Providing inoculum for Didymella bryoniae studies: the effect of light spectrum and storing at low temperature

The in vitro sporulation of Didymella bryoniae is of great importance for studies that require pure inoculum and in large quantities. Thus, the objectives of this study were to identify the best condition for D. bryoniae sporulation combining different light spectra (UV-A or UV-B light, white light, and continuous dark), with distinct culture media (PDA, V8, ML, and PDAB) and, to evaluate fungus’ survivability stored at -20°C over time. The fungus samples were only able to sporulate when subjected to the UV-B light treatment, regardless of the culture medium. The highest appearance of spores conidium type was observed in the PDAB medium, and the lowest production occurred in the ML medium. Reproductive structures, such as perithecia and pycnidia, were observed in all culture media. However, there was considerable variation in the amount of each structure between the different culture media. The ML and V8 media showed a greater number of perithecia and the PDA and PDAB media presented a greater proportion of pycnidia compared to perithecia. The storage duration at -20°C did not affect mycelial growth or mycelial growth rate. In conclusion, the UV-B light is essential for D. bryoniae in vitro sporulation. Moreover, the culture medium composition influences the type of fungal structure produced, as well as spores’ size and quantity. Freezing at -20°C is an efficient technique that can be used to store D. bryoniae for at least five months without loss of viability.

Variability in phosphite sensitivity observed within and between seven Phytophthora species

Phosphite is used to control and manage many phytophthora diseases in horticultural systems worldwide and natural ecosystems in Australia, Africa, New Zealand and parts of Northern America and Europe. Phosphite does not kill Phytophthora species, but inhibits growth while also stimulating host defence responses. Phytophthora species differ in their underlying tolerance to phosphite and isolates have been shown to acquire tolerance after prolonged exposure. Intra- and inter-specific variability in phosphite sensitivity is of interest to determine the efficacy and sustainability of phosphite for the treatment of phytophthora diseases, which continue to spread globally. Seven Phytophthora species were tested for their sensitivity to phosphite in vitro in a mycelial growth experiment. Phytophthora agathidicida was the species most sensitive to phosphite, being inhibited by 98.7% on average at the lowest phosphite treatment (15 µg/mL phosphite), followed by P. aleatoria, P. cinnamomi, P. pluvialis, P. multivora, P. kernoviae and P. citricola. Huge intraspecific variability was observed with P. kernoviae, which raises the question of whether diseases caused by P. kernoviae such as phytophthora needle blight of Pinus radiata could be managed effectively with phosphite. Further work is required to determine the phosphite sensitivity of different introduced and native Phytophthora species growing in key hosts and whether tolerance observed in vitro is also expressed in vivo.

Insecticidal and Antifungal Activities of Chemically-Characterized Essential Oils from the Leaves of Withania frutescens L.

The current study was conducted to investigate antifungal and insecticidal activities of essential oil extracted from the Moroccan Withania frutescens L. (EOW), and their chemical composition was profiled. To achieve this goal, EOW was extracted by the hydro-distillation method and their phytochemical constituents were characterized by gas chromatography-mass spectrometry analyses (GC-MS). Insecticidal activity was evaluated by use of four tests: contact toxicity, inhalation toxicity, and repellent tests. Antifungal activity was evaluated on Fusarium oxysporum f. sp. Ciceris (F. oxysporum) using different concentrations of EOW. GC/MS analysis revealed that EOW was rich in carvacrol (31.87%), thymol (30.08%), and camphor (9.13%). At a 1-µL/L dose, EOW exhibited mortality rates of 23.13 ± 1.07% and 24.41 ± 1.21% against Callosobruchus maculatus (C.maculatus) by inhalation and contact, respectively. Notably, EOW dose of 20 μL/L caused significant mortality rates of 95.1 ± 3.5% and 76.69 ± 1.71% by inhalation and contact, respectively. EOW exhibited an inhibitory effect on mycelial growth against the tested fungi F. oxysporum of 100% and 93.5 ± 1.1% for the 9 and 4.5 mg/mL doses, respectively. The reduced mycelial growth rate for F. oxysporum was recorded to be 0.3 ± 0.1 and 0.6 ± 0.1 mm/h for the EOW doses of 2.25 and 4.5 mg/mL, respectively. The outcome of the present work showed that EOW has a promising antifungal and insecticidal activity, and it can therefore be employed as a natural alternative insecticidal and mycocidal agent to replace the chemically-synthesized ones.

