Biological and proteomic analysis of a new isolate of the nematophagous fungus lecanicillium sp.

BACKGROUND: In our continuing search for biologically active natural enemies from North of Africa with special reference to Tunisian fungi, our teamwork screened fungi from different ecological habitats in Tunisia. Our previous study on the comparative effectiveness of filamentous fungi in the biocontrol of Meloidogyne javanica, a taxon (Lecanicillium) showed high potentiality against M. javanica. We undertook the present study to evaluate the ability and understand the mechanism of this fungal parasite as a biological control candidate against the root-knot nematode M. javanica. This study used in vitro bioassays with fungal filtrate cultures, scanning electron microscopy (SEM) observation, and isobaric tag for relative and absolute quantitation (iTRAQ) methodology to characterize the biological and molecular features of this fungus. RESULTS: The microscopic and SEM observation revealed that Lecanicillium sp. exhibited exceptional hyperparasitism against M. javanica eggs. The hyphae of this fungi penetrated the eggs, causing destructive damage to the outer eggshell. The exposure to five concentrations of Lecanicillium sp. filtrate cultures showed high inhibition of egg hatching, which increases depending on the exposure time; the best results are recorded at 50%, 75%, and 100% dilutions after seven days of exposure. The SEM observation of nematode-parasitized eggs and juveniles suggests that the production of lytic enzymes degrades the egg cuticle and fungal hyphae penetrate unhatched M.javanica juveniles. Forty-seven unique proteins were identified from the Lecanicillium sp. isolate. These proteins have signalling and stress response functions, bioenergy, metabolism, and protein synthesis and degradation. CONCLUSION: Collectively, Lecanicillium sp. had ovicidal potentiality proved by SEM and proteomic analysis against root-knot nematode' eggs. This study recommended applying this biological control candidate as a bio-agent on vegetable crops grown in situ.

Electricity generation and oxidoreductase potential during dye discoloration by laccase-producing Ganoderma gibbosum in fungal fuel cell.

Color chemicals contaminate pure water constantly discharged from different points and non-point sources. Physical and chemical techniques have certain limitations and complexities for bioenergy production, which motivated the search for a novel sustainable production approaches during dye wastewater treatment. The emerging environmental problem of dye decolorization has attracted scientist's attention to a new, cheap, and economical way to treat dye wastewater and power production via fungal fuel cells. Ganoderma gibbosum was fitted in the cathodic region with laccase secretion in the fuel cell. At the same time, dye water was placed in the anodic region to move electrons and produce power. This study treated wastewater using the oxidoreductase enzymes released extracellularly from Ganoderma gibbosum for dye Remazol Brilliant Blue R (RBBR) degradation via fungal-based fuel cell. The maximum power density of 14.18 mW/m2 and the maximum current density of 35 mA/m2 were shown by the concentration of 5 ppm during maximum laccase activity and decolorization of RBBR. The laccase catalysts have gained considerable attention because of eco-friendly and alternative easy handling approaches to chemical methods. Fungal Fuel Cells (FFCs) are efficiently used in dye treatment and electricity production. This article also highlighted the construction of fungal catalytic cells and the enzymatic performance of fungal species in energy production during dye water treatment.

The aggregation pheromone phenylacetonitrile: Joint action with the entomopathogenic fungus Metarhizium anisopliae var. acridum and physiological and transcriptomic effects on Schistocerca gregaria nymphs.

The combined use of entomopathogenic fungi and sublethal rate of chemical insecticides or other biological control agents have been proposed as an environmentally and sustainable strategy in the control of locust pests. In this paper, the quarter and the half of the recommended dose of Metarhizium anisopliae var. acridum (» and ½ Ma) and the aggregation pheromone (Phenylacetonitrile: PAN) were applied simultaneously and sequentially to Schistocerca gregaria fifth-instar nymphs. In addition, the physiological effects of PAN on locusts were assessed at the behavior, immune response, and biochemical level by evaluating for glutathione-S-transferase (GST), acetylcholinesterase inhibition (AChE), and malondialdehyde accumulation (MDA). Results showed that simultaneous application of PAN and the entomopathogenic fungus exhibited additive interaction. Synergistic interaction was also demonstrated when nymphs were exposed to PAN first, then treated with M. anisopliae var. acridum. Behavioral bioassay revealed that fifth-instar nymphs avoided the PAN odour and tended to remain away from the stimulus cup. In the choice assay, the pheromone significantly repelled the locusts at 2, 4, and 6 h of exposure which selected the PAN-free arena chamber. Moreover, treated nymphs become hyperactive and disoriented as evidenced by the cumulative distance travelled and the trajectory of locusts during the experiment. Immunological studies showed that PAN significantly decreased the differential haemocyte counts (prohemocytes and plasmatocytes) with a dose-response relationship. Data of biochemical analyzes showed that the PAN exposure reduced the activity of acetylcholinesterase and induced significantly the glutathione S-transferases and MDA concentration in the desert locust fifth-instar nymphs. Moreover, transcriptomic responses to the PAN exposure were evaluated using gene expression levels of CYP540 and GST. The transcript levels showed an up-regulation in GST expression level particularly in nymphs exposed for 4 and 6 h. A significant increase in CYP450 transcript level was also observed after 2 h of exposure, which decreased significantly after 4 and 6 h.

