Biological control of Podosphaera xanthii the causal agent of squash powdery mildew disease by upregulation of defense-related enzymes

Plant material and experimental design

Squash seeds (Cucurbita pepo L., cv. Eskandarani) were sown in a clay loam soil and grown under greenhouse conditions at Sakha Agricultural Research Station, Agricultural Research Center, Egypt. The experiment was conducted in a randomly complete block design (RCBD), with three replicated plots for each treatment. Squash seeds were cultivated at spacing of 60 cm between plants in rows and 80 cm between plant rows at the rate of three seeds “per hill.” The seedlings were thinned to one per hill after 15 days after sowing (DAS). Squash plants in all plots received all the recommended agricultural practices.

Tested treatments

The efficacy of the bio-agents (two fungal and five bacterial strains) comparing to the fungicide (Topas-100) for controlling squash powdery mildew disease was tested under greenhouse conditions. Bacillus subtilis, Bacillus pumilus, and Bacillus chitinosporus were previously isolated from the surface of healthy cucumber and squash leaves and identified according to Kamel (2003), while B. megaterium, Bacillus polymexa, and fungal strains (Trichoderma harzianum and Trichoderma viridi) were obtained from the Microbiology Department, Soil, Water and Environment Research Institute, Agriculture Research Center (ARC), Giza, Egypt. Trichoderma harzianum and T. viridi were grown on PDA medium for 10 days, then their spores and mycelial suspensions were separately prepared and adjusted to about 10⁷ spore ml⁻¹ with sterilized water, using a hemocytometer slide, while B. subtilis, B. polymexa, B. pumilus, B. chitinosporus, and B. megaterium were separately grown in nutrient liquid medium in 250-ml flasks and kept on an orbital shaker at 150 rpm for 3–4 days. The pellets of each bacterium were separately suspended in tap water, and number of cells was adjusted to 10⁹ cell ml⁻¹, using a hemocytometer slide. For comparison with the tested bio-agents, plants were sprayed by distilled water as a negative control (C). In addition, widely used fungicide, Topas-100® (10.0% Penconazole “w/v” [(R, S-1-(2-(2, 4-dichlorophenyl) -Q pentyl)-1H-1, 2, 4-triazole]), was sprayed, at the recommended dose of 0.25 ml l⁻¹ as a positive control.

Spore germination of Podosphaera xanthii as affected by tested bio-agents

Conidial spores of squash powdery mildew were obtained from young sporulating lesions. To avoid the old unviable conidia, the lesions were gently shaken by a glass rod, and 24 h later (as recommended by (Godwin et al. 1987), new conidia were deposited on glass slides according to Nair et al. (1962). Slides were previously cleaned by ethyl alcohol and air dried before covering with thin smears of 2% water agar, amended with filter-sterilized culture filtrate of the tested antagonist. Slides were placed on V-shaped glass rods in sterilized Petri-dishes, containing several layers of water-moistened filter papers. Slides with conidia were incubated at 25 °C for 24 h under continuous light (Reifschneider et al. 1985) before microscopically examined, at × 100 magnification, to determine the spore germination. Conidia were considered to have germinated if a germ tube, at least as long as the width, was produced (Menzies et al. 1991). Percentages of germination were calculated for 100 conidia on a slide. Three slides were examined for each treatment. Agar-free culture filtrate slides were used as a control treatment.

Pathogenic fungal inoculation

Natural infection with P. xanthii conidia, the causal agent of squash powdery mildew, was conducted under greenhouse conditions. Infected plants used as inoculum source (susceptible host Eskandarani) were uniformly inoculated by freshly collected conidia by placing heavy infected plants of squash which are sensitive to P. xanthii inoculation.

Disease assessment

The disease severity of squash leaves at 45 DAS was assessed as described by Descalzo et al. (1990). Nine random plants were used for each replicate. Severity of powdery mildew was estimated based on the percentage of affected leaf area. The mean of area under disease progress curve (AUDPC) for each replicate was calculated as follows (Pandey et al. 1989).

