Effects of UV-B radiation on the infectivity of Magnaporthe oryzae and rice disease-resistant physiology in Yuanyang terraces

Abstract

The traditional rice variety “Baijiaolaojing” was planted in Yuanyang terraces (1600 m altitude) under field conditions. The effects of enhanced UV-B radiation (0 kJ m⁻², 2.5 kJ m⁻², 5.0 kJ m⁻² and 7.5 kJ m⁻²) on the rice–Magnaporthe oryzae system were studied with respect to the Magnaporthe oryzae infection, the disease-resistance physiology of the rice and the rice blast disease condition. The results showed that under enhanced UV-B radiation, the infectivity of Magnaporthe oryzae was decreased, which could significantly inhibit its growth and sporulation. The activities of rice leaf disease-resistance-related enzymes (phenylalanine ammonia-lyase, lipoxygenase, chitinase and β-1,3-glucanase) were significantly increased under enhanced UV-B radiation. Following inoculation with Magnaporthe oryzae, levels of disease-resistance-related substances in the rice leaves were significantly increased. Among the results, it was found that leaves after UV-B radiation had a more significant resistance response. The level of UV-B irradiation showed a parabolic relationship with the rice blast index (r² = 0.85, P < 0.01; in the control group, r² = 0.88, P < 0.01). The disease index decreased with increase in irradiation. The DI was at a minimum with enhanced UV-B irradiance of 4 kJ m⁻²; thereafter, it increased with increasing irradiation. The enhanced UV-B radiation had a direct impact on the growth of rice and Magnaporthe oryzae, and it indirectly changed the rice–Magnaporthe oryzae system. UV-B radiation could reduce the harmful impact of rice blast.

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Introduction

Magnaporthe oryzae can affect rice leaves through the spread of conidia and causes the foliar fungal disease known as rice blast.¹ Rice blast occurs worldwide and severe rice blast can cause great losses of rice yields. The Magnaporthe oryzae rice infection process mainly includes spores attaching onto the host epidermis, spore germination, germ tube development, appressorium formation, growth in the host plant and the spread of spores.² Necrosis may occur on the surface of the infected leaves.

After rice has been infected with Magnaporthe oryzae, a defence system forms through pattern recognition receptors (PRR) identification and an immune system response.^3,4 The main physiological pathways to increase the disease resistance of rice include the hypersensitive response (HR),⁵ oxidative burst, defence-related protein accumulation, and phytoalexin synthesis and accumulation.⁶ During the infection process with Magnaporthe oryzae, an important component of the pathogenic fungal cell wall chitin can stimulate the plant defence response, promote the lipoxygenase (LOX) pathway and phenylalanine ammonia-lyase (PAL) pathway and synthesize pathogenesis-related proteins (PRPs).^7,8

Numerous studies have shown that UV-B radiation (280–315 nm) may also induce the plant to activate its defence system.^9–11 Enhanced UV-B radiation can induce plant cell ageing and apoptosis, reduce morbidity,^12–14 improve the activity of the plant phenylalanine ammonia-lyase (PAL) pathway and stimulate the production of polyphenolic compounds, improving the plant's resistance to stress.^15,16 The polyphenolics can absorb UV radiation, forming a defence barrier to prevent damage.^17–20 Plants can form a systemic functional defence system to prevent damage by light; they achieve this by altering physiological activities under field conditions in response to changing levels of UV-B radiation.²¹

When leaves infected by pathogenic fungi are exposed to UV-B radiation, the response of the fungal community on the surface of the leaves to UV-B radiation depends on the wavelength and irradiation of the incident light.²² The production, survival, propagation, invasiveness and virulence of the fungal conidia will be affected by UV-B radiation. In addition, the invasiveness and virulence of the pathogen are closely related to its photosensitivity.^23–26

In a field environment, changes in land-use (e.g., aridity, deforestation) and composition of ecosystems will affect the level of exposure of plants and other biota to UV-B radiation.^27,28 This in turn will affect the interaction between the plant and microorganisms.²⁹ In an agroecological system, the study of UV-B radiation has important theoretical and practical significance for the crop–pathogen interaction system.

Yuanyang terraces, a stable and sustainable rice field ecosystem, are planted in Yunnan Province.³⁰ They use a traditional rice variety. This has a stronger adaptive capacity to the environment and climate, as a result of long-term natural selection.³¹ Enhanced UV-B radiation can change the traditional Yuanyang terrace rice leaf morphology, plant N, P and K accumulation and transport, the physiological metabolism of stress-resistance substances and the antioxidant system, and thereby enhance its ability to resist adversity.^32–36 However, the effect of enhanced UV-B radiation on the Yuanyang terrace ecosystem rice–Magnaporthe oryzae, system is unclear. In this study, the traditional rice variety of the Yuanyang terraces, “Baijiaolaojing”, was selected as the research subject. The rice was planted in situ. In order to reveal the response mechanism of the rice–Magnaporthe oryzae system to enhanced UV-B radiation, and to provide a basis for predicting the effect of enhanced UV-B radiation on rice diseases, the progress of Magnaporthe oryzae infection was continuously observed, and the rice leaf physiological response analysed, following different UV-B irradiances (0 kJ m⁻², 2.5 kJ m⁻², 5 kJ m⁻² and 7.5 kJ m⁻²) and artificial inoculation with Magnaporthe oryzae.

Based on the phenomena mentioned above, a hypothesis was put forward that enhanced UV-B radiation would decrease the infectivity of Magnaporthe oryzae and induce the light defence physiology process, which, coupled with the disease-resistance process, would increase the disease resistance of the rice.

Materials and methods

Experimental conditions and local rice variety

The UV-B radiation rice field experimental station is located in Qingkou Village, Yuanyang County, Yunnan Province. It is the core area of Yuanyang terraced rice planting. A rice field at 1600 m altitude in the Yuanyang terraces was selected as the experimental station (23°7′15.8′′N, 102°44′45.6′′E). The physical and chemical properties of the experimental station soil were: the pH value of the soil was 6.58; the soil total N, total P and total K were 2.51 g kg⁻¹, 0.72 g kg⁻¹ and 5.98 g kg⁻¹, respectively, and the alkaline hydrolysis N, quick-acting P and K were 67.8 mg kg⁻¹, 20.3 mg kg⁻¹ and 150.8 mg kg⁻¹, respectively. The Yuanyang terrace local rice variety, “Baijiaolaojing”, was planted in situ (provided by the Agricultural Science Station in Yuanyang County). The rice variety has a 300-year history in the local area and is also the main cultivated variety at present.

