Crude toxin production and chemical control of Boeremia exigua

Abstract

Mung bean (Vigna radiata (L.) R. Wilczek) rotiform disease is caused by Boeremia exigua var. exigua which affects seriously its yields and quality. During its growth, B. exigua can produce toxins but less is known about it. The biological characters of B. exigua were studied. The ideal culture conditions of mycelial growth were pH 5–8, 25 °C, static, and continuous light for 27 days, mannitol can replace sucrose as the most favorable carbon source in the modified Czapek solution, moreover, the addition of inositol or Vitamin B2 was benefit. The ideal culture conditions of crude toxin production were pH 5–7, 25 °C, static, and continuous light for 21 days, the studies did demonstrate that Czapek medium resulted in high levels of toxin formation, while Malt extract and Richard medium resulted in low production levels of toxins, there were no toxins are produced in PD medium at all. Sucrose and glucose can be used as suitable carbon source for the production of toxins, moreover, the addition of inositol or Vitamin C can stimulate the crude toxin production of B. exigua. The crude toxin of B. exigua has good thermal stability, low sensitivity to various wavelengths of light. It is also shown that crude toxins could inhibit the germination and radicle elongation of mung bean, lead to necrotic spots on leaves and have a strong wilt effect on seedlings of mung bean. It also had different degrees of inhibition to other crops such as sorghum, string bean and so on. In order to better control the disease, laboratory toxicities of 21 fungicides were tested. it revealed that Prochlomz has the greatest inhibitory effect, followed by Prochlomz and Carbendazim.

Introduction

Mung bean (Vigna radiata L.) is a pulse consumed legume crop globally, especially in Asian countries and is valued not only for its high nutrient value but also for its medicinal value^1,2, (Hou et al. 2023). However, corresponding plant diseases caused by plant pathogenic microorganisms are also happened more frequently, and become the main problems in yield reduction. B. exigua, the pathogen which is responsible of mung bean rotiform disease, belongs to Ascomycota, Dothideomycetes, Pleosporales, Didymellaceae, the genus of Boeremia³. The pycnidium of the pathogen is dark brown, spherical or oblate, with round spores, generally scatters or aggregates on the leaf surface. Its conidium, colorless cylindrical, not pointed and arc-shaped at both ends, begins to be unicellular and forms a constricted or slightly constricted septum at its middle or posterior part⁴ (Fig. 1).

Fig. 1.

The pycnidium of B. exigua.

In the early stage of disease, yellow-green spots will form on the mung bean leaves, and then gradually becomes concentric whorls, finally causes leaf perforation. In the late stages of the disease, small black spots will form in whorls or scattered on the diseased spots. When the disease is severe, the leaves dry out and fall off, turn red, even black⁵ (Fig. 2). Like other pathogens, B. exigua also produces non-specific toxins, for example, A phytotoxic toxin is a polyketide compound called ascochitine⁶. Nevertheless, it is less known about the mechanisms of the toxins production. In this study, the growth conditions, toxin-producing influence factors, and biological activity of the crude toxins from B. exigua were studied. In order to provide a theoretical foundation for better disease prevention, fungicides control experiments were conducted.

Fig. 2.

The symptom of Mung bean rotiform disease induced by B. exigua.

Materials and methods

Reagent and culture medium

The reagents are as follows: Zineb 80%WP (WP: water power), Shenyang Pesticide Co., Ltd.; Mancozeb 50%WP, Limin Chemical Co., Ltd; Iprodione 50%WP, Rhdne-poulenc S.A.; Carbendazim 50%WP, Zhejiang Dongfeng Pesticide Factory; Chlorothalonil 75%WP, Novartis Agrochemical China Co., Ltd; Thiophanate-Methyl 70%WP, Zhejiang Dongfeng Pesticide Factory; Prochloraz 25%Ec, Germany Aigefu Bio.Technology Limited; Ronilan (Vinclozolin) 50%WP, BASF; Xintaisheng 80%WP, Limin Chemical Co., Ltd; Fengmi (nicosulfuron)6% WP, Shanxi Qinyan Agrochemical Trade Co., Ltd; Sandofan (Oxadixyl) 64%WP, Sandex Pesticide Factory; Daobingning (Carbendazim&Triadimefon) 30%WP, Yangzhou Dongbao Agrochemical Company; Tuobushuang (Thiophanate Methyl &Thiram) 50% WP, Jiangsu Xinyi Pesticide Factory; Triflumizole 30%WP, Japan Caoda Corporation Limited; Haolike (Tebuconazole) 43%SC(emu), Bayer AG; Difenoconazole 10%WG (syngenta), Nuohua Agrochemical Co., Ltd.; Zhenling (2%Validamycin & 8% Bacillus cereus) 10% SC, Shanghai Nongle Biological Products Co., LTD; DT Fungicide 30% SL (Soluble liquid), Qishi Chemical Research Institute Co., Ltd; Oligosaccharins 20% SL, Dalian Kaifei Chemical Co., Ltd; OS-oligosaccharin 0.5% SL, Beihai Guofa Marine Biology Co., Ltd; Likubisha 3%SC, Liaoning Dongfang Petrochemical Co., Ltd.

