that of WT. Consequently, the deletion mutant strains decreased utilization efficiency with which they utilized various nitrogen sources. The deletion mutants maintain responsiveness to osmotic stress and oxidative stress stimuli. Additionally, compared to WT strains, the deletion mutant strains exhibited differences in culture temperature tolerance, UV exposure response, and fungicide sensitivity. After cotton was infected with deletion strains conidial suspension, its disease index increased dramatically, while it gradually decreased after spraying with 2 mM glutamate in batches. With the increase of spraying times, the effect was more significant, and the disease index decreased by 18.95%-19.66% at 26 dpi. These results indicated that VdP5CDH regulates the pathogenicity of fungi and controls mycelia growth, melanin formation, conidia morphology, abiotic stress resistance, and the expression of infecting structure-related genes.

Compatibility study of Trichoderma sp. with Chemical Fungicides Commonly Used by Nepalese Farmers, Under In-Vitro Condition

The excessive use of synthetic fungicides has led to the emergence offungicide-resistant strains of pathogens, raising concerns about human health andenvironmental impact. Trichoderma spp., an endophytic and versatileopportunistic plant symbiont, has recently gained popularity as a biocontrolagent. Integrated use of Trichoderma with compatible fungicides gives betterdisease management and causes less harm to the environment in the long run.This study aimed to evaluate the compatibility of six fungicides commonly used byNepalese farmers at concentrations of 250, 500, and 1000 ppm with Trichodermasp. using the poisoned food technique in-vitro. The experiment was conducted ina completely randomized design with four replications for each treatment, anddata were taken at 24, 36, 48, 60, and 72 hours after incubation. Among thefungicides tested, Mancozeb was compatible at all concentrations from the startand exhibited the highest compatibility at 72 hours, with a growth inhibitionpercent of 0.39%. Metalaxyl+Mancozeb at 250 and 500 ppm, and Copperoxychloride at 250 ppm showed good compatibility. However, Carbendazim,Hexaconazole, and Carbendazim+Mancozeb resulted in 100% growth inhibition ofTrichoderma sp. throughout the experiment. While all treatments significantlyreduced mycelial diameter, Mancozeb and Metalaxyl+Mancozeb showed agradual decrease in growth inhibition percent over time, indicating increasedcompatibility. In contrast, Copper oxychloride demonstrated variable growthinhibition percent over time. So, the fungicides Mancozeb, Metalaxyl+Mancozeband Copper oxychloride at compatible concentrations can be used withTrichoderma sp. in integrated disease management to control soil and seed-bornepathogens.

QUANTITATIVE ANALYSIS OF CHITOSAN CONCENTRATION-DEPENDENT MODULATION OF PHYTOPATHOGENIC FUNGAL GROWTH

The study investigated the impact of varying concentrations of chitosan on fungal growth, measured by mycelial diameter, in a solid medium containing 2% malt extract agar (MEA). Concentrations of chitosan (0.05%, 0.10%, 0.15%, and 0.25%) were compared against a control group. The results demonstrated a notable decrease in fungal growth as chitosan concentration increased, indicating a linear relationship between fungal growth and chitosan concentration. The fungal mycelium covered the chitosan-amended medium within 7 days at lower (0.05%), while a prolonged duration (18-22 days) was required for growth at medium (0.10 and 0.15%) and higher concentrations (0.15 and 0.25%), compared to the control. Minimum inhibitory concentration (MIC) values for chitosan were determined at 0.25% for A. alternata, A. flavus, and S. rolfsii, while concentration > 0.25% was required to achieve complete inhibition in the growth of M. phaseolina. Principal component analysis (PCA) was conducted to elucidate fungal growth inhibition at different chitosan concentrations signifying fungal sensitivity to chitosan concentrations. A. alternata exhibited a more sensitivity, followed by S. rolfsii and A. flavus, to chitosan concentrations. The findings of this study will contribute valuable insights into the potential utility of chitosan nanoparticles as an antimycotic agent against these pathogenic fungi

