Inhibitory effects of Bacillus licheniformis BL06 on Phytophthora capsici in pepper by multiple modes of action

Biological Control xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Biological Control journal homepage: www.elsevier.com/locate/ybcon

Inhibitory effects of Bacillus licheniformis BL06 on Phytophthora capsici in pepper by multiple modes of action Ye Lia,b, Xiaoqian Fenga,b, Xiaoli Wangc, Li Zhengd, Hongxia Liua,b,

⁎

a

Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Integrated Management of Crop Diseases and Pests (Nanjing Agricultural University), Ministry of Education, China c Jiangsu Provincial Key Construction Laboratory of Probiotics Preparation, Huaiyin Institute of Technology, Huaian 223003, China d Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Biocontrol Phytophthora capsici Bacillus licheniformis Zoospore

Phytophthora capsici is a pathogenic oomycete that affects pepper cultivation and other commercial products, causing serious economic losses. Here, Bacillus licheniformis strain BL06 effectively reduced pepper Phytophthora blight severity. BL06 suppressed P. capsici mycelium growth by 70% in dual culture tests and induced excessive branching and significant lysis of P. capsici hyphae. The BL06 bacterial culture also inhibited in vitro sporangia development in a dose-dependent manner. Moreover, BL06 inhibited motility and displayed lytic activities against zoospores of P. capsici in a dose- and time-dependent manner. Indeed, the P. capsici infectiousness was reduced in host tissue treated by BL06, which is possibly associated with its ability to inhibit zoospore germination and germ tube growth in host tissues. Collectively, our results demonstrate the utility B. licheniformis BL06 as a potential biocontrol agent for the management of Phytophthora blight of peppers.

1. Introduction In China, the cultivated area of pepper plants (Capsicum annuum L.) is about 1.5341 million hm2, second to only to Chinese cabbage in land use (Tian et al., 2017). The output and health benefits of pepper plants outweigh those of Chinese cabbage given their relatively high vitamin C content, which provides several health benefits including the promotion of healthy blood circulation (Wang, 2017). Phytophthora blight of peppers, which was first described in 1922 in New Mexico bell pepper plants (Leonian, 1922), is a devastating soil-borne disease caused by the oomycete pathogen Phytophthora capsici that severely reduces pepper yield and quality (Mo, 2013). The pathogen has a broad host range and mostly infects tomato, bell pepper and chili pepper plants (Lamour et al., 2012). Under favorable environmental conditions, P. capsici is able to infect every part of the pepper plant allowing for rapid spread within a very short time (Meitz et al., 2010). P. capsici infection is characterized by root and crown rot, grayish brown water-soaked lesions on leaves, and black stem and fruit lesions (Kamoun et al., 2015; Ristaino and Johnston, 1999). Management strategies, such as application of chemical pesticides, rapidly and effectively reduce disease severity (Chen et al., 2016). While chemical pesticides are effective, they also kill useful insects and beneficial microorganisms and cause potential safety hazards to humans and the environment (Joo, 2005).

⁎

The systemic fungicide was once widely effective, however, its recurrent use and the genetic adaptation of P. capsici have led to resistant pathogen populations (Hausbeck et al., 2006). These resistant isolates may persist in the soil for years Therefore, there is a demand for environmentally friendly disease management strategies that are focused on biological control (Yau et al., 2013). In recent years, microorganisms have been used to control P. capsici, including Trichoderma harzianum (Ezziyyani et al., 2010), Penicillium spp. (Yan et al., 2008), Pseudomonas spp. (Khatun et al., 2018; Zohara et al., 2016), Bacillus spp. (Syed-Ab-Rahman et al., 2018) and Streptomyces spp. (Nguyen et al., 2015). Bacillus species have shown the greatest potential P. capsici growth suppression: Bacillus subtilis (Kim et al., 2012), Paenibacillus polymyxa (Kim et al., 2010), Brevibacillus laterosporus (Zhao et al., 2012), Bacillus cereus (Melnick et al., 2008) and Bacillus licheniformis (Ling et al., 2014). The biocontrol mechanism of B. licheniformis includes the production of antibiotics (Nigris et al., 2018), competition (Kim, 2007), induction of host resistance (Helepciuc et al., 2014; Ling et al., 2016) and host growth promotion (Syed-Ab-Rahman et al., 2019). B. licheniformis GL174 produced lipopeptide molecules that inhibited the mycelium growth in a variety of plant pathogens (Nigris et al., 2018). B. licheniformis K11 antagonized Fusarium oxyspoum (KACC 40037) and other phytopathogenic fungi in vitro, and produced siderophore, a ferric ion competitor (Kim, 2007). B.

Corresponding author at: College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China. E-mail address: [email protected] (H. Liu).

https://doi.org/10.1016/j.biocontrol.2020.104210 Received 15 October 2019; Received in revised form 3 January 2020; Accepted 27 January 2020 1049-9644/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Ye Li, et al., Biological Control, https://doi.org/10.1016/j.biocontrol.2020.104210

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

blight severity was rated on a scale of 0–5, where 0 = no symptoms, 1 = slightly withered leaves and brown spots on stems, 2 = disease over 30–50% of the whole plant, 3 = disease over 50–70% of the whole plant, 4 = disease over 70–90% of the whole plant, and 5 = whole plant death (Rajkumar et al., 2005). Each treatment included 36 pepper seedlings and the experiment was repeated three times.

licheniformis B40 stimulated defense mechanisms in Cucumis sativus L. plants, including activation of antioxidant enzymes and promotion of lignin production (Helepciuc et al., 2014). Similarly, after inoculation of cucumber roots with B. licheniformis TG116, levels of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine (PAL) in cucumber leaves increased (Ling et al., 2016). Bacillus amyloliquefaciens UQ154 and Bacillus velezensis UQ156 significantly promoted growth of seedlings, as measured by root length, total fresh weight, and seedling vigor (Syed-Ab-Rahman et al., 2019). Previous research on B. licheniformis BL06 revealed that this strain inhibited mycelial growth of several fungal plant pathogens in vitro (Wang et al., 2014). In this study, the objectives were to (i) assess the biocontrol efficacy of BL06 in detached leaf test and potted greenhouse conditions; (ii) test the effects of BL06 on mycelia growth, development of sporangia, zoospore germination, motility and lytic ability of zoospores in vitro. This study highlights a great potential in controlling P. capsici in pepper by B. licheniformis BL06 in the future.