The media is shown as follows. PD medium (g L⁻¹) was composed of 200 g potatoes, 20 g sucrose. PDA medium (g L⁻¹) was composed of 200 g potatoes, 20 g sucrose, 15 g agar. Mung bean decoction medium (g L⁻¹): 200 g mung beans were boiled with water for 30 min, and filtered with eight layers of gauze, then, 20 g of sucrose was added, water was added to the filtrate to 1000 ml. Malt extract medium (g L⁻¹) was composed of 5 g Malt paste powder, 0.1 g yeast extract. Czapek medium(g L⁻¹): NaNO₃ 3g, KH₂PO₄ 1g, MgSO_4·7H₂O 0.5g, KCl 5g, FeSO₄ 0.01 g, Sucrose 30 g, agarose 20 g. Richard medium (g L⁻¹): Sucrose 50 g, KNO₃ 10 g, KH₂PO₄ 5 g, MgSO₄ 2.5 g, FeCl₃ 0.02 g. All the chemicals and reagents used were of analytical grade.

Isolation of B. exigua

Diseased leaves of mung bean (mung bean L008, which picked at Peide Town, Mishan city, Heilongjiang province, China) were rinsed about 5 min under running water. Diseased spots of mung bean leave with a diameter of 2 ~ 3mm were cut with a scalpel, disinfected with 70% alcohol for 5 s, immersed in a 1:5 sodium hypochlorite aqueous solution for 2min, rinsed three times with sterile water, then picked pieces into PDA medium and cultured at 25 °C. The single colony on the PDA was selected and purified.

Cultivation and crude toxins filtrate obtainment of B. exigua

The pure mycelium with a diameter of 1 cm were harvested by punching a hole along the colony’s edge with punch, then three mycelium block was placed into 250 ml erlenmeyer flask containing 150 ml medium. After being kept at 25 °C for 20 days, the culture liquid was filtered using eight layers of dried gauze. The dry weight of the mycelium was determined by putting the dish in an electric heating oven with 85 °C until a constant weight. The culture filtrate was sterilized and kept in 4 °C for later use.

Concentration and purification of crude toxins liquid

The culture filtrate was boiled and concentrated to only one-third of its initial volume, then filtered through filter paper. crude toxins liquid was extracted using activated carbon column chromatography, then eluted with 40 ml of hot methanol three times. The eluted liquid was evaporated with rotary evaporator at 70 °C. At last, the concentrate of crude toxins liquid was diluted into solutions which contained 1%, 12.5%, 25%, 50% and 100% crude toxins and stored at 4 °C.

Radicle elongation inhibition rate of crude toxins on seed radicle of mung bean

Mung bean seeds were soaked in 60 °C water bath for about 2 min, then 20 seeds were positioned in the sterilizated petri dish which covered with two layers of filter paper. After 24 h of dark incubation at 25 °C, 6 ml of crude toxin was added into the petri dish, the length of the radicle was measured after 5 days. The growth inhibition rate of the radicle was calculated. The corresponding sterile culture medium was served as the control.

Effects of culture conditions on the growth and production of crude toxins of B. exigua

B. exigua mycelium growth rate and radicle elongation inhibition rate of Mung bean was used to determine the effects of different treatments on the growth and crude toxin production of B. exigua. each treatment was repeated for three times.

Five media were used to test the influence to growth and production of crude toxins of B. exigua, namely PD medium, Mung bean decoction medium, Malt extract medium, Czapek and Richard medium.

Effects of temperature: 150 ml Czapek medium Inoculated with equivalent hyphae of B. exigua was cultured in constant temperature incubators at 10 ℃, 15 ℃, 20 ℃, 25 ℃ and 30 ℃.