Impact of Temperature on the Bioactive Compound Content of Aqueous Extracts of Humulus lupulus L. with Different Alpha and Beta Acid Content: A New Potential Antifungal Alternative

Hops contain a wide variety of polyphenolic compounds with diverse antimicrobial properties. This study aimed to evaluate the impact of temperature on the bioactive components of samples of aqueous extracts of hops with different characteristics. A central compound rotating design model was used in order to obtain optimal conditions of temperature and extract concentration to inhibit Fusarium oxysporum and Alternaria solani. At intermediate temperatures according to the design of experiments, significant effects on antifungal activity were observed. The optimal conditions with antifungal activity were at a concentration of 160 mg/mL and a temperature of 65 °C to obtain mycelial diameters ≤ 25 mm. The bioactive compounds were shown in the FT-IR spectrum after each heat treatment of both samples; significant changes were observed in the bands between 2786 to 3600 cm−1 and 1022 to 1729 cm−1. The content of total phenols and flavonoids showed a concentration increase of 4.54 to 6.24 mg GAE/g and 6.21 to 8.12 mg QE/g from an initial evaluation temperature of 25 °C to 57.5 °C, respectively, benefited by the heating temperature, enhancing antifungal activity. However, when increasing the temperature ≥90 °C, a tendency to decrease the concentration of bioactive compounds was observed, probably due to their denaturation due to the effect of temperature and exposure time, being non-thermolabile compounds at high temperatures. These aqueous extracts are an alternative to effective natural antifungals.

Evaluation of the antifungal activity of propolis extracts from stingless bees on phytopathogenic fungi

The bees of the Meliponini tribe, known as stingless bees (SB), elaborate a distinct type of propolis, whose application potential is largely unexplored. Currently, there is some knowledge of the pharmacological activities of the propolis from Apis mellifera, however, little is known about the diversity and antimicrobial activity of Meliponini propolis, especially in relation to pathogens of agricultural importance. This study aimed to investigate the antimicrobial activity of the alcoholic extract of propolis from T. clavipes (Borá), S. bipunctata (Tubuna), and T. angustula (Jataí), comparing with the ethanolic extract from A. mellifera propolis. The autoclaved and non-autoclaved extracts were evaluated at the concentrations of zero, 1.6 %; 3.2 %; 6.4 %, and 12.0 % v/v. Fungal development was determined by measuring the mycelial diameter up to the fourteenth day of inoculation, with five replicates; the growth inhibition was calculated relative to the control. According to the results, the highest concentration of phenolic compounds and flavonoids was present in the ethanolic extract of A. mellifera propolis when compared to the SB extracts. The results indicated an inhibitory effect on the growth of the phytopathogens Colletotrichum gloeosporioides, Fusarium sp., Botryosphaeria sp., and Botrytis sp., mainly from 6.4 % and 12.0 % v/v when evaluated in the non-autoclaved medium, followed by the autoclaved medium. The fungus Botrytis sp. was the phytopathogen that suffered greater inhibition from 3.2 mL∙L-1, with the T. angustula propolis extract. The obtained results demonstrate that the alcoholic extracts of propolis from SB have the potential to control phytopathogenic fungi of agricultural interest.

Effect of hydroxypr1opylation on physical properties, antifungal and mycotoxin inhibitory activities of clove oil emulsions coated with chitosan

Fusarium graminearum is one of the most common fungi infecting grains and the production of mycotoxin, deoxynivalenol (DON), poses a serious threat to human and livestock health. Chitosan coated emulsion can be used as a highly effective antifungal agent. However, the poor solubility of chitosan leads to a longer ultrasonic time for preparing emulsion. In our work, hydroxypropyl chitosan with better water dispersion was prepared and used as a coating for clove oil emulsion, and the effect of hydroxypropylation on the physical properties, antifungal and mycotoxin inhibitory activities of chitosan coated clove oil emulsion was investigated. Compared with unmodified chitosan coated clove oil emulsion, hydroxypropyl chitosan coated clove oil emulsion had smaller particle size, lower viscosity, and its maximum reduction of the mycelial diameter and the minimum fungicidal concentration was 35% and 50%, respectively. These results indicated that hydroxypropylation could improve the physical properties and antifungal activity of chitosan coated clove oil emulsion to a certain extent. Surprisingly, after being cultured in barley medium for 7 d, the maximum reduction of DON and ergosterol accumulation was 87% and 86% respectively, showing that hydroxypropyl chitosan coated clove oil emulsion can inhibit the production of DON by mitigating the growth of F. graminearum. Moreover, clove oil emulsions coated with high molecular weight hydroxypropyl chitosan showed a stronger inhibitory effect on the accumulation of DON and ergosterol. This work can provide a reference for mitigating fungal growth and mycotoxin production in food by essential oil emulsions.

