Fumigation and contact activities of 18 plant essential oils on Villosiclava virens, the pathogenic fungus of rice false smut

Rice false smut (RFS), caused by Villosiclava virens, is an emerging devastating disease of rice panicles worldwide and produces yield loss and mycotoxin residues in rice. In this study, 18 plant essential oils (PEOs) were selected to evaluate antifungal activity via fumigation and contact methods against the mycelial growth and conidial germination of V. virens. The primary compositions of PEOs with stronger fungistatic activity were analyzed using gas chromatography (GC)-mass spectrometry (MS), and the changes in the mycelial morphology were observed using scanning electron microscopy (SEM). Antifungal tests showed that cinnamon bark oil and cinnamon oil had stronger fumigation and contact effects on V. virens than the other oils tested. The primary active composition in both cinnamon bark oil and cinnamon oil was trans-cinnamaldehyde, which exhibited contact activities with EC50 values of 2.13 and 35.9 μg/mL against mycelial growth and conidial germination, respectively. The hyphae surface morphological alterations caused by cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde included shriveling, vacuolation and exfoliation. In conclusion, cinnamon bark oil and cinnamon oil have the potential to prevent and control RFS, and trans-cinnamaldehyde is a promising natural lead compound for new fungicide discoveries to control RFS contamination and mycotoxin residues in rice.


Antifungal evaluation of 18 PEOs in vitro.
. In the fumigation activity assay, all the 18 PEOs showed inhibitory effects on the mycelial growth of V. virens at the concentration of 10 µL/L air Acorus tatarinowii rhizome oil, Angelia dahurica oil, cinnamon bark oil, cinnamon oil, Litsea cubeba oil, myrrh oil and thyme oil inhibited the mycelial growth completely, followed by peppermint oil, clove oil, holly oil and Forsythia suspansa oil that inhibited 74.1%, 72.1%, 67.7% and 60.4%, respectively. In contrast, Artemisia argyi oil, camphor oil, Ligusticum wallichii oil, myristica oil, Platycladus orientalis oil and tea seed oil showed less than 50% inhibition against mycelial growth of V. virens.
Alternatively, the inhibition of mycelial growth was 66.1% and 71.9%, respectively, after cinnamon bark oil and cinnamon oil treatment in the contact activity assay. L. wallichii oil, dalbergia wood oil and A. dahurica oil reduced only 32.6%, 26.9% and 24.5% of the mycelial growth, respectively. The other 13 PEOs showed lower than 20% inhibition.
Inhibitory effects of 18 PEOs on conidial germination. Effects of the fumigation and contact of the 18 PEOs on the conidial germination of V. virens are shown in Table 1. In the fumigation activity test, A. dahurica oil, A. tatarinowii rhizome oil, cinnamon bark oil, cinnamon oil L. cubeba oil and myrrh oil exhibited 100% inhibition on conidial germination, and thyme oil inhibited 86.5% of the conidial germination of V. virens at the concentration of 7.5 μL/L air. In the contact activity tests, only cinnamon bark and cinnamon oils completely inhibited conidial germination. However, the other 16 PEOs had no significant inhibitory effect on the conidial germination of V. virens.
The results of the EC 50 values of the PEOs with strong antifungal activity against the conidial germination of V. virens are shown in Table 2. Cinnamon bark and cinnamon oils showed the strongest inhibitory effect on the conidial germination in both the fumigation and contact activity assays with EC 50 values less than 0.5 µL/L air and 33.1 and 30.9 µg/mL, respectively. L. cubeba oil exhibited strong fumigant activity with EC 50 values less than 0.5 µL/L air but not good contact activity. In contrast, L. wallichii oil had moderate contact activity with an EC 50 value of 234 µg/mL but little fumigation activity.
