Host-delivered RNAi-mediated silencing using fusion cassettes of different functional groups of genes precludes Meloidogyne incognita multiplication in Nicotiana tabacum.

KEY MESSAGE: This study demonstrates multi-gene silencing approach for simultaneous silencing of several functional genes through a fusion gene strategy for protecting plants against root-knot nematode, Meloidogyne incognita. The ability of root-knot nematode (RKN), Meloidogyne incognita, to cause extensive yield decline in a wide range of cultivated crops is well-documented. Due to the inadequacies of current management approaches, the alternatively employed contemporary RNA interference (RNAi)-based host-delivered gene silencing (HD-RNAi) strategy targeting different functional effectors/genes has shown substantial potential to combat RKNs. In this direction, we have explored the possibility of simultaneous silencing of four esophageal gland genes, six plant cell-wall modifying enzymes (PCWMEs) and a serine protease gene of M. incognita using the fusion approach. In vitro RNAi showed that combinatorial gene silencing is the most effective in affecting nematode behavior in terms of reduced attraction, penetration, development, and reproduction in tomato and adzuki beans. In addition, qRT-PCR analysis of M. incognita J2s soaked in fusion-dsRNA showed perturbed expression of all the genes comprising the fusion construct confirming successful dsRNA processing which is also supported by increased mRNA abundance of five key-RNAi pathway genes. In addition, hairpin RNA expressing constructs of multi-gene fusion cassettes were developed and used for generation of Nicotiana tabacum transgenic plants. The integration of gene constructs and expression of siRNAs in transgenic events were confirmed by Southern and Northern blot analyses. Besides, bio-efficacy analyses of transgenic events, conferred up to 87% reduction in M. incognita multiplication. Correspondingly, reduced transcript accumulation of the target genes in the M. incognita females extracted from transgenic events confirmed successful gene silencing.

Identification, Characterization, and Evaluation of Nematophagous Fungal Species of Arthrobotrys and Tolypocladium for the Management of Meloidogyne incognita.

Root-knot nematodes belonging to the genus Meloidogyne are agriculturally important pests, and biocontrol strategies offer safer alternatives for their management. In the present study, two fungal species from Indian soils were identified as Arthrobotrys thaumasia and Tolypocladium cylindrosporum based on morphological characteristics and further confirmed using molecular markers. In vitro evaluation of A. thaumasia against M. incognita and Caenorhabditis elegans showed 82 and 73% parasitism, respectively, whereas T. cylindrosporum gave 65.2 and 57.7% parasitism, respectively. Similarly, culture filtrates of A. thaumasia caused 57.7 and 53.7% mortality of M. incognita and C. elegans, respectively, whereas T. cylindrosporum caused higher mortality of 87.3 and 64%, respectively. Besides, greenhouse evaluation of both fungi against M. incognita infecting tomato significantly reduced nematode disease burden reflecting parasitic success measured as the total number of galls, egg masses, eggs per egg mass, and derived nematode multiplication factor. Application of A. thaumasia and T. cylindrosporum reduced nematode multiplication factor by 80 and 95%, respectively, compared with control. General metabolite profiling of tested fungi using gas chromatography-mass spectrometry and ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry reported for the first time here showed presence of various volatile and non-volatile compounds with nematicidal activity, viz., trimethyl-heptadiene, methyl-hexadecanol, dodecadienal, decane, terpendole E, dodecane, acetamido-6-anthraquinone, and hexadecanol. Also, other compounds such as undecane, dibutyl-disulfide, octadecenal, paganin, talathermophilin, dactylarin, tolypyridone A, tolypyridone B, pyridoxatin, and destruxin were identified, reported in the literature to possess antibacterial, antifungal, and insecticidal properties. This is the first report of the occurrence of both fungi from India and pioneer demonstration of T. cylindrosporum for root-knot nematode management.