2.3. Antagonistic activity of BL06 against P. capsici in vitro 2.3.1. Dual culture assay To evaluate the antagonistic activity of BL06, a dual culture assay was performed. Fresh mycelial plugs (5 mm diameter) were cut from the margins of the P. capsici colony and were transferred to the center of V8 juice agar plates. Two pieces of sterile round filter paper containing 2 µL BL06 suspension (108 cfu/mL) were placed at a distance of 2.5 cm from the plugs. The colony diameter was calculated by measuring the average diameter in two perpendicular directions after 2, 4, and 6 days of incubation in a growth chamber at 25 °C. The percentage inhibition was calculated using the formula. I = (C − T)/(C − D) × 100. Where I: % inhibition, C: the colony diameter of the control (mm), T: colony diameter of the treatment (mm) and D: plug diameter (mm). Three replicates of each treatment were performed and the assays were repeated three times.

2. Materials and methods 2.1. Phytophthora capsici strain, bacterial strain and culture conditions The P. capsici strain was provided by Dr. G. Xuewen of the Nanjing Agricultural University. The BL06 strain (Bacillus licheniformis) was isolated from the rhizosphere of healthy cucumber in the Huaian, Jiangsu province. The P. capsici was cultured in the dark on 10% V8 juice agar medium (10% V8 juice, 0.1% CaCO3 and 1.5% agar) at 25 °C (Lamour et al., 2012). B. licheniformis BL06 was cultured on LuriaBertani (LB) agar (1% Tryptone, 0.5% Yeast extract, 1% NaCl and 1.5% agar) at 28 °C. The pepper plant variety was “Sujiao 5.”

2.3.2. Effects of BL06 treatment on P. capsici mycelium morphology To observe the effects of BL06 on P. capsici mycelium morphology, five mycelium plugs (5 mm in diameter) cut from the margins of an actively growing culture, were placed into 20 mL V8 juice agar in Petri dishes (9 cm × 9 cm). Exactly 100 µL BL06 bacterial culture grown on LB broth (108 cfu/mL) was added to the Petri dish and they were subsequently incubated in the dark at 25 °C for 3 days. A control group using LB liquid medium instead of bacterial culture was also included. After 3 days of incubation, the mycelium morphology was observed under a light microscope (OLYMPUS-DP72).

2.2. Biological control assay 2.2.1. Disease suppression by BL06 in detached leaves A detached leaf assay was used to analyze the biocontrol ability of BL06 bacterial culture on P. capsici. Detached leaves collected from healthy and fully expanded leaves of 4-week-old pepper plants were submerged in BL06 bacterial culture (108 cfu/mL). A 5 mm diameter mycelium plug from P. capsici was placed onto the abaxial surface of two opposing leaves (the third and fourth true leaves). Detached leaves were placed in Petri dishes and wet filter paper was placed at the bottom and top of the plate to maintain high relative humidity. The dishes were immediately sealed with parafilm and incubated in an artificial climate chamber (25 °C, 70% relative humidity for 16/8 light/ dark cycle). Disease phenotype and lesion diameter were measured and photographed 3 days post inoculation (dpi). The leaf disease index was calculated 3 dpi. Disease severity of Phytophthora blight was rated on a scale of 0–5, where 0 = no lesion, 1 = lesion area accounts for 1–10% of leaf area, 2 = lesion area accounts for 11–20% of leaf area, 3 = lesion area accounts for 21–30% of leaf area, 4 = lesion area accounts for 31–50% of leaf area, and 5 = the area of lesion is more than 50% of the leaf area (Peng et al., 2005). Each treatment consisted three replicates, each group consisted of 12 leaves, and the experiments were repeated two times.

2.3.3. Effects of BL06 on sporangia development and production To assess sporangia production, five mycelium plugs (5 mm in diameter) were cut from the margins of an actively growing culture and placed into Petri dishes (9 cm × 9 cm) containing 20 mL 10% V8 juice. After incubation at 25 °C for 3 days in the dark, the medium was removed and 20 mL sterile water containing 100 µL BL06 bacterial culture of different concentrations was added, and then put at 25 °C for 24 h in the dark to induce sporangia formation. Negative controls were treated with 100 µL LB broth. Sporangia on the mycelia tips were quantified using a light microscope. The number of sporangia were counted in three different visual fields (as one replicate). Three replicates were conducted for each treatment and the experiment was repeated three times. 2.3.4. Effects of BL06 on motility and lysis of P. capsici zoospores A previously described method (Islam et al., 2016) was used to analyze the effects of BL06 bacterial culture on of zoospore motility and lysis. The preparation method of zoospore suspension is as follows. Mycelium plugs (5 mm in diameter) were cut from the margins of an actively growing culture and placed into Petri dishes containing 20 mL 10% V8 juice. After incubation at 25 °C for 3 days in the dark, the culture medium was removed and 20 mL sterile water was added to induce sporangia formation. After incubation at 25 °C for 24 h in the dark, the sterile water in the Petri dishes was removed and sterile water was added again and placed in the refrigerator at 4 °C for 30 min. The release of the zoospores was stimulated by low temperature stress, and then placed at room temperature for 30 min. The zoospore suspension was attenuated to 105 zoospores/ml in sterile water through use of a hemacytometer. Samples (10 µL) of BL06 bacterial culture (108 cfu/mL) were directly added to 990 µL freshly prepared zoospore suspension (105 zoospores/mL) in a centrifuge tube and then quickly mixed.

