1. Introduction
Rosa roxburghii Tratt., an edible and medicinal fruit rich in vitamin C, flavonoids, superoxide dismutase (SOD) and various minerals, has high medicinal and nutritional values [
1,
2,
3,
4,
5]. Recently, the
R. roxburghii industry has developed rapidly in southwest China, especially in Guizhou Province, where the planting areas reached 170,000 hm
2 in 2020 [
3,
6]. Powdery mildew, caused by
Sphaerotheca sp., is the most serious disease regarding
R. roxburghii production [
1]. In Guizhou Province of southwest China, powdery mildew seriously affects the growth, yield, and quality of
R. roxburghii, and often causes 30~40% economic losses [
7]. Although some chemical fungicides (triadimefon, myclobutanil, azoxystrobin and tebuconazole) [
8] and conventional antibiotics (polyoxin and kasugamycin) [
9] are frequently used to control powdery mildew, their residuals inevitably affect the environment, wildlife, and human beings [
10]. Moreover, these chemicals and antibiotics easily generate resistance to pathogens with the increase in the use frequency [
11,
12]. Therefore, there is an urgent need to develop an alternative, cost-effective and environmentally friendly control strategy against powdery mildew of
R. roxburghii.
It is generally believed that natural products are mild and basically harmless compared with chemical fungicides and conventional antibiotics. Although this view is not completely accurate, it has been suggested as one of the reasons for the growing preference of natural products by consumers, and their increasingly popular use in agriculture [
13,
14]. For instance, Yan et al. [
8] reported that 6% ascorbic acid aqueous solutions could induce
R. roxburghii against powdery mildew with the control effect of 61.45%. Chitosan, a natural resource substance for sustainable agriculture, can be used as a resistance inductor and biofungicide for controlling plant diseases and as a promoter for enhancing plant growth [
15,
16,
17,
18,
19,
20]. In our previous study, the foliar application of 1.0~1.5% chitosan could effectively control powdery mildew of
R. roxburghii with an inducing control efficacy of 69.30~72.87%, and could notably induce the systemic disease resistance of
R. roxburghii, as well as reliably enhancing its photosynthesis, growth, yield, and quality [
7]. Although chitosan can be used as an effective, safe and economical inductor for controlling powdery mildew, its control effect is still relatively inferior. Thus, natural products enhancing chitosan against powdery mildew of
R. roxburghii are worthy of further exploration and development.
Allicin, an oxygenated sulfur natural compound, was isolated and identified from garlic in 1944 by Cavallito and Bailey [
21]. Since then, allicin has been widely used in agricultural plant protection and medical therapy due to its superior antimicrobial activity and ecofriendly advantage [
13,
14,
22,
23,
24,
25]. Allicin has a prominent reactivity, antioxidant activity and membrane permeability, and can undergo thiol–disulphide exchange reactions with free thiol groups of proteins in microorganisms [
13,
14,
26,
27,
28]. In agriculture, allicin has been demonstrated to have satisfactory bioactivity against many plant-pathogenic fungi, such as
Plectospherella cucumerina,
Botrytis cinerea,
Phytophthora infestans,
Xanthomonas axonopodis,
Magnaporthe grisea and
Alternaria brassicicola [
13,
29]. However, to date, there are no documentations available about the application of allicin for controlling powdery mildew of
R. roxburghii caused by
Sphaerotheca sp. Moreover, whether allicin can be used as an adjuvant to enhance chitosan against powdery mildew of
R. roxburghii. is worth further attention.
In this work, the bioactivity of allicin, chitosan and conventional antibiotics against Sphaerotheca sp. was firstly determined. Subsequently, the field control efficacy of the co-application of allicin and chitosan for powdery mildew of R. roxburghii was evaluated. Moreover, the effects of the co-application of allicin and chitosan on the powdery mildew resistance, growth, yield and quality of R. roxburghii were investigated. This study provides a green, cost-effective and environmentally friendly alternative strategy to conventional antibiotics for controlling powdery mildew of R. roxburghii.
