Screening Of Plant Essential Oils For Antifungal Activity Against Villosiclava Virens And Studies On Action Mechanisms

Villosiclava virens（Vv）,is the pathogenic fungus of rice false smut（RFS）.In recent years,RFS has become a devastating disease of rice panicles in rice-growing areas of the world due to the change of hybrid rice cultivars,farming practices,and climate.Plant essential oil（PEO）is an important source for developing fungicides with high efficiency and low risk.However,there are few reports about the antifungal activity and action mechanism of PEOs against Vv.In this study,18 PEOs were selected to evaluate antifungal activity on Vv and to explore the main active compound of 2 PEOs with stronger inhibitory effect and its action mechanism.The experimental results are as follows:Preliminary screening of 18 PEOs against Vv showed that cinnamon bark oil and cinnamon oil pose stronger antifungal activity than the other 16 PEOs and these two oils have the potential to prevent and control RFS.Total 18 plant essential oils（PEOs）were selected to evaluate antifungal activity against the mycelial growth and conidial germination of V.virens via fumigation and contact methods.Antifungal tests showed that cinnamon bark oil and cinnamon oil had stronger fumigation and contact effects on V.virens than the other oils tested.In the fumigation activity assay,the EC₅₀0 values（concentration of the tested substance causing a 50%inhibition against the mycelial growth or conidial germination compared with the control）of both cinnamon bark oil and cinnamon oil against mycelial growth and conidial germination of Vv were less than 0.5μL/L air.In the contact activity experiment,the EC₅₀0 values of cinnamon bark oil and cinnamon oil against mycelial growth were 4.28 and 4.47μg/mL,respectively.The EC₅₀0 values of cinnamon bark oil and cinnamon oil against conidial germination were 33.1 and 30.9μg/mL,respectively.The primary active component in both cinnamon oil and cinnamon bark oil was trans-Cinnamaldehyde（TC）.The compounds were analyzed using gas chromatography（GC）-mass spectrometry（MS）.These results showed that the relative content of TC was 71.32%and 71.27%,respectively,in the cinnamon oil and cinnamon bark oil.TC exhibited contact activity with an EC₅₀value of 2.13 and 35.9μg/mL against mycelial growth and conidial germination,respectively.The fumigation activity of TC against mycelial growth and conidial germination of Vv were less than 0.5μL/L air.The effect of cinnamon oil,cinnamon bark oil and TC（Three substances）on the morphology of mycelial growth was observed by using light microscopy.The fungal sporulation of Vv were determined.Results showed that three substances induced mycelial morphology abnormal,which showed in different forms（fracture,crimp or distortion）and inhibited the sporulation of Vv.The fungal growth of Vv is inhibited by TC through affect the cell structure of Vv hyphae and disturbing the metabolic enzymes’activity of the mitochondrion.（1）It can be seen in scanning electron microscopy,the abnormal phenomena caused by TC includes shriveling and vacuolation on the mycelial surface.（2）The ultrastructure of mycelial cell was characterized by liposomes increase and mitochondrial structural disorder after treatment with TC.It was observed by the transmission electron microscopy.（3）A serial of biochemical indicators of mitochondrial function of Vv,such as succinodehydrogenase（SDHase）,nicotinamideadeninedinucleotide-malicdehydrogenase（NAD-MDHase）and adenosine triphosphatase（ATPase）were determined by SDHase kit,NAD-MDHase kit,and ATPase kit to study the action mechanism of TC.The enzyme activity of SDHase and NAD-MDHase were reduced when treated with TC at the concentration of 2,4,8,15,30μg/mL.The activity of Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,Ca²⁺-ATPase and Mg²⁺-ATPase fluctuated as the U-Curve with the increase of TC concentration.When the concentration of TC was 4μg/mL,the inhibitive activity of TC on ATPase was the strongest among the tested concentration.（4）TC is potential to reduce the ustilaginoidins production in Vv by down-regulating the gene expression of UV-2086 gene.The relative expression quantity of UV-2086 gene in Vv detected by RT-PCR was significantly reduced after TC treatment at the concentration of 15μg/mL.The above results provided some experimental data and theoretical basis for the prevention and control of RFS by TC.