Effects of culture time: The pathogen was cultured in 150 ml Czapek medium at a biochemical incubator under all-day illumination at 25 ℃. From the 6th day after inoculation, 3 vials were taken from each treatment every 3 days for testing until the 35th day.

Effects of light: The different treatments were placed in the light biochemical incubator at 25 ℃. The treatment of total darkness was covered with a photographic jacket; the light and dark were alternately processed manually. The lighting treatment methods were full light (light:darkness = 24h:0h), alternating light and dark (light:darkness = 12h:12h) and full darkness (light:darkness = 0h:24h). The bioactivity of crude toxins and mycelia growth were determined after 20 days.

Effects of pH: Different treatments of pH was obtained with NaOH and HCl. After autoclave sterilization, the pH value of medium was measured, and the pathogen was inoculated and cultured at 25 ℃ for 10 days.

Effects of ventilation: 150 ml Czapek medium with equivalent hyphae (g) of B. exigua was placed in a shaking incubator (180 rpm/min) or an incubator with the same culture conditions for static culture at 25 ℃ for 10 days.

Effects of vitamin: equivalent VB1, VB2, VB6, Vc or inositol (g) was added into 150 ml Czapek medium at 25 ℃  for 15 days. Czapek medium without vitamin was used as control.

Effects of carbon sources: Czapek medium was used as the base medium, sucrose was replaced by equivalent different carbon sources (glucose, soluble starch, maltose, lactose, d-galactose, mannitol, sorbitol). All treatments were inoculated and cultured at 25 ℃ for 20 days.

Effects of toxins liquid to plant

Seedling needling method: Leaves of mung bean was punctured to make some small wound with insect needles, then dipped toxin liquid with sterile cotton on the leaves⁷, Czapek medium liquid and sterile water treatment were used as the controls. A glass cover was placed over the leaves for 1 day. The lesions on the leaves were checked after 15 days.

Seedling impregnation method: Mung bean seedlings were cleaned up with sterile water for 3times, then transferred to 50ml sterilized erlenmeyer flask which containing toxin liquid with different concentrations at 25 °C. As a control, the sterilized medium and water were used. The wilting index was used to represent the ability of toxins to cause seedling wilting activity. Level 0 are asymptomatic; level 1 involves the two cotyledons drooped and water lossed, but true leaves are asymptomatic; level 2 involves the two cotyledons drooped, water loss and yellow, at the same time, the true leaves drooped, water loss; level 3 involves not only the cotyledons, but the true leaves drooped and dried up; level 4 involves whole plant wilting and shedding of true leaves.

W, Wilting index; L, Wilting level value; n, number of seedlings at this wilting level; T, total seedling number.

Effects of toxins liquid to different plants: the tested plants were Vigna umbellata (Jingnong 5), Cucumis sativus L. (Dongling 120), Glycine max (Linn.) Merr. (Jiyu 653), mung bean, Capsicum annuum L (Sujiao 14), Phaseolus vulgaris L. (Shuangqing 12), Sorghum bicolor (Liaoza 12) and Oryza sativa L. (Jigeng 88). Seeds of the same size were placed in a small beaker containing toxin solution for 24 h, then thoroughly rinsed three times with sterile water. Next, thirty seeds of each plant were put in a petri dish (9 cm), covered with wet filter paper at 25 °C for 3 days. The rate of germination was examined.

Laboratory toxicity test of fungicides to B. exigua

Carbendazim powder was dissolved in 0.1 ml/L hydrochloric acid solution, then diluted with sterile water to achieve the required concentration; Prochloraz and Difenoconazole were dissolved in analytical pure acetone, then diluted with sterile water to achieve the required concentration; Other fungicides were prepared with sterile water. Fungicide preparation configuration equation:

A, required concentration of fungicide (μg/ml); B, active ingredient content of fungicides (%); V, the volume of fungicide (ml); X, the mass of weighed fungicide (g).

10 mL liquid fungicide was added into 90mL medium. When the sterilized PDA medium’s temperature reached 45–50 °C, medium was evenly distributed into four petri dish (9cm), then 10mm-diameter mycelium block was placed in the center of medium and cultured at 25 °C. After one day, the colony diameter was measured every 48 h using the cross-crossing method, and the inhibition rate of fungicides on the growth of mycelia was calculated. The medium containing no fungicides were served as the control (10 ml sterile water was added into 90mL medium). Inhibition rate was calculated using the following equation^8,9.

