Characterization and Evaluation of Metarhizium spp. (Metsch.) Sorokin Isolates for Their Temperature Tolerance

A field survey was done in teak (Tectona grandis F.) forests in South India to explore the entomopathogenic effect of Metarhizium anisopliae (Ascomycota: Sordariomycetes) against teak defoliator, Hyblaea puera (Lepidoptera: Hyblaeidae). About 300 soils and infected insect samples were collected during the survey and thirty-six fungal isolates were isolated from soil and insect samples and characterized. The fungi were cultured on PDAY with dodine and antibiotics. Generally, the EPF culture was incubated at 27 °C in darkness for 15 days. Virulence of the Entomopathogenic Fungi (EPF) ability to germinate under cold and heat temperatures was assessed in a culture impregnated with conidia. In the experiment, it was found that for the first time Metarhizium quizhouense, Metarhizium robertsii, and Metarhizium majus species caused significantly higher mortality to hosts. These isolates of M. anisopliae, M. robertsii, M. majus, and M. quizhouense were all considered to be effective virulent and environmentally adaptive. The Metarhizium isolates were recommended as effective bio-control agents through the field investigation of teak defoliator Hyblaea puera from South India forest. This study paves the way to utilize the indigenous isolates of EPF for the control of teak defoliator and to combat the pests thatare resistant to insecticide.

Efficacy of Beauveria bassiana and Beauveria pseudobassiana isolates against the pine processionary moth, Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera/Notodontidae)

Background The pine processionary moth, Thaumetopoea wilkinsoni Tams, 1926 (Lepidoptera/Notodontidae) is one of the most harmful insects that destroys pine ecosystems by feeding on pine leaves at its larval stage. Because of its urticating setae, the insect also causes severe skin reactions to animals and humans. Instead of chemical control, eco-friendly biological control methods are preferred to combat this species. Results The purpose of this study was to evaluate the efficacy of five different Beauveria bassiana Vuill, 1912 (Hypocreales/Cordycipitaceae) isolates (TR-SM-10, TR-SM-11, TR-SM-2, TR-SK-1 and TR-D-1) and one B. pseudobassiana Rehner & Humber (Hypocreales/Cordycipitaceae) isolate (TR-SM-1) against the fourth instar larvae of T. wilkinsoni under laboratory conditions. T. wilkinsoni larvae were collected from the Ondokuz Mayıs University Kurupelit Campus in Samsun, Turkey, in 2021, and the fourth instar larvae were used in the experiment. Two ml of spray of the six fungal isolates were applied to every ten larvae at each concentration (1 × 107 and 1 × 108 conidia ml−1). The experiment was carried out in five replicates per group, and the larvae were observed for 10 days. As a result, all isolates of B. bassiana caused 100% mortality at 1 × 108 conidia ml−1 concentration. B. pseudobassiana isolate also caused 100% mortality at both concentrations. At 1 × 107 conidia ml−1 concentration, the larvae treated with the B. pseudobassiana isolate (TR-SM-1) had the lowest LT50 (2.89 days) and LT90 values (4.79 days), while the larvae treated with TR-SM-10 isolate had the highest LT50 (5.65 days) and LT90 values (9.39 days). At 1 × 108 conidia ml−1 concentration, the larvae treated with TR-SK-1 isolate had the lowest LT50 (2.89 days) and LT90 values (4.79 days), while those treated with TR-SM-10 isolate had the highest LT50 (3.95 days) and LT90 values (8.15 days). Conclusion It has been recommended that the five different isolates of B. bassiana and B. pseudobassiana isolates were virulent to T. wilkinsoni larvae and can be used for biological control of T. wilkinsoni.

