Identification of locally isolated entomopathogenic Fusarium species from the soil of Changa Manga Forest, Pakistan and evaluation of their larvicidal efficacy against Aedes aegypti

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases. Current reliance of dengue vector control is mostly on chemical insecticides. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control through chemical insecticides. These chemical insecticides also have negative environmental impacts on animals, plants and human health. Myco-biocontrol agents are naturally occurring organisms and are found to be less damaging to the environment as compared to chemical insecticides. In the present study, entomopathogenic potential of local strains of fungi isolated from soil was assessed for the control of dengue vector. Local fungal isolates presents better alternative to introducing a foreign biocontrol strain, as they may be better adapted to environmental conditions of the area to survive and may have more entomopathogenic efficacy against target organism. Larvicidal efficacy of Fusarium equiseti and Fusarium proliferatum was evaluated against Aedes aegypti. Local strains of F. equiseti (MK371718) and F. proliferatum (MK371715) were isolated from the soil of Changa Manga Forest, Pakistan by using insect bait method. Larvicidal activity of two Fusarium spp. was tested against forth instar larvae of A. aegypti in the laboratory, using concentrations 105, 106, 107 and 108 conidia /ml. LC50 values for F. equiseti after 24h, 48h, 72h and 96h of exposure were recorded as 3.8x 108, 2.9x107, 2.0x107, and 7.1x106 conidia /ml respectively while LC50 values for F. proliferatum were recorded as 1.21x108, 9.6x107, 4.2x107, 2.6x107 conidia /ml respectively after 24h, 48h, 72h and 96h of exposure. The results indicate that among two fungal strains F. equiseti was found to be more effective in terms of its larvicidal activity than F. proliferatum against larvae of A. aegypti.

Taqman qPCR Quantification and Fusarium Community Analysis to Evaluate Toxigenic Fungi in Cereals

Fusarium head blight (FHB) is an economically important plant disease. Some Fusarium species produce mycotoxins that cause food safety concerns for both humans and animals. One especially important mycotoxin-producing fungus causing FHB is Fusarium graminearum. However, Fusarium species form a disease complex where different Fusarium species co-occur in the infected cereals. Effective management strategies for FHB are needed. Development of the management tools requires information about the diversity and abundance of the whole Fusarium community. Molecular quantification assays for detecting individual Fusarium species and subgroups exist, but a method for the detection and quantification of the whole Fusarium group is still lacking. In this study, a new TaqMan-based qPCR method (FusE) targeting the Fusarium-specific elongation factor region (EF1α) was developed for the detection and quantification of Fusarium spp. The FusE method was proven as a sensitive method with a detection limit of 1 pg of Fusarium DNA. Fusarium abundance results from oat samples correlated significantly with deoxynivalenol (DON) toxin content. In addition, the whole Fusarium community in Finnish oat samples was characterized with a new metabarcoding method. A shift from F. culmorum to F. graminearum in FHB-infected oats has been detected in Europe, and the results of this study confirm that. These new molecular methods can be applied in the assessment of the Fusarium community and mycotoxin risk in cereals. Knowledge gained from the Fusarium community analyses can be applied in developing and selecting effective management strategies for FHB.

Development of PCR-Based Race-Specific Markers for Differentiation of Indian Fusarium oxysporum f. sp. cubense, the Causal Agent of Fusarium Wilt in Banana

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish group of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races were isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genome sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The putative lineage-specific identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and non-pathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL−1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which are important for the effective management of the disease.

Revealing the diversity of Fusarium micromycetes in agroecosystems of the North Caucasus plains for replenishing the State Collection of Phytopathogenic Microorganisms of the All-Russian Scientific Research Institute of a Phytopathology

In order to prevent crop yield losses from the most dangerous and economically important pathogenic organisms, it is necessary not only to monitor the virulence gene pool, but also to study the nature of pathogen variability and determine the potential for the emergence of new genes and races. This requires centralized collections of fungal cultures characterized by a set of stable strains to provide for phytopathological, immunological, breeding, genetic, toxicological, parasitological and other studies. The State Collection of Phytopathogenic Microorganisms of the ARSRIP is the State Depository of Phytopathogenic microorganisms that are non-pathogenic to humans or farmed animals. Currently, it has more than 4,500 accessions of plant pathogenic strains of fungi, oomycetes, bacteria, viruses, phytoplasmas, and the collection is updated annually. For this purpose, the study of the inter- and intraspecific genetic diversity of genus Fusarium was carried out in agricultural systems of the Krasnodar Territory. In 2020, the State Collection of Phytopathogenic Microorganisms was supplemented with 13 strains of Fusarium fungi isolated from tissues of winter wheat plants collected in several locations of the Krasnodar region. The complex of Fusarium fungi revealed on winter wheat usually included Fusarium oxysporum, F. culmorum, F. lolii, F. graminearum, F. fujikuroi, F. sporotrichioides, etc. The effect of the preceding crop on the frequency of Fusarium species isolated from winter wheat was observed. After series cloning of collected isolates, 21 strains of different fungal species characterized by stable morphology traits and known pathogenic and phytotoxic properties were selected for collection replenishment. Significant differences in pathogenic activity were revealed between fungi belonging to either the same or different species; the manifestation of this activity varied from the absence of any effect of spore suspensions on seedling development to a complete inhibition of their growth. The phytotoxic activity towards wheat seedlings varied from medium to high. Species possessing a high intensity of phytotoxic activities are the most dangerous for wheat, since they promote accumulation of dangerous phytotoxins in plant tissues.