Production and bromatological analysis of the oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm.) grown with cocoa, banana, coconut and African palm husk substrates

Oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm. (1871) is a rich food source. It is cultivated on compost and plant waste material. Choosing adequate substrate is essential for oyster production as the substrate can change oyster production in terms of mass and metabolite composition. The different medium substrates for oyster production including T1 (PDA, potato-dextrose-agar), T2 (CCA: PDA + Cocoa Shell), T3 (APR: PDA + African Palm Rachis), T4 (BP: PDA + Banana Peel), T5 (CCO: PDA + Coconut Peel) were used. Based on mycelial diameter, CCO treatment was the best treatment with growth measures of 66.83 mm at 168 hours. CCA treatment with 164.13 g kg-1 yield had the highest production that was significantly different from other treatments. For APR treatment, trace production was observed. The bromatological analysis determined that the highest levels of crude total protein were obtained in CCO treatment (30.08 %) while CCA treatment exceeded significantly dry matter (94.05 %), ethereal extract (6.52 %), crude fiber (12.34 %), non-nitrogen matter (56.15 %) and titratable acidity (3.32 %). The substrates with more lignocellulosic compounds like banana and coconut residues are better for producing oyster with a higher percentage of total protein, while substrates that retain moisture like cocoa residues lead to an excellent production. It is recommended to keep fibrous residues moist constantly when they are used in oyster production because of their low absorbent capacity as they quickly lose moisture.

Enhancing the biomass and riboflavin production of Ashbya gossypii by using low-intensity ultrasound stimulation

The riboflavin (vitamin B2) produced by the fermentation of Ashbya gossypii is an essential nutrient for humans and animals. In this study, favorable low-intensity ultrasound (US) with a power of 210 W was applied to stimulate A. gossypii for 45 min at 33 kHz during the logarithmic phase of 48 h. Compared with those of the untreated A. gossypii, the extracellular riboflavin, total riboflavin, and biomass of the US-stimulated A. gossypii increased by 23.5%, 26.95%, and 71.99%, at the 168th h, respectively. The optimal US stimulation increased the mycelial membrane permeability, decreased the mycelial diameter, and changed mycelial morphology to promote mass transfer, causing the enhancement of biomass and riboflavin production. Furthermore, US stimulation also increased the reactive oxygen species generation of A. gossypii, which may be attributed to this fungus’ US-induced stress defense that enhanced riboflavin production. Therefore, combining US and fermentation is a feasible and efficient method for enhancing the production of riboflavin and other compounds.

A New Strategy to Improve Management of Citrus Mal Secco Disease Using Bioformulates Based on Bacillusamyloliquefaciens Strains.