Experimental design

The seedlings were grown in the rice field from March 16, 2015 and transplanted on May 14, 2015. Twenty-four plots were set as test points. The area of each plot was 390 cm × 225 cm. The interval was 50 cm. Ten rows of rice were planted in each plot, with 10 strains of rice per row. The line spacing was 15 cm. The row spacing was 30 cm. There was a rice seedling in every cluster. An isolation strip was established at the periphery. Polyethylene film, with a width of 1.5 m, was used to separate the plots. Conventional management was performed. Chemical fertilizers and pesticides were not used.

A lantern support adjustable for height was set up immediately above the rice. A 40 W UV-B lamp (wavelength 280–315 nm, Beijing Lighting Research Institute) was inserted, and the rice plants were put in place. A 0.13 mm cellulose acetate membrane was used to filter out the UV-C band light below 280 nm. The desired irradiances at the tops of the plants were achieved and detected by adjusting the lamp height along with the UV-B radiation tester (Beijing Normal University Photoelectric Instrument Factory). The enhanced UV-B radiation commenced on June 13th. Six plots were selected for enhanced radiation levels. The enhanced irradiation amounts were 0 kJ m⁻², 2.5 kJ m⁻², 5 kJ m⁻² and 7.5 kJ m⁻² (equivalent to increased UV-B radiation caused by 0%, 10%, 20% and 30% ozone layer attenuation, respectively, of the experimental field on a sunny summer day with 7 h radiation per d (10:00 AM–5:00 PM, except for rainy days)). The experiment was concluded on October 9^th and then the rice was harvested. In the natural-light group, the ultraviolet lamp holder alone was hung above the plants to ensure the consistency of natural light conditions between the treatment group and the control group. The irradiation was changed by adjusting the distance between the lamp and the plant material.

Cultivation and inoculation of Magnaporthe oryzae

Magnaporthe oryzae strain B6-4 was isolated from infected leaves of Yuanyang terrace “Baijiaolaojing”. The strain was cultured on oatmeal agar (28 °C, pure culture, for 7 d). The sample was cut into 2 mm mycelia, transferred to oatmeal agar (oatmeal – 50 g, saccharose – 20 g, agar – 16 g and distilled water – 1000 mL) and cultured for 10 d under 28 °C in the light. The spores were washed using sterile water. The concentration of the spore suspension liquid was adjusted to 3 × 10⁵ (spore) mL⁻¹ using a blood cell counting plate. After the rice had been exposed to UV-B radiation for 15 d, 3 plots were selected for each radiation level and the rice stem nodes were inoculated by needle tubing injection.

Determination of Magnaporthe oryzae infectivity-related indicators

At the time of inoculation with Magnaporthe oryzae (0 h), and 12 h after inoculation, five samples 4th leaves were randomly selected from each treatment group, placed on a glass slide containing aniline blue–glycerol–lactic acid solution and observed. Ten fields of view were randomly selected for each leaf. The total number of spores was recorded at 0 h. The number of germinated germ tubes was recorded at 12 h. The germination rate of the spores was calculated. At 32 h, 72 h and 120 h post-inoculation, a sample 4th leaf (a total of 5 leaves per plant) was selected randomly and removed. The spore amount was recorded at 32 h. The number of fungal colonies and scabs were recorded at 72 h and 120 h. After sparse greyish white mildew appeared on the leaf scab, 10 scabs were randomly labelled. The spores on the scab were carefully washed using sterile water and placed in a centrifuge tube. The volume was brought up to 0.5 mL. The sporulation quantity was measured using a blood counting plate 6 times for each treatment group, with 4 fields of views each time. The sporulation quantity was calculated by using the average value as the sporulation quantity for a single spore. The sporulation quantity was measured the next day and then every other day, and the measurements were stopped once the scabs had rotted away.³⁷

Determination of disease-resistant physiological indices of rice leaves

At the tillering stage of rice blast, the rice leaves of 24 plots were sampled. The second infected leaf from the bottom to the top and an uninoculated healthy leaf at the same position were selected. The leaf veins of the rice were removed. The specimens were washed with distilled water and dried with filter paper. The middle segment of the leaf was cut into 0.5 cm pieces. Liquid nitrogen was added and the leaf was ground into a powder.

Measurement of PAL activity was determined by adding 3 mL of 0.1 mol L⁻¹ boric acid buffer (pH = 8.8, containing 5 mmol L⁻¹ β-mercaptoethanol and 0.1 g L⁻¹ PVP) to 0.300 g of rice leaf. The mixture was ground in an ice-bath and centrifuged at 10 000g for 10 min in a refrigerated centrifuge at 4 °C. 20 μL of supernatant was collected, and 780 μL of 0.1 mol L⁻¹ boric acid buffer (pH = 8.8) and 200 μL of 0.02 mol L⁻¹ phenylalanine were added. The reaction mixture was incubated in a water bath at 30 °C for 0.5 h, and the change in the OD₂₉₀ value was measured using an ultraviolet–visible spectrophotometer (UV-5800, Shanghai Metash instruments Co., Ltd, Shanghai). One unit of activity represented a change in the optical density at 290 nm of 0.1 unit in 1 min in a 1 mL reaction system containing 1 mg of tissue protein.³⁸

Measurement of LOX activity. 4.5 mL of 50 mmol L⁻¹ buffer (containing 1.33 mmol L⁻¹ EDTA and 1% PVPP in PBS, pH = 7.8) were added to 0.300 g of rice leaf, which was then ground in an ice-bath and centrifuged at 16 000g for 20 min in a refrigerated centrifuge at 4 °C. The reaction mixture comprised 280 μL of reaction liquid (40 mL of 0.1 MPBS, pH of 7.0, containing 200 μL of Tween20 and 40 μL of linoleic acid). 20 μL of enzyme extract was added to start the reaction, and the optical density values at 234 nm were recorded at 15 s and 75 s using an ultraviolet–visible spectrophotometer (UV-5800). One unit of activity represented a change in optical density of 0.001 unit per min at 25 °C per mg of protein.³⁹

