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Bacillus subtilis DZSY21 isolated from the leaves of Eucommia ulmoides oliv. was labeled by antibiotic marker and found to effectively colonize the leaves of maize plant. Agar diffusion assays and biocontrol effect experiments showed that strain DZSY21 and its lipopeptides had antagonistic activity against Bipolaris maydis, as well as high biocontrol effects on southern corn leaf blight caused by B. maydis. Using MALDI-TOF-MS analysis, we detected the presence of antimicrobial surfactin A, surfactin B, and fengycin in the strain DZSY21. Signaling pathways mediated by DZSY21 were analyzed by testing the expression of key plant genes involved in regulation of salicylic acid (SA) or JA/ET pathways, the defense-related genes PR1 and LOX were concurrently expressed in the leaves of DZSY21-treated plants; this corresponded to slight increase in the expression level of PDF1.2 and decreases in ERF gene transcription levels. The results indicated an induced systemic response that is dependent on the SA and jasmonic acid (JA) pathways. Thus, we hypothesized that the strain DZSY21 inhibits B. maydis by producing antifungal lipopeptides and activating an induced systemic response through SA- and JA-dependent signaling pathways. This work describes a mechanism behind reduced disease severity in plants inoculated with the endophytic bacteria DZSY21.
从杜仲叶片中分离的枯草芽孢杆菌 DZSY21 经抗生素标记后可有效定殖于玉米植株叶片。琼脂扩散实验和生物防治效果实验表明，菌株 DZSY21 及其脂肽对马氏双孢蘑菇具有拮抗活性，并对马氏双孢蘑菇引起的南方玉米叶枯病具有较高的生物防治效果。通过 MALDI-TOF-MS 分析，我们检测到 DZSY21 菌株中存在抗菌素表面活性素 A、表面活性素 B 和芬吉素。通过检测参与调控水杨酸（SA）或 JA/ET 通路的关键植物基因的表达，分析了 DZSY21 介导的信号通路；在 DZSY21 处理的植物叶片中，防御相关基因 PR1 和 LOX 同时表达；这与 PDF1.2 的表达水平略有上升和 ERF 基因转录水平下降相对应。结果表明，诱导的系统反应依赖于 SA 和茉莉酸（JA）途径。因此，我们假设菌株 DZSY21 通过产生抗真菌脂肽，并通过依赖 SA 和 JA 的信号途径激活诱导的系统反应，从而抑制 B. maydis。这项工作描述了接种了内生细菌 DZSY21 的植物的病害严重程度降低的机制。

Introduction 导言

Epidemics of southern corn leaf blight in maize are caused by Bipolaris maydis; these infections are regarded as one of the most destructive foliar diseases due to their extensive damage to crop yield and quality (Wang et al., 2015). Traditionally, fungicides and resistant cultivars have been used to control this disease in the field. Some fungicides, such as Chlorothalonil, Carbendazim, and Thiophanate-methyl wettable powders are effective in preventing disease (Akgül and Mirik, 2008); however, the use of chemical fungicides pollutes the environment and catalyzes the development of fungicide-resistant strains (Bajwa et al., 2003). Therefore, there is an interest in technologies that would reduce dependency on chemical pesticides. Biological pest control, including the use of microorganisms to control plant diseases, offers an attractive alternative that would alleviate many of the negative impacts of chemicals.
玉米南方叶枯病是由Bipolaris maydis引起的；由于对作物产量和质量的广泛破坏，这些感染被认为是最具破坏性的叶面病害之一（Wang等人，2015年）。传统上，田间使用杀菌剂和抗病品种来控制这种病害。一些杀菌剂，如百菌清、多菌灵和甲基硫菌灵可湿性粉剂可有效预防病害（Akgül 和 Mirik，2008 年）；然而，化学杀菌剂的使用会污染环境并催化抗杀菌剂菌株的发展（Bajwa 等人，2003 年）。因此，人们对能减少对化学杀虫剂依赖的技术很感兴趣。生物病虫害控制，包括使用微生物控制植物病害，提供了一种有吸引力的替代方法，可减轻化学品的许多负面影响。

Endophytic bacteria are micro-organisms that colonize healthy plant tissue without causing any apparent symptoms or diseases to the host (Arnold et al., 2003). These strains could exert several beneficial effects on host plants, including conferring resistance against different biotic and abiotic stresses (Kharwar et al., 2008), inducing resistance to plant pathogens, and producing beneficial bioactive substances. There is interest in the use of endophytic bacteria for the biological control of plant diseases (Nejad and Johnson, 2000; Verhagen et al., 2004; Compant et al., 2005). So far, a variety of endophytes had been reported to confer protection against bacterial and fungal pathogens (Lodewyckx et al., 2002; Sessitsch et al., 2004). However, not enough is known about the mechanisms by which endophytic bacteria confer benefits. Understanding the mechanisms of biocontrol is critical to improving the efficacy of and implementing the use of biocontrol agents. The object of this study was to understand the inhibitory mechanisms by endophytic bacteria confer protection against disease.
内生细菌是在健康植物组织中定植的微生物，不会对宿主造成任何明显的症状或疾病（Arnold 等人，2003 年）。这些菌株可对寄主植物产生多种有益影响，包括对不同的生物和非生物胁迫产生抗性（Kharwar 等人，2008 年），诱导对植物病原体的抗性，以及产生有益的生物活性物质。人们对利用内生细菌进行植物病害生物防治很感兴趣（Nejad 和 Johnson，2000 年；Verhagen 等人，2004 年；Compant 等人，2005 年）。迄今为止，已有报道称多种内生菌可对细菌和真菌病原体产生保护作用（Lodewyckx 等人，2002 年；Sessitsch 等人，2004 年）。然而，人们对内生细菌产生益处的机制了解得还不够。了解生物防治的机制对于提高生物防治剂的功效和实施使用至关重要。本研究的目的是了解内生细菌对病害的抑制机制。