The major constituent of PEOs with higher antifungal activities and its contact activity against V. virens. Major constituent of PEOs with higher inhibitory activity. The major compounds of the cinnamon bark and cinnamon oils were analyzed using GC-MS and were listed in Table 3, along with their retention indices, compound names and relative contents. As presented in Fig. 1, the chemical components in cinnamon (Fig. 1A) and cinnamon bark (Fig. 1B) oils were satisfactorily separated under the chromatographic and MS conditions. The natural components identified from the cinnamon bark and cinnamon oils represented 71.47% and 71.46% of the total oils, respectively. The most abundant compound in both the cinnamon bark and cinnamon oils was trans-cinnamaldehyde. Its relative contents were 71.32% and 71.27%, respectively, in the two oils. These results were consistent with previous studies 15,28,29 . In addition, eucalyptol was found in the two oils, while o-cymene was identified solely in the cinnamon oil, and camphene was detected in the cinnamon bark oil.
Antifungal activity of trans-cinnamaldehyde on V. virens. The fumigation and contact activity of trans-cinnamaldehyde on V. virens was shown in Table 2. The EC 50 values of trans-cinnamaldehyde against mycelial growth and conidial germination were both less than 0.5 µL/L air in the fumigation activity assay, and 2.13 µg/ mL and 35.9 µg/mL in the contact activity assay, respectively.  Table 2. EC 50 value of ten substances tested on. V. Virens under two conditions. Pearson χ 2 statistic with P values indicating the goodness-of-fit for data to the expected probit response mode. "-" indicates that this essential oil has such low or high activity that the EC 50 value cannot calculated or χ 2 cannot be calculated with this method.

Plant essential oils
www.nature.com/scientificreports www.nature.com/scientificreports/ Mycelial morphology alteration of V. virens. SEM micrographs were used to determine the effect of cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde on the surface morphology of the mycelia after seven days treatment at the concentrations of 0.5 μL/L air and 2 µg/mL by fumigation and contact activity methods, respectively. As shown in Fig. 2, the hyphae exposed to the chosen concentrations of the vapor of the PEOs or grown on toxic media showed degenerative changes in the hyphal morphology in comparison to the thick, elongated, smooth surfaced hyphae in the control plates ( Fig. 2A,a). From the aspects of the substances tested, the cinnamon bark oil induced the mycelia surface rough and ruptured, but cinnamon oil caused the mycelial shriveling, applanate and membrane injury. As shown in Fig. 2(D,d), trans-cinnamaldehyde aggravated the mycelial abnormalities of collapsing and blistering.
Fungal sporulation difference of V. virens. Sporulation of V. virens was significantly affected by the tested substances (cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde) under both fumigation and contact conditions. The results of fungal sporulation difference was shown in Table 4. Compared to the traditional contact method, fumigation method exhibited stronger inhibitory effect on the conidium sporulation. The fumigation treatment of trans-cinnamaldehyde completely inhibited the conidium sporulation of V. virens. Among the three tested substances, trans-cinnamaldehyde had the highest sporulation inhibition in the fumigation treatment method and cinnamon oil had the lowest inhibition under both treatment methods.

Discussion
PEOs are one of the important sources of novel fungicides 30,31 . In this study, we evaluated the fumigation and contact activities of 18 PEOs against V. viren. Among the 18 PEOs, cinnamon bark and cinnamon oils showed the strongest fumigation and contact activities against the mycelial growth and conidial germination of V. virens. In the fumigation assay, 6 PEOs, including A. dahurica oil, A. tatarinowii rhizome oil, cinnamon bark oil, cinnamon oil, L. cubeba oil and myrrh oil completely inhibited the mycelial growth and conidial germination of this pathogen, and the other 12 PEOs had moderate inhibitory effects. In the contact activity test, cinnamon bark and cinnamon oils showed stronger antifungal activity on the mycelial growth and conidial germination than the other 16 PEOs. It is interesting that L. cubeba oil had stronger fumigation activity while L. wallichii oil showed stronger contact activity against mycelial growth and conidial germination of V. virens. Similar researches have been reported that volatile phase effects of oregano, rosemary and lavender essential oils were more effective on fungal growth of P. infestans and B. cinerea than contact  Table 3. Natural major compositions of cinnamon bark oil and cinnamon oil. www.nature.com/scientificreports www.nature.com/scientificreports/ phase effect 29,32 . However, some other PEOs showed the contrary antifungal activity by two methods. For example, thyme, clove and cinnamon proved to be better contact effect than fumigation activity against P. roqueforti, P. corylophilum, Eurotium. repens and A. flavus. The components of PEOs and species of fungi are the main influence factors on the inhibitory effect 33 . The results of this study indicated that cinnamon bark and cinnamon oils had the strongest antifungal effects on V. virens, with EC 50 values of 4.28 μg/mL and 33.1 μg/mL, respectively, against mycelial growth and 4.47 μg/mL and 34.6 μg/mL, respectively, against conidial germination, which were lower than the values generally reported 14,32 . In previous studies, cinnamon bark oil exhibited strong contact toxicity against toxigenic and foodborne fungi, and cinnamon oil had contact inhibitory effect on wood-rot fungi 19,20,34,35 . Alternatively, since cinnamon bark and cinnamon oils are both from the C. cassia plant, which is commonly known as cinnamon, consumer acceptance is less of a problem because the US Food and Drug Administration (US FDA) lists cinnamon as a generally recognized as safe substance GRAS), and it is used in the fields of food, Chinese medicine, cosmetic and other materials around the world 17,35,36 . A comparison of these results revealed that cinnamon bark and cinnamon oils have the potential to prevent and control RFS.
