Identification and physiological activity of (methoxymethyl)triphenylphosphonium chloride as a new phytotoxin isolated from Rhizoctonia solani AG-3 TB

doi:10.21203/rs.3.rs-2876341/v1

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Identification and physiological activity of (methoxymethyl)triphenylphosphonium chloride as a new phytotoxin isolated from Rhizoctonia solani AG-3 TB

https://doi.org/10.21203/rs.3.rs-2876341/v1

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Rhizoctonia solani as a cosmopolitan fungus is the causative agent of many crop diseases and leads to significant economic losses in crop production. To explore the toxin structure and its physiological activity of R. solani AG-3 TB, high performance liquid chromatography (HPLC), infrared absorption spectrum (IR) and nuclear magnetic resonance spectrum (NMR) were required. Here, the compound (methoxymethyl)triphenylphosphonium chloride (MMC) with the molecular formula C₂₀H₂₀ClOP was purified and identified from R. solani AG-3 TB. The pure compound MMC treated at 20 µg/mL, 50 µg/mL, and 100 µg/mL can cause obviously necrosis on leaves, increase active oxygen species (AOS), decrease chlorophyll content, and damage cellular structure. The results enrich the understanding of toxin compounds for R. solani as well as provide valuable insights for toxicology of R. solani AG-3 TB.

Biological sciences/Biochemistry

Biological sciences/Chemical biology

Biological sciences/Microbiology

Biological sciences/Molecular biology

Biological sciences/Plant sciences

(Methoxymethyl)triphenylphosphonium chloride (MMC)

Rhizoctonia solani AG-3 TB

Phytotoxin

Pathogenic mechanism

Toxin compound

Rhizoctonia solani can cause serious diseases on the stem, leaves and roots of many plants. It is divided into 14 subgroups based on its special genetic and biological characteristics, including AG-1 to AG-13 and a bridging isolate AG-BI (Taheri et al., 2011), among which, R. solani AG-1 IA and AG-3 PT subgroups cause black scurf, and stem canker on the stem and roots of rice and potato, respectively (Salamone and Okubara., 2020), while yellow-brown lesions with concentric rings can be induced on leaves of tobacco by R. solani AG-3 TB subgroups, which was known as tobacco target spot disease (Wu et al., 2012; Elliott et al., 2008). Tobacco target spot disease was first recorded in the United States, and it had been occurred subsequently in South Africa, Italy, and Canada (Lucas et al, 1975; Meyer et al., 1990; Kuninaga et al., 2000; Reeleder., 1996). In 2012, dark brown lesions were discovered in tobacco field in Kuandian and Fengcheng Counties, Dandong City of Liaoning in northeast China, which was identified to be tobacco target spot disease (R. solani AG-3 TB) by physiological and molecular method (Wu et al., 2012). This disease has been discovered in Guizhou, Yunnan, and Sichuan provinces and the area of tobacco field has gradually expanded (Xu et al., 2018; Xu et al, 2021).

R. solani, as a common pathogen in nature, can inhibit growth of plant and reduce crop yield. In the middle of the 20th century, carboxylic acid was isolated and identified to be the main toxin compound in R. solani AG-1 IA, such as succinic acid, phenylacetic acid (PAA) and furan acid (Aoki., 1963). Furthermore, m - hydroxy and o – hydroxy - phenylacetic acid that cause wilting of plant and other toxic effects, are two potentially toxic compound separated from R. solani AG-4 (Mandava et al., 1980). 3-methylthiopropionic acid (3-MTPA) isolated from R. solani AG-3 PT can destroy the cell membrane and cause cytoplasmic breakage of potato (Kankam et al., 2016). In our previous study, we have revealed that 3-methoxyphenylacetic acid (3-MOPAA) is an important toxin compound during R. solani AG-3 TB infection and its pure compound at different concentrations of 1 mg/mL, 2 mg/mL, 4 mg/mL can cause necrotic lesion on leaves (Li et al., 2023).