2.2.2. Potted plant greenhouse experiment The pepper seeds were germinated for 4 days, planted on seedling trays and cultivated under controlled greenhouse conditions at 28 °C, 70% relative humidity and a 16/8h light/dark cycle. When pepper seedlings produced 4–5 true leaves, they were transplanted into pots with 4 seedlings per pot. Two days after transplantation, 150 mL BL06 bacterial culture (108 cfu/mL) was applied to drench the seedling roots. For negative controls, 150 mL LB broth was applied. Four days after application, P. capsici was inoculated with 2 mL 105 cfu/mL zoospore suspension near the roots. The disease index and biocontrol effects were calculated 4, 5, and 6 days after pathogen challenge. Phytophthora 2

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

Instead of bacterial culture, 10 µL LB broth was added to the negative control samples. Zoospore motility was observed under a light microscope at 1000x magnification and measures of motility and lysis were recorded over time. Three replicates were conducted for each treatment and the experiment was repeated three times. 2.3.5. Effects of BL06 on zoospore germination The following procedure was used to investigate the effects of BL06 bacterial culture on zoospore germination. Tubes containing 990 µL zoospore suspensions were vortexed for 90 s to induce encystment and 10 µL BL06 bacterial culture (108 cfu/mL) was added directly to the suspension and quickly mixed. For negative control samples, 10 µL LB broth was added in place of bacterial culture. Then 20 µL mixed solution was pipetted onto a glass cover slip and cyst germination was observed under a light microscope at 200× magnification after incubation for 2, 4, and 6 h at 28 °C in the dark. Cysts were scored as germinated if the germ tube length equaled or exceeded the cyst diameter (10 μm). The percentage inhibition was calculated using the formula. I = G/T. Where I: % inhibition, G: the percentage of germinating zoospores and T: the total number of zoospores. At least 300 cysts were examined for each sample, three replicates were conducted for each treatment, and the experiment was repeated twice. Fig. 1. Effects of BL06 on the disease symptoms developed on detached leaves infected by P. capsici. (A) Detached leaves were submerged in BL06 bacterial culture (108 cfu/mL), LB was used as a Control. A 5 mm diameter mycelium plug from P. capsici was placed onto the abaxial surface, disease phenotype and (B) lesion diameter on the inoculated detached leaves were measure at 3 dpi. Error bars represent the standard error of the mean of three independent replicates. Double asterisk represents an extremely significant difference (P < 0.01).

2.4. Effects of BL06 on zoospore virulence To assess the effects of BL06 bacterial culture on P. capsici zoospore virulence, inoculation assays were performed on detached pepper leaves. A final BL06 concentration of 105 cfu/mL was selected to inhibit BL06-induced zoospore lysis. Samples (10 µL) of BL06 bacterial culture was added directly to 10 mL zoospore suspension and quickly mixed. For negative controls, 10 µL LB broth was added in place of bacterial culture. Then 20 µL mixed solution was applied to one half of the leaf back surface. The control solution was added to the other half of the leaf back surface. The inoculated leaves were incubated in the dark at 25 °C in 80% humidity for 4, 24, and 48 h. The leaves were boiled in 96% ethanol for 10 min and stained with trypan blue solution (lactic acid:glycerol:trypan blue:alcohol:sterilized water = 1:1:1:4:1) for 2 min, and rinsed and decolorized with 96% ethanol until the background was clear (Safdar et al., 2017). To examine the mechanism of reduced zoospore virulence, the process of zoospore germination, tube formation, and mycelial expansion in leaf tissues was observed under a light microscope. Leaf tissues around the inoculation sites were excised and also examined under a light microscope. Lesion-associated germ tubes and growth of mycelia in the leaf tissues were monitored and photographed. Three replicates were conducted for each treatment and the experiment was repeated two times.

Table 1 The control effect of P. capsici on detached leaves when treatment with BL06. Treatment Control-LB BL06

A

Disease index B

100 ± 0a 29.44 ± 1.93b

Biocontrol effect (%) – 70.56

A Detached leaf was submerged in BL06 bacterial culture (108 cfu/mL), LB was used as a Control. A 5 mm diameter mycelium plug from P. capsici was placed onto the abaxial surface, lesion diameter on the inoculated detached leaves were measure at 3 dpi. B Data are means ± SD. Different letters in the same column indicate significant difference at P < 0.05 level according to the least significant difference test (LSD).

while only 29.44% of BL06 treated samples were diseased. Therefore, the overall biocontrol effect of BL06 on Phytophthora blight was 70.56% (Table 1).

2.5. Statistical analyses 3.1.2. BL06 as a biocontrol against pepper Phytophthora blight Disease index measures were used to assess the efficacy of BL06 as a biocontrol against Phytophthora blight. Peppers treated with BL06 bacterial culture and later inoculated with P. capsici zoospores displayed significantly lower disease index at 4, 5, and 6 dpi compared to controls (control efficacy 68.74%, 52.78% and 47.27%, respectively, Table 2).

All treatments were repeated at least three times and data were analyzed with DPS 15.10 software. 3. Results 3.1. Biological control assay 3.1.1. Detached leaf inoculation The detached leaf inoculation assay was used to assess the biocontrol efficacy of BL06 bacterial culture on P. capsici virulence. Detached leaves were infiltrated with BL06 and control LB broth and exposed to a 5 mm diameter mycelium plug and incubated at 25 °C. After 48 h, characteristic water-soaked lesions were observed on control samples, however, the BL06 treated leaves displayed significantly smaller lesions (3.63 cm vs. 1.72 cm diameter, Fig. 1A, B). At 3 dpi, 100% of control samples displayed evidence of disease,

3.2. Antagonistic activity of BL06 against P. capsici in vitro The antagonistic activity of BL06 against P. capsici was tested to explore the mechanisms of BL06 disease inhibition. The radial growth of P. capsici mycelium toward the BL06 was remarkably stunted compared to the untreated control in the dual culture plate assay (Fig. 2A). BL06 inhibited P. capsici mycelium growth more than 70% when cultured for 6 days (Fig. 2B). To determine the effects of BL06 on 3

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

of sporangia was inhibited relative to controls (Fig. 3A). BL06 with the concentration of 1 × 105 cfu/mL significantly reduced P. capsici sporangia production. A few and few sporangia were produced when BL06 concentration was increased to 1 × 106 and 1 × 107 cfu/mL separately (Fig. 3B). These results demonstrated that this inhibitory effect is dosedependent.

Table 2 The disease index of phytophthora blight of pepper after root treatment with BL06. Treatment

A

Disease index 4d

Control-LB BL06

53.33 ± 2.89 a 16.67 ± 2.89b

B

5d

6d

63.33 ± 2.89 a 30 ± 5b

77.67 ± 7.64 a 40 ± 8.66b

A

3.4. Effects of BL06 bacterial culture on P. capsici zoospore motility and lysis

5

Pepper plants were inoculated with P. capsici by applying 2 mL zoospores (10 cfu/mL) into pepper seedlings roots. Phytophthora blight was rated based on a scale of 0–5 as described in Section 2. B Data are means ± SD. Different letters in the same column indicate significant difference at P < 0.05 level according to the least significant difference test (LSD).