Bacillus species are among the most common endophytic bacteria (Bacon and Hinton, 2002; Choudhary and Johri, 2009), and there are many reports describing the ability of Bacillus spp. to suppress several important plant pathogens (Melo et al., 2009). In recent years, Bacillus spp. have been used as a biocontrol agent to protect crops against plant diseases, and provide an alternative to chemical fungicides (Augustine et al., 2010; Yang et al., 2012; Zhou et al., 2014). The main biocontrol mechanisms of Bacillus spp. are considered to be the production of antibiotics (direct) (Ongena et al., 2005; Ongena and Jacques, 2008), such as lipopeptides which aroused great attention for suppressing the growth of fungal pathogens and stimulating the innate immunity of plant system against various pathogens (Ongena et al., 2007; Romero et al., 2007; Raaijmakers et al., 2010), the competition for ecological niches (direct) (Compant et al., 2005), or the induction of systemic resistance (ISR) in host plants (indirect) (Van Loon and Bakker, 2006; Saravanakumar et al., 2007). However, the effectiveness of endophytes as biological control agents (BCAs) is dependent on efficient colonization of the plant environment. The extent of endophytic colonization in host plant organs and tissues reflects the ability of bacteria to selectively adapt and compete in those specific ecological niches.
芽孢杆菌是最常见的内生细菌之一（Bacon 和 Hinton，2002 年；Choudhary 和 Johri，2009 年），许多报告都描述了芽孢杆菌属抑制几种重要植物病原体的能力（Melo 等人，2009 年）。近年来，芽孢杆菌属已被用作一种生物防治剂来保护作物免受植物病害的侵害，并为化学杀菌剂提供了一种替代品（Augustine 等人，2010 年；Yang 等人，2012 年；Zhou 等人，2014 年）。芽孢杆菌的主要生物防治机制被认为是生产抗生素（直接）（Ongena 等人，2005 年；Ongena 和 Jacques，2008 年），如脂肽，它在抑制真菌病原体的生长和刺激植物系统的先天免疫力以对抗各种病原体方面引起了极大的关注（Ongena 等人，2007 年；Romero 等人，2014 年）、2007；Romero 等人，2007；Raaijmakers 等人，2010）、竞争生态位（直接）（Compant 等人，2005）或诱导宿主植物产生系统抗性（ISR）（间接）（Van Loon 和 Bakker，2006；Saravanakumar 等人，2007）。然而，内生菌作为生物防治剂（BCA）的有效性取决于对植物环境的有效定殖。内生菌在寄主植物器官和组织中的定殖程度反映了细菌在这些特定生态位中选择性适应和竞争的能力。

Eucommia ulmoides oliv. was a medicinal plant in southern China, which was known for hosting several metabolites having medicinal property (Matsuda et al., 2006; Zhang et al., 2014), and many studies on it had been carried out in terms of some products of secondary metabolism (Matsuda et al., 2006; Zhang et al., 2014). However, reports on the antagonistic endophytic bacteria isolated from the E. ulmoides oliv. and their potential to promote plant disease resistance were relatively few. In this study, the Bacillus subtilis DZSY21 isolated from the leaves of E. ulmoides oliv. exerts a strong antifungal effect on B. maydis. On that basis, we investigated the inhibition mechanisms of the biocontrol strain DZSY21, including direct antagonism and induced systematic resistance, as well as the ability of DZSY21 to colonize maize leaves. This work provides a theoretical basis for the use of DZSY21 as a replacement for pesticides and supplements.
杜仲（Eucommia ulmoides oliv.）是中国南方的一种药用植物，因含有多种具有药用价值的代谢产物而闻名（Matsuda 等人，2006 年；Zhang 等人，2014 年），对其二次代谢的一些产物也进行了许多研究（Matsuda 等人，2006 年；Zhang 等人，2014 年）。然而，从橄榄溃疡埃希氏菌（E. ulmoides oliv.）中分离出的拮抗内生细菌及其促进植物抗病潜力的报道相对较少。在本研究中，从 E. ulmoides oliv.叶片中分离出的枯草芽孢杆菌 DZSY21 对 B. maydis 有很强的抗真菌作用。在此基础上，我们研究了生防菌株 DZSY21 的抑制机制，包括直接拮抗和诱导系统抗性，以及 DZSY21 在玉米叶片上的定殖能力。这项工作为利用 DZSY21 替代杀虫剂和补充剂提供了理论依据。