Measurement of chitinase activity. 0.100 g of rice leaf was used. Liquid nitrogen was added to grind it into a powder and 1 mL of acetate extraction buffer (0.05 mol L⁻¹, pH 5.0) was then added, which was followed by centrifugation at 12 000 rpm for 20 min at 4 °C. To 0.4 mL of the supernatant, 0.2 mL of acetate extraction buffer (0.05 mol L⁻¹, pH 5.0) and 0.4 mL of colloidal chitin solution (1%) were then added. The mixture was kept in a 37 °C water bath for 1 h, followed by centrifugation at 5000 rpm for 10 min. The N-acetyl glucosamine content in the supernatant was then measured. To 0.4 mL of supernatant, 0.2 mL of saturated borax solution was added and the mixture was kept in a boiling water bath for 7 min; after cooling, 0.2 mL of glacial acetic acid and 0.2 mL of 1% paradimethylaminobenzaldehyde (DMAB) solution was added and the reaction mixture was kept in a 37 °C water bath for 15 min. The optical density of the solution was measured at 585 nm with an ultraviolet–visible spectrophotometer (UV-5800). One enzyme activity unit was defined as the amount of enzyme required to decompose chitin to produce 1 mg of N-acetyl glucosamine per h per g of tissue.⁴⁰

Measurement of β-1,3-glucanase activity. 100 μL of crude enzyme liquid was used, and an equal volume of 0.05 mol L⁻¹ sodium acetate solution (pH = 5.0, and containing 5 mg mL⁻¹ of laminarin) was added. The mixture was then kept at 37 °C for 1 h. Then, 600 μL of DNS was added, and the mixture was kept at 100 °C for 5 min. After cooling, the mixture was diluted 20 times with distilled water. The absorbance at 540 nm was then measured using an ultraviolet-visible spectrophotometer (UV-5800), and the reducing sugar content was determined by comparison with a standard curve. One enzyme activity unit represented the production of 1 mg of reducing sugar by 1 g of tissue in 1 h; enzyme solution boiled for 5 min was used as the control.⁴¹

Measurement of silicon content. Rice plant samples were baked at 70 °C for more than 7 d, ground, sieved with a 60-mesh sieve, baked at 60 °C for 2 d, cooled and mixed. The sample was rapidly weighed. A 100 mg sample was placed in a high-pressure-resistant plastic pipe, and 3 mL of 50% NaOH solution was added; it was then shaken on an oscillating shaker, sterilized in an autoclave for 20 min and transferred to a 50 mL volumetric flask using a funnel. The constant volume was adjusted with distilled water. The sample was shaken upside down 10 times. A 1 mL sample was drawn into a 50 mL volumetric flask, and 30 mL of 20% tartaric acid was rapidly added. Then, 10 mL of ammonium molybdate solution (54 g L⁻¹, pH = 7.0) was added and mixed, and 5 min later 5 mL of 20% tartaric acid was quickly added. 1 mL of reducing reagent was then rapidly added. Finally, the volume was brought to 50 mL with 20% glacial acetic acid. The colorimetric assay was performed at 650 nm 30 min later (UV-5800). The absorbance value was read, and the silicon content was calculated.⁴²

Measurement of flavonoid content. A 0.10 g sample was removed using a punch and placed in a test tube, and the sample was extracted for 30 min with 10 mL of acidified methanol (methanol : water : HCl = 79 : 20 : 1). The absorbance at 305 nm was measured using an ultraviolet–visible spectrophotometer (UV-5800).⁴³ The flavonoid content was calculated.

Measurement of the total phenol content. A 0.1 g sample was placed in a test tube and the sample was extracted for 24 h with 10 mL of acidified methanol (methanol : water : hydrochloric acid = 7 : 1 : 2). The absorbance was measured with an ultraviolet–visible spectrophotometer (UV-5800). The total phenol content was calculated.⁴⁴

Effects of UV-B radiation on rice blast

One week after inoculation with Magnaporthe oryzae, the disease index (DI) of rice blast was observed. In accordance with the international rice disease level classification standard (IRRI),⁴⁵ the DI was divided into 10 levels according to the scab area. Each level represented the corresponding value. The number of rice leaves and DI were investigated every 5 days.

Data analysis and statistics

The data were processed using Microsoft Excel 2010 and drawn using Origin9.0. The data were analysed using the statistical software SPSS 19 Duncan test. P < 0.05 indicated that the difference was significant.

Results

Effect of enhanced UV-B radiation on Magnaporthe oryzae

The results of the UV-B radiation enhancement treatment indicated an obvious weakening of the pathogenicity of the Magnaporthe oryzae inoculation. The germination rate of the spores was significantly inhibited by UV-B radiation. The numbers of appressoria, fungal colonies and scabs were lower than those of the rice leaves under natural light conditions. Both 2.5 kJ m⁻² and 5.0 kJ m⁻² of UV-B radiation could significantly inhibit the formation of appressoria on the rice leaves (Table 1). The number of fungal colonies on the rice leaves significantly decreased after UV-B radiation. Both 5.0 kJ m⁻² and 7.5 kJ m⁻² of UV-B radiation could significantly inhibit the number of scabs and fungal colonies.

Table 1 Effects of enhanced UV-B radiation on the growth of Magnaporthe oryzae

UV-B radiation (kJ m^−2 d^−1)	Growth indicator
	Germination rate of the spores (%)	Appressorium	Fungal colony	Scab
Values are the average ± standard error. Different letters within the same column indicate significant differences between treatments (P < 0.05).
Ambient light	44.8 ± 2.6^a	140.0 ± 8.8^a	29.0 ± 2.3^a	21.0 ± 2.5^a
Enhanced 2.5	29.0 ± 2.5^b	91.0 ± 4.7^b	27.0 ± 2.6^ab	21.0 ± 1.5^a
Enhanced 5.0	21.9 ± 1.6^c	86.0 ± 2.7^b	23.0 ± 2.3^b	14.0 ± 0.9^b
Enhanced 7.5	32.7 ± 3.5^b	134.0 ± 3.4^a	23.0 ± 1.5^b	16.0 ± 1.2^ab

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The sporulation quantity of the rice leaves under natural light was significantly higher than of those treated with UV-B radiation. The three levels of UV-B radiation (2.5, 5.0 and 7.5 kJ m⁻²) reduced the sporulation quantity of the leaf scabs by 13.8%, 41.4% and 31%, respectively. The sporulation quantity of the rice leaves was lowest after 5.0 kJ m⁻² UV-B treatment, which was significantly different (P < 0.05) (Fig. 1).