Screening and Identification of Indigenous Entomopathogenic Fungal Isolates from Agricultural Farmland Soils in Nile Delta, Egypt

The compound negative impact of insect pests attacking agricultural ecosystems includes (i) direct yield losses from damaged crops, (ii) the economic cost of the attempt to prevent these losses and (iii) the negative short- and long-term hazard effects of chemical pesticides on human and environmental health. Entomopathogenic fungi (EMPF) are a group of microorganisms that represent the natural enemies of a number of crop pests, presenting an opportunity to harness their evolutionary fine-tuned relationship with their insect hosts as biocontrol agents in integrated pest management programs. The aim of this study was to establish an indigenous EMPF collection via the Galleria mellonella (greater wax moth) entrapment method from the soils of Nile Delta, Egypt. Obtained insect associated fungal isolates were bio-assayed for pathogenicity against the serious pest Spodoptera litura and Tenebrio molitor, and the seven outperforming isolates were selected for molecular identification and thermotolerance assay. Based on ITS sequence analysis and phylogeny, selected isolates were identified as Beauveria bassiana (four isolates), Metarhizium anisopliae (two isolates) and one isolate of Cordyceps javanica. The obtained results demonstrated (i) the efficacy of using insect baiting coupled with molecular identification and pathogenicity screening to isolate EMPF to control insect pests, and (ii) the availability of indigenous virulent EMPF in Nile Delta’s soil, which can be exploited for the development of sustainable crop protection strategies.

POTENTIAL OF ENDOPHYTIC FUNGI DERIVING FROM ASIATIC PENNYWORTH TO PRODUCE ANTIOXIDANTS

<p>Asiatic pennyworth is a medicinal plant that contains triterpenoids, saponin, flavonoids, and tannins which possess antioxidants. Endophytic fungi from the plant could produce a similar compound; therefore, antioxidants could be made in the laboratory if the fungi are isolated. This study aimed to evaluate the potential of endophytic fungi isolated from Asiatic pennyworth to produce antioxidants. The study used 34 endophytic fungal isolates from Asiatic pennyworth accessions of Malaysia (17 isolates) and Bengkulu, Indonesia (17 isolates) collected by the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development. The fungi were propagated in a potato dextrose broth medium, then mycelia mats and filtrates were separated and then freeze-dried. The antioxidant activities were first tested with 1,1-diphenyl-2- picrylhydrazyl (DPPH) using thin layer chromatography (TLC), then UV-Vis spectrophotometry λ517 nm with five variations of concentration. Results showed all 34 fungal isolates have antioxidant activities based on a yellowish-white color change after applying 0.002% DPPH solution of the TLC method and IC50 value of the UV-Vis spectrophotometer. The highest antioxidant activity was shown by <em>Aspergillus austroafricanus </em>MB 1 (IC50 = 12.08 ppm) from Bengkulu accession and <em>A. oryzae </em>MM 13 (IC50 = 10.29 ppm) from Malaysia accession. <em>A. austroafricanus </em>MB 1 produced more antioxidant compounds (seven) than <em>A. oryzae </em>MM 13 (six). The antioxidant compounds produced by both endophytic fungi included in the group of flavonoids, fatty acids, and carboxylic acids. The research implies that <em>A. austroafricanus </em>MB 1 and <em>A. oryzae </em>MM 13 could be further developed as sources of antioxidants.</p>

Degradation of polystyrene by tropical bacterial and fungal isolates

Plastic waste is one of the major contributing factors to the growth of municipal solid wastes globally. It is a threat to public health and negatively impact the aquatic and terrestrial animals due to its persistence in the environment. Bacterial and fungal isolates were tested for their ability to degrade polystyrene. The cultures were grown on mineral salts medium supplemented with polystyrene as the sole carbon source in 250 mL conical flasks. The polystyrene reduced from 1 g (week 0) to 0.7g (week 7). Statistical analysis of variance (one-way ANOVA) shows a significant difference between the reduction in weight of polystyrene in the experimental flask compared to the control (P<0.05). Gas chromatography mass spectrometry analysis of biodegradation products shows that some toxic polystyrene constituents such as O-xylene, cis-9-hexadecenal and 3-phenol pentadecyl were removed.