To explore the possible active constituent of cinnamon bark oil and cinnamon oil, the major components of cinnamon bark oil and cinnamon oil were analyzed, and its contact activity against mycelial growth and conidial germination was evaluated. The photographs of cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde with light microscope of V. virens hyphae can be seen in Supplementary Information in Figs S1 and S2. The results revealed that trans-cinnamaldehyde was the most abundant compound in both cinnamon bark and cinnamon oils and had a higher inhibitory effect on the mycelial growth than that of cinnamon bark and cinnamon oils. Similarly, several research papers reported that the fungicidal activity of cinnamon oil on wood-rot fungi and plant pathogenic fungi was attributed to its primary component, trans-cinnamaldehyde 14,19 . Many studies indicated that cinnmaldehyde could be used as an excellent candidate to control plant diseases caused by Rhizoctonia. solani, F. oxysporum, A. flavus, C. gloeosporioides, A. citrii and B. cinerea 14,20,37 . The results indicated that trans-cinnamaldehyde could be studied further to develop an eco-friendly and acceptable fungicide to protect rice from RFS and mycotoxin contamination.   www.nature.com/scientificreports www.nature.com/scientificreports/ The SEM micrographs indicated that the mycelia shrank and became applanate, exuvial and blistery after treatment with cinnamon bark oil and cinnamon oil. Soylu et al. found that Origanum essential oil caused the hyphal morphology of B. cinerea to degenerate, resulting in cytoplasmic coagulation, vacuolations, hyphal shriveling and protoplast leakage 16 , and Xu et al. also reported that trans-cinnamaldehyde induced the hyphae collapse and fracture of A. alternata 27 . As shown in the SEM graphs, cinnamon bark oil was more toxic than cinnamon oil, which might be related to the contents of camphene, eucalyptol, and o-cymene, since different chemical compounds stimulated or inhibited the mycelial growth alone or together 38 . The most serious disruption to the hyphae caused by trans-cinnamaldehyde indicated that it was the primary active compound in the cinnamon bark and cinnamon oils. Different abnormal phenomena might be responsible for the different treatments. As the previous study, fumigation impacts of PEOs on fungal structures might reflect effects of the volatiles emitted by oils on surface mycelial development and/ or the perception/transduction of signals involved in the switch from vegetative to reproductive development 16 . The contact activity of PEOs may be impacted by interactions with the matrix components, which resulted in mycelial injury 39 . This result further confirmed that cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde inhibited mycelial growth by destroying the surface structure of the mycelia and provided some reference and a basis to further explore the action mechanism of PEOs against V. virens. The specific mechanism on gene expressions and protein activities of cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde against hyphae surface morphological alterations of V. virens remained unclear and needs to be studied.