Materials and Methods 材料与方法

Endophytic Bacterial Strain and Phytopathogen
内生细菌菌株和植物病原体

The leaves of E. ulmoides oliv., which were collected at Anhui Agricultural University (31°86′ N and 117°25′ E) in the Anhui Province of China, were firstly washed in running water, and dipped in 70% ethanol for 1 min and then treated with 1% sodium hypochlorite for 10 min. The samples were then washed several times with sterilized distilled water, the final wash was spread plated onto nutrient agar plate (g/L; peptone 5, beef extract 2, yeast extract 3, sodium chloride 5 and agar 18, pH 7.0) and cultivated at 28°C for 3 days as a sterility check. Samples were discarded if the growth was detected in the sterility check samples after 3 days (Tiwari et al., 2010; Sun et al., 2013).
在中国安徽省安徽农业大学（北纬31°86′，东经117°25′）采集的橄榄叶（E. ulmoides oliv.），先用流动水冲洗，再用70%乙醇浸泡1分钟，然后用1%次氯酸钠处理10分钟。然后用灭菌蒸馏水多次洗涤样品，最后将洗涤液平铺到营养琼脂平板（克/升；蛋白胨 5、牛肉浸膏 2、酵母浸膏 3、氯化钠 5 和琼脂 18，pH 值 7.0）上，在 28°C 下培养 3 天，作为无菌检查。如果 3 天后在无菌检查样本中检测到生长，则丢弃样本（Tiwari 等人，2010 年；Sun 等人，2013 年）。

For the isolation of endophytic bacterium DZSY21, 1 g of leaf tissues were fully grinded to homogenate in 9 ml sterilized distilled water in a mortar, 100 μL of the extract was taken and serial diluted up to 10^-3 dilution. Then, 100 μL was plated onto nutrient agar plate with three replications. The plates were incubated at 28–30°C for 48–72 h. Each bacterium, as evident from their colony morphology was transferred to fresh nutrient agar medium plates to establish pure culture of endophytic bacterium. Lastly, endophytic bacterium DZSY21 was obtained and identified as Bacillus subtilis, the sequence data has been submitted to GenBank (accession No. KP777560). The strain was grown at 30°C for 24 h in beef-protein medium (beef extract 3 g/L, peptone 10 g/L, NaCl 5 g/L, pH 7.0–7.2). The cell density was adjusted to approximately 10⁸ CFU/mL in sterile distilled water for use.
为了分离内生细菌 DZSY21，用研钵将 1 克叶片组织在 9 毫升灭菌蒸馏水中充分研磨匀浆，取 100 μL 浸提液，稀释至 10 ^-3 稀释度。然后，将 100 μL 培养到营养琼脂平板上，三次重复。将菌落形态明显的每种细菌转移到新鲜的营养琼脂培养基平板上，以建立内生细菌的纯培养。最后，内生细菌 DZSY21 被鉴定为枯草芽孢杆菌，其序列数据已提交至 GenBank（登录号：KP777560）。该菌株在 30°C 的牛肉蛋白培养基（牛肉提取物 3 克/升，蛋白胨 10 克/升，氯化钠 5 克/升，pH 值 7.0-7.2）中生长 24 小时。细胞密度在无菌蒸馏水中调节到约 10 ⁸ CFU/mL 以备使用。

The target phytopathogenic fungal strain, B. maydis, was obtained from the School of Plant Protection at Anhui Agricultural University. The strain was maintained on potato dextrose agar medium (potato 200 g/L, dextrose 20 g/L, agar 15 g/L). Conidia of B. maydis was induced on niblet culture (niblet 80 g, H₂O 10 mL) incubated at 28°C for 10 days under a 12-h of light/dark cycle. The cultures were washed in sterile water and the mycelia were filtered through four layers of gauze to obtain conidia suspension. The concentration of the conidia suspension was adjusted to approximately 10⁵ CFU/mL using a hemocytometer.
目标植物病原真菌菌株 B. maydis 从安徽农业大学植物保护学院获得。该菌株在马铃薯葡萄糖琼脂培养基（马铃薯 200 克/升，葡萄糖 20 克/升，琼脂 15 克/升）上培养。麦地那龙线虫的分生孢子被诱导在小麦培养基上（小麦 80 克，H ₂ O 10 毫升），在 28°C 下培养 10 天，光照/黑暗周期为 12 小时。用无菌水清洗培养物，用四层纱布过滤菌丝体，得到分生孢子悬浮液。用血细胞计数器将分生孢子悬浮液的浓度调整为约 10 ⁵ CFU/mL。