Due to the special genetic and physiological characteristics of R. solani, the pathogenic mechanism remains unclear. Therefore, exploration of pathogenic toxin compound and pathogenic mechanism become an important direction to control the disease spread. According to our previous study, we separated a compound, 3-methoxyphenylacetic acid (3-MOPAA), and another potential pathogenic compound from R. solani AG-3 TB. Here, we further identified and clarified the pathogenic novel compound, (methoxymethyl) triphenylphosphonium chloride (MMC), produced by R. solani AG-3 TB based on high-performance liquid chromatography (HPLC), infrared absorption spectroscopy (IR), and nuclear magnetic resonance spectroscopy (NMR). And the pure compound MMC at different concentrations of 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL can induce necrotic lesions on leaves and damage normal physiological activity of plant. This result enriches the research on toxin of R. solani as well as providing new direction for in-depth exploration of R. solani.

Isolation, and purification of toxin compound of R. solani AG-3 TB

According to our previous study, the radicle elongation and lesion diameter of tobacco was inhibited by the crude toxin extraction from R. solani AG-3 TB, and the toxin extraction was separated into four compounds by thin-layer chromatography, among which, the compound mixture 3 had strong pathogenicity to leaves of tobacco (Li et al., 2023). Then, this compound 3 was detected by HPLC. The seven compound peaks were tested and the retention times were 1.62 s, 1.785 s, 2.043 s, 2.382 s, 3.366 s, 4.233 s, and 6.071 s, respectively (Fig. 1a). Then, the preparative HPLC was used to collect these compound peaks, and their pathogenicity were determined, among which, compound Ⅰ can cause the larger lesion diameter on leaves (0.868 ± 0.198) (Li et al., 2023). Then the compound Ⅰ was collected by preparative HPLC, and the single peak can be detected (Fig. 1b).

Structure identification of the toxin compound

The infrared absorption spectroscopy (IR) was used to identify the toxin compound structure. According to IR spectrum, -C-H telescopic vibration and C-H bending vibration was observed at 2990 cm^-1, 2952 cm^-1, 2845cm^-1,1465 cm^-1, and 1439cm^-1, which indicated that - CH₂ - and - OCH₃ were existed in the structure. The result indicated that benzene ring structure, especially monosubstituted benzene, was identified in this structure on the basis of = C-H telescopic vibration, benzene ring skeleton C = C, and = C-H out of plane bending vibration determined at 3044 cm^-1, 1622 cm^-1, 1586 cm^-1, 1483 cm^-1, 752 cm^-1, 721 cm^-1, 691 cm^-1. At ཞ1120 cm^-1, ⁺P-C telescopic vibration was existed, while at 1096cm^-1, C-O-C telescopic vibration were observed, therefore, the ⁺P-Ar and dialkyl ether (ROR’) were included in this structure. According to the result of IR, monosubstituted benzene, ROR’, ⁺P-Ar, - CH₂ - and - OCH₃ were contained in the toxin component (Table 1, Fig. 2A).

Table 1

The element composition of toxin compound by IR.
Wave number of absorption peak (cm^− 1)	Vibration type	Group	Absorption peak intensity
3400	=C-H Telescopic vibration	=C-H	m
2990, 2952, 2845	-C-H Telescopic vibration	Saturated -C-H	s, m, s
1622, 1586, 1483	Benzene ring skeleton C = C	Benzene ring	m, s, s
1465	C-H Bending vibration	-CH2-	s
1439	C-H Bending vibration	-OCH3	s
~ 1120	C-O-C Telescopic vibration	Dialkyl ether ROR’	s
1096	⁺P-C Telescopic vibration	⁺P-Ar	s
752, 721, 691	=C-H Out of plane bending vibration	Monosubstituted benzene	s, s, s
1234	O-H Bending vibration	Carboxylic acid (-COOH)	s
881, 793, 704	=C-H Out of plane bending vibration	1,3- disubstituted benzene	s, s, s

According to mass spectrometry, the mass to charge ratio of the M⁺ peak of the toxin sample was 307.1 (Fig. 2B), and molecular weight of phosphate cation in the compound was 307. Based on the percentage content of elements C, H, and O, the number ratio of elements C, H, and O in the compound was calculated to 20:20:1 (Table S1). Combining with the result of IR, the molecular formula of toxin compound was consistent with C₂₀H₂₀ClOP.