I, inhibition rate; d_c, colonial diameter of control; d_e, colonial diameter in the experimental group.

According to the above formula, using the logarithm of each drug concentration as the x-axis (x) and the probability value of inhibition rate as the y-axis (y), the toxicity regression equation y = ax + b was derived, and the correlation coefficient (r) and EC50 concentration value of the fungicide’s effective inhibition were calculated.

Statistical analysis

All the experiments were done in triplicate. The data was statistically analyzed using SPSS 25.0 software. To measure differences between of means, LSD test was used. All experiments with significant differences were detected at P = 0.05 or 0.01 level.

Results

Effects of culture conditions on the growth and production of crude toxins from B. exigua

The type of medium had a significant effect on the growth of B. exigua and production of crude toxins (Table 1). The influence of medium to mycelial yields were the same as the production of crude toxins. Not only Czapek culture medium conducive to the growth of pathogens, but it was also conducive to the crude toxin production, so it was chosen as the crude toxin production medium. Secondly, mycelium production and production of crude toxins was higher in media of Richard than all other media tested. As for PD medium, it was suitable for mycelium growth, whereas did not promote production of crude toxins.

Table 1.

Effects of media on the growth and production of crude toxins from B. exigua.

Medium	Mycelium dry weight (mg)	Radicle elongation inhibition rate (%) crude toxins to (%)	Significance of difference (P = 0.05)
PD	511	0.0	a
Green bean decoction	490	28.9	b
Malt extract	654	31.7	b
Richard	692	32.6	b
Czapek	700	56.0	c

In the tested temperature range, the biomass and radicle elongation inhibition rate increased with the degree of temperature, which ranged from 10 to 30 ℃, and then decreased slightly. the optimal temperature for the growth of mycelia was 25 °C, and the inhibition rate of the toxin on the radicle growth of mung bean was as high as 65 percent (Fig. 3).

Fig. 3.

Effects of culture temperatures on the growth and crude toxin production from B. exigua.

Different culture days had apparent effect on the growth and production of crude toxins. The results indicated that B. exigua began to produce crude toxins on the 6th day of culture, with the highest level of crude toxin production occurring on the 21st day, then, the crude toxin activity began to decrease. On the 27th day, the mycelial growth of the pathogens was at its peak (808 mg), thereafter, the mycelial dry weight decreased (Table 2).

Table 2.

Effects of culture days on the growth and production of crude toxins from B. exigua.

Time of cultivation (d)	Mycelial dry weight (mg)	Radicle elongation inhibition rate (%)	Significance of difference
			0.05	0.01
21	790	58.6	a	A
24	798	57.7	a	A
18	770	54.7	ab	AB
15	645	51.3	b	B
12	530	49.2	b	B
27	808	49.0	b	B
30	800	48.2	c	B
9	180	44.8	c	B
6	130	36.2	d	C

Light had obvious effects on the growth and crude toxin production of B. exigua. Continuous light was favorable, the radicle elongation inhibition rate was 57.2% under these conditions and mycelial growth was at its highest. However, the impact of complete darkness or alternately light and dark was not conducive to the mycelia growth of B. exigua or crude toxin production (Table 3).

Table 3.

Effects of light conditions on the growth and production of B. exigua.

Light	Mycelium dry weight (mg) ()( (mg/flask)	Radicle elongation inhibition rate (%)	Significance of difference
			0.05	0.01
Continuous light	665	57.2	a	A
Alternating light and dark	640	46.3	b	B
All dark	590	39.6	c	C

According to Table 4, the mycelial dry weight of B. exigua was heavier under static culture than oscillating culture, and the toxins activity of filtrate was also greater under static culture than oscillating culture. The radicle elongation inhibition rate under static culture was 57.2%, whereas it was only 43.7% under oscillating culture.

Table 4.

Effects of ventilation level on the growth and crude toxin production of B. exigua.

Mode of cultivation	Mycelium dry weight (mg)	Radicle elongation inhibition rate (%)	Significance of difference
			0.05	0.01
Static	645	57.2	a	A
Oscillating	600	43.7	b	B
Sterile water (CK)	0	0.0	c	C

Table 5 showed that the variation in pH influenced the production of crude toxins from B. exigua. The highest production ability of crude toxins was at pH9.96, the inhibition rate was 45%, whereas the inhibition rate was less than 30% when the pH was below 5 or above 8. Different pH values had a significant effect on the mycelial growth of pathogens. B. exigua grew most easily when the pH value of the culture medium was at 5–8.