Extraction of Lipopeptides
提取脂肽

DZSY21 was grown in Landy medium (Glucose 20 g/L, L-glutamic acid 5 g/L, MgSO₄ 0.5 g/L, KCl 0.5 g/L, KH₂PO₄ 1 g/L, FeSO₄⋅6H₂O 0.15 mg/L, MnSO₄ 5.0 mg/L, CuSO₄⋅5H₂O 0.16 mg/L, pH 7.0) at 30°C and 180 rpm in a shaker for 38 h. After centrifugation (5000 rpm, 15 min), and cell-free supernatants (adjust to pH 2.0 by with 6 M HCl) were incubated overnight at 4°C. The acid precipitate was collected by centrifugation (8000 rpm for 10 min) and extracted twice with methanol. The methanol extracts of acid precipitate were lipopeptides and the pH was adjusted to 7.0 with NaOH (2 M). The lipopeptides were concentrated using a vacuum freeze drier, and the dried material was dissolved in a suitable volume of methanol for further analysis.
DZSY21 在 Landy 培养基（葡萄糖 20 g/L，L-谷氨酸 5 g/L，MgSO ₄ 0.5 g/L，KCl 0.5 g/L，KH ₂ PO ₄ 1 g/L，FeSO ₄ ⋅6H ₂ O 0.15 mg/L，MnSO ₄ 5.离心（5000 转/分，15 分钟）后，将无细胞上清液（用 6 M HCl 调至 pH 值 2.0）在 4°C 下培养过夜。通过离心（8000 rpm，10 分钟）收集酸沉淀，并用甲醇提取两次。酸沉淀的甲醇提取物为脂肽，用 NaOH（2 M）将 pH 值调至 7.0。使用真空冷冻干燥机浓缩脂肽，并将干燥的材料溶解在适当体积的甲醇中，以作进一步分析。

Antifungal Assays of the Strain DZSY21 and its Lipopeptides
菌株 DZSY21 及其脂肽的抗真菌试验

The inhibitory effect of endophytic bacterium DZSY21 on the growth of B. maydis was evaluated by the plate dual-culture method (Kunova et al., 2016). Firstly, a 5-mm mycelium disk cut from a 5-day-old culture of B. maydis was placed on the center of Petri dish, and cultivated for 3 days in advance, then strain DZSY21 from 2-day-old culture was streaked across approximately 2.5 cm away from the disk in the center of the plates. Water was used as negative control. The plates were continued to incubate at 28°C for 4 days. The inhibitory activity of treatment was carried out using the following formula, where DC = radius of control, and DT = radius of fungal colony with treatment. The experiments were repeated in triplicate and the data presented here were the averages of three experiments.
采用平板双培养法（Kunova et al.）首先，将从培养 5 天的 B. maydis 菌丝体上切下的 5 毫米菌丝盘放在培养皿中央，并提前培养 3 天，然后将培养 2 天的菌株 DZSY21 从培养皿中央距菌丝盘约 2.5 厘米处横穿培养皿。水作为阴性对照。平板继续在 28°C 下培养 4 天。处理的抑制活性按下式计算：DC = 对照组的半径，DT = 处理后真菌菌落的半径。实验一式三份，此处给出的数据为三次实验的平均值。

$$\text{Growth inhibition}(\%) = \frac{DC-DT}{DC} \times 100\%$$

The antifungal activity of lipopeptides was evaluated by disk diffusion assay (Bauer et al., 1966). Firstly, the disk of B. maydis was placed on the center of Petri dish and incubated for 3 days in advance, then filter paper disks (5 mm) were switched with 300 μg of lipopeptides and placed in approximately 2.5 cm away from the disk, a methanol switched disk was used as the control. The plates were continued to incubate at 28°C for 4 days. Antifungal activity was determined by observing the inhibition of fungal growth around the disk. Then the fungal mycelia treated with the DZSY21 and its lipopeptides was examined under a microscope.
脂肽的抗真菌活性通过盘扩散试验进行评估（Bauer 等人，1966 年）。首先，将 B. maydis 的圆盘放在培养皿中心并提前培养 3 天，然后将滤纸圆盘（5 毫米）与 300 μg 的脂肽调换，并放在离圆盘约 2.5 厘米处，用甲醇调换的圆盘作为对照。平板继续在 28°C 下培养 4 天。通过观察盘周围真菌生长的抑制情况来确定抗真菌活性。然后在显微镜下观察经 DZSY21 及其脂肽处理的真菌菌丝。