First report of Pratylenchus vulnus associated with apple in Tunisia.

The root-lesion nematode of the genus Pratylenchus Filipjev (1936) has a worldwide distribution and cause severe production constraints on numerous important crops. In 2013-14, during a survey of the apple nurseries and orchards in center of Tunisia (Kairouan, Zaghouan, Monastir and Kasserine), 70 different roots and soil samples were collected. The populations of root-lesion nematode were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observation of females and males demonstrated the occurrence of Pratylenchusd vulnus on apple trees. The ribosomal DNA D2-D3 expansion segments of the 28S rRNA and of the Pratylenchus populations were PCR amplified and sequenced. The sequences were compared with those of Pratylenchus species in the GenBank database with high similarity (99%). This comparison reconfirmed the morphological identifications. Phylogenetic studies placed those populations with P. vulnus. This is the first report of P. vulnus infecting apple in Tunisia.The root-lesion nematode of the genus Pratylenchus Filipjev (1936) has a worldwide distribution and cause severe production constraints on numerous important crops. In 2013-14, during a survey of the apple nurseries and orchards in center of Tunisia (Kairouan, Zaghouan, Monastir and Kasserine), 70 different roots and soil samples were collected. The populations of root-lesion nematode were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observation of females and males demonstrated the occurrence of Pratylenchusd vulnus on apple trees. The ribosomal DNA D2-D3 expansion segments of the 28S rRNA and of the Pratylenchus populations were PCR amplified and sequenced. The sequences were compared with those of Pratylenchus species in the GenBank database with high similarity (99%). This comparison reconfirmed the morphological identifications. Phylogenetic studies placed those populations with P. vulnus. This is the first report of P. vulnus infecting apple in Tunisia.

Phytochemical characterization of forest leaves extracts and application to control apple postharvest diseases.

The study investigated the antifungal and phytochemical properties of three forest plants (Eucalyptus globulus, Pistacia lentiscus, and Juniperus phoenicea) against apple diseases caused by Colletotrichum gloeosporioides and Alternaria alternata. The determination of the total polyphenol and flavonoid contents in the three aqueous extracts of studied plants showed that E. globulus exhibited the highest contents than those of P. lentiscus and J. phoenicea. Furthermore, the three studied extracts showed very appreciable antioxidant activity with decreasing order: E. globulus, P. lentiscus, and J. phoenicea. The phytochemical analysis showed different common phenolic acids in the three studied plants namely: quinic acid, gallic acid, chlorogenic acid, and caffeoylquinic acid as well as other flavonoids mainly quercetin and catechin. The results of the current study demonstrated that the fungistatic activity of E. globulus EO (4 and 2 µl/ml) seemed to be the most effective under laboratory conditions with an inhibition zone diameter above 16 mm. However, the poisoned food technique indicated that the aqueous extract (80%) and the essential oil (4 µl/ml) of E. globulus exhibited the highest mycelial growth (> 67%) and spore germination (> 99%) inhibition. Preventive treatments with essential oils (4 µl/ml) and aqueous extracts (80%) applied to apple fruits inoculated with A. alternata and C. gloeosporioides resulted in the lowest lesion diameter (< 6.80 mm) and disease severity index (< 15%) and the most favorable inhibitory growth (> 85.45%) and protective potentials (> 84.92%). The results suggest that E. globulus has a brilliant future in the management of anthracnose and Alternaria rot of apple and provide a basis for further studies on its effects under field conditions.