Molecular characterization and vegetative growth of pathogenic seed-borne fungus, Curvularia lunata of tomato and its in vitro control measures

Present studies were conducted to isolate and identify the seed-borne pathogenic fungus from the selected tomato variety through morphological and molecular techniques based on the sequencing of internal transcribed spacer (ITS) region of 18S rDNA. According to the colony and conidial features, the fungus was identified as Curvularia sp. The obtained ITS sequencing showed above 99% similarity with Curvularia lunata in the NCBI database. The sequence of the fungus was deposited in NCBI GenBank under the accession number: ITS, MH382879.1. Besides, the phylogenetic tree further confirmed the taxonomic position of the studied fungus. Growth characteristics of the fungus on nine different fungal culture media were evaluated, in which Honey peptone agar, Carrot agar, Potato sucrose agar, and Kauffman’s agar were found the most suitable. The maximum vegetative growth of the fungus was recorded at 30°C temperature and pH conditions. The bio-control potential of five different antagonists against the studied fungus was assessed, in which Trichoderma harzianum showed the better performance to restrict mycelial growth. Three ethanolic plant extracts were also evaluated, in which Lowsonia inermis L. exhibited above 60% mycelial growth inhibition of the fungus. Among three tested fungicides, Tilt 250 EC was found as an excellent fungicide to inhibit mycelial growth of C. lunata under in vitro conditions. Int. J. Agril. Res. Innov. Tech. 11(2): 124-132, Dec 2021

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density

Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi to apply the methods used for unicellular organisms such as yeast and bacteria.

Response to Fungicides of Colletotrichum spp. Isolated from Red Peppers in Sunchang, Korea

In August 2020, 377 anthracnose strains were isolated from anthracnose-infected peppers collected from 25 farms in Sunchang-gun, Jeollabuk-do. Inhibition rate of mycelial growth of 11 pepper anthracnose fungicides registered in Korea was investigated for 62 strains selected by region and the degree of susceptibility to each fungicide was investigated. As a result of the fungicide susceptibility test of anthracnose to the fungicide, no resistant strains were observed in fluazinam, prochloraz manganese, and benomyl, but resistant strains appeared in at least three areas for other fungicides, and all strains in all regions were resistant to dithianon. Through this, it was confirmed that the fungicide resistance was expressed in the strain group due to the continuous treatment of the fungicide in some areas. By region, resistant strains to seven pesticides appeared in Sunchang-eup and Paldeok-myeon, and resistant strains to six pesticides appeared in Geumgwa-myeon, Bokheung-myeon, Ssangchi-myeon, Yudeung-myeon, and Pungsan-myeon. There was no significant correlation between fungicide registration time and emergence of resistant strains.

ANTIFUNGAL ACTIVITY OF CHITOSAN AGAINST Rhizopus stolonifer

These days instead of chemical pesticides, various natural alternatives have been used for the management of post-harvest diseases. This research was carried out to evaluate the antifungal potential of different chitosan concentrations viz., 5, 10, and 15 mg/µl against Rhizopus stolonifer by the agar dilution and well diffusion methods. The evaluation was carried out after seven days of incubation by measuring the inhibition of R. stolonifer mycelial growth. Results of the study revealed that the chitosan had a significant inhibitory effect on mycelial growth and maximum mycelial growth inhibition was reported at the 10 and 15 mg/µl concentrations and these two treatments are not significantly different. Based on these results, the best concentration (15 mg/µl) was further evaluated by the well diffusion technique. The average inhibition zones formed by the higher concentration was 25mm. Results of the study suggested that chitosan can be used for the management of post-harvesting diseases of tomatoes.