Fig. 1 Effects of enhanced UV-B radiation on sporulation quantity. The results are given as the mean ± SE (n = 3). Different letters indicate significant differences between treatments (P < 0.05).

The study found that UV-B irradiation was significantly negatively related to leaf surface scabs, the number of fungal colonies and sporulation. A very significant positive correlation was found with sporulation quantity, scabs and fungal colonies (P < 0.01).

Response of the rice leaves to enhanced UV-B radiation

Enhanced UV-B radiation influenced the PAL activity of rice leaves infected by Magnaporthe oryzae (Fig. 2A). The PAL activity of rice leaves showed an upward trend with an increase in the UV-B radiation. Moreover, the PAL activity of the rice leaves significantly increased after inoculation. The PAL activity was highest at 5 kJ m⁻² of enhanced UV-B radiation. The LOX activity of the rice leaves was significantly increased at 5.0 kJ m⁻² UV-B enhanced UV-B radiation (Fig. 2B), but significantly decreased at 7.5 kJ m⁻² UV-B. The activities of chitinase (Fig. 2C) and β-1,3-glucanase (Fig. 2D) were significantly increased after the enhanced UV-B radiation (r = 0.739, P < 0.01 and r = 0.766, P < 0.01). In addition, infection with Magnaporthe oryzae also caused an increase in pathogenesis-related protein activity.

Fig. 2 Effects of enhanced UV-B radiation on enzyme activities related to disease resistance in rice leaves infected by Magnaporthe oryzae. (A) PAL; (B) lipoxygenase; (C) chitinase; (D) β-1,3-glucanase. Bars indicate standard errors of the mean ± 3 replications. Significance levels are indicated by ** and * representing P < 0.01 and P < 0.05, respectively.

The flavonoid, total phenol and silicon contents in rice leaves were all altered after UV-B radiation (Table 2). 5.0 kJ m⁻² UV-B radiation could significantly increase the flavonoid and total phenol contents. The silicon content of the rice leaves after UV-B radiation was significantly higher than that with natural light, and reached a maximum at 7.5 kJ m⁻² UV-B radiation. The flavonoid and total phenol contents in infected leaves after UV-B radiation were significantly higher than under natural light. The increased content of flavonoids was at a maximum after 5.0 kJ m⁻² UV-B radiation. The silicon content in the infected leaves was significantly increased with an increase in irradiation.

Table 2 Effect of enhanced UV-B radiation on the contents of different compounds in rice leaves

UV-B radiation (kJ m^−2 d^−1)	Control			Inoculation
	Flavonoids (A305 per g FW)	Total phenol (μg cm^−2)	Silicon (mg g^−1)	Flavonoids (A305 per g FW)	Total phenol (μg cm^−2)	Silicon (mg g^−1)
Values are the average ± standard error (n = 3). Different letters within the same column indicate significant differences between treatments (P < 0.05).
Ambient light	0.35 ± 0.03^c	51.73 ± 2.37^c	37.84 ± 0.41^c	0.44 ± 0.03^c	54.77 ± 2.1^c	34.11 ± 0.11^d
Enhanced 2.5	0.38 ± 0.05^c	56.17 ± 4.56^bc	55.63 ± 0.33^b	0.53 ± 0.03^b	60.30 ± 1.37^b	43.00 ± 0.50^c
Enhanced 5.0	0.72 ± 0.04^a	65.00 ± 0.20^a	55.63 ± 0.20^b	0.85 ± 0.05^a	68.07 ± 1.72^a	49.87 ± 0.41^b
Enhanced 7.5	0.61 ± 0.05^b	60.07 ± 1.05^ab	65.25 ± 0.34^a	0.82 ± 0.03^a	63.33 ± 1.31^b	60.28 ± 0.20^a

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Effect of enhanced UV-B radiation on rice blast disease index

The enhanced UV-B radiation changed the incidence and DI of rice blast (Table 3). Through the investigation of the incidence and DI of the rice tillering stage, we found that UV-B radiation significantly inhibited the occurrence of rice blast and reduced the severity of the disease. Treatment with 5.0 kJ m⁻² of UV-B radiation produced the maximum degree of inhibition, significantly higher than that produced by the other two levels. After 2.5 kJ m⁻² and 7.5 kJ m⁻² of UV-B radiation, the incidence was very similar, though in each case significantly higher than under natural light. After enhanced UV-B radiation, the DI of rice blast was significantly lower than under natural light. The difference among the three enhanced radiation treatment groups was not significant.

Table 3 Effect of enhanced UV-B radiation on the incidence and DI of rice blast

UV-B radiation (kJ m^−2 d^−1)	Control		Inoculation
	Incidence (%)	DI (%)	Incidence (%)	DI (%)
Values are the average ± standard error (n = 3). Different letters within the same column indicate significant differences between treatments (P < 0.05).
Ambient light	11.39 ± 2.82^a	12.47 ± 0.92^a	33.02 ± 1.22^a	27.60 ± 2.42^a
Enhanced 2.5	6.25 ± 1.68^b	5.23 ± 1.45^c	26.34 ± 1.36^b	13.88 ± 0.25^c
Enhanced 5.0	6.20 ± 1.55^b	4.52 ± 1.14^c	19.00 ± 1.14^c	15.16 ± 0.74^c
Enhanced 7.5	10.41 ± 4.60^a	9.62 ± 1.60^b	26.64 ± 1.55^b	19.67 ± 2.37^b

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Correlation analysis between enhanced UV-B radiation and rice blast disease index

A curve-fitting analysis of the DI of Magnaporthe oryzae-inoculated and uninoculated rice after UV-B irradiation was performed (Fig. 3). The rice blast DI showed a parabolic relationship with the radiation level. It first decreased and then increased. The DI was at a minimum with enhanced UV-B irradiance of 4 kJ m⁻².