Colonization Studies on Maize Leaves
玉米叶上的定殖研究

To study the ability of DZSY21 to colonize maize leaves, the Bacillus subtilis DZSY21 was labeled by antibiotic marker (Chen et al., 1995; Nautiyal et al., 2002; Bennett et al., 2003; Wang et al., 2010a). Firstly, the sensitive concentrations of the strain DZSY21 to antibiotics were analyzed, and sensitive concentrations of the strain DZSY21 to kanamycin and chloramphenicol were 7.5 and 1 μg/mL, respectively. On that basis, the DZSY21^Kan was obtained by transferring colonies to LB medium agar plates containing increasing concentrations of kanamycin (Serva; 5, 10, 15, 20, 25, 50, 100, 150, and 200 μg/mL), then the DZSY21^Kan was transferred to LB medium agar plates containing increasing concentrations of chloramphenicol (0.5, 1, 5, 10, and 15 μg/mL) and fixed concentration of kanamycin (200 μg/mL), lastly, the double-resistance strain DZSY21^Kan,chl was obtained. The stability and antagonistic effect of the DZSY21^Kan,chl were tested by 20 sub-cultures on LB agar with kanamycin (200 μg/mL) and chloramphenicol (15 μg/mL), incubating for 48 h at 30°C, the stability of the DZSY21^Kan,chl was determined by comparing the number of CFUs after the last subculture, the antagonistic effect was evaluated through the plate confrontation method. And the DZSY21^Kan,chl was characterized by amplification and sequencing of a partial sequence of the 16S rDNA gene. The DZSY21^Kan,chl were stored at 4°C on LB with kanamycin (200 μg/mL) and chloramphenicol (15 μg/mL).
为了研究 DZSY21 在玉米叶片上的定殖能力，对枯草芽孢杆菌 DZSY21 进行了抗生素标记（Chen 等，1995；Nautiyal 等，2002；Bennett 等，2003；Wang 等，2010a）。首先，分析了菌株 DZSY21 对抗生素的敏感浓度，发现菌株 DZSY21 对卡那霉素和氯霉素的敏感浓度分别为 7.5 和 1 μg/mL。在此基础上，将菌落转移到含有递增浓度卡那霉素（Serva；5、10、15、20、25、50、100、150 和 200 μg/mL）的 LB 培养基琼脂平板上，得到 DZSY21 ^Kan ，然后将 DZSY21 ^Kan 转移到含有递增浓度氯霉素（0.5、1、5、10、15 μg/mL）和固定浓度的卡那霉素（200 μg/mL），最后得到双抗菌株DZSY21 ^Kan,chl 。在LB琼脂上用卡那霉素（200 μg/mL）和氯霉素（15 μg/mL）进行20次亚培养，在30℃下培养48 h，检测DZSY21 ^Kan,chl 的稳定性和拮抗作用，通过比较最后一次亚培养后的CFU数量确定DZSY21 ^Kan,chl 的稳定性，通过平板对抗法评估其拮抗作用。而 DZSY21 ^Kan,chl 的特征则是通过 16S rDNA 基因部分序列的扩增和测序来确定的。将 DZSY21 ^Kan,chl 在 4°C 下保存在含有卡那霉素（200 μg/mL）和氯霉素（15 μg/mL）的 LB 中。

To determine whether the strain could colonize plant leaves, maize leaves inoculated with the suspensions (1 × 10⁸ CFU/mL) of the double-resistance strain DZSY21^Kan,chl (50 mL per plant) and incubated for 24 h, maize leaves treated with sterile water were used as a control (50 mL per plant). The samples were collected at 24 h post-inoculation, they were treated and observed by transmission electron microscopy at biotechnology center of Anhui Agricultural University.
为了确定该菌株是否能在植物叶片上定植，将双抗菌株DZSY21 ^Kan,chl 的悬浮液（1 × 10 ⁸ CFU/mL）接种玉米叶片（每株50 mL），培养24 h后，用无菌水处理玉米叶片作为对照（每株50 mL）。接种后 24 小时采集样品，处理后在安徽农业大学生物技术中心进行透射电镜观察。

To analyze the population density of the strain in maize leaves, at the pumping stage of maize plant, suspensions of the double-resistance strain DZSY21^Kan,chl were adjusted to 10⁸ CFU/mL in sterile distilled water, and 50 mL/plant was applied to the maize leaves in each plant. Maize leaves treated with sterile water were used as a control (50 mL per plant). Leaves were harvested at 1, 3, 5, 7, 10, 15, 20, 25, and 30 days after inoculation, and the population of the DZSY21^Kan,chl inside the mesophyll tissue was estimated. The maize leaves were weighed, cut into small pieces, surface sterilized with 0.1% corrosive sublimate for 1 min, treated with 1% sodium hypochlorite for 10 min, and washed several times with sterilized distilled water. The sterilized plant material was trimmed, ground, and diluted with phosphate buffer saline (PBS) (sodium chloride 8 g/L, potassium chloride 0.2 g/L, disodium hydrogen phosphate 1.44 g/L, and potassium dihydrogen phosphate 0.24 g/L; pH 7.4) up to a dilution factor of 10^-3. 100 μL of each dilution was plated on LB medium containing kanamycin (200 μg/mL) and chloramphenicol (15 μg/mL). The plates were counted after 48 h incubation at 30°C. This experiment was conducted in duplicate.
为了分析菌株在玉米叶片中的种群密度，在玉米植株的抽雄期，将双抗菌株 DZSY21 ^Kan,chl 的悬浮液在无菌蒸馏水中调至 10 ⁸ CFU/mL，然后将 50 mL/ 株施用于每株植株的玉米叶片。用无菌水处理的玉米叶片作为对照（每株 50 mL）。在接种后 1、3、5、7、10、15、20、25 和 30 天收获叶片，并估算叶肉组织内 DZSY21 ^Kan,chl 的数量。称取玉米叶片，切成小块，用 0.1% 腐蚀性亚硝酸盐表面灭菌 1 分钟，再用 1% 次氯酸钠处理 10 分钟，然后用灭菌蒸馏水清洗数次。将灭菌后的植物材料修剪、研磨，并用磷酸盐缓冲盐水（PBS）（氯化钠 8 g/L、氯化钾 0.2 g/L、磷酸氢二钠 1.将每个稀释液 100 μL 培养在含有卡那霉素（200 μg/mL）和氯霉素（15 μg/mL）的 LB 培养基上。）在 30°C 孵育 48 小时后对平板进行计数。本实验重复进行。