Epimerization of Deoxynivalenol by the Devosia Strain A6-243 Assisted by Pyrroloquinoline Quinone

Deoxynivalenol (DON) is a secondary metabolite produced by several Fusarium species that is hazardous to humans and animals after entering food chains. In this study, by adding cofactors, the Devosia strain A6-243 is identified as the DON-transforming bacteria from a bacterial consortium with the ability to biotransform DON of Pseudomonas sp. B6-24 and Devosia strain A6-243, and its effect on the biotransformation process of DON is studied. The Devosia strain A6-243 completely biotransformed 100 μg/mL of DON with the assistance of the exogenous addition of PQQ (pyrroloquinoline quinone) within 48 h and produced non-toxic 3-epi-DON (3-epi-deoxynivalenol), while Pseudomonas sp. B6-24 was not able to biotransform DON, but it had the ability to generate PQQ. Moreover, the Devosia strain A6-243 not only degraded DON, but also exhibited the ability to degrade 3-keto-DON (3-keto-deoxynivalenol) with the same product 3-epi-DON, indicating that DON epimerization by the Devosia strain A6-243 is a two-step enzymatic reaction. The most suitable conditions for the biodegradation process of the Devosia strain A6-243 were a temperature of 16–37 °C and pH 7.0–10, with 15–30 μM PQQ. In addition, the Devosia strain A6-243 was found to completely remove DON (6.7 μg/g) from DON-contaminated wheat. The results presented a reference for screening microorganisms with the ability of biotransform DON and laid a foundation for the development of enzymes for the detoxification of mycotoxins in grain and its products.

Volatile Organic Compound Profile Fingerprints Using DART–MS Shows Species-Specific Patterns in Fusarium Mycotoxin Producing Fungi

Fungal volatile organic compounds (VOCs) are low-molecular weight fungal metabolites that have high vapor pressure at ambient temperatures and can function as airborne signals. Here, we report a VOC study of several different species of Fusarium. Direct analysis in real time mass spectrometry (DART–MS) was applied for non-invasive VOC fingerprinting of Fusarium isolates growing under standardized conditions. A large number of ions were detected from the headspaces of the Fusarium species sampled here. Ions were detected with distinctively high concentrations in some species. While there were few VOCs produced by only one species, the relative concentrations of VOCs differed between species. The methodology has potential for convenient detection and identification of Fusarium contamination in agricultural commodities.

The occurrence of Fusarium species in white spears of Asparagus officinalis

The aim of the study was to determine the species composition of fungi of the Fusarium genus found on white Asparagus officinalis spears, depending on the presence or absence of disease symptoms, age of the plantation, date of harvest and the place from which samples were collected for mycological analysis. Additionally, the pathogenicity of selected Fusarium spp. isolates was determined. A. officinalis L. was cultivated for white spears. The research was conducted on the German dioecious cultivar ‘Eposs’. Samples of A. officinalis spears for tests were collected from two plantations. Six species of fungi of the Fusarium genus were identified in the asparagus spears: F. culmorum, F. equiseti, F. oxysporum, F. proliferatum, F. solani, and F. fujikuroi. Among the Fusarium species colonizing Asparagus officinalis the greatest threat is F. oxysporum. Always there are more isolates in the spears with the symptoms, in epidermis. The late harvest date favors the development of fusariosis. This means that the spears harvested at the latest date (late June) are the most heavily colonised by fungi. The isolates of fungi of the Fusarium genus collected from the spears exhibit pathogenicity against A. officinalis plants.

4-O-Glucosylation of Trichothecenes by Fusarium Species: A Phase II Xenobiotic Metabolism for t-Type Trichothecene Producers

The t-type trichothecene producers Fusarium sporotrichioides and Fusarium graminearum protect themselves against their own mycotoxins by acetylating the C-3 hydroxy group with Tri101p acetylase. To understand the mechanism by which they deal with exogenously added d-type trichothecenes, the Δtri5 mutants expressing all but the first trichothecene pathway enzymes were fed with trichodermol (TDmol), trichothecolone (TCC), 8-deoxytrichothecin, and trichothecin. LC-MS/MS and NMR analyses showed that these C-3 unoxygenated trichothecenes were conjugated with glucose at C-4 by α-glucosidic linkage. As t-type trichothecenes are readily incorporated into the biosynthetic pathway following the C-3 acetylation, the mycotoxins were fed to the ΔFgtri5ΔFgtri101 mutant to examine their fate. LC-MS/MS and NMR analyses demonstrated that the mutant conjugated glucose at C-4 of HT-2 toxin (HT-2) by α-glucosidic linkage, while the ΔFgtri5 mutant metabolized HT-2 to 3-acetyl HT-2 toxin and T-2 toxin. The 4-O-glucosylation of exogenously added t-type trichothecenes appears to be a general response of the ΔFgtri5ΔFgtri101 mutant, as nivalenol and its acetylated derivatives appeared to be conjugated with hexose to some extent. The toxicities of 4-O-glucosides of TDmol, TCC, and HT-2 were much weaker than their corresponding aglycons, suggesting that 4-O-glucosylation serves as a phase II xenobiotic metabolism for t-type trichothecene producers.