The structure of toxin compound was deduced by nuclear magnetic resonance (NMR), and ¹H-NMR results (Fig. 3A, Table S2) revealed that four groups of peaks were identified and their integral ratio (from low field to high field) was 3:12:2:3, the total number of protons was 20. According to the results of chemical shift values and COSY and HMBC experiments (Fig. 3C, D), the structure of toxin compound had a spin system composed with 15 aromatic protons on 3 monosubstituted benzenes (δ 7.81 (3H, m)、7.65 (6H, m), and 7.62 (6H, m)), which were 3H₆、6H₄, and 6H₅, respectively. On the base of δ5.24 (2H, d) and 3.49 (3H, s) values, they were two groups of isolated protons, which were H₂ and H₁, among which, H₂ was split into two peaks by the coupling splitting of P ion.

The results of the carbon spectrum, displacement table and HSQC two-dimensional spectrum (Table S3, Fig. 3B, E) indicated that 2 saturated C (δ 65.4 (d) and 62.0 (d)) were observed in structure, which were C₂ and C₁ and they were split into two peaks by the coupling splitting of P ion. There were three kinds of 15 aromatic tertiary C according to the value of δ130.1 (d), 133.8 (d), and 135.5 (d), which were 6C₅, 6C₄, and 3C₆, and they were isolated by the coupling splitting of P ion. One kind of 3 unsaturated seasons C was detected at δ 115.9(d), they were 3C₃, which were split into two peaks by the coupling splitting of P ion. According to the results of NMR, the structure of toxin compound was consistent with (methoxymethyl)triphenylphosphonium chloride (MMC) (C₂₀H₂₀ClOP) (Fig. 3A).

Pathogenicity of pure compound MMC on N. tabacum

The pure compound MMC at the concentration of 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL was treated on leaves, and the lesion diameter was measured to clarify its pathogenicity and virulence. The results indicated that MMC can cause necrosis around the inoculation point on leaves and the lesion diameter were 0.175 ± 0.120 cm, 0.263 ± 0.057 cm, 0.366 ± 0.072 cm, 0.425 ± 0.276 cm, and 0.506 ± 0.185 cm, respectively, after treated by 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL of MMC (Fig. 4, Table 2).

Table 2

The lesion diameter treatment by pure compound MMC with different concentrations.
Compound Treatment	5 µg/mL	10 µg/mL	20 µg/mL	50µg/mL	100µg/mL	CK
Lesion diameter (cm)	0.175 ± 0.120	0.263 ± 0.057	0.366 ± 0.072	0.425 ± 0.276	0.506 ± 0.185	0.00

Effects of pure compound MMC on the production of active oxygen species (AOS)

In order to clarify effect of MMC (5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL) on production of active oxygen species (AOS) on leaves, the production rate of superoxide anion O²⁻ and content of hydrogen peroxide (H₂O₂) were determined (Fig. 5A). The results showed that production rate of O²⁻ in leaf tissue was increased at 24 h and 48 h and decreased at 72 h after treated by MMC. In addition, the production rate of O²⁻ was declined at 72 h, which the number of rates were 0.082 µmol๒g^− 1๒min^− 1 and 0.104 µmol๒g^− 1๒min^− 1 treated by 50 µg/mL and 100 µg/mL of MMC. The production rate of O²⁻ treated with different concentrations revealed that production rate of O²⁻ treatment by 20 µg/mL, 50 µg/mL, and 100 µg/mL of MMC were 2.01, 2.25, and 3.09 folds higher compared with the control at 24 h.

