Induction of secondary metabolites by endophytic fungi inphaseolus vulgaris for liriomyza leafminer and fall armyworm management.

Abstract

The Liriomyza leafminer flies (LMF) are invasive pests that attack several horticultural crops one of which is the common bean Phaseolus vulgaris. Endophytic fungi have been shown to deter performance, oviposition and feeding of these Liriomyza flies in beans. However, the clear infection mechanism or pathways of inoculated fungal endophytes against LMF has not been well established. This study, therefore, investigated the induction of active compounds in the endophytically colonized P. vulgaris plants, as defense mechanism against herbivorous insects. Two fungal isolates Beauveria bassiana (G1LU3) and Hypocrea lixii (F3STI) were used for artificial seeds inoculation. Inoculation was done by soaking P. vulgaris seeds in these fungal conidial suspensions prior to planting. Colonization of the different plant parts was assessed to confirm the endophytic property of the inoculated fungi. Volatiles emitted from endophytically colonized P. vulgaris were collected into adsorbent Super-Q traps and evaluated using gas chromatography combined with mass spectrometry (GC-MS). Liquid extraction was conducted with methanol and dichloromethane solvents followed by chromatographic analysis. Methanol and dichloromethane extracts of endophytically colonized plants were screened for their efficacy against leafminer fly, Liriomyza huidobrensis Blanchard and Fall Armyworm (FAW), Spodoptera frugiperda larvae using leaf dipping method and topical application, respectively. The two isolates successfully colonized the entire host plant (roots, stems, and leaves) with significant variation (F = 19.22, Df = 6, P = 0.01) between fungal isolates and the controls. The results also showed qualitative differences in the volatile profiles between the control P. vulgaris plants, endophytically colonized plants and insect damaged plants. The volatile blend from the control P. vulgaris plants consisted mainly of the following compounds: meta cresol (52) (35%) and para cresol (53) (64%). The most abundant emissions from the insect damaged plants included terpinen- 4-ol (59) (28%) and benzaldehyde dimethyl acetal (61) (21%). The H. lixii inoculated plants consistently released m-cresol (52) (72%) and p-cresol (53) (13%). The most abundant emissions from H. lixii colonized plants with insect damage included benzaldehyde dimethyl acetal (61) (6%), butylated hydroxytoluene (63) (4%) and methyl salicylate (76) (3%). Beauveria bassiana colonized plants released the highest number of volatiles with the most abundant including benzaldehyde dimethyl acetal (61) (6%), butylated hydroxytoluene (63) (5%), ()- γ-bisabolene (86) (4%), methyl salicylate (76) (4%) and 4,8,12-trimethyl-1,3(),7(),11-tridecatetraene (87) (4%). The most abundant volatiles detected from B. bassiana colonized plants with insect damage include benzaldehyde dimethyl acetal (61) (16%) and butylated hydroxytoluene (63) (4%). Qualitative and quantitative differences were also reported between solvent extracted compounds detected from control P. vulgaris plant extracts and fungal extracts. All extracts except the B. bassiana fungal extract contained the hexadecenoic acid methyl ester (92) (18%) and 9,12- octadecanoic acid methyl ester (93) (13%). Among the extracts’ compounds, the most abundant included 9,12,15-octadecatrienoic acid methyl esters (94) (70%) and 1-hydroxy-4- methylanthraquinone (102) (58%). The bioassay results showed significant differences between the liquid crude extracts from endophytically colonized plants and the controls plants with regard to the effects on pupation of 2nd instar LMF larvae (F = 4.33, Df = 6, P = 0.03) and adult Liriomyza flies emergence of LMF pupae (F = 5.4, Df = 6, P = 0.02). The survival of the 1st instar FAW larvae dipped into the methanolic endophytically colonized plant extracts was also significantly reduced (F = 3.7, Df = 8, P = 0.001) as compared to the controls. The extracts of B. bassiana inoculated plants were the most lethal to FAW larvae with median lethal time (LT50) of 4.42 days. Profiled compounds have previously been identified in plant extracts and volatiles with activities including insect repellence, predator attractant and insecticidal properties. Colonization of the host plant P. vulgaris therefore triggers production of compounds for defense against herbivorous insects including the leafminer and fall armyworm. The compounds deter feeding and oviposition of the pest through insect repellence and predator attraction. The toxic compounds also infect the pest through effects on its physiology. However, the identities of compounds were based on comparisons of mass spectra available from an MS library. Therefore, some of the identifications may be tentative especially for volatiles with trans-cis isomers. This study demonstrated the high potential of endophytic fungi H. lixii and B. bassiana, to induce mainly specific defense compounds in the common bean P. vulgaris that can be used against the Liriomyza leafminers and Fall Armyworm.