1. Introduction
Cucumber (
Cucumis sativum L.) is one of the most important vegetable crops grown under protected cultivation worldwide, including Saudi Arabia [
1]. Saudi Arabia has a production rate of 1.27 tons/ha and an area under cultivation of 11,764 hectares, yielding 149,074 tons [
1]. Cucumbers can be cultivated year-round in regulated environments, so there is much interest in greenhouse production of the vegetable. In addition, loans and subsidies are offered for the construction of greenhouses and polyhouses. Vegetables are being grown more frequently in greenhouses as the demand for organic produce rises. Under a safe environment, a grower can make a respectable profit. As compared to vegetables cultivated in the usual manner, plants grown in polyhouses are more frequently organic and offer health benefits [
2].
Cucumber plants are frequently attacked by pathogens that can decrease production and cause significant losses [
3]. One of the most common fungal diseases is powdery mildew, which is caused by
Podosphaera xanthii (Castagne) U. Braun & Shishkoff. It is a serious disease that causes significant damage to the whole plant, including the leaves, fruits, and stems, under greenhouse and field conditions [
4]. Powdery and downy mildews on cucumber can cause losses reaching 30–80% of yield [
5].
Control of such diseases depend on the use of fungicidal treatment, but repeated use of fungicides in the control of plant disease has some problems, such as the development of tolerance to the fungicides used [
6]. Additionally, the use of fungicides to treat plant diseases can pollute the environment and increase the level of hazardous compounds in the human food supply [
7]. For this reason, researchers are searching for alternative methods to control such diseases, e.g., cultivation of resistant cultivars and biological control by various materials [
8].
Increasing concerns for public health have encouraged researchers to find environmentally safe strategies to control plant diseases. Successful biological control methods for mildews caused by fungal and bacterial antagonists have been used under greenhouse conditions, such as the use of
Trichoderma spp. and certain bacteria species.
Pseudomonas fluorescens and
Bacillus subtilis reduced powdery mildew of cucumber [
9,
10,
11]. The efficiency of biological control depends on the potential of beneficial antagonistic microorganisms. However, further efforts are required to understand the mechanisms underlying the biological control of powdery mildew.
Application of
Bacillus spp. as a biotic inducer has been used to control plant diseases [
12]. These bacteria have a positive effect on plant diseases directly or indirectly; directly by production of auxins or promoting plant growth along with indirect growth effects by reduction or killing of the pathogen via production of antibiotics or toxins and/or secondary metabolite production [
13]. The use of
Bacillus spp. can induce systemic resistance in plants after treatment. It can enhance the activation of peroxidase (PO) and polyphenol oxidase (PPO), which are involved in plant-induced systemic resistance (ISR) [
14]. Additionally, it can increase the total phenol content (TPC) of tannins and flavonoids [
15]. Biological control of powdery mildew diseases has been achieved by various bioagents under greenhouse and field conditions [
16]. In the present study, we attempted to use culture filtrate and cell suspension to reduce disease symptoms before or after inoculation. The aim of this study was to assess the effectiveness of
Bacillus spp. against cucumber powdery mildew under both in vitro and in vivo conditions, as well as the effect of those treatments on the induction of PO, PPO, and phenolic compounds.
4. Discussion
In the present study, the effects of 19 isolates from the cucumber rhizosphere were tested in vivo against
P.
xanthii spore germination. Only seven isolates were able to reduce spore germination to various degrees. Of the seven isolates, two showed great reduction of the spore germination (KAUBL1 and PST13) and were selected for further experiments. The antifungal effects of bacterial isolates can be due to the production of direct inhibitory substances such as hydrogen cyanide, hydrolytic enzymes (amylase, cellulase pectinase, and protease), and siderophores or antibiotics [
13]. Present results showed that both biagents
(B. licheniformis and
B. aerius) produced amylase, cellulase pectinase and protease. These findings are consistent with those of Elsisi [
14], who demonstrated the ability of the bioagents to prevent the germination of powdery mildew conidial spores maybe due to hydrolytic enzymes. According to research by Sarhan et al. [
33], the culture filtrate of the examined bioagents, including
B. subtilis,
P. polymyxa, and
S. marcescens, significantly reduced the germination of
P. xanthii conidia in vitro.
The in vitro and in vivo investigations revealed that both of the bioagents examined significantly reduced the severity of the disease. Highly reductions of disease severity were achieved by treatment of
B. licheniformis as a cell suspension and
B. aerius strain as culture filtrate 45.3 and 77.3%, respectively, two days before inoculation. Additionally, they increased the fresh and dry weight and number of leaves per plant as compared to the control. It is well known that bioagents are effective methods of treating a number of fungal diseases, such as cucumber powdery mildew [
33]. In general, antibiosis, competition, mycoparasitism, and induced resistance are among the mechanisms associated with biological control of phytopathogenic fungi [
34].