The content of hydrogen peroxide (H₂O₂) in leaves indicated that H₂O₂ content treated by MMC at 48 h was higher than other treatment groups, among which, H₂O₂ content was 0.298 µmol/g treated by 50 µg/mL of MMC, which comparison with the control was 3.55 folds higher. In addition, H₂O₂ content in leaves treated for 72 h was lower than those leaves treated for 24 h or 48 h, but H₂O₂ content in leaves during this period was still significantly higher than control group. The H₂O₂ content produced in leaves significantly changed treated by MMC, among which, the H₂O₂ contents in leaves were 0.163 µmol/g, 0.169 µmol/g, 0.217 µmol/g, and 0.298 µmol/g, respectively, treated by 5µg/mL, 10µg/mL, 20µg/mL, and 50µg/mL of MMC inoculation at 48 h (Fig. 5B).

Therefore, AOS content in leaves can be affected by pure compound MMC, especially, the higher concentration of MMC can cause serious impact and changed the content of H₂O₂ and O²⁻ in leaves.

Effects of pure compound MMC on chlorophyll content

Chlorophyll is the main component for capturing the light in photosynthesis. In the study, the chlorophyll content in leaves treated with different concentrations of MMC was decreased significantly (Table 3). The decline ratio of chlorophyll content was not obvious treated by 5 µg/mL ~ 20 µg/mL of MMC. But the decline ratio of chlorophyll content treated by 50 µg/mL and 100 µg/mL of MMC were 42.35% and 56.13%, respectively. Therefore, the result revealed that chlorophyll content can be damage by pure compound MMC identified from R. solani AG-3 TB, and the higher concentration of MMC can be produced, the large damage of the chlorophyll content can be caused.

Table 3

Chlorophyll content on *N. tabacum* treatment by pure compound MMC
Concentration (µg/mL)	Decline ratio of chlorophyll content (%)	Difference significance
Concentration (µg/mL)	Decline ratio of chlorophyll content (%)	5 (%)	1 (%)
CK	0	e	E
5	2.59	e	E
10	12.74	d	D
20	21.98	c	C
50	42.35	b	B
100	56.13	a	A

Effects of pure compound MMC on cell structure

In order to clarify the toxicity of phytotoxin of R. solani AG-3 TB to mesophyll cell structure of leaves, the structure of chloroplast, mitochondria in leaves were observed treatment by 50 µg/mL of MMC. The result indicated that comparing with control results of leaves treated with sterile water (Fig. 6a, b), the structure of chloroplast and mitochondrial cell began to split treated by 50 µg/mL of MMC at 12 h (Fig. 6c, d). Moreover, the cytoplasmic wall was seriously separated, and the structure of chloroplast membrane and lamellar were completely disappeared after leaves treated by MMC for 48 h (Fig. 6e, f).