4. Discussion
Previous findings have demonstrated that allicin could effectively inhibit the growth of
Plectospherella cucumerina,
Botrytis cinerea,
Phytophthora infestans,
Xanthomonas axonopodis,
Magnaporthe grisea and
Alternaria brassicicola, etc. [
13,
29], and chitosan had antifungal activity against various fungal pathogens [
15,
16,
32,
33,
34,
35]. The results here show that 5% allicin ME displayed outstanding toxicity against
Sphaerotheca sp., with an EC
50 value of 148.65 mg kg
−1, which was 2.80-, 1.24- or 6.95-fold higher than chitosan, 3% polyoxin WP or 6% kasugamycin WP, respectively. This work extended the antimicrobial spectrum of allicin. Although chitosan exhibited a relatively inferior toxicity against
Sphaerotheca sp., its EC
50 value was still 2.48-fold higher than that of 6% kasugamycin WP. Moreover, the control effect of powdery mildew of
R. roxburghii by allicin + chitosan was 85.97%, which was significantly (
p < 0.01) higher than 76.70% of allicin, 70.93% of chitosan and 60.23% of polyoxin, respectively. Chitosan can trigger plant defense responses by inducing a variety of defense-related reactions [
16,
34,
35,
36,
37,
38]. Our previous results show that the inducing control effect of 1.0~1.5% chitosan against
Sphaerotheca sp. was 69.30~72.87% [
7]. In this study, the co-application of allicin and chitosan significantly (
p < 0.01) enhanced the control effect of powdery mildew in
R. roxburghii compared with allicin, chitosan or conventional antibiotic polyoxin alone. This suggests that allicin and chitosan had a notably synergetic effect in the control of powdery mildew of
R. roxburghii. The effective control effect of allicin + chitosan was probably derived from the superior antimicrobial activity of allicin, as well as the excellent antimicrobial and induced resistance effect of chitosan.
The inducing of disease resistance is an effective agricultural practice for controlling plant diseases [
39,
40]. Pro and soluble sugar are important regulators of cell permeability, MDA is an important indicator of membrane lipid peroxidation and flavonoid is an important disease-resistant substance, as well as SOD and PPO being defense enzymes associated with plant disease resistance [
38,
40]. Many studies have also shown that chitosan could induce increases in sugar, Pro, flavonoid, polyphenolics and lignin in the plant and boost its defense enzyme activity, thereby enhancing its disease resistance [
16,
30,
31,
32,
34,
35,
36,
37,
38,
39,
40]. Our previous results also indicate that the foliar application of 1.0~1.5% chitosan significantly (
p < 0.01) increased Pro, soluble sugar and flavonoid contents, as well as SOD and POD activities of
R. roxburghii leaves, and decreased their MDA [
7]. The present results show that as compared with polyoxin or control, allicin + chitosan, allicin and chitosan could effectively increase Pro, soluble sugar, and flavonoid of
R. roxburghii leaves, and enhance their SOD and PPO activities, as well as reduce their MDA, which is consistent with the above studies. Moreover, the enhancing or inhibiting effects of allicin + chitosan on Pro, soluble sugar, flavonoid and MDA contents, as well as SOD and PPO activities of
R. roxburghii leaves were higher than those of allicin or chitosan alone. These results emphasize that the co-application of allicin and chitosan was more helpful in improving the disease resistance of
R. roxburghii, and an obviously synergetic effect of allicin and chitosan was available.
Chlorophyll is an essential pigment for plant photosynthesis, and photosynthesis is the physiological basis of plant growth and development. Chitosan can promote plant growth and development by enhancing the photosynthetic rate by increasing chlorophyll content [
16]. Our previous results show that foliar application of 0.5~1.5% chitosan effectively enhanced the photosynthetic rate, the content of chlorophyll a, chlorophyll b, and chlorophyll a + b of
R. roxburghii leaves [
7]. In this work, the co-application of allicin and chitosan more effectively promoted the chlorophyll and photosynthetic rate of
R. roxburghii leaves compared with allicin, chitosan or polyoxin alone. This is closely related to the synergistic effect between allicin protecting plant leaf organs from pathogens and chitosan promoting plant growth. The growth and development of
R. roxburghii determine its fruit yield and quality. Chitosan can also promote plant growth by activating the auxin and cytokinin signal transduction and gene expression, as well as increasing the nutrient intake [
16,
41]. Our previous results also indicate that the foliar application of 1.0~1.5% chitosan notably improved yield and quality of
R. roxburghii fruits [
7]. The present results indicate that the co-application of allicin and chitosan effectively enhance
R. roxburghii fruit growth and yield formation. Moreover, vitamin C, soluble solid, soluble sugar, total acidity and SOD activity of
R. roxburghii fruits treated by allicin + chitosan was significantly (
p < 0.05) higher than that of treatments by allicin or chitosan alone. These notable effects were probably derived from their division of labor; allicin can protect
R. roxburghii from pathogen infection and chitosan can induce the disease resistance of
R. roxburghii, which guarantee the healthy growth of
R. roxburghii plants.
At present, increasing attention has been focused on natural products as effective fungicides for controlling plant fungal disease, with high efficacy, nontoxicity and low food safety risks [
24,
42]. Therefore, natural products as an alternative to traditional antibiotics have been recognized by the public. Allicin, extracted from garlic, is used for daily consumption, and chitosan is a natural, nontoxic substance widely used in food, cosmetics and other fields. Moreover, the safe interval period (from April 29 to September 2, more than 120 days) of
R. roxburghii was very long. Thus, the food safety risks caused by allicin or chitosan are almost nonexistent. This study highlights that the co-application of allicin and chitosan can be used as a green, cost-effective and environmentally friendly alternative approach to conventional antibiotics for controlling powdery mildew of
R. roxburghii and enhancing its resistance, growth, yield and quality.