Table 5.

Effects of pH levels on crude toxin production and growth B. exigua.

pH	Radicle elongation inhibition rate (%)	Mycelial dry weight (mg)
3.1	28	475
4.16	29	525
4.99	29	650
5.87	37	780
6.96	45	660
8.12	35	586
8.86	24	315

Carbon sources had significant effects on the production of crude toxins (Table 6). The most suitable carbon sources for the mycelial growth of B. exigua was not the most suitable carbon sources for the production of crude toxins. B. exigua grew most rapidly on mannitol-containing medium, and its mycelia dry weight was up to 823 mg. While B. exigua grew slowest on the sorbitol-containing medium, its mycelia dry weight was only 350 mg. Only 28.4% of the radicle growth of mung bean was inhibited at this conditions. Sucrose was best for the production of crude toxin, and the inhibitory rate of radicle growth of mung bean was 59.8%. mannitol had the similar effect with sucrose to the production of crude toxin.

Table 6.

Effects of carbon sources on crude toxin production and growth from B. exigua.

Carbon source	Mycelium dry weight (mg)	Radicle elongation inhibition rate (%)	Significance of difference difference
			0.05	0.01
Sucrose (CK)	710	59.8	a	A
Glucose	686	59.6	a	A
Mannitol	823	56.0	a	AB
Maltose	652	50.3	b	B
Lactose	610	48.8	b	B
D-Galactose	590	40.9	b	B
Starch	562	31.9	c	C
Sorbitol	350	28.4	c	C

Sucrose and soy protein were important for B. exigua vegetative growth and production of crude toxinss, which were supported by higher inhibitory rate and mycelial biomass. As shown in Table 7, inoitol was the most suitable compound for the production of crude toxins and mycelium growth, with an inhibitory rate of radicle elongation 74.8 percent and mycelium dry weight as high as 610 mg. Vc was also advantageous to the production of crude toxins with the 69.9% inhibitory rate. VB₂ had little effect on the crude toxin production.

Table 7.

Effects of vitamins on crude toxin production and growth from B. exigua.

Vitamins	Mycelium dry weight (mg)	Radicle elongation inhibition rate (%)	Significance of difference
			0.05	0.01
Inositol	610	74.8	a	A
Vitamin C	570	69.9	b	B
Vitamin B1	562	58.8	c	C
Czapek Medium without vitamins	600	58.7	c	C
Vitamin B2	610	56.8	c	C

Biological activity of crude toxins

Radicle elongation determination

As mentioned above, the results showed that the pathogens could produce toxins and toxins could inhibit the growth of mung bean radicle.

Seedling needling method

By means of acupuncture, 1 mL of toxin-soaked cotton was inserted into the wound, 3 days later, lesions on the treated portions of the leaves were seen. Whereas the control of blank culture medium without microbes hadn’t any lesions, the results demonstrated that toxic liquid could form necrosis patches on leaves of mung bean (Fig. 4).

Fig. 4.

Seedling needling effect on seeding of mung bean.

Seedling impregnation method

The effect of crude toxins is associated with its concentration. B. exigua caused mung bean seedlings to wilt severely. When seedlings were exposed with varying concentrations of toxic solution, the degree of wilting with increased with time and the index of wilting increased with concentration (Table 8). This is consistent with the findings of Liu et al.¹⁰. Of the five concentration investigated, 100% crude toxins were the most influential with the highest wilting index (100%) observed at 96 h. where the two controls had no any wilting influence to seedlings of mung bean.

Table 8.

The wilt effect of toxin on mung bean seedlings.

Concentration of crude toxins	Wliting index (%)
	9 h	24 h	36 h	48 h	72 h	96 h
100%	25.0	33.3	37.5	66.6	79.2	100.0
50%	20.8	29.2	37.5	62.5	75.0	87.5
25%	16.6	25.0	29.2	54.2	66.6	87.5
12.5%	9.5	20.8	25.0	33.3	50.0	66.6
1%	0.0	0.0	12.5	16.6	25.0	33.3
Czapek medium (ck)	0.0	0.0	0.0	0.0	0.0	0.0
Sterile water (ck)	0.0	0.0	0.0	0.0	0.0	0.0

Effects of crude toxins on seed germination of different plants

The effect of crude toxins on seed germination of several plants was investigated, the results indicated that B. exigua had obvious inhibitory effects on all different plants. The germination inhibition rate to the seed of Vigna umbellata was the highest, followed by Cucumis sativus, Glycine max and Mung bean. while that of Sorghum bicolor seed was the lowest (Table 9).