Due to the special multi-nuclear and multi‐subgroup genetic characteristics and differentiation background of R. solani, it is relatively difficult to explore on pathogenicity in depth. However, the pathogenic factors including toxin compounds, effectors, and enzyme genes were confirmed during R. solani AGs infection. The toxin compounds of carboxylic acid in R. solani isolated and identified from AGs, such as phenylacetic acid (PAA), o‐hydroxy‐phenylacetic acid (o‐OH‐PAA), m‐hydroxy‐ phenylacetic acid (m‐OH‐PAA), and 3‐methylthi- opropionic Acid (3‐MTPA), were confirmed to be the main pathogenic toxin compound (Aoki., 1967; Kankam et al., 2016; Adachi et al., 1988; Bartz et al., 2013). Moreover, 3-methoxyphenylacetic acid (3-MOPAA) produced by AG-3 TB subgroup can cause the chlorosis of leaves at 24 hpi, and expression levels of crucial enzyme genes in PAA synthesis pathway were increased dramatically (Li et al., 2023; Li et al., 2022). In this study, MMC was identified to be an important toxin compound secreted by AG-3 TB using HPLC, IR, and NMR. At present, there is no relevant report that MMC as the secondary metabolites produced by microorganisms, however, it is interesting that this compound plays a key role in Wittig reaction as Wittig reagent, which is a valuable method for synthesis for aldehydes and ketones transform to olefins. During in Wittig reaction, the aldehyde condensed with MMC, the carbon-carbon single bond was transformed to the olefins with carbon-carbon double bond, such as in the raction of 7-acylamino-6,7-dideoxy-galactohepto-pyranoses, intermediate aldehyde was condensed with the Wittig reagent (methoxymethyl)triphenylphosphonium chloride, and a homologous heptodialdo-1,5-pyranose was afforded after hydrolysis (Wünsch., 2003). In addition, Wittig reaction also play an important role in insect pheromone, among which, in the synthesis of production for rice stem borer pheromone, MMC was used for Wittig reaction to obtain the intermediate product of 13 cis-1-methoxyoctadec-1,13-diene (Schlosser., 1970; Schlosser et al., 1985; Xiao et al., 2019). Therefore, we supposed that MMC in R. solani AG-3 TB may be an important reaction intermediate for producing some kind of double bond alkene, such as synthesis of insect pheromone.

Active oxygen species (AOS) plays several key roles involved in disease resistance, including the hypersensitive response in the plant-pathogen incompatible interactions, limit the entry and transmission of pathogens by cell wall reinforcements or directly killing pathogens, and activate host defense signaling pathways (Levine et al., 1994; Chamnongpol et al., 1998; Dat et al, 2000). The different H₂O₂ kinetics in AOS can activate defensive genes, induce programmed cell death, and promote the production of salicylic acid (SA). In the process of interaction between plants and pathogens, the toxin with low concentration usually has a powerful destructive role in damaging plants even harm human health (Nieto et al., 2018; Schafer, 1994). However, some reported that fungal secondary metabolites have a closely relation to the oxidative stress of plants, among which, H₂O₂ plays a crucial role in regulating the production of toxic factors. For instance, the specific Aspergillus flavus strains grew well in the medium containing 0 to 50 mM H₂O₂ and increasing H₂O₂ concentrations in the media resulted in elevated aflatoxin production in toxigenic isolates (Fountain et al., 2015). Moreover, the content of moniliformin (MON) and fumonisins (FBs) in corn can be effectively controlled after H₂O₂ treatment, which highlighted the direct impact of H₂O₂ on the stability of mycotoxins (Ferrigo et al., 2021). In this study, the content of AOS (H₂O₂ and O²⁻) was increased obviously treatment by MMC identified from R. solani at increased concentrations. This result revealed that MMC can induce the H₂O₂ production to stimulate plant defense, thereafter reducing the damage of the toxin to mesophyll tissue. At present, it is uncertain that the production of MMC identified from R. solani AG-3 TB has a direct relationship with AOS production in plants, but this compound may be a toxic compound to damage tissue.

Photosynthesis is critical for the synthesis of organic substances in plants. The solar energy and carbon dioxide can be absorbed by chlorophyll, which positively correlated with photosynthesis in plants (Suzuki et al., 1997; Eggink et al., 2001; Melis., 2009). Most toxic compounds can cause yellowing of leaves and reduce the chlorophyll content of plant leaves, such as higher MTPA concentrations decreased chlorophyll content of potato based on the data of the luminous energy and electron transfer efficiency (Kankam et al., 2016). In addition, the cell structure is a complex environment, which can ensure the self-regulation and order-progress of life. The cell membrane and cytoplasm fracture corroborating can be caused by high levels of toxin concentration. Particularly, mycotoxin of many necrotrophic pathogenic fungi cause cell death to facilitate the absorption of the nutrients (Friesen., 2008). Based on the result of the study, pure MMC identified in R. solani AG-3 TB may play a key contributor for producing disease spot and it also was an important toxin compound for spread of leaf chlorosis.