Phytochemical and antimicrobial analyses of Plectranthus amboinicus leaf extracts

The present study is to evaluate the phytochemical constituents and antimicrobial activity of Plectranthus amboinicus leaf extract using petroleum ether, methanol and water as solvents. The antimicrobial activity of P. amboinicus leaf extracts were examined against the selected bacterial and fungal isolates namely Escherichia coli, Staphylococcus aureus and Aspergillus niger, Candida albicans using agar well diffusion method. FT –IR analysis was performed to identify the presence of functional groups in the methanol extract. The results of the study revealed the presence of distinctive active secondary metabolites in the tested leaf extract. The results revealed that the methanol extract exhibited maximum antimicrobial activity against the tested bacterial and fungal isolates when compared with aqueous and petroleum ether extracts. The FT-IR analysis reveals the presence of functional groups such as hydroxyl, amines, alkynes, ketones and carboxylic acid in the methanolic extract of P. amboinicus. Thus, P. amboinicus leaves are effective against the selected microbes and recommends that the plant derived phytochemicals are comparatively safer than synthetic alternative, thereby contributing insightful remedial benefits for the treatment of diseases.

Screening of halotolerant microfungi isolated from hypersaline soils of Algerian Sahara for production of hydrolytic enzymes

The Algerian Sahara contains numerous hypersaline ecosystems including salt lakes in which the fungal diversity has not been characterized. The abundance and diversity of soil microofungi in three salt lakes in southeastern Algeria was investigated together with their profiles of hydrolytic enzyme. Fungal population size and relative abundance were determined in about 75 soil samples by plate count. From 69 fungal isolates, 46.38% were Aspergillus, 20.29% were Penicillium and 11.59% belonging to Cladosporium genus. The sixty-nine isolates have been studied at different constant temperatures and salinities. All fungal isolates are halotolerant or halophiles with the ability to grow at 50°C. The screening for extracellular halophilic enzymes at 40°C showed that 69.57% of the isolates were able to produce at least two types of the screened enzymes. Protease was the most abundant enzyme detected in 60.87% of the total isolates. The results obtained of all the growth tests indicate the adaptability of fungal isolates tested to the extreme conditions and their possible utilisation as producers of halophilic-active hydrolytic enzymes.

Morphological and Molecular Analysis of Fungal Species Associated with Blast and Brown Spot Diseases of Oryzae sativa

Fungal diseases; blast, and brown spot in rice incur severe yield losses worldwide. Blast is caused by Magnaporthe oryzae, while Bipolaris oryzae is reported as the main causal organism of brown spot. Both diseases cause leaf lesions which are difficult to be differentiated by symptomatology until the late stages. Early detection and differentiation of the lesions would help the adoption of disease management strategies specific to the pathogen and will prevent the native impact on the quality and quantity of rice yields. This study was conducted in the Northern Province of Sri Lanka over five consecutive rice cultivating seasons to characterize the causal fungi of rice blast and brown spot diseases by morphological and molecular means and to develop a visual guide to differentiate the two diseases. Disease incidence was recorded in 114 fields from 2017 to 2019, and fungal isolates associated with lesions of both the diseases were cultured and subjected to morphological and molecular characterization. Competitive growth interaction between M. oryzae and the more common individual fungal isolates of the brown spot lesions, was evaluated. Fungal metagenomics analysis was conducted for the fungal spp. isolated from brown spot lesions. A suppression of blast accompanied by an increased incidence of brown spot disease was observed during the study period. M. oryzae was confirmed to be the causal organism of the blast while over 20 species of fungi were identified to be associated with brown spot lesions through morphological, molecular studies, and metagenomics analyses. Fungal ITS region sequencing revealed considerable genetic variation in the highly conserved region of DNA sequences of blast and brown spot fungal isolates. B. oryzae, Curvularia, and Microdochium species were commonly isolated from brown spot lesions. In vitro competitive growth interaction among the fungal isolates revealed growth suppression of M. oryzae by the fungal isolates associated with the brown spot lesions. Similarly, it can be speculated that the abundance and severity of blast in the field may have an influence on brown spot associated fungi. A simple visual guide was developed to differentiate blast and brown spot lesions. The findings would be highly useful in the timely management of these major fungal diseases affecting rice.