Table 9.

Effects of toxins on seed germination of different plants.

Plant	Germination inhibition rate (%)
Vigna umbellata	71.2
Cucumis sativus	65.7
Glycine max	51.7
Mung bean	51.7
Capsicum annuum	45.7
Phaseolus vulgaris	44.8
Oryza sativa	42.5
Sorghum bicolor	42.0

Laboratory toxicity test of fungicides to B. exigua

Fungicides effects to B. exigua at 1000 or 500 μg/mL

Fungicides had different effects at 1000 μg/mL. The control effect of 11 chemical fungicides which reached 100 percent, was utilized in the following test. Other fungicides were disused at the following test (see supplement material).

Fungicides had different effects at 500 μg/mL. Based on the inhibitory impact, they were separated into two groups: the first group consisted of Carbendazim, Thiophanate Methyl Compound (Mixture) and Difenoconazole (syngenta), all of which had inhibitory rates of 100 percent. The second group included Ronilan (Vinclozolin), Rovral (Iprodione), Thiophanate-Methyl, etc., whose inhibition rate was less than one hundred percent, and no further testing were done (see supplement material).

Inhibitory effect at 250 or 50 μg/mL

The results demonstrated that when the concentration of the fungicide was lowered to 250 g/mL, the inhibition rate of half of the fungicides on the mycelia growth of B. exigua remained extremely high, such as Carbendazim, Thiophanate Methyl, Prochloraz and Difenoconazole (syngenta), which remained at 100 percent (seesupplement material), while the other fungicides were disused at the following test.

When the concentration of each fungicide reduced to 50 μg/mL, the results revealed that the inhibition rate of fungicides on the mycelium also decreased. Difenoconazole is the fungicide with the highest inhibitory rate, reaching 50.5%. Inhibition rate of Carbendazim and Prochloraz was 47.3 and 46.7, separately (Table 10). Thiophanate Methyl Compound had the lowest inhibition rate (32.4%), so it was discontinued and the first three fungicides were chosen for the down-step test.

Table 10.

Inhibitory effects of fungicides at 50 µg/mL.

Fungicides	7d. colony diameter (cm)	Rate of inhibition (%)	Significance of difference
			0.05	0.01
Difenoconazole	3.9	50.5	a	A
Carbendazim	4.1	47.3	b	B
Prochlomz	4.2	46.7	c	C
Thiophanate Methyl	5.3	32.4	d	D
ck	7.0	0.0	c	E

Inhibitory effects of three fungicides at different concentrations

The experiment demonstrated that the inhibitory effect of Difenoconazole had a positive correlation with the increase of concentration, although there was no significant difference between the inhibitory effect at 35 μg/mL, 50 μg/mL, 60 μg/mL, and 75 μg/mL at P = 0.05 level. Inhibition rate was the highest at 63%, when the concentration of the drug is 75 μg/mL, which was at least 25% more than that in 15 μg/mL (Table 11).

Table 11.

Inhibitory effect of Difenoconazole at different concentrations.

Concentration (µg/mL)	4d. colony diameter (cm)	Rate of inhibition (%)	Significance of difference
			0.05	0.01
0	4.0	0.0	a	A
15	2.5	37.7	b	B
25	1.9	52.1	b	BC
35	1.8	54.8	c	BC
50	1.7	57.3	c	BC
60	1. 6	60.0	c	C
75	1.5	63.1	c	C

The rate of inhibition of Prochlomz at different concentrations had a significant level influence at P = 0.05 level, except the difference between 65 μg/mL and 80 μg/mL was not significant. 100 μg/mL Prochlomz had the best inhibit effect (63%), followed was the concentration of 80 μg/mL (Table 12). Inhibit effect at 25 μg/mL was the lowest, but the inhibition rate can also reach 35.1%.

Table 12.

Inhibitory effect of Prochlomz at different concentrations.