The effectiveness of biological commercial products based on Bacillus amyloliquefaciens strains was evaluated through in vitro and in vivo experiments against Plenodomus tracheiphilus. The activity of bacterial cells, volatile organic compounds (VOCs), and culture filtrates of bacteria were tested in vitro against different isolates of P. tracheiphilus. Afterwards, the virulence of these isolates was evaluated on Citrus volkameriana plants to select the most virulent isolate to use in the in vivo experiments. To evaluate the effectiveness of products, C. volkameriana seedlings were pre-treated, twice with biological products and once with standard fungicides, before pathogen inoculation. Moreover, in order to determine the endophytic ability of the bacteria, the population density within the treated citrus stem was determined. Comprehensively, bacterial cells, filtrates, and VOCs were able to significantly reduce the average mycelial diameter of P. tracheiphilus, with some variability according to pathogen isolate. In planta experiments showed that the biological products on average were less effective than fungicides, although all formulates were able to significantly reduce disease incidence and symptom severity, except B. amyloliquefaciens strain D747 (Amylo-X) for symptom severity (SS) 20 days after inoculation. Bacteria were re-isolated from the internal woody tissue of treated plants, showing strong endophytic ability. This work is important as commercial biological products based on B. amyloliquefaciens strains could represent a promising and sustainable alternative for the integrated management of mal secco disease.

Variability and Virulence Analysis of Aspergillus niger Isolates Causing Collar Rot of Groundnut

Background: Collar rot of groundnut (Arachis hypogaea L.) caused by Aspergillus niger is a significant constraint in groundnut cultivation and responsible for huge economic losses in India including Rajasthan. Methods: By surveying of eight major groundnut growing districts of Rajasthan, India, one representative Aspergillus niger isolate from each district was established (ANBK-01= Bikaner, ANCH-02= Churu, ANDA-03= Dausa, ANJP-04= Jaipur, ANJL-05= Jalore, ANJD-06= Jodhpur, ANNG-07= Nagaur and ANSK-08= Sikar) for studying variability in the pathogen as well as to know the response of groundnut varieties to the highly virulent isolate. The colony and spore characteristics were observed for cultural and morphological variability. For resistance response to the disease, ten varieties (M-13, RG-633-9, RG-382, Girnar-2, RG-604, RG-578, Gajraj 10, RG-510, RG-632-1 and RG-644) were evaluated in the field for two consecutive years against a highly virulent Aspergillus niger (ANJP-04) isolate. Result: Our investigations cleared that all the isolates were showed cultural and morphological variability such as shape, colour and size of colony and size of conidia, conidiophores and columella. Isolate (ANJP-04) collected from Khejroli village of Chomu tehsil in Jaipur district showed maximum mycelial growth, conidia diameter, length and diameter of conidiophores and length and diameter of columella, early sporulation and found most virulent as it produced higher disease incidence (54.43%). Ten released varieties of groundnut in the field conditions, revealed that none of the variety was found completely free from the disease whereas RG-644, M-13 and RG-510 were found resistant while RG-604, Girnar-2, Gajraj-10 and RG-632-1 were found moderately resistant and rest were found susceptible to highly susceptible to the disease. Conclusively, it can be finalized that famers may cultivate these resistant varieties in areas where collar rot is a severe constraint. The conclusion of this study can also be utilized to screen varieties/genotypes of groundnut against highly virulent isolate for sustainability of breeding material to the disease effectively.

APPLICATION OF NANOPARTICLES FOR THE CONTROL OF Colletotrichum gloeosporioides CAUSING ANTHRACNOSE DISEASE OF CHILI (Capsicum frutescens L.)

Chili (Capsicum annum L.) was one of the most important seasoning constituents of the cuisines of many countries in the world and it was ranked the fourth major crop cultivated globally. However, Anthracnose disease caused by Colletotrichum gloeosporioides has affected chili production worldwide, especially in tropical and subtropical regions. It is necessary to have greatly effective disease management. Therefore, this study had examined the antifungal effect of some nano solutions on C. gloeosporioides in both in vitro and in vivo conditions. The results clearly demonstrated that silver nano, copper nano, and silver-copper nano solutions had inhibited the spore germination and the growth of C. gloeosporioides. Inhibitory effects were 100% mycelial diameter at the solutions with concentrations of 125 ppm silver nano or 75 ppm copper nano or 50 ppm mixed silver-copper nano. The medium with the concentration of 75 ppm silver nano or 25 ppm copper nano or 12.5 ppm silver-copper nano solution completely inhibited spore germination of C. gloeosporioides. In vivo experiment, the silver-copper solutions with a concentration of 50 ppm could control the growth of C. gloeosporioides in chili fruits the findings were that there were only 13.3% disease fruits, and the inhibition efficiency was 86.7%.

