张国辉, 李荣玉, 吴小毛, 李 明

(1. 贵州大学 作物保护研究所，贵阳 550025；2. 贵州省山地农业病虫害重点实验室，贵阳 550025；3. 凯里学院 大健康学院，贵州 凯里 556000)

Rice (Oryza sativa L.) is an important food crop grown under worldwide，one of the major classes of organisms that cause plant disease are phytopathogenic fungi. Over the past decades, the actions of these organisms worldwide have caused millions of dollars[1-2],The diseases are spread worldwide, but its occurrence and severity vary by year, location and environmental conditions[3-5]. Curvularia leaf spot,caused mainly by Curvularia lunata, is a widespread plant disease in China. C. lunata could cause the seeds of the rice to lose the germination capacity and cause the seedling blight and leaf spot of the rice. In recent years, directional host selection by the pathogen, which likely results in the virulence differentiation in pathogen population, is widely reported[6-7]. This need further enough study establish a foundation for the effective managment of C.lunata.

Management of leaf spot is difficult because the pathogen is seed-borne. To date, management approaches have mainly focused on the use of synthetic chemicals and resistant rice varieties[8-10]. However,their efficacy can be limited by the rapid development of resistance to these chemicals, which applied as either seed dressing or spray, such as benomyl,benomyl + copper sulfate, probenazole, thiabendazole and pyroquilon fungicides, have been used in different rice growing countries to manage rice disease[11].

Many plant diseases caused by fungi have been controlled by plant extracts[12-16]. Plants produce a high diversity of natural products with a prominent function against pathogens on the basis of their toxic effect on growth and reproduction of microbes[17].Thus, the exploration of plant resources for their antifungal potential against pathogens is important for the sustainable and ecofriendly management of pathogens. Moreover, Prisana et al. reported that all the Streptomyces species inhibited the growth of C.lunata[18]. The application of the Streptomyces angustmyceticus NR8-2 spore suspension and cellfree culture filtrate reduced the disease severity index of leaf spots[18].

Aloe is rich in a variety of active ingredients,including anthraquinones, polysaccharides, active enzymes, amino acids and organic acids, antibiotics,and saponins[19-20]. Among these, anthraquinone compounds and anthraquinone derivatives have strong inhibitory action toward many fungi and bacteria[21]. Aloesin is an anthraquinone compound with a chemical structure of 2-acetyl-8-D-pyrrolidone-7-hydroxy-5-methylparaphthone that has antihepatitis, neutralizing toxin, anti-tumor, and leukemia effects[22]. Aloe gel is the most widely recognized herbal medicine in the world today because it contains a wide range of enzymes and provides preservative,anti-inflammatory, and anti-fungal benefits[23]. The main focus of this study was to evaluate the bioactivity of aloesin and aloe gel against C. lunata.

1 Materials and methods

1.1 Plant material and fungal material

Aloesin (95%) was obtained from Xi’an Yunyue Biotechnology Co., Ltd. Aloe gel (100%) was acquired from Yunnan Wan Green Biological Co.,Ltd. C. lunata was obtained from the Agricultural Products Quality Safety Laboratory of the College of Agriculture, Guizhou University. C. lunata was cultured on the Potato Sucrose Agar (PSA) at 28 ℃for 2-3 d; filter paper (6 mm in diameter) was sterilized at 121.3 ℃ for 20 min.

1.2 Inhibition effect of aloesin against C. lunata by filter paper method

The anti-fungal activity against C. lunata was tested by the disc diffusion method, and clear zones appeared around the treatment discs were recorded[24-25].But we were slightly modified. First, C. lunata discs were placed in the center of PSA plates. Next, filter paper containing three different plant extracts at three different concentrations (300, 200, or 100 mg/mL)was also placed around the fungal discs. In addition ,all experiments were repeated in triplicate and all plates were incubated at 28 ℃ for 2-3 d. Meanwhile,inhibition effect was assessed using conidial suspensions. A hemocytometer was used to adjust the conidial concentration to 3 × 104conidia/mL. After the medium solidified, conidial suspensions (0.1 mL)were then placed in treated PSA plates, filter papers of various concentrations were added to each dish. All experiments were repeated in triplicate and all plates were then incubated at 28 ℃ for 2-3 d.The width and the diameter of the inhibition zone were then measured, and the average value of the three treatments was taken.