Biocontrol Assays 生物控制试验

Plot experiments were carried out in the teaching practice base of Anhui Agricultural University in July 2015. Maize seedlings (Chang 7-2, F1) were grown in the soil with planting depth of 10 cm. The soil was tilled twice before pre-sterilized (3% sodium hypochlorite solution) maize seeds were planted. At the pumping stage, the maize plants were divided into four plots, and 40 plants were present in one plot. The efficacy of DZSY21 in suppressing southern corn leaf blight was evaluated by applying lipopeptides (1 mg/mL) and DZSY21 suspensions (1 × 10⁸ CFU/mL) by spraying the leaves of the maize plants (50 mL per plant). Sterile water and 50% carbendazim wettable powder 600 times liquid were used as the negative and positive controls, respectively. The conidial suspension of B. maydis (1 × 10⁵ CFU/mL) was applied to the leaves via the same mechanism 24 h after application of the treatment; the plants were then moistened with a humidifier for 12 h. Each treatment was replicated 40 times and the experiment was repeated three times. The disease severity was recorded 4, 6, 8, 10, and 15 days after challenge with the pathogen according to a rating scale of 1–9 scales with 1 being the most resistant and 9 being dead (Balint-Kurti et al., 2007), and the Disease index and disease reduction were calculated according to the formulas below.
2015 年 7 月在安徽农业大学教学实习基地进行了小区试验。玉米幼苗（长 7-2，F1）生长在种植深度为 10 厘米的土壤中。在播种预先消毒（3%次氯酸钠溶液）的玉米种子前，土壤翻耕两次。在抽穗期，玉米植株被分成四个小区，每个小区 40 株。通过向玉米植株叶片喷洒脂肽（1 mg/mL）和 DZSY21 悬浮液（1 × 10 ⁸ CFU/mL）（每株 50 mL），评估了 DZSY21 抑制南方玉米叶枯病的功效。无菌水和 50% 多菌灵可湿性粉剂 600 倍液分别作为阴性和阳性对照。在施用处理剂 24 小时后，通过相同的机制将麦地那龙线虫分生孢子悬浮液（1 × 10 ⁵ CFU/mL）施用到叶片上；然后用加湿器湿润植株 12 小时。每个处理重复 40 次，实验重复三次。病原体侵染后 4、6、8、10 和 15 天，根据 1-9 级评分标准记录病害严重程度，1 级为最抗病，9 级为死亡（Balint-Kurti 等人，2007 年），并根据以下公式计算病害指数和病害减轻程度。

$$\begin{array}{c}\text{Disease index} = \frac{\Sigma ({\text{d}}_{\text{i}}\times {\text{l}}_{\text{i}})}{\text{L}\times \text{N}} \times 100\\ \text{Disease reduction} = ({\text{I}}_{\text{0}}-{\text{I}}_{\text{i}}) \times 100\%\end{array}$$

where, d_I = represents for the grade of disease severity, l_I = the number of leaves at different grades of disease, L = the number of all investigated leaves, N = the highest grade of disease severity, I₀ = the disease index of control, and I_I = the disease index of different treatment groups. As a control, sterile water was spread on leaves.
其中，d = 病害严重程度等级，l = 不同病害等级的叶片数，L = 所有调查叶片数，N = 最高病害等级，I ₀ = 对照组的病害指数，I = 不同处理组的病害指数。作为对照，在叶片上涂抹无菌水。

Molecular Mass of DZSY21 Lipopeptides Determined by MALDI-TOF
通过 MALDI-TOF 测定 DZSY21 脂肽的分子质量

Lipopeptides were analyzed for surfactin, iturin, and fengycin using MALDI-TOF-MS. A sample (1 mg/mL) was diluted 10x with 100% methanol, and data was acquired in positive reflector mode from 800 to 4000 m/z. The analysis was performed at biotechnology center of Anhui Agricultural University.
利用 MALDI-TOF-MS 分析了脂肽中的表面活性素、伊图肽和芬吉肽。样品（1 mg/mL）用100%甲醇稀释10倍，在正反射模式下获得800-4000 m/z的数据。分析在安徽农业大学生物技术中心进行。