Bioactive molecules produced by specific fungi are known as secondary metabolites (natural products), which are crucial determinants in fungal development and metabolism and actively shape interactions with other organisms (Keller., 2018). Some secondary metabolites produced by most pathogenic fungi have harmful damage for host plants. Herein, MMC is a new phytotoxin compound isolated from R. solani AG-3 TB, which can decrease the chlorophyll content and damage the cellular structure. According to the content of AOS (H₂O₂ and O²⁻) in leaves, the defense system of plant may be stimulated by MMC. However, the toxicological mechanism, synthetic pathway and related pathogenic genes of this toxin substance are still unclear. Collectively, our results provide valuable insights into toxin compound in R. solani and potential pathogenic mechanism of the toxin compound in plant was validated.

Isolation, purification and identification the structure of toxin compound

In our previous study, the toxin extraction produced by R. solani AG-3 TB YC-9 strain was obtained with activated carbon adsorption extraction, and the purified toxin compound was tested by Thin-Layer Chromatography (TLC) (Li et al., 2023). Then the toxin sample was prepared by KBr compression method. The 1.8 mg of toxin sample and KBr was ground into powder and made into a sheet. The infrared absorption peak was determined by the Nicolet 6700 Fourier transform spectrometer (Thermo fisher Company, USA).

The mass charge ratio (m/z) of the toxin sample was obtained by the combined quadrupole Orbitrap mass spectrometer (Q Exactive™). The distilled water was used as mobile phase solvent, and ESI (-) ionization mode was adopted. The toxin sample was measured by the German Elemental Verio MICRO cube element analyzer. The sample was decomposed, quantified, and transformed, finally the percentage content of C, H, O and N was detected. The toxin structure was analyzed by ¹H-NMR and ¹³C-NMR spectra.

Pathogenicity of pure compound MMC

The MMC with 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL were used for inoculation on leaves of Nicotiana tabacum (Var.: NC89), the sterile water was used as control. The leaves were cultured at 28℃ for 3 d and the lesion diameters were measured. Each treatment was tested three times, and each treatment was performed on 10 tobacco plants.

The Active oxygen species (AOS) on N. tabacum inoculation by MMC

The MMC solution of 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL was injected into the leaves according to the infiltrating method (Armon, 1949). The treated leaves was cultured in a dish soaked with three layers of sterile gauze at 28℃, and the samples were collected at an interval of 8 h.

The leaf of 0.05g was dissolved with 1 mL phosphate buffer solution of 56 mmol/L and fully pestled. The extraction was centrifuged at 4 ℃, 12000 rpm for 8 min, and 1mL of 1 mol/L light amine hydrochloride (pH7.8) was dissolved 0.5 mL supernatant incubated for 1 h at 25 ℃, 1 mL of 17 mmol/L p-aminobenzene sulfonic acid and 1 mL of 7 mmol/L 1-naphthylamine were added and cultured in 25 ℃ for 25 min, then OD₅₃₀ number of this mixture was measured. The amount of different substance in the standard solution (n) was measured as NO²⁻ standard curve, and the absorbance was measured as the ordinate and the amount of NO^{2 –} was used as abscissa. According to the chemical reaction equation (µ mol·g^− 1·min^− 1), the production rate of superoxide ions (O^{2 −}) was calculated.

The H₂O₂ production in leaves was conducted according to constant volume colorimetry method (Patterson et al., 1984). The 0.05 g of leaf tissue was attrited evenly by 2 mL of precooled acetone. The exaction was centrifuged at 4 ℃, 12000 rpm, for 15 min. The 1mL of supernatant was absorbed as the sample extraction solution, and 1 mL of 5% titanium sulfate and 1 mL of concentrated ammonia water was added into extraction solution. The supernatant was removed, the precipitate was washed by acetone (3–5 times) and dissolved by 4 mL H₂SO₄ (2 mol/L). The solution constant volume colorimetry (415 nm) was measured.