BL06 bacterial culture was added to a suspension of P. capsici zoospores and zoospore motility and lysis were monitored (Fig. 4). Microscopy revealed that in presence of BL06, zoospore motility rapidly diminished. All affected zoospores ceased movement 1 min after treatment and subsequently lysed within 10 min of treatment. Two min post treatment, the edge of one side of the cell membrane appeared transparent and the zoospores gradually became circular. Five min post treatment, the zoospores expanded outward from thinning membrane to form a vesicle and the granulated intracellular contents moved into the vesicles. At 7–10 min post treatment, cell lysis occurred and the intracellular contents fragmented and dispersed into the surrounding water. There was a dose dependent relationship between BL06 concentration and degree of zoospore lysis. At a BL06 concentration of 1 × 107 cfu/mL, zoospore lysis was near complete in 2 min (Fig. S1). The zoospores treated with LB broth were elliptical and kept in a swimming state for 10 min after continuous observation. Under control conditions, the zoospores appeared elliptical maintained a swimming state for the 10 min observation period. The direction and route of swimming was irregular for this group.

mycelium morphology, the mycelium was observed and found that BL06 disrupted normal polar hyphae growth by inducing excessive branching (Fig. 2C, D). Hyphae lysis was also observed with BL06 treatment (Fig. 2E). Together, these results indicate that BL06 antagonizes P. capsici growth by inducing excessive branching and lysis of hyphae in vitro.

3.3. Inhibitory effects of BL06 bacterial culture on P. capsici sporangia development and production To further investigate the mechanisms of BL06 disease inhibition, the effects of BL06 on sporangia production were evaluated. When normal mycelia were treated with BL06 bacterial culture, the formation

Fig. 2. Antagonistic activity of BL06 against P. capsici in vitro. (A) Interactions between antagonistic BL06 and P. capsici in dual culture assay onV8 juice agar. (B) Inhibitory rate of BL06 on mycelium growth of P. capsici. The inhibition of mycelium growth (%) = (colony diameter of the control – colony diameter of the treatment)/(colony diameter of the control − plug diameter) × 100. Error bars represent the standard error of the mean of three independent replicates. (C–E) Mycelium morphology of (C) LB and (D–E) BL06 bacterial culture on P. capsici was observed under a microscope. LB liquid medium was used as a Control. Bar = 1 mm. 4

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

Fig. 3. Inhibitory effects of BL06 bacterial culture on sporangia development and production of P. capsici. The mycelia of P. capsici and BL06 bacterial culture of different concentration were cultured in sterilized water for 24 h, LB was used as a Control. (A) The formation of sporangia was observed under a microscope. Bar = 100 μm. (B) The number of zoosporangia formed on tips of mycelia was counted. Error bars represent the standard error of the mean of three independent replicates. Double asterisk represents an extremely significant difference (P < 0.01).

Fig. 4. Effects of bacterial culture on motility and lysis of P. capsici zoospores. The final concentration of BL06 was 106 cfu/mL by mixing BL06 bacterial culture with zoospores suspension, LB was used as a Control. The motility and lysis of zoospores was observed under a light microscope. Bar = 10 μm.

displayed a zoospore germination rate of 81.18%, compared to 5.28% for BL06 treated samples (Table 3). The inhibition rate of BL06 on zoospore germination was 90.54%, 93.24% and 93.5% (2, 4, and 6 h, respectively). The germ tubes of zoospores that successfully germinated under BL06 treatment were shorter compared to those observed in the

3.5. Inhibitory effects BL06 bacterial culture on P. capsici zoospore germination BL06 bacterial culture significantly suppressed P. capsici zoospore germination and germ tube growth. After 6 h, the control group

Table 3 Inhibitory effect of B. licheniformis BL06 bacterial culture on zoospores germination of P. capsici. Treatment

A

Zoospore germination Germination rate (%) 2h

Control-LB BL06

48.63 ± 0.57a 4.6 ± 0.76b

D

B

Inhibition rate (%)

C

4h

6h

2h

4h

6h

65.86 ± 1.18a 4.45 ± 0.9b

81.18 ± 4.89a 5.28 ± 0.93b

– 90.54

– 93.24

– 93.5

A Control–LB:LB liquid medium was used as a control; BL06: bacterial culture at the concentration of 1 × 106 cfu/mL. Zoospores were encysted by vortexing for 90 s and cysts were mixed with BL06 bacterial culture. 20 μL of mixed solution were pipetted onto a cover glass, the germination of cysts were observed under a light microscope after incubation at 28 °C for 2, 4 and 6h in darkness. B Germination rate (%) = germination cyst numbers/total cysts × 100. C Inhibition rate (%) = (germination rate of control – germination rate of BL06)/germination rate of control × 100. D Data are means ± SD. Different letters in the same column indicate significant difference at P < 0.05 level according to the least significant difference test (LSD).

5

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

Fig. 5. Effects of BL06 bacterial culture on the P. capsici zoospore germination at 6 h post treatment. Zoospores were encysted by vortexing for 90 s and cysts were mixed with BL06 bacterial culture, LB liquid medium was used as a Control. 20 μL of mixed solution were pipetted onto a cover glass and cocultured at 28 °C. Photographs of cysts germination was taken under a light microscope at 6 h post treatment. C, cyst; G, germinated cyst. Bar = 100 μm.

control group (Fig. 5).