AUDPC = D [1/2(Y₁ + Y_k) + (Y₂ + Y₃ + ……. . + Y_k − 1)]

where D = time interval; Y₁ = first disease severity; Y_k = last disease severity; Y₂, Y₃, and Y_k−1 = intermediate disease severity.

Biochemical assays of antioxidant enzymes

For enzyme assays, 0.5 g of leaf material collected after 15 days from treatment with bio-agents were homogenized at 0–4 °C in 3 ml of 50 mM TRIS buffer (pH 7.8), containing 1 mM EDTA-Na₂ and 7.5% polyvinylpyrrolidone. The homogenates were centrifuged (12,000 rpm, 20 min, 4 °C), and the total soluble enzyme activities were measured spectrophotometrically in the supernatant (Hafez et al. 2014a). All measurements were carried out at 25 °C, using the model UV-160A spectrophotometer (Shimadzu, Japan). The enzymes’ assay was tested three times. Activity of catalase (CAT) was determined spectrophotometrically according to Aebi (1984). Polyphenol oxidase (PPO) activity was determined according to the methods described by Malik and Singh (1980). Changes in the absorbance at 495 nm were recorded every 30 s intervals for 3 min. Enzyme activity was expressed as the increase in absorbance min⁻¹ g⁻¹ fresh weight. Activity of peroxidase (POX) was directly determined of the crude enzyme extract, according to a typical procedure proposed by Hammerschmidt et al. (1982). Changes in absorbance at 470 nm were recorded at 30 s intervals for 3 min. Enzyme activity was expressed as increase in absorbance min⁻¹ g⁻¹ fresh weight.

Microscopic examination

The infected leaves, treated by the most effective bio-agents, were selected to investigate the growth status of powdery mildew fungus after 24 h of treatment. Control plants were microscopically observed for comparison and for any differences on the tested pathogen P. xanthii. For scanning electron microscopy (SEM), sections (0.5 cm) were taken from untreated and treated leaves. The samples were prefixed in mixture of 2.5% glutaraldehyde and baraformaldehyde at room temperature for 24 h. The fixative was washed three times by phosphate buffer solution (pH 7.2–7.4). The specimens were postfixed in osmium tetroxide (1% w/v in phosphate buffer 0.07 M, pH 7.2) at room temperature for 1.5 h. After washing with phosphate buffer solution three times, the samples were hydrated in ethanol series (30 and 50% for 30 min, and 70% for 24 h), then dried at critical point of CO₂ (Balzers CPD-020) and covered with gold (30 nm) in a sputter coater (Balzers SCD-040). The specimens were examined and photographed by the TESLA BS-300 electron microscope. In all processes, SEM observations and photography were carried out at Electron Microscope Unit, Fac. of Medicine, Tanta Univ., Egypt.

Chlorophyll content and plant growth assessment

Plant growth parameters were determined in the experimental plants at 60 DAS. Total chlorophyll contents, using the SPAD-501 portable leaf chlorophyll meter (Minolta Corp) for greenness measurements in the fifth apical fully expanded leaf (Yadava 1986). Average number and weight of fruits per plant were measured by harvesting fruits at marketable size. The squash fruits from each replicate of each treatment were collected twice a week, from 45 to 90 DAS, and the accumulated yield was expressed as the number and weight of fruits per plant. Leaf area was determined, using the dry weight method. The leaves of a plant were cleaned from dust and then representative 10 disks were taken, using a test tube known for its area (1.76 cm²). The leaf area was calculated, using the following formula:

Leaf area = (weight of leaves ×   10 n)/weight of 10 disks

where n = the area of one disk.

Statistical analysis

The data were subjected to statistical analysis by ANOVA, using wasp software (Web Agriculture Stat Package). The values presented are the means of all measurements, and comparisons of means were determined by Duncan’s multiple range tests, at P ≤ 0.05 (Gomez and Gomez 1984).