Study of Plant Defense Response
植物防御反应研究

The lower leaves (i.e., the first leaf to the fourth leaf) of axenic maize plants from nine different leaf periods were covered with DZSY21 (5 mL per leaf of 10⁸ CFU/mL) and cultivated in the greenhouse (30–35°C) under a 12-h-light and 12-h-dark interval. Control plants were covered with 5 mL (per leaf) of sterile water instead of the bacterial cell suspension. Then the bacterized and non- bacterized leaves from the upper part of the plants (i.e., the sixth leaf to the ninth leaf) were harvested at 12, 24, 36, 48, and 60 h for RNA extraction. Forty-five plants were present in each treatment, nine maize plants were harvested and divided into three replicates at different period, and each sample was mixtures of the upper leaves of the three maize plants.
用 DZSY21（每片叶 5 mL，10 ⁸ CFU/mL）覆盖九个不同叶期的腋生玉米植株的下部叶片（即第一片叶至第四片叶），并在温室（30-35°C）中以 12 小时光照和 12 小时黑暗间隔进行培养。对照植物用 5 mL（每片叶子）无菌水代替细菌细胞悬浮液。然后分别在 12、24、36、48 和 60 小时收获植株上部（即第六片叶至第九片叶）的带菌叶片和未带菌叶片，以提取 RNA。每个处理有 45 株玉米植株，在不同时期收获 9 株玉米植株并分成 3 个重复，每个样品是 3 株玉米植株上部叶片的混合物。

Total RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The DNase-treated RNA was reverse-transcribed using M-MLV reverse transcriptase (Invitrogen). The primer pairs used for the qRT-PCR analysis were designed according to parameters established for the Primer3Plus program (Untergasser et al., 2007; Klosterman et al., 2011) (Table 1). Expression of pathogenesis-related protein 1 (PR1), defensin (PDF1.2), lipoxygenase (LOX), and ethylene response factor (ERF) were monitored by qRT-PCR in plants in response to DZSY21 treatment. The candidate reference genes actin was identified as the most stable gene and was used as an endogenous control in qRT-PCR analysis. qRT-PCR was performed on an ABI 7300 Real-Time System (Applied Biosystems). Each reaction contained 12.5 μL of 2× SYBR Green Master Mix reagent (Applied Biosystems), 400 nM of gene-specific primers, and 1.5 μL of diluted cDNA sample (final volume 20 μL). The thermocycle conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60 °C for 60 s. After the PCR was complete, a melting curve was generated to analyze the specificity for each gene by increasing the temperature from 60 to 95°C. Three replicates were performed for each gene. Results are based on the average of triplicates, and the standard deviation of the mean is shown.
根据生产商的说明，使用 Trizol 试剂（Invitrogen 公司）提取总 RNA。经 DNase 处理的 RNA 使用 M-MLV 逆转录酶（Invitrogen 公司）进行逆转录。用于 qRT-PCR 分析的引物对是根据 Primer3Plus 程序设定的参数设计的（Untergasser 等人，2007 年；Klosterman 等人，2011 年）（表 1）。通过 qRT-PCR 技术监测了 DZSY21 处理后植株中病原相关蛋白 1（PR1）、防御素（PDF1.2）、脂氧合酶（LOX）和乙烯反应因子（ERF）的表达情况。候选参考基因肌动蛋白被确定为最稳定的基因，并在 qRT-PCR 分析中用作内源对照。qRT-PCR 在 ABI 7300 实时系统（Applied Biosystems）上进行。每个反应包含 12.5 μL 2× SYBR Green Master Mix 试剂（Applied Biosystems）、400 nM 基因特异性引物和 1.5 μL 稀释的 cDNA 样品（最终体积 20 μL）。热循环条件为PCR 完成后，将温度从 60°C 升至 95°C，生成熔解曲线以分析每个基因的特异性。每个基因进行三次重复。结果基于三重复的平均值，并显示平均值的标准偏差。

TABLE 1 表 1

TABLE 1. Primer sequences used for quantitative polymerase chain reactions.
表 1.用于定量聚合酶链反应的引物序列。

Statistical Analysis 统计分析

The data of all experiments was analyzed by analysis of variance (ANOVA). When ANOVA showed treatment effects (P < 0.05), the least significant difference test (LSD) and Duncan’s multiple range test (for maize) were applied to make comparisons among the means. The statistical package DPS ver. 9.50 was used for all analyses.
所有实验数据均通过方差分析（ANOVA）进行分析。当方差分析显示处理效应（P < 0.05）时，采用最小显著性差异检验（LSD）和邓肯多重范围检验（玉米）进行均值比较。所有分析均使用 DPS 9.50 版统计软件包。9.50 版的统计软件包进行分析。

Results 成果

Antifungal Activity of the Strain DZSY21 and Its Lipopeptides
菌株 DZSY21 及其脂肽的抗真菌活性

Lipopeptides are cyclic, low molecular weight antimicrobial compounds, which are mainly composed of a 7–10 amino acid hydrophilic head linked with a hydrophobic fatty acid tail (Cai et al., 2013). Bacillus species have strong antimicrobial properties and are known to produce a structurally diverse group of antimicrobial lipopeptides, including surfactin, iturin, and fengycin families (Wang et al., 2010b; Cai et al., 2013).
脂肽是一种环状、低分子量的抗菌化合物，主要由一个 7-10 个氨基酸的亲水性头部和一个疏水性脂肪酸尾部连接而成（Cai 等人，2013 年）。芽孢杆菌具有很强的抗菌特性，已知可产生一组结构多样的抗菌脂肽，包括表面活性素、iturin 和 fengycin 家族（Wang 等人，2010b；Cai 等人，2013）。