LB broth (Fig. 6A). Pepper leaves inoculated with zoospores and BL06 did not show any lesions at 48 hpi, whereas typical disease lesions spread throughout control inoculated leaves. These results indicate that BL06 treated zoospores were less infectious to their host. To understand the mechanism underlying zoospore pathogenicity loss, zoospore behavior was monitored at different time points post

3.6. BL06 bacterial culture reduced the infectiousness zoospores. To determine whether BL06 influences the infectiousness of zoospores, leaves were inoculated with zoospores mixed with either BL06 or

Fig. 6. BL06 bacterial culture reduced the infection abilities of zoospores. BL06 bacterial culture was directly added to zoospores suspension and then quickly mixed, an equal amount of LB was added to the Control treatment. 20 μL of mixed solution were pipetted onto the back of the leaves. The inoculated leaves were incubated in the dark at 25 °C and 80% humidity for 4, 24 and 48 h. (A) The leaves were stained with trypan blue solution (B) and subjected to microscopic analysis. Bar = 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

permeability (Jung and Kim, 2005). The B. subtilis AH18A strain, which promotes plant growth and antagonizes P. capsici, uses a cellulase enzyme to degrade the fungal cell wall of P. capsici (Woo et al., 2006). Under field conditions, the sporangia of P. capsici detach from the sporangiophore and are dispersed by wind, rain and irrigation water. Sporangia germinate indirectly by producing zoospores, which readily infect roots or above ground plants tissues with sufficient free moisture (Ristaino and Johnston, 1999). Volatile organic compounds produced by Pseudomonas species act by impeding P. infestans mycelial growth and sporangia germination (Bailly and Weisskopf, 2017). Furthermore, the cyclic lipopeptide massetolide A, produced by Pseudomonas fluorescens, significantly reduces sporangium formation (Mortel et al., 2009). Therefore, an ideal biocontrol agent should impact mycelia, sporangia and zoospores. In our study, BL06 exhibited a strong inhibitory effect on sporangia development, zoospore motility, lysis and germination in vitro. Similar effects have been linked to compounds produced by other bacterial species. An antimicrobial peptide, produced by Brevibacillus laterosporus strain A60, demonstrated an inhibitory effects of different P. capsici life cycle stages, including mycelial growth, sporangia formation and cystospore germination (Zhao et al., 2012). The volatile organic compound, 1-undecene, produced by Pseudomonas bacterial strains, significantly reduced P. infestans sporangium formation in a dose-dependent manner (Lukas et al., 2015). Similarly, a dose-dependent effects of BL06 on inhibition of sporangia development were noted. Given their role in infection initiation, zoospores represent an important target in the control of Phytophthora blight of peppers (Islam et al., 2016; Tani and Judelson, 2006). Zoospore motility is critical for the virulence and disease expansion of oomycetes (Dong et al., 2004; Latijnhouwers et al., 2010). In the present study, BL06 rapidly impaired motility and induced P. capsici zoospore lysis in a dose- and time-dependent manner. Similarly, Islam et al. (2016) found that macrotetrolides from Streptomyces species inhibited motility and induced lysis of P. capsici, Plasmopara viticola and Aphanomyces cochlioides zoospores, and provided evidence for enhanced ATPase activity and ATP hydrolysis as a biocontrol mechanism. Previous reports have shown that bacterial metabolites have diverse effects on zoospore motility and lysis of P. capsici. Four antibiotics, pamamycin, oligomycin A, oligomycin B and echinosporin, were isolated from Streptomyces strains B8496 and B8739 (Dame et al., 2016). The biosurfactant orfamide, a type cyclic lipopeptides (CLPs), and putisolvin-like biosurfactants were identified in Pseudomonas species (Ma et al., 2016; Marco et al., 2009). Four new anti-fungal, non-cytotoxic cyclic lipopeptides were isolated from the marine-derived bacterium Bacillus subtilis strain 109GGC020 (Tareq et al., 2015). In vivo disease suppression may involve a comparable mechanism (Zohara et al., 2016). The significant inhibitory effects of BL06 on sporangial development and on the motility and lysis of zoospores warrant further experiments aimed at purification and identification of the bioactive metabolites. Cyst germination is required for subsequent germination into host tissues (Safdar et al., 2017). Measures to control of spore or conidia germination target the conversion of fungus to its tissue-invasive form (Waldorf, 1989; Zhu et al., 2016). Several in vitro studies have reported inhibition of P. capsici zoospore germination by different Bacillus species: Paenibacillus polymyxa (Kim et al., 2010), Bacillus licheniformis K11 (Kim, 2007), Bacillus subtilis (Kim et al., 2012) and Brevibacillus laterosporus (Zhao et al., 2012). Few studies have considered the effects of Bacillus species on P. capsici zoospore germination rate and germ tube growth in plant host leaves. In the present study, BL06 reduced the infectiousness P. capsici zoospores by inhibiting zoospore germination and germ tube growth in host tissues. BL06 may also inhibit the early stages of zoospore infection. The PcSDA1 (Zhu et al., 2016), PcAvh1 (Chen et al., 2019), PcNLP (Feng et al., 2014) genes, which regulate zoospore germination, are rapidly induced at the upon P. capsici infection. In summary, our study demonstrated that B. licheniformis BL06