Romero et al. [
35] mentioned that the
Bacillus species can used to control powdery mildew on melon (
Podosphaera fusca), they suggested that the bacteria antagonistic can reduced percentage the infection through inhibition of spore germination by production of antifungal compounds.
The greenhouse results showed that cucumber plants treated with both isolates either as a cell suspension or culture filtrate showed a significant reduction in disease severity two days before or after inoculation with the pathogen, and treatment two days before was better than two days after inoculation. These results are in line with a previous study by Punja et al. [
36], who observed that
Bacillus spp. can be used to control cucumber powdery mildew disease. This reduction of the disease may be due to the bioagent’s ability to reduce spore germination as well as to enhance plant growth [
37,
38]. Recently, several studies have investigated the effect of various bioagents, such as
Bacillus spp. for controlling airborne pathogens, e.g., powdery mildew disease [
39,
40,
41]. El-Sharkaway et al. [
42] found that spraying cucumber plants with
Pseudomonas fluorescens and
B.
subtilis significantly reduced the disease severity of cucumber powdery mildew. According to research by Punja et al. [
36], the severity of the powdery mildew disease was reduced when
B. subtilis was applied to greenhouse cucumber plants as a preventative or eradicative treatment.
Using PGPR to induce resistance in plants is an important method of suppressing plant diseases caused by fungi or bacteria [
20,
42,
43]. Induced resistance in plants is directly linked to the accumulation of phenolic compounds and/or induction of defense-related enzymes PO, PPO, LOPX and PAL [
15]. The results presented here indicated that cucumber plants treated with
B. licheniformis as CS caused the highest increase in antioxidant enzyme activities and TPC two days before or after infection with the pathogen. The reduction in disease severity of cucumber powdery mildew is associated with increased phenolic compounds and antioxidant enzymes PO and PPO [
42]. In addition, Elsisi [
14] demonstrated that the reduction of powdery mildew of squash under greenhouse conditions is correlated with increased defense-related enzymes PPO and PO as well as TPC in squash plants.
The current findings demonstrated that both bioagents used as cell suspension or culture filtrate could induce plant systemic resistance by PO and PPO activities, thus aiding in the management of cucumber powdery mildew. The prevention and control of disease by bacterial bioagents may occur by a variety of mechanisms [
44], including host resistance induction [
45], ecological place and nutrient competition, or production of antibacterial substances and colonization ability [
46,
47,
48].
The stimulation of phenolic compounds is directly interconnected with disease resistance as well as plant resistance against fungal plant pathogens [
47]. TPC was increased in treated cucumber plants compared to the infected and healthy control plants. The accumulation of phenolic compounds at the infection site has been linked to the restriction of pathogen development because such compounds are toxic to pathogens [
48]. A change in the pH of plant cell cytoplasm due to an increase in phenolic acid content may also increase resistance, thereby inhibiting pathogen development [
43,
49]. In the present study, treatment with bacterial bioagents resulted in an increased accumulation of phenolic compounds in response to infection by the pathogen.
It has been reported that after inoculation with biocontrol bacteria or other non-biological factors, the levels of oxidase activities, such as POD and PPO, are increased, thereby inducing resistance to pathogen invasion and expansion [
50]. PO activity was significantly increased in infected plants treated with all bacteria. These results suggest that endophytic bacteria promote cucumber plant growth by increasing defense-related PO enzymes. Several investigators have reported that the enhancement of PO activity is associated with resistance of plants to fungal, bacterial and viral pathogens [
51]. The highest PPO activity was obtained by treatment with both bioagents in the present study. These results are in agreement with those reported by Esmaeili [
52]. The importance of PPO activity in plant disease resistance probably stems from its ability to oxidize phenolic compounds to quinines, which are often more toxic to microorganisms than the original phenols [
51,
53].