Comparative Effectiveness of Filamentous Fungi in Biocontrol of Meloidogyne javanica and Activated Defense Mechanisms on Tomato.

The nematicidal potential of five filamentous fungi as biological control agents (BCAs) against the root-knot nematode (RKN), Meloidogyne javanica, infecting tomato was assessed in vitro and in pot experiments. The five promising native taxa, namely Trichoderma longibrachiatum, T. harzainum, T. asperellum, Lecanicillium spp., and Metacordyceps chlamydosporia, were selected to compare their effectiveness against both chemical (Mocap, 10% ethoprophos) and biological (abamectin) nematicides on M. javanica reproduction indices and plant growth parameters. The stimulation of defense mechanisms was assessed by monitoring changes in the enzymatic activities of the polyphenol oxidase (PPO), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), lipid peroxidation (MDA), phenols, and proteins content of tomato roots. The laboratory assays revealed that T. longibrachiatum, M. chlamydoporia, and Lecanicillium spp. seemed to be the most effective under laboratory conditions, with more than 60% of juvenile mortality. The egg infection rate was above 62%, and the egg hatching rate was below 32%. The direct parasitism by the five taxa was confirmed by scanning electron microscope observation. The results of this study found a similar parasitism mechanism for T. longibrachiatum, T. harzianum, and M. chlamydosporia, where their hyphae and spores adhered to the M. javanica juveniles cuticle layer and formed trapping rings around them. The pot experiment results showed that T. harzianum and Lecanicillium spp. enhanced the plant growth parameters. Trichoderma longibrachiatum, abamectin, and the ethoprophos-based nematicides effectively decreased the reproduction rates of the nematode. The Trichoderma species and M. chlamydosporia significantly reduced the gall index and female fecundity of RKN. The treatment with BCAs and chemical nematicides involved a significant increase in the antioxidant activities of nematode-infected plants. The ethoprophos and fungal treatments decreased the MDA and total phenols content compared with the nematode-infested seedlings. This paper analyzes the advancements made towards the effective and efficient biocontrol of M. javanica using different fungal taxa, especially T. longibrachiatum and M. chlamydosporia, and the implications of these advancements for sustainable agriculture and food security.

Biological control of wilt disease complex on tomato crop caused by Meloidogyne javanica and Fusarium oxysporum f.sp. lycopersici by Verticillium leptobactrum.

The efficacy of Verticillium leptobactrum isolate (HR1) was evaluated in the control of root-knot nematode and Fusarium wilt fungus under laboratory and greenhouse conditions. Five concentrations of V. leptobactrum (HR1) isolate were tested for their nematicidal and fungicidal activities against Meloidogyne javanica and Fusarium oxysporum f.sp. lycopersici in vitro. Laboratory trials showed that mycelium growth inhibition of Fusarium wilt fungus was correlated to the increase of the concentration of culture filtrate. All dilutions showed efficiency in reducing the growth of Fusarium oxysporum f.sp. lycopersici. The greatest nematicidal activity was observed at 50, 75, and 100% filtrate dilutions. The egg hatching percentage reached 42%, and the juvenile's corrected mortality registered 90% for the above treatments. In greenhouse experiment, the biocontrol agent fungus enhanced significantly tomato growth components (height and weight of plant and root). The multiplication rate of root-knot nematode and the Fusarium wilt disease incidence declined significantly with soil application of V. leptobactrum as with chemical treatments. The isolate HR1 was efficient to control wilt disease complex caused by M. javanica and Fusarium oxysporum f.sp. lycopersici.

Biological control of Meloidogyne javanica on tomato with Dazitol® and soil solarization.

Pot and greenhouse trials were conducted for the management of root-knot nematode, Meloidogyne javanica, infestation in tomato. Growth parameters, gall index, soil, and root nematode populations were measured to assess the effect of a novel bio-pesticide (Dazitol®), made from mustard oil and oleoresin of Capsicum, on plant growth and nematode reproduction. Data generated within the pot experiment showed that the tested bio-pesticide did not improve plant growth, but it reduced significantly root-knot nematode damage resulting in a decrease in gall index and root (91%) and soil (62%) population of M. javanica compared with untreated plants. The greenhouse experiment showed that Mocap® and Dazitol® decreased nematode incidence significantly (P < 0.05) on tomato. The result of this study suggested that the best nematode control was obtained by combining soil solarization with chemical or botanical nematicides as an integrated pest management approach.