inoculation. In control leaves, the zoospores transformed into cysts and germinated into germ tubes at 4 hpi and hyphae multiplied rapidly within the leaf tissues from 24 to 48 hpi (Fig. 6B, upper panel). At 24 hpi, the pathogen possessed infectious hyphae and mycelia spread to seemingly healthy leaf tissues, marking the biotrophic stage of infection. At 48 hpi, a large number of mycelium were found within leaf tissue and the typical necrotic lesions were formed. By contrast, only a small number of BL06 treated zoospores transformed into germinating cysts with germ tube formation at 4 hpi (Fig. 6B, lower panel). At 24 hpi, germ tubes appeared short in length and failed to penetrate into the host tissues. No infectious hyphae were formed. At 48 hpi, mycelia spread was limited and hyphae were distributed over the surface. The mycelia that did form under BL06 treatment were not able to establish successful infection and could not form lesions within 48 hpi. These results indicate that BL06 reduced the infectiousness of P. capsici zoospores by inhibiting zoospore germination and germ tube growth in host tissues. 4. Discussion A bacterial strain (B. licheniformis BL06) with antagonistic activity against various fungal pathogens including P. capsici was identified previously. In the present study, the BL06 biocontrol efficacy was further assessed by evaluating the disease index in inoculated detached leaves and potted greenhouse plants. The results demonstrated that BL06 reduced the severity of pepper Phytophthora blight disease. Furthermore, our efforts to monitor the effects BL06 bacterial culture on different P. capsici life cycle stages and assessments of pathogen infectiousness showed that different modes of action contribute to the BL06 antifungal mechanism. Detached leaf inoculation assays are typically used to assess the effects of oomycete pathogen infection (Melnick et al., 2011; Rajkumar et al., 2005; Wang et al., 2013) and the ability of endophytes to antagonize disease initiation and progression (Khatun et al., 2018). In present study, BL06 treatment prevented disease development and demonstrated a strong Phytophthora blight biocontrol effect (70%). Under greenhouse conditions, P. capsici infection caused stem lodging and leaf abscission in pepper plant seedlings. These results indicated that root irrigation with BL06 effectively reduced the disease index in pepper plant seedlings. The BL06 inhibitory effect may result from its ability to restrict of P. capsici expansion. Shenqinmycin, a novel antibiotic with inhibitory effects on mycelium growth, displays 67.98% efficacy against Sclerotinia sclerotiorum (Zheng et al., 2011). Bacillus methylotrophicus TA-1 exhibited a strong antifungal growth effects on Fusarium graminearum mycelium and the corresponding greenhouse experiment showed significantly reduced disease incidence and disease index (Cheng et al., 2019). Further studies are required to confirm the ability of BL06 to control pepper Phytophthor a blight under field conditions. The ability of Bacillus species to alter P. capsici hyphae has been widely reported. For example, Ling et al. showed that B. licheniformis TG116 treatment resulted in deformed P. capsici mycelia (Ling et al., 2014). In the present study, BL06 induced excessive branching and remarkable lysis of P. capsici hyphae. Similar lysis of Phytophthora spp. hyphae was observed with B. velezensis UQ156 (Syed-Ab-Rahman et al., 2018) and B. amyloliquefaciens Y1 (Jamal and Kimkilyong, 2015). B1, B10 and B17, which were identified as Pseudomonas spp., significantly inhibited hyphal growth through induction of excessive branching, swelling and cellular disintegration of P. capsici (Zohara et al., 2016). Several mechanisms of bacterial biocontrol have been demonstrated. Secondary metabolites produced by Bacillus species affect mycelia morphology (Liu et al., 2005; Xu et al., 2007). Treatment with B. amyloliquefaciens BPD1 lipopeptides results in deformed and cancerous conidia and hyphae, similar to that observed with fengycin treatment (Liao et al., 2016). The antibiotic KL39, purified from B. megaterium, causes P. capsici hyphae swelling by altering cell membrane 7

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

controls pepper Phytophthora blight caused by P. capsici in detached leaves and under potted greenhouse conditions. Results from in vitro experiments identified the antagonistic mechanism, which encompassed inhibition of mycelial growth, sporangia development, motility and germination of zoospores. The infectiousness of zoospores was also reduced by BL06 in host tissues, likely due to its ability to inhibit zoospore germination and germ tube growth. Future investigations should identify and characterize specific bioactive BL06 compounds as biocontrol measures for Phytophthora blight of peppers under field conditions.

