Volatile DMNT Directly Protects Plants Against Plutella Xylostella By Disrupting The Peritrophic Matrix Barrier In Insect Midgut

In agriculture,insect pest is one of the primary reasons for the reduction of crop yield and quality.Plutella xylostella(P.xylostella)is an important lepidopteran pest that feeds mainly on cruciferous vegetables and crops.Due to its strong gnawing ability and short reproductive cycles,it is easy to cause pest outbreaks and seriously endanger agricultural production.At present,the treatment of P.xylostella is mainly relying on chemical pesticides.However,too much application of chemical pesticides has brought various problems such as environmental pollution and residues endangering human health,and more importantly,pests are becoming resistant to pesticides during long time of field application.Therefore,the development of new natural insect-resistant methods is particularly important.In the process of evolution,plants have formed a variety of ways to resist herbivorous insects.The secretion of poisonous compounds to kill herbivorous insects is an important means of plant defense.Previous studies have shown that plants indirectly resist the infestation of pests by synthesizing terpenoid DMNT,but whether DMNT has a direct role on insects is not yet clear.This article takes the lepidopteran pest P.xylostella as the research object to explore the toxic effect and mechanism of plant terpenoid DMNT.The main results are as follows:(1)DMNT has significant influence on the growth and reproduction of P.xylostella.Selective feeding experiments found that Arabidopsis plants overexpressing the PEN1 gene have a repellent effect on P.xylostella.According to existing literature reports,Arabidopsis plants overexpressing the PEN1 gene can produce large amounts of terpene compounds DMNT.DMNT in vitro smear experiment shows that DMNT has a direct repellent effect on the larvae of P.xylostella.The feeding experiment of DMNT shows that DMNT has a direct toxic effect on P.xylostella larvae.(2)DMNT seriously affects the intestines of P.xylostella larvae.Starving the larvae of P.xylostella or feeding DMNT found that the larvae that consumed DMNT died earlier than the larvae that were hungry,indicating that the larvae were killed by DMNT rather than starvation.The larval intestinal enzyme activity experiment found that the lipase activity was significantly down-regulated,indicating that the larval intestinal digestion ability was weakened after eating DMNT.The "Smurf" experiment found that the proportion of larvae with intestinal leakage increased significantly after consuming DMNT.Observing the structure of the periphagus under the body microscope,it was found that the structure of the periphagus in the intestine of the larvae after eating DMNT was thin and loose.The HE-stained sections of the larval intestines found that the periphagus in the larvae intestines eroded after eating DMNT.Transmission electron microscope observation of the intestinal cross section obtained similar results.These results indicate that the periphagous membrane in the larvae's intestine is destroyed after eating DMNT.(3)DMNT treatment damages the Peritrophic metrix partly through downregulation of Px Mucin expression.RT-PCR analysis found that the expression of Px Mucin gene in the larval intestines after consuming DMNT decreased significantly,indicating that DMNT can significantly inhibit the expression of mucin like gene.RNAi experiments found that the survival rate,body weight and pupation rate of larvae after Px Mucin silence were significantly reduced,and the periphagus in the intestine became thinner.These results indicate that the damaging effect of DMNT on the periphagus is achieved by inhibiting the expression of Px Mucin.(4)The toxic effect of DMNT on P.xylostella requires the assistance of intestinal microbesThe feeding experiment of antibiotics found that antibiotics had no effect on the growth and development of larvae.The HE stained sections showed that the periphagous membrane structure in the intestine of the larvae after taking antibiotics was intact,indicating that the integrity of the periphatic membrane in the intestine of P.xylostella larvae does not require intestine Road microorganisms exist.At the same time,the experiment of feeding antibiotics and DMNT found that the survival rate and pupation rate of P.xylostella larvae were significantly increased compared with feeding DMNT,and the HE stained sections showed that the periphagous membrane structure of the larvae fed DMNT and antibiotics at the same time was still damaged.These results indicate that the toxic effect of DMNT on P.xylostella larvae is not caused by the damage of the periphagous membrane itself,but requires the assistance of intestinal microorganisms.Gram-stained sections fed with DMNT and Enterococcus faecalis(E.f)at the same time showed that E.f entered the intestinal cells after the destruction of the periphagus membrane structure,indicating that the death of the larvae was caused by the bacteria attacking the intestinal wall cells.(5)DMNT affects the homeostasis of the intestinal flora of P.xylostella larvae.A 16 S r DNA sequencing showed that the intestinal flora of the larvae was disordered after consuming DMNT: the Proteobacteria and Firmicutes changed greatly,and the abundance of Enterococcus sp in Firmicutes was increased by 85 times.These results indicate that DMNT can cause disturbance of the intestinal flora of the larvae.Therefore,we propose that the midgut microbiota assists DMNT in killing P.xylostella.These findings may provide a novel approach for plant protection against P.xylostella.