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

**Background:** Root-knot nematodes (RKNs), particularly *Meloidogyne javanica*, are globally distributed polyphagous pests that cause severe yield and quality losses in crops, especially in Mediterranean countries. Chemical nematicides are effective but expensive, harmful to human health, and disruptive to ecosystems. Biological control using nematophagous fungi offers a safer alternative. The genus *Lecanicillium* (formerly *Verticillium*) has demonstrated biocontrol capabilities against various plant pests and nematodes. This study aimed to evaluate the nematicidal potential of a new Tunisian *Lecanicillium* sp. isolate (OM169327) against *M. javanica* and to understand its mechanisms through scanning electron microscopy (SEM) and proteomic analysis using iTRAQ methodology.

**Methods:** The *Lecanicillium* sp. isolate was obtained from Tunisian soil and identified morphologically and molecularly via 28S rDNA sequencing and phylogenetic analysis. Culture filtrates were prepared by growing the fungus in potato dextrose broth for 15 days, then filtering through Whatman paper and a 0.45 μm Millipore filter. Five concentrations (10%, 25%, 50%, 75%, and 100% v/v) were tested against *M. javanica* eggs and J2 juveniles. Egg-hatching inhibition was assessed after 7 days, and juvenile mortality after 3 days, with five replicates per treatment. SEM was used to observe fungal parasitism of eggs. For proteomics, mycelial proteins were extracted, digested with trypsin, and analyzed by 1D-nano LC ESI-MS/MS on a Triple TOF 5600 system. Proteins were identified using Mascot against fungal databases with a 95% confidence threshold and 1% FDR.

**Key Results:** Phylogenetic analysis confirmed the isolate as *Lecanicillium* sp. with 98% affinity to *L. carpophylum* and *Lecanicillium* sp. (MK732148). SEM revealed that fungal hyphae adhered to, penetrated, and destroyed *M. javanica* eggs, with spore production inside eggs. All tested filtrate concentrations significantly inhibited egg-hatching and increased J2 mortality compared to controls. The 100% concentration achieved 53.99% egg-hatching inhibition and 90.21% net J2 mortality. The 50% and 75% concentrations were similarly effective, with 48.58% and 52.94% egg-hatching inhibition, and 90.68% and 89.36% net J2 mortality, respectively. Proteomic analysis identified 47 unique proteins, of which 22 were unambiguously identified. Key proteins included endochitinase (a cell-wall degrading enzyme), choline dehydrogenase, actin, hsp70-like protein, ATP synthase subunit beta, and various hypothetical proteins. The identified proteins were functionally classified into metabolism, energy production, signalling and stress response, and protein synthesis/degradation groups.

**Clinical Implications:** This study provides strong experimental evidence that *Lecanicillium* sp. (OM169327) is a promising biological control agent against *M. javanica*. The fungus acts through multiple mechanisms: direct parasitism via hyphal penetration of eggs, production of hydrolytic enzymes (endochitinase, choline dehydrogenase) that degrade the eggshell, and likely competition for nutrients and induction of host plant resistance. The high efficacy of culture filtrates (up to 91% J2 mortality) suggests that secreted metabolites and enzymes are key to its nematicidal activity. The authors recommend applying this bio-agent to vegetable crops in situ as part of integrated pest management strategies. Further studies are needed to characterize the hypothetical proteins and understand their roles in pathogenicity.