Mukhtar, M.S., Tome, D.F.A., Tor, M., Van den Ackerveken, G., McDowell, J., Daayf, F., Fry, W.E., Lindqvist-Kreuze, H., Meijer, H.J.G., Petre, B., Ristaino, J., Yoshida, K., Birch, P.R.J., Govers, F., 2015. The Top 10 oomycete pathogens in molecular plant pathology. Mol. Plant Pathol. 16, 413–434. Khatun, A., Farhana, T., Sabir, A.A., Smn, I., West, H.M., Rahman, M., Islam, T., 2018. Pseudomonas and Burkholderia inhibit growth and asexual development of Phytophthora capsici. Z. Naturforsch. C.73, 123–135. Kim, S.-D., 2007. Confirmation of Non-Siderophore antifugal substance and cellulase from Bacillus licheniformis K11 containing antagonistic ability and plant growth promoting activity. Korean. Soc. Life Sci. 17, 983–989. Kim, S.G., Jang, Y., Kim, H.Y., Koh, Y.J., Kim, Y.H., 2010. Comparison of microbial fungicides in antagonistic activities related to the biological control of phytophthora blight in chili pepper caused by Phytophthora capsici. Plant Pathol. J. 26, 340–345. Kim, Y.-K., Hong, S.-J., Ki, S.C., Min-Jeong, K., Jee, H.-J., 2012. Functional analysis of Bacillus subtilis isolates and biological control of red pepper powdery mildew using Bacillus subtilis R2–1. Res. Plant Dis. 18, 201–209. Lamour, K.H., Mudge, J., Gobena, D., Hurtado-Gonzales, O.P., Schmutz, J., Kuo, A., Miller, N.A., Rice, B.J., Raffaele, S., Cano, L.M., Bharti, A.K., Donahoo, R.S., Finley, S., Huitema, E., Hulvey, J., Platt, D., Salamov, A., Savidor, A., Sharma, R., Stam, R., Storey, D., Thines, M., Win, J., Haas, B.J., Dinwiddie, D.L., Jenkins, J., Knight, J.R., Affourtit, J.P., Han, C.S., Chertkov, O., Lindquist, E.A., Detter, C., Grigoriev, I.V., Kamoun, S., Kingsmore, S.F., 2012. Genome sequencing and mapping reveal loss of heterozygosity as a mechanism for rapid adaptation in the vegetable pathogen Phytophthora capsici. Mol. Plant Microbe Interact. 25, 1350–1360. Latijnhouwers, M., Ligterink, W., Vleeshouwers, V.G., Van, W.P., Govers, F., 2010. A Galpha subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans. Mol. Microbiol. 51, 925–936. Leonian, L.H., 1922. Stem and fruit blight of peppers caused by Phytophthora capsici sp. nov. Phytopathology 12 (9), 401–408. Liao, J.H., Chen, P.Y., Yang, Y.L., Kan, S.C., Hsieh, F.C., Liu, Y.C., 2016. Clarification of the antagonistic effect of the lipopeptides produced by Bacillus amyloliquefaciens BPD1 against Pyricularia oryzae via in situ MALDI-TOF IMS analysis. Molecules 21, 1670. Ling, L.J., Feng, L., Lei, L., Nan, H.E., Ding, L., 2016. Induction of defense-related enzymes in cucumber roots by Bacillus licheniformis Strain TG116. J. Northwest Norm. Univ. 1, 100–104. Ling, L.J., Lei, L., Feng, L., Nan, H.E., Ding, L., 2014. Isolation and identification of endophytic bacterium TG116 from Typhonium giganteum and its antimicrobial characteristics. J. Northwest Univ Nat Sci. 5, 91–97. Liu, W.C., Pan, H.Y., Jing-Hui, X.I., Hong-Yu, L.I., Guo, Y.L., Wang, X.M., 2005. Biocontrol effects of some antifungal strains from Bucillus against pathogens of wheat head blight. J. Trit. Crops. 25 (4), 95–100. Lukas, H., Denise, B.N., Ulrike, G., Aurélien, B., Stefan, S., Laure, W., 2015. Pseudomonas strains naturally associated with potato plants produce volatiles with high potential for inhibition of Phytophthora infestans. Appl. Environ. Microbiol. 81, 821–830. Ma, Z., Geudens, N., Kieu, N.P., Sinnaeve, D., Ongena, M., Martins, J.C., Höfte, M., 2016. Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species. Front. Microbiol. 7, 382. Marco, K., Ha, T., Raaijmakers, J.M., 2009. Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267. J. Appl. Microbiol. 107 (2), 546–556. Meitz, J.C., Linde, C.C., Thompson, A., Langenhoven, S., Mcleod, A., 2010. Phytophthora capsici on vegetable hosts in South Africa: distribution, host range and genetic diversity. Australas. Plant Path. 39 (5), 431–439. Melnick, R., Zidack, N., Ba, Maximova S., Guiltinan, M., Backman, P., 2008. Bacterial endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod rot of cacao. (Special Issue: Endophytes.). Biol. Control. 46 (1), 46–56. Melnick, R.L., Suárez, C., Bailey, B.A., Backman, P.A., 2011. Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases. Biol. Control. 57 (3), 236–245. Mo, X., 2013. Interactions between plant growth promoting rhizobacteria (Pgpr) and squash plants the role of Pgpr-Mediated suppression of phytophthora blight. Univ. Fla. 1, 14–15. Mortel, J.E., De, Van, Ha, T., Francine, G., Raaijmakers, J.M., 2009. Cellular responses of the late blight pathogen Phytophthora infestans to cyclic lipopeptide surfactants and their dependence on G proteins. Appl. Environ. Microbiol. 75 (15), 4950–4957. Nguyen, X.H., Naing, K.W., Lee, Y.S., Kim, Y.H., Moon, J.H., Kim, K.Y., 2015. Antagonism of antifungal metabolites from Streptomyces griseus H7602 against Phytophthora capsici. J. Basic Microbiol. 55 (1), 45–53. Nigris, S., Baldan, E., Tondello, A., Zanella, F., Vitulo, N., Favaro, G., Guidolin, V., Bordin, N., Telatin, A., Barizza, E., Marcato, S., Zottini, M., Squartini, A., Valle, G., Baldan, B., 2018. Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera. BMC Microbiol. 18 (1), 133. Peng, H.X., Liu, B.W., Wei, L.I., 2005. Biological characteristic and identification method of pepper resistance to Phytophthora capsici in Sichuan. J. Yunnan Agric. Univ. 20 (1), 140–144. Rajkumar, M., Lee, W.H., Lee, K.J., 2005. Screening of bacterial antagonists for biological control of Phytophthora blight of pepper. J. Basic Microbiol. 45 (1), 55–63. Ristaino, J.B., Johnston, S.A., 1999. Ecologically based approaches to management of phytophthora blight on bell pepper. Plant Dis. 83 (12), 1080–1089. Safdar, A., Li, Q., Shen, D., Chen, L., He, F., Wang, R., Zhang, M., Mafurah, J.J., Khan, S.A., Dou, D., 2017. An LRR receptor kinase regulates growth, development and pathogenesis in Phytophthora capsici. Microbiol. Res. 198, 8–15. Syed-Ab-Rahman, S.F., Carvalhais, L.C., Schenk, P., 2018. Identification of soil bacterial isolates suppressing different Phytophthora spp. and promoting plant growth. Front.

CRediT authorship contribution statement Ye Li: Methodology, Investigation, Writing - original draft. Xiaoqian Feng: Validation. Xiaoli Wang: Formal analysis. Li Zheng: Conceptualization, Visualization. Hongxia Liu: Resources, Data curation, Supervision, Writing - review & editing, Funding acquisition. Acknowledgments This work was supported by National Natural Science Foundation of China (31571992) and the opening foundation of Jiangsu Provincial Key Construction Laboratory of Probiotics Preparation (JSYSZJ2018002). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biocontrol.2020.104210. References Bailly, A., Weisskopf, L., 2017. Mining the volatilomes of plant-associated microbiota for new biocontrol solutions. Front. Microbiol. 8, 1638. Chen, X., Zhang, Y., Li, H., Zhang, Z., Sheng, G., Li, Y., Xing, Y., Huang, S., Tao, H., Kuan, T., Zhai, Y., Ma, W., 2019. The RXLR effector PcAvh1 is required for full virulence of Phytophthora capsici. Mol. Plant Microbe Interact. 32 (8), 986–1000. Chen, Y.-Y., Chen, P.-C., Tsay, T.-T., 2016. The biocontrol efficacy and antibiotic activity of Streptomyces plicatus on the oomycete Phytophthora capsici. Biol. Control. 98, 34–42. Cheng, X., Ji, X., Li, J., Qi, W., Ge, Y., Qiao, K., 2019. Characterization of antagonistic Bacillus methylotrophicus isolated from rhizosphere and its biocontrol effects on maize stalk rot. Phytopathology 109 (4), 571–581. Dame, Z.T., Islam, M.T., Helmke, E., Von, T.A., Laatsch, H., 2016. Oligomycins and pamamycin homologues impair motility and induce lysis of zoospores of the grapevine downy mildew pathogen, Plasmopara viticola. FEMS Microbiol. Lett. 363 (16), fnw167. Dong, W., Latijnhouwers, M., Jiang, R.H.Y., Meijer, H.J.G., Govers, F., 2004. Downstream targets of the Phytophthora infestans Gα subunit PiGPA1 revealed by cDNA-AFLP. Mol. Plant Pathol. 5, 483–494. Ezziyyani, M., Requena, M.E., Egea Gilabert, C., Candela, M.E., 2010. Biological control of phytophthora root rot of pepper using Trichoderma harzianum and Streptomyces rochei in combination. J. Phytopathol. 155, 342–349. Feng, B.Z., Zhu, X.P., Fu, L., Lv, R.F., Storey, D., Tooley, P., Zhang, X.G., 2014. Characterization of necrosis-inducing NLP proteins in Phytophthora capsici. BMC Plant Biol. 14 (1), 126. Hausbeck, M.K., Gevens, A.J., Cortright, B., 2006. Integrating cultural and chemical strategies to control Phytophthora capsici and limit its spread. CABI. 2006, 427–435. Helepciuc, F.E., Mitoi, M.E., Manole-Paunescu, A., Aldea, F., Brezeanu, A., Cornea, C.P., 2014. Induction of plant antioxidant system by interaction with beneficial and/or pathogenic microorganisms. Rom. Biotech. Lett. 19, 9366–9375. Islam, M.T., Laatsch, H., Von, T.A., 2016. Inhibitory effects of macrotetrolides from Streptomyces spp. on zoosporogenesis and motility of peronosporomycete zoospores are likely linked with enhanced ATPase activity in mitochondria. Front. Microbiol. 7, 1824. Jamal, Q., Kimkilyong, 2015. Isolation and biocontrol potential of Bacillus amyloliquefaciens Y1 against fungal plant pathogens. Korean. J. Soil Sci. Fertil. 48, 485–491. Joo, G.J., 2005. Production of an anti-fungal substance for biological control of Phytophthora capsici causing phytophthora blight in red-peppers by Streptomyces halstedii. Biotechnol. Lett. 27, 201–205. Jung, H.K., Kim, S.D., 2005. An antifungal antibiotic purified from Bacillus megaterium KL39, a biocontrol agent of red-pepper Phytophthora-blight disease. J. Microbiol. Biotechnol. 15, 1001–1010. Kamoun, S., Furzer, O., Jones, J.D.G., Judelson, H.S., Ali, G.S., Dalio, R.J.D., Roy, S.G., Schena, L., Zambounis, A., Panabieres, F., Cahill, D., Ruocco, M., Figueiredo, A., Chen, X.R., Hulvey, J., Stam, R., Lamour, K., Gijzen, M., Tyler, B.M., Grunwald, N.J.,