To evaluate the inhibitory effect on reproductivity of V. virens. The sporulation difference of V. virens was conducted. As shown in the Fig. 3, cinnamon bark oil, cinnamon oil and trans-cinnamaldehyde reduced the fungal sporulation yields significantly compared with the normal sporulation yield (control). Among the three tested substances, cinnamon bark oil possessed strongest contact effect (Fig. 3B) and trans-cinnamaldehyde showed the strongest fumigation effect (Fig. 3A) on the sporulation of V. virens, which is not accordance with the antifungal effect of the tested substances against the conidial germination of V. virens. The reason for different antifungal effect on germination and sporulation haven't been reported before and the different compounds in both PEOs and the interaction between chemicals and conidium might be the important factors and need further study.
In summary, cinnamon bark oil and cinnamon oil showed obvious fumigation and contact activity against the mycelial growth, conidial germination and sporulation of V. virens, and their primary active compound was trans-cinnamaldehyde. The SEM observations revealed that the hyphae were shriveled and exfoliated after trans-cinnamaldehyde treatment. The sporulation was significantly decreased after fumigation with trans-cinnamaldehyde. This study demonstrated that cinnamon bark and cinnamon oils possess the potential to be used as a bio-fungicide to prevent and control RFS, and trans-cinnamaldehyde is a very promising natural lead compound for the discovery of high efficacy and low risk fungicides. Up till now it has not been reported that any plant essential oils can be used to prevent and control RFS in the greenhouse or fields. It is an important topic to evaluate the efficay of cinnamon bark oil and cinnamon oil against this disease in fields in further studies.  Supplementary Fig. S3).

Determination of the fumigation and contact activities of 18
PEOs on mycelial growth. The contact activity of the PEOs was determined using the toxic medium method. First, the stock solution was made by adding 0.04 g PEOs to 10 mL acetone. Second, a 100 μL stock solution of individual PEO was immediately added to the 20 mL sterile PDA medium at 45-50 °C and mixed thoroughly, which was designated toxic medium at the concentration of 20 μg/mL. When the EC 50 values (the concentration of the tested substance causing a 50% inhibition against the mycelial growth compared with the control) of the PEOs with higher antifungal activity and individual components were determined, appropriate volumes of the stock solution of the PEOs and compounds were adjusted to a series of concentration gradients using acetone. The concentrations tested were 0.250 to 200 μg/ mL. The control received the same quantity of acetone mixed with PDA. The 20 mL toxic medium was poured onto three aseptic 6-cm plastic Petri dishes. V. virens was inoculated immediately by plating a 5 mm diameter disc of the fungus cut with a sterile cork borer from the edge of actively growing cultures on PDA plates in the center of each plate. The Petri dishes were incubated in the dark at 28 °C.
The fumigation activity was determined as described by Soylu et al. 40 with some modifications. Petri dishes (60 × 12 mm, which offer 28 mL air spaces after the addition of 6 mL PDA medium) were used and inoculated with a 5-mm diameter disc of V. virens in the center. A 0.28 μL aliquot of the PEOs was added to the inner surface of the lid of the Petri dishes, and the final concentration was 10 μL/L air (the EC 50 values of PEOs with higher antifungal activity and chemicals were determined by pipetting different amounts of the substances tested (0.5 to 6 μL) into the inner surface of the lid of the Petri dishes). The control group was prepared without oil. The plate was immediately sealed with parafilm to prevent any leakage of the PEOs. Each concentration was tested in triplicate. The 5-mm diameter mycelial disc cut from a 15-day-old colony of V. virens grown on PDA plates and placed upside down in the center of the Petri dishes.
After 7 days of incubation at 28 °C in the dark, the colony growth diameter (mm) was measured using a 150-mm digital caliper. The growth inhibition was calculated using the formula 41  where: DC, DT -average diameter (mm) of the fungal colony of the control and the treatment, respectively. Each test was repeated three times.  www.nature.com/scientificreports www.nature.com/scientificreports/ Determination of the fumigation and contact activities of 18 PEOs on conidial germination.
