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.

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 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.

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.

First Report of the Spiral Nematode Rotylenchus incultus (Nematoda: Hoplolaimidae) from Cultivated Olive in Tunisia, with Additional Molecular Data on Rotylenchus eximius.

Spiral nematode species of the genus Rotylenchus have been reported on olive (Olea europaea L.) in several Mediterranean countries (Castillo et al., 2010; Ali et al., 2014). Nematological surveys for plant-parasitic nematodes on olive trees were carried out in Tunisia between 2013 and 2014, and two nematode species of Rotylenchus were collected from the rhizosphere of olive cv. Chemlali in several localities of Tunisia (Tables 1,2 [Table: see text] [Table: see text] ). Twenty-two soil samples of 3 to 4 kg were collected with a shovel from the upper 50 cm of soil from arbitrarily chosen olive trees. Nematodes were extracted from 500 cm3 of soil by centrifugal flotation method (Coolen, 1979). Specimens were heat killed by adding hot 4% formaldehyde solution and processed to pure glycerin using the De Grisse's (1969) method. Measurements were done using a drawing tube attached to a Zeiss III compound microscope. Nematode DNA was extracted from single individuals and PCR assays were conducted as described by Castillo et al. (2003). Moderate-to-low soil populations of these spiral nematodes were detected (5.5-11.5, 1.5-5.0 individuals/500 cm3 of soil, respectively). This prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Morphological and molecular analyses of females identified these species as Rotylenchus eximius Siddiqi, 1964, and Rotylenchus incultus Sher, 1965. The morphology of R. eximius females (five specimens studied) was characterized by having a hemispherical lip region clearly off set, with four to five annuli, body without longitudinal striations, lateral fields areolated in the pharyngeal region only, stylet 32 to 36 µm long, and broadly rounded tail. The morphology of R. incultus females (51 females and 16 males; Table 2) was characterized by a hemispherical lip region with the basal annulus subdivided by irregular longitudinal striations, with three, rarely four annuli; stylet 21.5 to 27.5 µm long, female tail hemispherical with terminus regularly annulated; phasmids anterior to anus level (3-6 annuli above). The morphology of the isolated nematodes agreed with previous descriptions of R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005) and R. incultus (Sher, 1965; Castillo and Vovlas, 2005; Vovlas et al., 2008), respectively. A single individual was used for DNA extraction. Primers and PCR conditions used in this research were specified in Cantalapiedra-Navarrete et al. (2013), and a single amplicon of 800, 1,100, and 450 bp was obtained and sequenced for D2 to D3, ITS1, and cytochrome c oxidase subunit 1 (coxI), respectively. Sequence alignments for D2 to D3 (KX669231-KX669233), ITS1 (KX669238-KX669240), and coxI (KX669244-KX669245) from R. eximius, showed 99% to 97%, 98% to 94%, 93% similarity to other sequences of R. eximius deposited in GenBank (EU280794-DQ328741, EU373663-EU373664, JX015401-JX015402, respectively). Similarly, D2 to D3 (KX669234-KX669237), ITS1 (KX669241-KX669243), and coxI (KX669246-KX669249) sequence alignments from R. incultus, showed 99%, 99% to 95%, 99% to 90% similarity, respectively, to other sequences of R. incultus deposited in GenBank (EU280797, EU373672-EU373673, JX015403, respectively). The best fitted model of DNA evolution was obtained using jModelTest v. 2.1.7 (Darriba et al. 2012) with the Akaike information criterion. BI analyses were performed under the general time reversible (GTR) with invariable sites and a gamma-shaped distribution of substitution rates (GTR + I + G) model for ITS1 and coxI. Phylogenetic analyses of ITS1 and coxI using Bayesian inference (BI) placed R. eximius and R. incultus from Tunisia in subclades that included all R. eximius and R. incultus sequences deposited in GenBank (Fig. 1Fig. 1Phylogenetic relationships within Rotylenchus species found in Tunisia and other species from GenBank. Bayesian 50% majority rule consensus trees as inferred from ITS1 and coxI sequences alignments under the GTR + I + G model. Posterior probabilities more than 0.70 are given for appropriate clades. Newly obtained sequences in this study are in bold. Scale bar = expected changes per site.), which agrees with previous results (Cantalapiedra-Navarrete et al., 2013). Morphology, morphometry, and molecular and phylogenetic data obtained from these samples were consistent with R. eximius and R. incultus identification. To our knowledge, this is the first report of R. incultus in Tunisia. Consequently, all these data suggest that spiral nematode species of the genus Rotylenchus are predominant in olive as previously reported in other Mediterranean areas (Ali et al., 2014).