1.3 Inhibition rate of mycelium growth against C. lunata by aloe gel

A series of sterile water containing different concentrations of aloe gel (10, 20, 40, 80, 160, 320 and 640 mg/mL) were prepared. Next, PSA(40℃)was melted in an electric furnace, after which 1 mL aliquots of test solution were mixed with 9 mL PSA agar and added to 90 mm culture dishes. The test compounds were present at concentrations of 1, 2, 4,8, 16, 32 and 64 mg/mL. In addition, untreated media was used as a CK. A piece of fungus disc was placed in each plate with five treatments (1, 2, 4, 8, 16, 32 and 64 mg/mL) and CK. Three piece of fungus disc was placed in each plate, and each treatment was performed in quadruplicate C. lunata was incubated at 28 ℃ for 5 d, and the colony diameters of the tested fungus were recorded[26]. SPSS 19.0 was used for analysis. The percent inhibition of fungal growth was estimated according to the Ogbeborand Adekunle[26]methods as formula (1).

In formula (1): I means mycelial inhibition, %;Dcmeans mycelial growth diameter in control, mm;Dtmeans mycelial growth diameter in treatment, mm;Ddmeans mycelia discs diameter, mm.

1.4 Microscopic examination

The spore morphology and germination of C.lunata in water were observed using a US3 multifunction digital microscope (400 × ). C. lunata was also observed in mixtures with aloesin and aloe gel. For clear observation, 0.05% aniline blue staining was conducted. In addition, the spore morphology and germination of C. lunata in aloesin and aloe gel were observed and recorded. The spore morphology and germination of C. lunata in water were observed using a US3 multifunction digital microscope (400 × ).C. lunata was also observed in 200 mg/mL aloesin and aloe gel. In addition, the spore morphology and germination of C. lunata in 200 mg/mL aloesin and aloe gel were observed and recorded. A piece of fungus disc was placed in each plate with three treatments.

2 Results and Analysis

2.1 Inhibition effect of aloesin against C. lunata by filter paper method

After 24 h, the filter paper with 300 mg/mL aloesin showed the best antimicrobial activity and could produce an obvious 3.37 mm inhibition zone(Fig.1a) that could persist for 40 h (Fig.1b). However,the filter paper with 100 and 200 mg/mL aloesin had low antimicrobial activity and did not produce obvious inhibition zones, and instead, only made colony color fade, and these inhibitory effects remained after 6 days (Fig.1c). The filter paper method showed that 300 mg/mL aloesin led to an obvious inhibitory band (Φ = 51.27 mm) against 3 ×104cfu/mL spores (Fig.1d).

2.2 Inhibitory effect of aloe gel on C. lunata mycelium growth

At the same concentration, the inhibitory effect was enhanced with time (2-5 d). As the aloe gel concentration increased, the rate of inhibition increased, too. Specifically, 64 mg/mL aloe gel showed the lowest inhibition rate of 9.25% at 2 d and the highest of 56.21% at 5 d, representing an increase in inhibition of 46.96% (Fig. 2).

2.3 Microscopic examination

2.3.1 Inhibition effect of aloesin against conidial germination The in-vitro antimicrobial activity of aloesin against C. lunata spores revealed that 200 mg/mL aloesin could cause both or one end of the C.lunata spores to break or rupture and 200 mg/mL aloesin also cause C. lunata spores to rupture, the broken ends could not germinate to produce germ tubes, and the unbroken ends could still germinate(Fig.3).

C. lunata spores could germinate at 4 h on onion skin. Appressoria development of C. lunata after 3 d with onion skin. After 4 d, a large number of appressoria were produced. C. lunata spores could germinate in water without appressorium produced.After 6 d, a new round of C. lunata spores were produced (Fig. 4).

After 22 d, the C. lunata spores on onion skin were essentially unchanged in response to 160 mg/mL aloesin. In addition, there were no appressoria produced, only a small amount of spores germinated,mycelial growth was weak, and the average diameter of hypanthium was 1.25 μm (Fig.5), which was weaker than control (3.75 μm).