Fig. 3 Curve-fitting analysis of the DI of Magnaporthe oryzae-inoculated and uninoculated rice after UV-B irradiation. The figure shows the relationship between rice blast disease index (DI) and UV-B radiation treatments in inoculated and uninoculated (control group) rice stem nodes. The formulas in the figure caption show one-variable curve-fitting quadratic equations (y = c + bx + ax²), along with their squared correlation coefficients (r²). The r² value is the measure of the degree of the linear relationship between DI and UV-B irradiation.

Discussion

Enhanced UV-B irradiation could reduce the infectivity of Magnaporthe oryzae. Spore germination, scabs, fungal colonies and sporulation were inhibited after the irradiation of rice leaves, and the colony sporulation ability was inhibited to varying degrees. UV-B radiation can induce the chemical mutation of pathogenic fungi DNA, change molecular structure and bipolymer composition,⁴⁶ cause cell death, affect the normal physiological activity of pathogenic fungi and directly damage the pathogens of plant exposed tissues.⁴⁷

A previous study found that the growth and sporulation of Magnaporthe oryzae grown on a medium exposed to 0 to 7.5 kJ m⁻² UV-B radiation were significantly inhibited.⁴⁸ Different proportions of the spores could not germinate and died. This led to the inhibition of fungal growth and sporulation. This result suggests that enhanced UV-B radiation is harmful to Magnaporthe oryzae. It confirmed the hypothesis that UV-B radiation reduced the infectivity of Magnaporthe oryzae. Similar results were obtained in previous research on Metarhizium anisopliae and Puccinia striiformis.^26,49 In this study, the growth and spore production of Magnaporthe oryzae under the enhancement of 7.5 kJ m⁻² UV-B radiation was significantly higher than with the 5 kJ m⁻² treatment. This result showed that the germination of spores was also influenced by the plant's innate resistance, and that high UV-B irradiation can weaken the disease resistance of rice. The UV-B radiation used also caused impaired pathogen sporulation. The enhanced UV-B radiation could promote Colletotrichum acutatum sporulation, which was related to the tolerance of pathogens to UV-B radiation.⁵⁰

When UV-B radiation is enhanced, the plant will directly detect the UV-B photons through the photoreceptor UVR8 (resistance locus UV 8) and form a systemic defence system to alleviate the damage caused by UV-B radiation. Exposure of rice to high amounts of UV radiation can result in a change in the phenylpropanoid pathway, increased activity of phenylalanine ammonia-lyase and increased synthesis of flavonoids that absorb UV-B radiation, which can protect the leaf tissues from damage.⁵¹ This finding was consistent with the results of a study of the pathogen's effect on the phenylpropanoid pathway in rice leaves. With the enhancement in UV-B radiation, the PAL activity of inoculated leaves was significantly increased, which was reflected in an increase in the synthesis and accumulation of phenolic substances in rice leaves (flavonoids and total phenols). This response could not only resist the UV-B radiation stress but also inhibited the pathogen infection in the incubation period.⁵²

The finding that LOX activity in rice leaves increased with enhanced UV-B irradiation was consistent with findings from other research.⁵³ In addition, LOX also plays an important role in rice immunity.⁵⁴ Hydroperoxides, oxygen radicals and other substances produced by LOX-catalysed membrane lipid peroxidation are involved in signal transduction. This can speed up the rapid ageing and death of unhealthy leaves, effectively inhibit the expansion of pathogenic fungi and improve the disease resistance of rice.⁵⁵ In this experiment, 5 kJ m⁻² enhanced UV-B radiation significantly increased the front-line defence against pathogen-induced injury. This could improve the resistance of plants, or it could speed up the ageing and death of infected leaf tissues. Dead cells constitute a physical barrier, reduce the availability of water and nutrients, speed up the pathogen cell death and reduce the incidence of disease. It appears that the tolerance of rice to disease decreased under 7.5 kJ m⁻² of UV-B radiation. The finding that the tolerance decreased was consistent with a study that showed that, when exposed to UV-B radiation, rice expended energy for growth at the same time as inducing defence mechanisms, causing a shortage of energy for growth in the partially weakened areas and resulting in an increased incidence of disease.⁵⁶ This difference might be related to the rice growth environment, degree of tolerance to UV-B radiation and adaptation to the environment.

To test the hypothesis that UV-B radiation could induce the light defence physiology process coupled with the disease-resistant process, we evaluated the effect of UV-B radiation on PRPs, which could enhance the active resistance of rice. It has been widely discussed that chitinase and β-1,3-glucanase, as the PRPs, enhance fungal resistance in rice.^57,58 The enhancement of the activities of chitinase and β-1,3-glucanase in rice leaves caused by Magnaporthe oryzae was promoted by pretreatment with UV-B radiation. Compared with natural light, after the Magnaporthe oryzae was inoculated, the activities of PAL, LOX, chitinase and β-1,3-glucanase defence enzymes substantially increased with enhanced UV-B radiation. Following the absorption of UV-B radiation photons by the UVR8 photoreceptor, defence-related hormones could be generated through gene transcription,²⁸ and thereby lead to the production of defensive phenolic compounds and PRPs.^59,60

Enhanced UV-B radiation can influence the infectivity and sporulation of Magnaporthe oryzae, alleviate the disease and reduce the spread and diffusion of rice blast. UV-B radiation can stress the rice, induce the formation of a defence system and indirectly improve the resistance of rice. In this study, the enhanced UV-B radiation was within the tolerance range of the plants, which would not affect the normal physiological activities of the plants, but could improve the sensitivity of the plants’ resistance to stress so that they could quickly respond to pathogen infections. The combination of pathogenic fungi and UV-B radiation changed the disease incidence and disease index of the traditional rice variety in a Yuanyang terrace. It had an inevitable connection with the fitness of “Baijiaolaojing” to the environment. UV-B radiation occupies only a very small proportion of the solar spectrum (less than 1%). The irradiation attenuates sharply through canopy obstruction.⁶¹ The rice is dominant in the rice–Magnaporthe oryzae system. Under UV-B radiation stress, the free radicals increased, and they were continuously eliminated through the antioxidase system to maintain the normal physiological indices.³⁶ UV-B radiation can change the composition and content of root exudates of rice,⁶² change the environment of rice roots, reduce the fungal microorganisms in the soil and reduce the occurrence of disease.⁶³ Therefore, the tolerance of a rice variety to UV-B radiation is the main factor in determining disease resistance.