Effect of tested bio-agents on conidial germination of P. xanthii and disease measurements of mildewed squash plants

Results presented in Table 1 indicated that all treatments significantly inhibited the conidial germination of P. xanthii than in the control. The highest efficiency over control (98.53%) was recorded by B. megaterium and fungicide, followed by B. pumilus and B. polymexa (95.21 and 90.75%, respectively). The disease symptoms of squash plants infected with the powdery mildew pathogen and treated with fungal and bacterial bio-agents under greenhouse conditions were suppressed and inhibited, similarly to the fungicide treatments, as compared with control (Fig. 1). Control plants showed the typical disease symptoms, as a leaf surface was covered by a visible white, talcum-like, powdery mass that composed of mycelia and conidia of the causal agent. Results presented in Table 2 revealed that treated plants, four times, with the tested bio-agents reduced area under disease progress curve (AUDPC) and disease severity (DS %) compared with control. Maximum reduction in DS (%) over control was found in plants sprayed with suspension of B. subtilis (64.72%) and B. pumilus (64.44%), followed by plants treated with B. megaterium, T. harzianum, T. viridi, and B. chitinosporus (56.68, 43.61, 38.79, and 33.2%, respectively), while B. polymexa achieved the lowest reduction in DS% compared with other bio-agents. The fungicide (Topas-100) caused considerable reduction in DS% (62.61%). Similarly, B. subtilis and B. pumilus were the most effective bio-agent treatments for reducing AUDPC of squash powdery mildew (225.61 and 226.87, respectively) than the control (643.54), while there was insignificant difference of AUDPC in plants treated with T. harzianum, T. viridi, and B. chitinosporus. Obtained results are in agreement with the findings of many researchers who demonstrated that the bio-agents that were used in this study were effective for controlling powdery mildew disease (Kamel 2003; García-Gutiérrez et al. 2013 and Tanaka et al. 2017). Previously, many studies demonstrated that Bacillus spp. and Trichoderma sp. made major contributions to the control of plant diseases, particularly powdery mildew (El-Sharkaway et al. 2014 and Sawant et al. 2017).

Treatments	Concentration	Germination (%)	Efficiency (%)
Control	–	64.91 a	0.00
Fungicide	0.25 ml l−1	0.95 c	98.53
T. harzianum	107 spore ml−1	17.24 b	73.44
T. viridi	107 spore ml−1	13.79 bc	78.75
B. subtilis	109cfu	11.97 bc	81.55
B. chitinosporus	109 cfu	9.23 bc	58.05
B. pumilus	109 cfu	3.11 bc	95.21
B. megaterium	109 cfu	0.95 c	98.53
B. polymexa	109 cfu	6.00 bc	90.75
LSD 0.05		15.66	–

Values are means of three replicates of each treatment

Means values for each parameter followed by different letters are significantly different (P ≤ 0.05), according to Duncan’s multiple range test

Treatments	Diseases severity (DS %)					AUDPC
	Days after application				Efficiency %
	Zero time	5 days	10 days	15 days
Control	33.24 a	46.23 a	58.95 a	78.25 a	–	643.54 a
Fungicide	12.26 fg	18.13 ef	23.11 d	27.44 e	64.93	244.31 fg
T. harzianum	17.11 de	26.14 d	33.93 c	44.87 cd	42.65	364.05 e
T. viridi	22.43 bc	29.15 cd	34.89 c	46.02 cd	41.18	391.29 de
B. subtilis	10.04 g	14.15 f	21.54 d	30.83 e	60.61	225.61 g
B. chitinosporus	25.24 b	31.92 bc	38.24 c	49.18 cd	37.15	429.44 cd
B. megaterium	14.66 ef	20.92 e	24.02 d	36.41 de	53.46	277.31 f
B. pumilus	9.09 g	16.12 ef	20.93 d	30.24 e	61.35	226.87 g
B. polymexa	20.52 cd	29.11 cd	38.01 c	55.49 bc	29.08	442.94 c
LSD0.05	3.91	5.00	7.05	13.39	–	45.84