2.3.2 Inhibition effect of aloe gel against C. lunata conidial germination Most C. lunata spores in CK could germinate in 6 h, the germinating of C. lunata were colorless and tubular, and the sporoduct length was 96.64-106.78 μm (16.11-17.80 μm/h). However,in aloe gel, the germinating C. lunata were colorless and sporoduct growth was very slow (Fig.6). The sporoduct length was 3.31-26.28 μm with in 6 h(0.55-4.38 μm/h) and the inhibitory rate was 75.39-96.58%. Because the sporoduct growth was slow, the total mycelium quantity was less than that of the CK.

3 Conclusions and Discussion

The pathogenic fungus C. lunata causes rice production serious loss, biological control technology to reduce the use of chemical fungicides has received a great deal of attention. Studies were conducted to determine the effects of aloesin and aloe gel on the control of C. lunata. Microscopic examination showed that there was an obvious inhibitory effect of aloesin against C. lunata spores. The spores of C.lunata were damaged and reduced the number of germinating spores and hyphae, only a small amount of spores could germinate in aloesin. Aloesin can inhibit appressorium, while the presence of melanin is a necessary condition for germination of C. lunata[27].Aloesin had long-term and stable inhibitory effects. In the aloesin and aloe gel treatments, spores were ruptured at one or both ends, leading to direct death of the spores. Aloesin and aloe gel have the potential for use as biocontrol materials, indicating that it is necessary to continue screening and add other components to improve the inhibitory effect as a biocontrol material. The inhibition effect of aloe was similar to that of antibiotics, but its function was superior[25].

Lynch et al. reported that aloesin may be useful as a functional food ingredient[28]. There were no toxicologically or statistically significant changes in body weight gain or in feed and water consumption and the no-observed-adverse-effect level was considered to be 1 000 mg/kg body weight/day, the highest dose tested, supporting the potential use of aloesin as a functional food ingredient[28]. Based on these advantages, further studies on the use of aloe to induce bacteriostasis of plant pathogens and the preservation of agricultural products are necessary. In this study, aloesin and aloe gel in aloe were studied,and their inhibitory effects on plant pathogenic microorganisms were evaluated. However, this study investigated the antimicrobial effects of specific components of A. vera against C. lunata. Chemical preservatives as traditional methods were often used for preservation, many of which are harmful to health.Therefore, it is important to identify new and highly efficient natural active substance to improve food safety. Moreover, consumers are increasingly choosing products with natural preservatives[29-30].Aloe active ingredients are nontoxic to humans and have been shown to have good efficacy. Therefore,they have the potential for use in the research and development of non-toxic and harmless food preservatives.

This study was reported that aloesin effectively inhibited mycelia growth, conidia germination, and that aloe gel effectively inhibited mycelia growth of the rice pathogen (C. lunata). The results presented herein supported that aloesin and aloe gel mostly control spore germination and inhibit spore formation,thereby effectively preventing spore germination and reducing the production of blast spots. The current findings suggest that the extracts from most of the tested plants possess antifungal properties and can be used as seed and foliar treatments against the pathogen C. lunata. These plant extracts are safe and eco-friendly when compared to synthetic chemicals.However, additional studies are required to identify and characterize the active antifungal compounds in the extracts of these plants, as well as their roles in Curvularia leaf spot control.

Overall, this is the first report of antifungal activity of aloesin and aloe gel against C. lunata. The results presented herein provide a basis for the investigation of biological control and food preservation of rice fungus. Fungicide application and planting resistant varieties are considered the two main methods to control disease[31]. Recently, the increasing environmental and economic concerns caused by synthetic fungicides have led to increased efforts to produce safer agricultural products and develop new and safer antifungal agents such as plant based essential oils and botanical extracts to combat fungal plant diseases[32]. The tendency towards application of natural products and botanical extracts as safer antimicrobial agents against plant pathogens has recently increased[33].

Conflicts of Interest: The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

Acknowledgment: We would like to thank Dr.Liang-sheng Hu from Shantou University, China, for his advice and guidance.