The fumigation and contact inhibitory activities of the PEOs on conidial germination were conducted as described by Soylu et al. 42 with slight modification. A spore suspension (10 6 conidia/mL) of V. virens was prepared from fermentative potato sucrose broth (PS) as described by Lu et al. 43 . A suspension of 80 μL was spread onto the surface of the PDA medium or toxic medium as described in 2.3. The treated concentrations of the PEOs were 7.5 μL/L air for the fumigation activity experiment and 40 μg/mL for the contact activity test. Different concentrations of PEOs with higher antifungal activity (0.5 to 7.5 μL/L air in the fumigation activity test and 32 to 260 μg/mL in the contact activity assay) were applied for the determination of EC 50 values. The plates were incubated at 28 °C until the conidial germination in the control reached >90% (10-12 h, depending on the rate of germination of V. virens). Conidial germination was defined as the point at which the germ tube length exceeds the short diameter of the spore. The percentage of conidial germination was estimated under a microscope (Olympus BX51, Tokyo, Japan).
Each test was repeated three times. The percent inhibition was calculated as follows: where Gc and Gt represent the germination rate in control and treated Petri dishes, respectively.
Analysis of the primary natural components of PEOs with higher antifungal activity. The primary natural components of the strongest PEOs were determined using a gas chromatograph Agilent 7890 A interfaced with a QP2010 5975 C mass spectrometer (Shimadzu). An Rtx-5MS capillary column (30 m × 0.25 mm internal diameter, 0.25 μm film thickness) was used. The column temperature was maintained at 40 °C for 3 min and programmed to 200 °C at a rate of 10 °C/min. The carrier gas was helium with a flow rate of 1 mL/min. The MS were collected at 70 eV and a mass range of 30-550 amu (scan time 0.3 s). Qualitative identification of the compounds of the PEOs was based on retention indices relative to n-alkanes and computer matching with the NIST14. LIB (National Institute of Standards and Technology) in combination with published data. Quantitative analysis of the individual natural major component was based on the peak area normalization measurement, and the relative content of each component was calculated using the % peak area.

Mycelial morphology observation of V. virens.
Using a clean blade, the square fungal block (5 mm × 5 mm × 2 mm) was cut from the fungal colony incubated in 2.3. The fungal block was dipped into 3.5% glutaraldehyde in 0.1 mol/L phosphate buffer at room temperate for 48 h, and the mycelial block was washed with sterile water four times to rinse off the glutaraldehyde in 30 min. The mycelia were fixed with 1% OsO 4 for 2 h. The samples were dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) for a period of 20 min in each series and finally treated with 100% ethanol. The samples were dried in a drying apparatus (Leica EM CPD030, Germany) up to the critical point with CO 2 . The fixed material was mounted on stubs using double-sided carbon tape and coated with gold/palladium in a sputter coater system in a high-vacuum chamber (Hitachi MC1000, Japan) for 1 min at 6 mA. The samples were examined, and digital images were captured using an SU 8010 SEM at an accelerating voltage of 12 kV.
Fungal sporulation difference of V. virens. The sporulation of V. virens was tested according to the method described by Sun et al. 44 with some modification. A spore suspension (10 7 conidia/mL) of V. virens was prepared from fermentative potato sucrose broth (PS) as described in 4.4. A suspension of 50 μL was spread onto the surface of the PDA medium or toxic medium as described in 2.3. The treated concentrations of the PEOs were 0.5 μL/L air for the fumigation activity experiment and 35 μg/mL for the contact activity test. Plates were sealed with parafilm and incubated at 28 °C and cultured for 10d for sporulation. Fungal spores of each Perti-dish were dislodged with appropriate amount of distilled water and suspended by vigorous mixing for 30 s. The number of spores was determined with a hemacytometer under a microscope (magnification 100×). Each combination of medium and conidial suspension was replicated three times.
Statistical analysis. The data were analyzed by a one-way analysis of variance (ANOVA) using the SPSS program (IBM SPSS Statistics 22.0). The measurement of the difference was determined using Scheffe's multiple range test. Values of P < 0.05 were considered statistically significant. Assays were performed in triplicate, and the results were expressed as the mean (±SE). The EC 50 values were calculated using probit analysis.

Data Availability
Te datasets generated and analysed during the current study are not publicly available due to possible valorisation of the data but are available from the corresponding author on reasonable request.