First report of Fusarium oxysporum f. sp. cubense Tropical Race 4, causing Fusarium wilt in Cavendish bananas in Peru

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), the causal agent of Fusarium wilt of banana (FWB), is currently the major threat to the banana industry worldwide (Dita et al. 2018). Restricted to South Asia for more than 20 years, Foc TR4 has spread in the last years to the Middle East, Mozambique, and Colombia (García-Bastidas et al. 2019; https://pestdisplace.org/embed/news/map/disease/11). The incursion of Foc TR4 in Colombia increased awareness and prevention efforts across Latin American and the Caribbean (LAC). However, new Foc TR4 outbreaks in LAC countries were considered a matter of time. In April 2021, banana (Musa spp., Cavendish, AAA) plants (30% of incidence) showing typical symptoms of FWB, such as leave yellowing, wilting and vascular discoloration were observed in one farm (about 1 ha) located in Querecotillo, Peru (4°43’54.84”S 80°33’45.00”W). Mycological analyses of samples (pseudostem strands) collected from 10 symptomatic plants were performed as described by Dita et al. (2010). These analyses revealed a continuous presence of fungal colonies identified as Fusarium oxysporum species complex. Molecular diagnostics targeting two different genome regions (Dita et al 2010; Li et al. 2013) identified nine of these isolates as Foc TR4. These results were further confirmed by qPCR analyses using the commercial Clear Detections TR4 kit. The genome of four single-spore isolates (PerS1, PerS2, PerS3 and PerS4) was sequenced using the Illumina platform (MiSeq Kit, 2x151 bp Paired-End). The strain PerS4 was also sequenced using Oxford Nanopore (FLOW-MIN111; R10.3 chemistry) as described by Lopez-Alvarez et al., (2020). The generated draft assembly yielded 533 contigs for a size of 47 Mbp (BioProject: PRJNA755905), which is comparable with sizes of previously reported Foc TR4 strains (Asai et al. 2019; García-Bastidas et al. 2019; Maymon et al. 2020; Warmington et al. 2019; Zheng et al. 2018). The sequence assembly showed high contiguity (94.9%) and high similarity (95.48%) with the high-quality genome sequence of the Foc TR4 isolate ‘UK0001’ (Warmington et al. 2019). Further analyses to identify the presence/absence of full sequences for the putative effector genes (Secreted In Xylem - SIX) and their allelic copies, also revealed that the SIX genes profile of the strains isolated from Querecotillo matched with previously reported Foc TR4 isolates (Czislowski et al. 2017). Pathogenicity tests with three isolates and water controls were performed as described by Dita et al. (2010), using five Cavendish plantlets per treatment. Four weeks after the inoculation typical external and internal symptoms of FWB were observed only in the inoculated plants. Fungal isolates recovered from inoculated plants tested positive for Foc TR4 when analyzed with PCR diagnostics as mentioned above. No fungal isolates were recovered from water-control plants which did not show any symptoms. Altogether, our results confirm the first incursion of Foc TR4 in Peru. Currently, Foc TR4 has the phytosanitary status of a present pest with restricted distribution in Peru and it is under official control of the National Plant Protection Organization – SENASA. Reinforced prevention and quarantine measures, disease monitoring and capacity building to detect, contain and manage eventual new outbreaks of Foc TR4 are strongly encouraged across LAC banana producing countries, especially for those bordering Peru with larger banana plantations, such as Ecuador and Brazil.