In this study, with an enhancement in UV-B radiation in the moderate range, the rice disease resistance increased because of the adjustment of physiological activities. Meanwhile, UV-B radiation inhibited the pathogenic fungi and alleviated the rice blast. When the irradiation exceeded the tolerance range of rice, the rice responded to the dual stresses of pathogen and UV-B radiation, and the growth energy distribution was unbalanced. The growth energy was insufficient, so that the protective barrier was more vulnerable, the antioxidant system was damaged and the success rate of the pathogen infection increased. However, the inhibiting effect of UV-B radiation on the Magnaporthe oryzae pathogenicity showed a threshold. When the irradiation exceeded the threshold, the damage to the pathogen was weakened. However, it could damage the resistivity of the host plant, eventually worsening the disease condition. In addition, studies have found that UV-B radiation treatment and then pathogen inoculation can increase the susceptibility of the plant. The reason is that the UV-B radiation injury favours the infection of pathogenic fungi.⁶⁴ However, we did not reach similar conclusions, which might have been linked to the characteristics of the dominant rice variety of Yuanyang traditional rice, which is a product of considerable genetic diversity and natural selection. The effects of dual stress on the grain production of rice requires further analysis.

Conclusions

The study found that enhanced UV-B radiation changed the survival interaction between traditional Yuanyang terrace rice and Magnaporthe oryzae. Under a certain radiation level, it could reduce the infectivity of Magnaporthe oryzae, improve the disease resistance of rice leaves, indirectly change the rice–Magnaporthe oryzae system and reduce the occurrence of rice blast.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 31460141, 31760113 and 41565010), The Innovation Team for Farmland Non-pollution Production of Yunnan Province (2017HC015) and Yunnan Agricultural University Innovation Foundation for Postgraduate (2015ykc35).