Effect of Sodium Selenite on the Metabolite Profile of Epichloë sp. Mycelia from Festuca sinensis in Solid Culture

Selenium (Se) is an essential micronutrient with many beneficial effects for humans and other living organisms. Numerous microorganisms in culture systems enrich and convert inorganic selenium to organic selenium. In this study, Epichloë sp. from Festuca sinensis was exposed to increasing Na2SeO3 concentrations (0, 0.1, 0.2, 0.3, and 0.4 mmol/L) in Petri dishes with potato dextrose agar (PDA) for 8 weeks. Epichloë sp. mycelia were immediately collected after mycelial diameters were measured at 4, 5, 6, 7, and 8 weeks of cultivation, respectively. Gas chromatography-mass spectrometer (GC-MS) analysis was performed on different groups of Epichloë sp. mycelia. Different changes were observed as Epichloë sp. was exposed to different selenite conditions and cultivation time. The colony diameter of Epichloë sp. decreased in response to increased selenite concentrations, whereas the inhibitory effects diminished over time. Seventy-two of the 203 identified metabolites did not differ significantly across selenite treatments within the same time point, while 82 compounds did not differ significantly between multiple time points of the same Se concentration. However, the relative levels of 122 metabolites increased the most under selenite conditions. Specifically, between the 4th and 8th weeks, there were increases in 2-keto-isovaleric acid, uridine, and maltose in selenite treatments compared to controls. Selenium increased glutathione levels and exhibited antioxidant properties in weeks 4, 5, and 7. Additionally, we observed that different doses of selenite could promote the production of carbohydrates such as isomaltose, cellobiose, and sucrose; fatty acids such as palmitoleic acid, palmitic acid, and stearic acid; and amino acids such as lysine and tyrosine in Epichloë sp. mycelia. Therefore, Epichloë sp. exposed to selenite stress may benefit from increased levels of some metabolite compounds.

Efficacy assessment of Pantoea jilinensis D25 fermentation broth against Botrytis cinerea

Tomato gray mold, caused by Botrytis cinerea, is one of the most devastating diseases affecting tomato plants. In this study, Pantoea jilinensis D25 fermentation broth (PJFB) was used to inhibit B. cinerea growth. The optimum fermentation time was 7 days. PJFB antifungal activity decreased when PJFB was heated to ≥60°C, whereas it was the strongest at pH 8. Moreover, ultraviolet treatment for a long time reduced its antifungal effect. PJFB, applied at 10 % (v/v), could inhibit B. cinerea growth by inhibiting its mycelial diameter, conidial production, and mycelial dry weight with the inhibition rates of 57.3 %, 51.2 %, and 78.6 %, respectively. PJFB could also increase conductivity and malondialdehyde (MDA) content in B. cinerea. Furthermore, PJFB inhibited B. cinerea on not only the leaf tissues but also the seedlings of tomato. The secondary metabolites in PJFB were determined through gas chromatography–mass spectrometry. Through laboratory toxicity analysis, we found that the major compounds in PJFB were 1,2-benzenedicarboxylic acid bis(2-methylpropyl) ester (14.3098 %), 2,4-di-tert-butylphenol (3.8036 %), 2,6,10,14-tetramethyl pentadecane (2.9947 %), and 1,3-dimethyl naphthalene (1.8586 %).

Isolation and identification of pathogen causing damping off at seedling stage of Trollius chinensis