Concentration (µg/mL)	4th day mean colony diameter (cm)	Rate of inhibition (%)	Significance of difference
			0.05	0.01
0	4.0	0.00	a	A
25	2.6	35.1	b	B
30	2.4	40.2	c	C
50	2.3	42.56	cd	C
65	2.1	47.1	d	CD
80	1.7	57.8	d	D
100	1.6	60.3	e	D

Table 13 indicated that the inhibitory impact of carbendazim at various concentrations had a significant level influence at P = 0.05 level or 0.1 level. The concentration at 60 μg/mL supported significantly more mycelium inhabitation than all other concentration. Followed was 50 μg/mL, 30 μg/mL and 20 μg/mL. 25 μg/mL carbendazim has the lowest inhibitory effect occurring with 17.5%, which was one third less than the effect at 60 μg/mL.

Table 13.

Inhibitory effect of Carbendazim at different concentrations.

Concentration (µg/mL)	4th day mean colony diameter (cm)	Rate of inhibition (%)	Significance of difference
			0.05	0.01
0	4.0	0.0	a	A
10	3.3	17.5	b	B
20	3.0	25.0	c	D
30	2.6	35.0	d	C
50	2.2	45.0	e	E
60	1.9	52.5	f	F

Inhibitory effect of three fungicides at different times

The concentration of Difenoconazole was set as 80 μg/mL. The results demonstrated that there was no significant change in the growth amount of mycelia of B. exigua on the drug-containing medium over the treatment period, indicating that the inhibitory effect of the fungicide on B. exigua was more durable and effective. As the chemical is a fungicide with internal absorption, it can be treated at the earliest stages of disease development (Table 14).

Table 14.

Inhibitory effect of three fungicides at different time.

Time (d)	Net growth of mycelium in medium with 80 µg/mL Difenoconazole (cm)	Net growth of mycelium in medium with 80 µg/mL Prochlomz (cm)	Net growth of mycelium in medium with 60 µg/mL Carbendazim (cm)
11	0.1	0.5	0.4
9	0.2	0.4	0.4
7	0.1	0.4	0.5
5	0.3	0.3	0.5
3	0	0.3	0.4

The concentration of Prochlomz used in the experiment was 80 μg/mL. The results demonstrated that the net growth of mycelium raised with time. As the inhibitory activity of the fungicide declined with time, therefore, it is advisable to use at the earliest stage of the disease for enhancing its application effect (Table 14).

The concentration of Carbendazim used in the experiment was 50 μg/mL. Table 14 showed that carbendazim had a highly substantial inhibitory impact at various intervals, average growth rate of every 48 h was the same basically, and this effect was also long-lasting. Because Carbendazim was a kind of endothermic fungicide, long lasting effect is its advantage, which can protect plants for a long time through their internal circulation and distribution, it can be absorbed by the roots, stems, and leaves of plants, enter the plant body, transmit to the top, and can be transmitted to other parts of the plant. They can also kill pathogens or prevent external pathogens from invading. Zhu and Duan⁵ determined that carbendazim can be sprayed to control mung bean rotiform disease.

Regression equation of three fungicides

The fastest growth time of B. exigua on PDA medium was between the fourth and sixth days, hence the fifth day was chosen for the production of toxins regression curve. regression equation of Difenoconazole was Y = 0.044 + 0.285X, with EC50 = 40.04 μg/mL, regression equation of Prochlomz was Y = 0.348 + 0.380X, with EC50 = 95.50 μg/mL, regression equation of Carbendazim was Y = − 0.371 + 0.476X, with EC50 = 67.76 μg/mL. (X represents the concentration of the fungicide, and Y represents the logarithm of the inhibition rate of the mycelium growth of B. exigua at different concentrations of the fungicide). Testing the above three toxicity regression equations, the F values of Prochlomz, Difenoconazole and Carbendazim were 138.2272, 17.16162 and 44.23251, respectively, which were higher than those of F_0.05 (0.000299, 0.014402, and 0.006928), and the difference reached a significant level, indicating that overall regression equation was correct. The EC50 value of Difenoconazole were the lowest, followed by Carbendazim and Prochlomz indicated that the inhibitory effect of Difenoconazole on B. exigua was most obvious.