8

Biological Control xxx (xxxx) xxxx

Y. Li, et al.

Woo, S.M., Jung, H.K., Kim, S.D., 2006. Cloning and characterization of a cellulase gene from a plant growth promoting rhizobacterium, Bacillus subtilis AH18 against phytophthora blight disease in red-pepper. Microbiol. Biotechnol. Lett. 34 (4), 311–317. Xu, L., Xu, L., Li, L., Zou, Y.F., Zhao, C.Q., Zhang, W.G., Liu, Z.Y., 2007. Inhibition effect of Bacillus subtilis on the growth of Botryosphaeria dothidea. J. Fungal. Res. 5 (3), 165–168. Yan, M., Chang, Z.Z., Zhao, J.T., Zhou, M.G., 2008. Antifungal activity of Penicillium striatisporum Pst10 and its biocontrol effect on Phytophthora root rot of chilli pepper. Biol. Control. 44 (1), 24–31. Yau, J.A., Dianez, F., Marín, F., Carretero, F., Santos, M., 2013. Screening and evaluation of potential biocontrol fungi and bacteria foliar endophytes against Phytophthora capsici and Phytophthora parasitica on pepper. J. Food Agric. Environ. 11 (2), 490–495. Zhao, J., Guo, L., Zeng, H., Yang, X., Yuan, J., Shi, H., Xiong, Y., Chen, M., Han, L., Qiu, D., 2012. Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60. Peptides 33 (2), 206–211. Zheng, W.J., Shu, X.U., Zhou, M.G., 2011. Preliminary study on biological activity of shenqinmycin against Sclerotinia sclerotiorum. Chinese. J. Pesticide Sci. 13 (1), 28–32. Zhu, C., Yang, X., Lv, R., Li, Z., Ding, X., Tyler, B.M., Zhang, X., 2016. Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology, mycelial growth and plant infection. Mol. Plant Pathol. 17 (3), 369–387. Zohara, F., Akanda, M.A.M., Paul, N.C., Rahman, M., Islam, M.T., 2016. Inhibitory effects of Pseudomonas spp. on plant pathogen Phytophthora capsici in vitro and in planta. Biocatal. Agric. Biotechnol. 5, 69–77.

Plant Sci. 9, 1502. Syed-Ab-Rahman, S.F., Xiao, Y., Carvalhais, L.C., Ferguson, B.J., Schenk, P.M., 2019. Suppression of Phytophthora capsici infection and promotion of tomato growth by soil bacteria. Rhizosphere 9, 72–75. Tani, S., Judelson, H., 2006. Activation of zoosporogenesis-specific genes in Phytophthora infestans involves a 7-nucleotide promoter motif and cold-induced membrane rigidity. Eukaryot. Cell 5 (4), 745–752. Tareq, F.S., Hasan, C.M., Lee, H.S., Lee, Y.J., Lee, J.S., Surovy, M.J., Islam, M.T., Shin, H.J., 2015. Gageopeptins A and B, new inhibitors of zoospore motility of the phytopathogen Phytophthora capsici from a marine-derived bacterium Bacillus sp. 109GGC020. Bioorg. Med. Chem. Lett. 25 (16), 3325–3329. Tian, J., Zhan, Y., Sun, Q., He, Y., Zhang, Z., Yang, L., Sun, X., Peng, Y., Chen, W., Kang, J., 2017. A new super-long line pepper variety-general series pepper. Changjiang Veg. 1, 17–18. Waldorf, A.R., 1989. Pulmonary defense mechanisms against opportunistic fungal pathogens. Immunol Series. 47, 243–271. Wang, J.E., Liu, K.K., Li, D.W., Zhang, Y.L., Zhao, Q., He, Y.M., Gong, Z.H., 2013. A novel peroxidase CanPOD gene of pepper is involved in defense responses to Phytophtora capsici infection as well as abiotic stress tolerance. Int. J. Mol. Sci. 14 (2), 3158–3177. Wang, Q., 2017. Occurrence conditions and control of phytophthora blight of peppers. Friends Farmers Get Rich 24, 61. Wang, Z., Wang, Y., Li, Z., Yang, X., Liu, H., Guo, J., 2014. Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10. Biochem. Biophys. Res. Commun. 454 (1), 48–52.

9