Values are means of three replicates of each treatment

Means values for each parameter followed by different letters are significantly different (P ≤ 0.05), according to Duncan’s multiple range test

Gilardi et al. (2008) and El-Sharkaway et al. (2014) indicated that Bacillus sp. was used for controlling cucurbit powdery mildew. B. subtilis achieved the highest reduction of “DS %.” This reduction may be attributed to the role of B. subtilis as plant growth-promoting rhizobacteria (PGPR) that is widely applied to the management of many crop diseases (Liu et al. 2014). Antibiotic compounds such as chitinase enzyme can hydrolyze the cell walls of fungi; siderophores and other antibiotics can prevent the development of pathogens (Wang and Chang 1997). B. chitinosporus, B. polymexa, and B. pumilus have been shown to possess antifungal activity against the plant pathogens Fusarium oxysporum and Pythium spp. (Axelrood et al. 1996). Trichoderma sp. acted synergistically for reducing the disease severity of squash powdery mildew disease by producing antifungal compounds (El-Kot and Derbalah 2011). EL-Naggar et al. (2012) found that spraying cucumber plants with T. viridi caused significant reduction in the disease of powdery mildew than in the control treatment. These results are in accordance with the results of Nada (2002) who proved that spraying plants with bio-agents was beholden effective in the programs of integrated pest management (IPM) for controlling plant diseases, especially those caused by obligate parasites such as powdery mildew. Abd-El-Moity et al. (1997) found that spraying onion and garlic plants grown under field conditions with a suspension of T. harzianum showed a significant reduction in incidence and severity of downy mildews. The fungicide (Topas-100) was effective against powdery mildew, as it is known that systemic fungicides are more efficient in management of many fungal diseases, including cucumber powdery and downy mildews (Abada et al. 2009).

Microscopic examination of the mildewed squash leaves treated with B. subtilis and T. harzianum

The light and SEM examination of squash leaves in control plants showed a complete fungal growth of P. xanthii (Fig. 2a). The fungal mycelia developed full coalescence and conidiophores in their right form. In addition, conidial chains were in natural form and size. However, the squash leaves treated with B. subtilis (Fig. 2b) and T. harzianum (Fig. 2c) exhibited morphological anomaly such as atrophy in conidiophore and hypha of the pathogen and hydrolysis in conidia spores. Thus, the fungus can no longer develop conidiophores and conidia spores.

Effect of different bio-agents on chlorophyll content and growth traits of mildewed squash plants

Impact of bio-agents on yield parameters of mildewed squash plants

Effect of bio-agents on the activity of defense-related enzymes in mildewed squash plants

Results illustrated in Fig. 3 revealed that the tested bio-agents and fungicide (Topas-100) significantly upregulated the activity of defense-related enzymes compared with untreated plants. The highest level of CAT activity was recorded in plants treated with B. pumilus and B. megaterium, followed by T. harzianum, B. chitinosporus, B. subtilis, T. viridi, B. polymexa, and then fungicide, while the highest level of POX activity was recorded when squash plants were treated by B. polymexa, followed by B. pumilus. PPO activity significantly increased in all tested treatments, particularly T. harzianum, compared with control, except the treatment with B. megaterium. These results are in agreement with the results of El-Sharkaway et al. (2014) who found that treatment of cucumber plants with bio-agents led to activation of antioxidant enzymes such as peroxidase and polyphenol oxidase, and significantly decrease the disease severity of downy and powdery mildew in cucumber plants. Resistance might be correlated to the production of oxidative enzymes in the treated healthy and infected plant tissues (El-Sharkaway et al. 2014; Hafez et al. 2016 and Hafez et al. 2017).