Trollius chinensis is a traditional Chinese medicinal material in China, the wild resource of T. chinensis are now exhausted, and commercial medicinal T. chinensis mainly depends on artificial cultivation. As one of the most severely happened diseases at the seedling period, damping off has been a serious threaten to the breeding of T. chinensis seedlings. However, no related research have been reported so far. So, the authors collected damping-off samples of T. chinensis in 2018 from seedling breeding nursery in Guyuan, Hebei province, and carried out study on taxonomic identification of the pathogen. Damping off occurs in the T. chinensis production area from mid-May to late June every year. At the beginning, brown lesions were observed on the basal stem, then the lesions circumferential expanded and constricted, and finally resulted in the fall and death of T. chinensis seedlings. Pathogenic isolate was growing rapidly on the PDA medium, well developed aerial mycelia were grey white at first, then turned brown gradually, and a great number of small dark brown sclerotia were developed in the middle and periphery of the colony. Mycelial diameter of the pathogen was about 7 to 10 μm, near right angle or acute angle branches, near branches with septa, branches and septa with constriction. After the healthy T. chinensis seedlings were inoculated by pathogenic isolate, damping-off was observed soon, and the symptom was as same as those observed in the field. Through homogenous blast, the rDNA-ITS sequence of the pathogenic isolate shown 99.49% to 99.84% homology with Rhizoctonia solani, R. solani AG-1 IC mycelium anastomosis group and Thanatephorus cucumeris, the sexual type of Rhizoctonia. Furthermore, obvious mycelial anastomosis phenomena were observed when the pathogenic isolate and R. solani AG-1 IC strain were confronting cultured. Based on the results above, the pathogenic isolate causing damping off of T. chinensis was identified as R. solani AG-1 IC mycelial anastomosis group. RESULTS:: in the present work have important significance for further research on basic biology of the pathogen and integrated control of damping off causing by it on T. chinensis.

Biosynthesis of Silver Nanoparticles Using Onion Endophytic Bacterium and Its Antifungal Activity against Rice Pathogen Magnaporthe oryzae.

Biosynthesis of silver nanoparticles (AgNPs) using endophytic bacteria is a safe alternative to the traditional chemical method. The purpose of this research is to biosynthesize AgNPs using endophytic bacterium Bacillus endophyticus strain H3 isolated from onion. The biosynthesized AgNPs with sizes from 4.17 to 26.9 nm were confirmed and characterized by various physicochemical techniques such as Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), UV-visible spectroscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) in addition to an energy dispersive spectrum (EDS) profile. The biosynthesized AgNPs at a concentration of 40 μg/mL had a strong antifungal activity against rice blast pathogen Magnaporthe oryzae with an inhibition rate of 88% in mycelial diameter. Moreover, the biosynthesized AgNPs significantly inhibited spore germination and appressorium formation of M. oryzae. Additionally, microscopic observation showed that mycelia morphology was swollen and abnormal when dealing with AgNPs. Overall, the current study revealed that AgNPs could protect rice plants against fungal infections.

Evaluación in vitro de métodos contra Botrytis cinerea

Aunque se dispone de métodos experimentales para el control de enfermedades fúngicas en el cultivo de fresa, entre ellos el químico, orgánico y biológico, no se descarta otros métodos de bajo costo como sustancias homeopáticas, que no se han evaluado contra Botrytis cinerea. El objetivo del presente estudio fue comparar tratamientos convencionales y sustancias homeopáticas, en condiciones in vitro, en el crecimiento micelial de B. cinerea como referencia previa a evaluación in vivo.Tratamientos: medio PDA, fungicida sintético Switch® (Fludioxonil, Ciprodinil) 1 g L-1, aceite esencial de Tagetes lemmonii (1%), extracto comercial de Larrea tridentata (2 mL L-1), sustancias homeopáticas, arsénico 6 CH y nosode de Botrytis 7 CH y prueba dual con Trichoderma harzianum. Variables registradas: diámetro del micelio, velocidad de crecimiento y porcentaje de inhibición micelial; en la prueba dual se midió días al primer contacto hifal y tipo de antagonismo. Los datos se sometieron a análisis de varianza y prueba de comparación múltiple de medias (Tukey, p≤ 0.05). Switch® y aceite de Tagetes inhibieron el crecimiento micelial en 100%, con extracto de Larrea la inhibición fue de 65.8%, T. harzianum inhibió 36.2% y el contacto hifal se dio al sexto día (antagonismo tipo II), controlando al patógeno, las sustancias homeopáticas nosode 7 CH y arsénico 6 CH inhibieron al hongo en 31.2 y 11.8%, respectivamente. El efecto del nosode 7 CH fue diferente (p≤ 0.05) del testigo, pero el arsénico homeopático no lo fue.