Conclusion and discussion

There are few studies on rotiform disease of mung bean currently. Zhang et al. conducted only a preliminary study on the physiological response caused by the infection of mung bean with B. exigua. She discovered that after mung bean was infected, the activities of peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase increased and then decreased¹¹. In 2018, Michel et al., first reported that B. exigua var. exigua could cause stem and leaf spot on common speedwell in Switzerland. In 2021, Wang et al., first reported that leaf spot was associated with B. exigua on white clover in China. At the same time, its secondary metabolites have been demonstrated various bioactivities. A number of sesquiterpenes, depsidones, cytochalasans and diaryl ethers have been reported from this fungus with cytotoxic and anti-inflammatory properties^12–14. However, there are few studies on the toxin production of B. exigua from mung bean rotiform disease currently. Toxins are a crucial pathogenic factor of phytopathic fungi that affect the subcellular and biological processes of the host¹⁵. Because of the significance of toxins in the pathogenic process of pathogens, understanding the mechanisms of toxin production is of utmost importance.

Results in this study demonstrated that the growth and crude toxin production of B. exigua were affected by many factors. Czapek medium was the most suitable whether it is for growth of mycelium or production of toxins. The ideal culture conditions of mycelial growth were pH 5–8, 25 °C, static, and continuous light for 27 days, mannitol can replace sucrose as the most favorable carbon source in the modified Czapek solution for growth of mycelium, moreover, the addition of inositol or Vitamin B2 in medium was benefit for higher levels of mycelial growth. Richard medium had the similar effect to the mycelial growth like Czapek medium.

The ideal culture conditions of crude toxin production were pH 5–7, 25 °C, static, and continuous light for 21 days, the studies did demonstrate that Czapek medium resulted in high levels of toxin formation, while Malt extract and Richard medium resulted in low levels of production of toxins, PD medium produced significantly lower mycelial growth compared to other medium, and there were no toxins are produced at all. Sucrose and glucose can be used as suitable carbon source for the production of toxins, moreover, the addition of inositol or Vitamin C can stimulate the development of crude toxin of B. exigua. The crude toxin has good thermal stability, low sensitivity to various wavelengths of light, so it can still maintain most of the activity after high temperature autoclave sterilization, which is conducive to the preservation of the toxin.

It is also shown that crude toxins of B. exigua could inhibit the germination and radicle elongation of mung bean. The results were similar to findings of Fu et al., on the which were produced by Ustilaginoidea virens (2017)¹⁶. It was found that by increasing the concentration of ustiloxins, the inhibition rate increased. Crude toxins of B. exigua led to necrotic spots on leaves and had a strong wilt effect on seedlings of mung bean. It also had different degrees of inhibition to other crops such as sorghum, string bean and so on. Further studies are needed to purify and analyze the pathogenic mechanism and structure of the toxin.

Some studies were done to investigate the issue of mung bean disease control¹⁷. Cao et al., studied systematically the prevention and treatment methods of mung bean rotiform disease symptom (2008)¹⁸. Wang et al., demonstrated that spraying 47% Garrinon wettable powder 700 times solution, and 40% good time suspension 500 times solution once every seven to ten days for a total of two to three times can also achieve a strong control effect (2013)¹⁹.

In order to better control the disease, we conducted research to screen out the best fungicide for inhibiting the growth of B. exigua. 11 fungicides exhibiting a 100% Inhibitory action at a dose of 1000 μg/mL were selected for further testing. At last, three fungicides were chosen for the down-step test. The three fungicides with the best inhibition effect were tested at various concentrations and times, and the regression equation of the effect of concentration on the value of hyphal growth inhibition rate was determined. Experiments revealed that the inhibitory effect of Difenoconazole reached a significant level at various concentrations and times. The inhibitory effect of different concentrations of Prochloraz reached a significant level, and at the concentration of 100 μg/mL, the inhibitory effect of different time reached a very significant level. However, the inhibitory effect decreased with the extend of time. Therefore, it is recommended to spray at the early stage of the disease. The analysis of the toxicity regression curve of the inhibition effect of the three fungicides reveals that Prochlomz has the greatest inhibitory effect, followed by Prochlomz and Carbendazim.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Pengpai Zhang: Methodology, Writing-original draft. Wenjing Zhang: Resources, Writing-original draft. Shihong Wang and Honglin Zhang: Data curation, software. Mengke Zhu,Yongmei Sun and Wu Die : Writing-review & editing. Haiyan Zhang: Investigation.

Funding

This study was supported in part by grants from Undergraduate Innovation and Entrepreneurship Training Program of Henan University, National Undergraduate Innovation and Entrepreneurship Training Program. (No.202310475098), Key Research and Development Projects of Henan Province (221111110200) , Science and Technology Program of Xizang Autonomous Region (QYXTCXZX—SNS—2022—1).

Data availability

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

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-79371-6.