Notes and references

1. M. C. Fisher, D. A. Henk, C. J. Briggs, J. S. Brownstein, L. C. Madoff, S. L. McCraw and S. J. Gurr, Emerging fungal threats to animal, plant and ecosystem health, Nature, 2012, 484(7393), 186–194.
2. R. Galhano and N. J. Talbot, The biology of blast: Understanding how Magnaporthe oryzae, invades rice plants, Fungal Biol. Rev., 2011, 25(1), 61–67.
3. S. T. Chisholm, G. Coaker, B. Day and B. J. Staskawicz, Host-microbe interactions: shaping the evolution of the plant immune response, Cell, 2006, 124(4), 803–814.
4. J. D. G. Jones and J. L. Dangl, The plant immune system, Nature, 2006, 444(7117), 323–329.
5. K. Hayashi, Y. Fujita, T. Ashizawa, F. Suzuki, Y. Nagamura and Y. Hayano-Saito, Serotonin attenuates biotic stress and leads to lesion browning caused by a hypersensitive response to Magnaporthe oryzae penetration in rice, Plant J., 2016, 85(1), 46–56; C. L. Ballaré, M. M. Caldwell, S. D. Flint, S. A. Robinson and J. F. Bornman, Effects of solar ultraviolet radiation on terrestrial ecosystems. Patterns, mechanisms, and interactions with climate change, Photochem. Photobiol. Sci., 2011, 10(2), 226–241.
6. Y. Nishizawa, S. Mochizuki, N. Yokotani, T. Nishimura and E. Minami, Molecular and cellular analysis of the biotrophic interaction between rice and Magnaporthe oryzae –– Exploring situations in which the blast fungus controls the infection, Physiol. Mol. Plant Pathol., 2016, 95, 70–76.
7. E. W. Chehab, J. V. Perea, B. Gopalan, S. Theg and K. Dehesh, Oxylipin pathway in rice and Arabidopsis, J. Integr. Plant Biol., 2007, 49(1), 43–51.
8. J. Wan, X. C. Zhang, D. Neece, K. M. Ramonell, S. Clough, S. Y. Kim, M. G. Stacey and G. Stacey, A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis, Plant Cell, 2008, 20(2), 471–481.
9. D. I. Lytvyn, A. I. Yemets and Y. B. Blume, UV-B overexposure induces programmed cell death in a BY-2 tobacco cell line, Environ. Exp. Bot., 2010, 68(1), 51–57.
10. Y. Yao, Z. Xuan, Y. Li, Y. He and H. Korpelainen, Effects of ultraviolet-B radiation on crop growth, development, yield and leaf pigment concentration of tartary buckwheat (Fagopyrum tataricum) under field conditions, Eur. J. Agron., 2006, 25(3), 215–222.
11. X. X. Gao, Z. H. Gao, J. J. Chen, Y. Q. Zu and Y. Li, Effects of enhanced UV-B radiation and inoculated Magnaporthe grisea on three physiological indexes in seedling leaf of two cultivars of Oryza sativa, J. Plant Resour. Environ., 2010, 19(1), 56–62.
12. Y. Li, Y. Q. Zu, L. L. Bao and Y. M. He, The responses of spatial situation, surface structure characteristics of leaves and sensitivity of two local rice cultivars to enhanced UV-B radiation under terraced agricultural ecosystem, Acta Physiol. Plant., 2014, 36(10), 2755–2766.
13. M. T. Charles, K. Tano, A. Asselin and J. Arul, Physiological basis of UV-C induced resistance to Botrytis cinerea in tomato fruit. V. Constitutive defence enzymes and inducible pathogenesis-related proteins, Postharvest Biol. Technol., 2009, 51(3), 414–424.
14. E. W. Chehab, C. Yao, Z. Henderson, S. Kim and J. Braam, Arabidopsis touch-induced morphogenesis is jasmonate mediated and protects against pests, Curr. Biol., 2012, 22(8), 701–706.
15. E. S. Mccloud and M. R. Berenbaum, Stratospheric ozone depletion and plantinsect interactions: Effects of UVB radiation on foliage quality of Citrus jambhiri for Trichoplusia ni, J. Chem. Ecol., 1994, 20(3), 525–539.
16. Y. Li, X. X. Gao, Z. H. Gao, Y. M. He, J. J. Chen and Y. Q. Zu, Effect of enhanced UV-B radiation and inoculated blast isolate (Magnaporthe grisea) on phenylalanine ammonial-yase activity and flavonoid content in seedlings of two rice cultivars, Chin. J. Eco-Agric., 2010, 18(4), 856–860.
17. Y. Dolzhenko, C. M. Bertea, A. Occhipinti, S. Bossi and M. E. Maffei, UV-B modulates the interplay between terpenoids and flavonoids in peppermint (Mentha× piperita L.), J. Photochem. Photobiol., B, 2010, 100(2), 67–75.
18. I. Eichholz, S. Huyskens-Keil, A. Keller, D. Ulrich, L. W. Kroh and S. Rohn, UV-B-induced changes of volatile metabolites and phenolic compounds in blueberries (Vaccinium corymbosum L.), Food Chem., 2011, 126(1), 60–64.
19. J. Singh, S. Gautam and P. A. Bhushan, Effect of UV-B radiation on UV absorbing compounds and pigments of moss and lichen of Schirmacher Oasis region, East Antarctica, Cell. Mol. Biol., 2012, 58(1), 80–84.
20. M. Schreiner, J. Martínez-Abaigar, J. Glaab and M. Jansen, UV-B induced secondary plant metabolites, Opt. Photonik, 2014, 9(2), 34–37.
21. P. V. Demkura, G. Abdala, I. T. Baldwin and C. L. Ballaré, Jasmonate-dependent and -independent pathways mediate specific effects of solar ultraviolet B radiation on leaf phenolics and antiherbivore defense, Plant Physiol., 2010, 152(2), 1084–1095.
22. Y. Feng, Y. Q. Zu, H. Y. Chen and Y. Li, Effects of enhanced UV-B radiation on growth, physiology and athogenicity of Alternaria alternate, Plant Prot., 2010, 36(4), 70–74.
23. A. Idnurm, S. Verma and L. M. Corrochano, A glimpse into the basis of vision in kingdom Mycota, Fungal Genet. Biol., 2010, 47(11), 881–892.
24. A. Idnurm, Light sensing in Aspergillus fumigatus highlights the case for establishing new models for fungal photobiology, mBio, 2013, 4(3), e00260–e00213.
25. S. M. Yu, G. Ramkumar and Y. H. Lee, Light quality influences the virulence and physiological responses of Colletotrichum acutatum causing anthracnose in pepper plants, J. Appl. Microbiol., 2013, 115(2), 509–516.
26. P. Cheng, Z. Ma, X. Wang, C. Wang, Y. Li, S. Wang and H. Wang, Impact of UV-B radiation on aspects of germination and epidemiological components of three major physiological races of Puccinia striiformis f. sp. tritici, Crop Prot., 2014, 65(4), 6–14.
27. C. E. Williamson, R. G. Zepp, R. M. Lucas, S. Madronich, A. T. Austin, C. L. Ballare, M. Norval, B. Sulzberger, A. F. Bais, R. L. McKenzie, S. A. Robinson, D. P. Häder, N. D. Paul and J. F. Bornman, Solar ultraviolet radiation in a changing climate, Nat. Clim. Change, 2014, 4(6), 434–441.
28. C. L. Ballaré, M. M. Caldwell, S. D. Flint, S. A. Robinson and J. F. Bornman, Effects of solar ultraviolet radiation on terrestrial ecosystems. Patterns, mechanisms, and interactions with climate change, Photochem. Photobiol. Sci., 2011, 10(2), 226–241.
29. M. M. Caldwell, J. F. Bornman, C. L. Ballaré, S. D. Flint, L. O. Björn, A. H. Teramura, G. Kulandaiveluf and M. Tevini, Terrestrial ecosystems, increased solar ultraviolet radiation, and interactions with other climate change factors, Photochem. Photobiol. Sci., 2007, 6(3), 252–266.
30. X. H. He, Y. Sun, D. Gao, F. G. Wei, L. Pan, C. W. Guo, R. Z. Mao, Y. Xie, C. Y. Li and Y. Y. Zhu, Comparison of agronomic traits between rice landraces and modern varieties at different altitudes in the paddy fields of Yuanyang terrace, Yunnan province, J. Resource. Ecol., 2011, 2(1), 46–50.