Ultrasound enhanced production of mycelia and exopolysaccharide by Agaricus bitorquis (Quél.) Sacc. Chaidam.

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC), is a kind of rare edible macrofungi with a variety of biological ingredients, especially its polysaccharides. However, the low yield limits the popularity and promotion of rare edible macrofungi as well as its macrofungi polysaccharides. Hence, developing a positive and effective cultivation method is of great importance. Herein, an efficient ultrasonic (US) stimulation strategy was developed to improve mycelial growth and exopolysaccharides (EPS) biosynthesis from ABSC in submerged cultivation without light. A time design was employed to illustrate the effect of various process parameters including duration, starting point and times of US irradiation on ABSC productivity. 5min US treatment for once upon ABSC after fermentation for 48h could significantly improve EPS production and mycelia growth by above 26% and 15.03%, respectively. Furthermore, six times of 5min US treatment could make the amount of EPS reach 218.78±17.09mg/g, which was 2.52-fold higher than that of the control. Moreover, the enhanced effect induced by US was further expounded by fermentation kinetics. Besides, the US treatment could increase mycelia permeability, change structure and reduce mycelial diameter to promote mass transfer, resulting in the improvement of EPS production and mycelia accumulation. The results demonstrated that the present proposed US intensification approach could be useful to boost up the fermentation of ABSC, which possibly applied to yield increase and fermentation product acquisition of macrofungi.

Characterization of the Metabolic Response of Streptomyces clavuligerus to Shear Stress in Stirred Tanks and Single-Use 2D Rocking Motion Bioreactors for Clavulanic Acid Production.

Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ®) and stirred tank bioreactor (STR), respectively. The CELL-tainer® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer® the mycelial diameter increased considerably (average diameter 2.27 in CELL-tainer® vs. 1.44 µm in STR). This suggests that CA production may be affected by a lower surface-to-volume ratio which would lead to lower diffusion and transport of nutrients, oxygen, and product. The present study shows that there is a strong correlation between macromorphology and CA production, which should be an important aspect to consider in industrial production of CA.

The multivariate statistical selection of fungal strains isolated from Neoteredo reynei, with the high hydrolytic potential to deconstruct cellulose.

The aim of this study was to select isolated filamentous fungi, naturally occurring in the digestive tract of Neoteredo reynei, with high potential to hydrolyze cellulolytic biomass. The selection of the fungi strains, which produce cellulases, was performed by adding carboxymethylcellulose (CMC), microcrystalline cellulose (MCC) or glucose in the media containing peptone and yeast-extract. In this case, the glucose was used as a reference standard for the growth of the mycelia. The abilities of the fungal strains, to hydrolyze cellulose, on the solid media (MCC or CMC), were evaluated. Two methods were used: the congo-red and the speed of mycelial growth. The measurements of the diameters were performed at 24 h intervals, and the speed of the mycelial growth was calculated after 72 h of cultivation. The molecular and morphological identification of the fungi were applied to the isolated strains. Statistical analysis, including ANOVA and Tukey test, and multivariate analysis were applied as tools to select the strains. Twelve strains were isolated and the results of the identification were 2 strains of Hypocrea lutea, 2 strains of Trichoderma reesei, 2 strains of Aspergillus niger; 2 genera of Aspergillus sp., 2 genera of Trichoderma sp., 1 genus of Fusarium sp. and 1 genus of Gliocadium sp. The discrimination analysis methods such as HCA (Hierarchical Cluster Analysis) and PCA (Principal Component Analysis) indicated three strains with the highest potential to hydrolyze cellulose. In this study, the selection strategy was successful, resulting in the classification of strains from the genera Trichoderma and Hypocrea. This is the first time that this kind of studying was applied to select the potential of the cellulolytic fungal strains, isolated from N. reynei, using the methods of the growth of the mycelial diameter and the statistical discrimination.