Hydrogen peroxide content in leaf tissue (µmol/g Fw) = (C×Vo)/(Fw×Vt)

C - the sample on the standard curve of H₂O₂ concentration (µmol/L)

Vo - volume of sample extract (mL)

Vt - volume of supernatant used for determination (mL)

FW - weight of leaf tissue (g)

The chlorophyll content on N. tabacum treatment by MMC

The leaves were put into 7 mL of pure toxin solution with different concentrations of 5 µ g/mL, 10 µ g/mL, 20 µ g/mL, 50 µ g/mL, and 100 µ g/mL and cultured at 28 ℃, light for 3 d. Then, 0.2 g of leaf tissue was cut into pieces and the mixture of acetone: ethanol (V/V = 1:1) (10 mL) was infiltrated and incubated for 24 h in dark condition. When the color of leaves was observed from green to white, the OD₆₄₅ and D₆₆₃ were measured. The leaves in sterile water were used as the blank control, and each treatment was repeated three times.

Chlorophyll content in leaf tissue (Co) = [(20.29×OD₆₄₅ + 8.05×OD₆₆₃) ×V] / (1000×W)

V- volume of chlorophyll acetone extract (mL)

W- Fresh weight of leaf tissue (g)

The cell structure on N. tabacum treatment by MMC

The 20 µL MMC of 50 µg/mL was injected into leaves of tobacco (Var.: NC89). The inoculated leaves were treated in 25 ℃ in dark condition. Then leaves were washed with distilled water (2–3 times), cut it to 2 mm × 1 mm tissue, and dissolved with 2.5% glutaraldehyde solution at 4 ℃ for 24 h. The 70%, 80% ethanol solutions, as well as 85%, 95% and 100% acetone were used respectively for 15 min dehydration, and the samples were embedded with EPon812 resin. The tissue was stained with 2% uranyl acetate lead citrate, and the ultrastructural changes of tobacco mesophyll cells were observed by transmission electron microscopy (TEM).

The present research study indicated that (methoxymethyl)triphenylphosphonium chloride (MMC) was a new toxin compound identified from Rhizoctonia solani AG-3 TB. And the pure MMC with 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, and 100 µg/mL can cause obviously necrosis on leaves, increase active oxygen species (AOS), decrease chlorophyll content, and damage cellular structure. The results enrich the structure of toxin from R. solani and provide the new direction for exploration of toxicology.

Author contributions

Conceptualization, C.Z. and Y.W.; methodology, X.L., C.X.; software, X.L., H.H.; formal analysis, X.L., B.L.; resources, X.L., L.J., and M.A.; data curation, X.L.; writing—original draft preparation, X.L., Y.X., S.G., and M.A.; writing—review and editing, M.A., X.L.; supervision, Y.W.; project administration, C.Z. and Y.W.; funding acquisition, Y.W. All authors have read and agreed to the published version of the manuscript.

Conflict of interest statement

The authors declare no conflict of interest.

Fundings

This work was financially supported by the Catastrophe Mechanism and Key Technologies for Main Leaf Spot Prevention and Control (SCYC202113), and the National Natural Science Foundation of China (No. 32172454).

Sample Availability

Samples of the compounds are available from the authors.

Ethics approval

No use of human or animal samples or tissues in this study. The tobacco used in this study complied with all local, national or international guidelines and legislation.

Data availability

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

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Identification and physiological activity of (methoxymethyl)triphenylphosphonium chloride as a new phytotoxin isolated from Rhizoctonia solani AG-3 TB

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Abstract

Figures

Introduction

Results

Structure identification of the toxin compound

Effects of pure compound MMC on the production of active oxygen species (AOS)

Effects of pure compound MMC on chlorophyll content

Effects of pure compound MMC on cell structure

Discussion

Materials and methods

Isolation, purification and identification the structure of toxin compound

Pathogenicity of pure compound MMC

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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