31. J. J. Lin, J. B. Li, L. Liu, X. Li, H. F. Li, X. H. He, S. S. Zhu, C. Y. Li and Y. Y. Zhu, Genetic diversity of Magnaporthe grisea of Hani terrace from Yuanyang county in Yunnan, Acta Phytopathol. Sin., 2009, 39(1), 43–51.
32. L. L. Bao, Y. M. He, Y. Q. Zu, Y. Li, G. X. Gao and Y. Z. He, Effects of enhanced UV-B radiation on the leaf morphology and anatomical structure of two local rice varieties in Yuanyang terraced fields, Yunnan Province of Southwest China, Chin. J. Ecol., 2013, 32(4), 882–889.
33. C. Liu, Y. M. He, Y. Q. Zu, Y. Li and Y. F. Tang, Effects of enhanced UV-B radiation on N accumulation of two traditional rice colonies in Yuanyang terraces under field conditions, J. Agro-Environ. Sci., 2013, 32(8), 1493–1499.
34. Y. Q. Zu, C. Liu, Y. M. He and Y. Li, Effects of two-year concessional enhance UV-B radiation on P and K accumulation and heterogeneous distribution of 2 traditional rice varieties in Yuanyan terraces, J. Ecol. Environ. Sci., 2015, 24(8), 1259–1265.
35. Y. M. He, F. D. Zhan, Y. Q. Zu, H. Y. Chen and Y. Li, Effects of UV-B radiation on the contents of silicon, flavonoids and total phenolic of two local rice varieties in Yuanyang terrace under field conditions, J. Agro-Environ. Sci., 2013, 32(8), 1500–1506.
36. Y. M. He, F. D. Zhan, Y. Q. Zu, C. Liu and Y. Li, Effect of elevated UV-B radiation on the antioxidant system of two rice landraces in paddy fields on Yuanyang terrace, Int. J. Agric. Biol., 2014, 16(3), 585–590.
37. H. C. Huang, Study on Mechanism of nitrogen-induced susceptibility of rice to rice blast, Yunnan Agriculture University, Kunming, 2014.
38. G. Engelsma, On the mechanism of the changes in phenylalanine ammonia-lyase activity induced by ultraviolet and blue light in gherkin hypocotyls, Plant Physiol., 1974, 54(5), 702–705.
39. J. F. Todd, G. Paliyath and J. E. Thompson, Characteristics of a membrane-associated lipoxygenase in tomato fruit, Plant Physiol., 1990, 94(3), 1225–1232.
40. J. L. Reissig, J. L. Storminger and L. F. Leloir, A modified colorimetric method for the estimation of N-acetylamino sugars, J. Biol. Chem., 1955, 217(2), 959–966.
41. A. Ippolito, A. Elghaouth, C. L. Wilson and M. Wisniewski, Control of postharvest decay of apple fruit by Aureobasidium pullulans and induction of defense responses, Postharvest Biol. Technol., 2000, 19(3), 265–272.
42. W. M. Dai, K. Q. Zhang, B. W. Duan, C. X. Sun, K. L. Zheng, R. Cai and J. Y. Zhuang, Rapid determination of silicon content in rice (Oryza sativa), Chin. J. Rice Sci., 2005, 19(5), 460–462.
43. R. M. Mirecki and A. H. Teramura, Effects of ultraviolet-B irradiance on soybean: V. The dependence of plant sensitivity on the photosynthetic photon flux density during and after leaf expansion, Plant Physiol., 1984, 74(3), 475–480.
44. V. G. Kakani, K. R. Reddy, D. Zhao and W. Gao, Senescence and hyperspectral reflectance of cotton leaves exposed to ultraviolet-B radiation and carbon dioxide, Physiol. Plant., 2004, 121(2), 250–257.
45. IRRI, Standard evaluation system for rice(SES), International Rice Research Institute, Los Banos, Philippine, 2002.
46. R. P. Rastogi, A. W. Richa, A. Kumar, M. B. Tyagi and R. P. Sinha, Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair, J. Nucleic Acids, 2010, 592980.
47. B. M. Wu, K. V. Subbarao and A. H. C. Van Bruggen, Factors affecting the survival of Bremia lactucae sporangia deposited on lettuce leaves, Phytopathology, 2000, 90(8), 827–833.
48. Y. Zhao, Y. Q. Zu and Y. Li, Effects of enhanced UV-B radiation on growth and sporulation quantity of blast isolate Magnaporthe grisea, J. Agro-Environ. Sci., 2010, 29(S1), 1–5.
49. G. U. L. Braga, S. D. Flint, C. D. Miller, A. J. Anderson and D. W. Roberts, Both solar UVA and UVB radiation impair conidial culturability and delay germination in the entomopathogenic fungus Metarhizium anisopliae, Photochem. Photobiol., 2001, 74(5), 734–739.
50. H. D. Menezes, N. S. Massola Jr., S. D. Flint, G. J. Silva, L. Bachmann, D. E. N. Rangel and G. U. L. Braga, Growth under visible light increases conidia and mucilage production and tolerance to UV-B radiation in the plant-pathogenic fungus Colletotrichum acutatum, Photochem. Photobiol., 2015, 91(2), 397–402.
51. C. S. Buer, N. Imin and M. A. Djordjevic, Flavonoids: new roles for old molecules, J. Integr. Plant Biol., 2010, 52(1), 98–111.
52. E. S. Mccloud and M. R. Berenbaum, Stratospheric ozone depletion and plantinsect interactions: Effects of UVB radiation on foliage quality of Citrus jambhiri, for Trichoplusia ni, J. Chem. Ecol., 1994, 20(3), 525–539.
53. J. K. Sung, S. H. Lee, S. Y. Lee, M. B. Shim, T. W. Kim and B. H. Song, Antioxidants stimulated by UV-B radiation in rice seedling, Korean J. Crop Sci., 2004, 49(2), 116–120.
54. S. S. Marla and V. K. Singh, LOX, genes in blast fungus (Magnaporthe grisea) resistance in rice, Funct. Integr. Genomics, 2012, 12(2), 265–275.
55. H. Ohta, K. Shida, Y. L. Peng, I. Furusawa, J. Shishiyama, S. Aibara and Y. Morita, A lipoxygenase pathway is activated in rice after infection with the rice blast fungus Magnaporthe grisea, Plant Physiol., 1991, 97(1), 94–98.
56. M. R. Finckh, A. Q. Chavez, Q. Dai and P. S. Teng, Effects of enhanced UV-B radiation on the growth of rice and its susceptibility to rice blast under glasshouse conditions, Agric., Ecosyst. Environ., 1995, 52(2–3), 223–233.
57. G. Sridevi, C. Parameswari, N. Sabapathi, V. Raghupathy and K. Veluthambi, Combined expression of chitinase and β-1,3- glucanase genes in indica, rice (Oryza sativa, L.) enhances resistance against Rhizoctonia solani, Plant Sci., 2008, 175(3), 283–290.
58. S. Karmakar, K. A. Molla, P. K. Chanda, S. N. Sarkar, S. K. Datta and K. Datta, Green tissue-specific co-expression of chitinase, and oxalate oxidase 4, genes in rice for enhanced resistance against sheath blight, Planta, 2016, 243(1), 115–130.
59. P. V. Demkura, G. Abdala, I. T. Baldwin and C. L. Ballaré, Jasmonate-dependent and -independent pathways mediate specific effects of solar ultraviolet B radiation on leaf phenolics and antiherbivore defense, Plant Physiol., 2010, 152(2), 1084–1095.
60. C. Wasternack and B. Hause, Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany, Ann. Bot., 2013, 111(6), 1021–1058.
61. C. L. Ballaré, C. A. Mazza, A. T. Austin and R. Pierik, Canopy light and plant health, Plant Physiol., 2012, 160(1), 145–155.
62. Y. M. He, F. D. Zhan, Y. Li, Y. Q. Zu and M. Yue, Effect of enhanced UV-B radiation on methane emission in a paddy field and rice root exudation of low-molecular-weight organic acids, Photochem. Photobiol. Sci., 2016, 15(6), 735–743.
63. C. H. Kong, P. Wang, Y. Gu, X. H. Xu and M. L. Wang, Fate and impact on microorganisms of rice allelochemicals in paddy soil, J. Agric. Food Chem., 2008, 56(13), 5043–5049.
64. A. B. Orth, A. H. Teramura and H. D. Sisler, Effects of ultraviolet-B radiation on fungal disease development in Cucumis sativus, Am. J. Bot., 1990, 77(9), 1188–1192.

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