Screening of the endophytic strain DZSY21 for antifungal activity by the dual culture assay method showed antifungal activity by inhibition of fungal colony growth. After being cultured for 7 days, the diameter of the colony of the B. maydis treated with the strain DZSY21 was roughly 3 cm (Figure 1B), while the colony of B. maydis treated with water was basically full of Petri dish (Figure 1A). The strain DZSY21 exhibited antifungal activity against B. maydis with inhibition 61.70%.
通过双重培养检测法筛选内生菌株 DZSY21 的抗真菌活性，结果显示其具有抑制真菌菌落生长的抗真菌活性。培养 7 天后，用菌株 DZSY21 处理的麦地那龙线虫菌落直径约为 3 厘米（图 1B），而用水处理的麦地那龙线虫菌落基本布满培养皿（图 1A）。菌株 DZSY21 对麦地那龙线虫具有抗真菌活性，抑制率为 61.70%。

FIGURE 1 图 1

FIGURE 1. Antifungal activity of Bacillus subtilis DZSY21 and its lipopeptides: (A) the colony of Bipolaris maydis treated with water incubated for 7 days; (B) the colony of B. maydis treated with the strain DZSY21 incubated for 7 days; (C) filter paper disks (5 mm) were switched with 300 μg lipopeptides (a, filter paper disk switched with methanol; b, filter paper disk switched with lipopeptides); (D) microscopic images showing mycelium morphology of B. maydis treated with water; (E) microscopic images showing deformation in DZSY21 treated culture (c is the expansion and rupture of hyphae), and (F) microscopic images showing deformation in lipopeptides treated culture (d and e are the expansion and rupture of hyphae, respectively).
图 1.枯草芽孢杆菌 DZSY21 及其脂肽的抗真菌活性：（A）用清水处理的 Bipolaris maydis 菌落，培养 7 天；（B）用菌株 DZSY21 处理的 B. maydis 菌落，培养 7 天；（C）滤纸盘（5 毫米）换上 300 μg 脂肽。菌株 DZSY21 处理的菌落，培养 7 天；（C）滤纸盘（5 毫米）与 300 μg 脂肽对换（a，滤纸盘与甲醇对换；b，滤纸盘与脂肽对换）；（D）显微镜图像显示 B. maydis 菌丝形态；（E）用甲醇处理的菌落；（F）用 DZSY21 处理的菌落；（G）用甲醇处理的菌落；（H）用 DZSY21 处理的菌落；（G）用 DZSY21 处理的菌落。(D) 显微图像显示用水处理的 B. maydis 菌丝形态；(E) 显微图像显示经 DZSY21 处理的培养物的变形（c 为菌丝的扩张和破裂），(F) 显微图像显示经脂肽处理的培养物的变形（d 和 e 分别为菌丝的扩张和破裂）。

The isolated endophytic strain DZSY21 was grown in Landy medium for 38 h in order to induce secretion of antifungal lipopeptides. Cell-free supernatant was collected by centrifugation (5000 rpm, 15 min) at 4°C, the acid precipitate was obtained from cell-free supernatant by adding concentrated HCl to reduce pH at 2 and incubated overnight at 4°C, the methanol extracts of acid precipitate were lipopeptides. The lipopeptides were assayed against the fungal pathogen B. maydis by disk diffusion assay. After being cultured for 7 days, the lipopeptides from DZSY21 exhibited antifungal activity against B. maydis, there was an obvious inhibition belt between the fungal colony and the disk with lipopeptides (Figure 1C-b), while the disk switched methanol was covered with hyphae of B. maydis (Figure 1C-a), the disk switched methanol had not antifungal activity against B. maydis. Microscopic examination of affected mycelia showed the DZSY21 could cause the mycelium inflation (Figure 1E-c), and some mycelia treated with lipopeptides were swelled, contorted (Figure 1F-d) and broken (Figure 1F-e). Meanwhile, the mycelium in the control plates (i.e., only treated with methanol) were smooth, vimineous, and evenly grown (Figure 1D).
将分离出的内生菌株 DZSY21 在 Landy 培养基中培养 38 小时，以诱导其分泌抗真菌脂肽。在 4°C 下离心（5000 转/分，15 分钟）收集无细胞上清液，加入浓盐酸使 pH 值降至 2，并在 4°C 下培养过夜，从无细胞上清液中获得酸沉淀，酸沉淀的甲醇提取物即为脂肽。脂肽对真菌病原体 B. maydis 的抗性通过盘扩散试验进行检测。培养 7 天后，DZSY21 的脂肽对麦地那龙线虫具有抗真菌活性，真菌菌落与含有脂肽的盘之间有明显的抑制带（图 1C-b），而调换甲醇的盘上布满了麦地那龙线虫的菌丝（图 1C-a），调换甲醇的盘对麦地那龙线虫没有抗真菌活性。显微镜下观察受影响的菌丝体，发现 DZSY21 能使菌丝膨胀（图 1E-c），一些用脂肽处理的菌丝体膨胀、扭曲（图 1F-d）和断裂（图 1F-e）。而对照平板（即仅用甲醇处理的平板）中的菌丝则光滑、细长、生长均匀（图 1d）。

Ability of DZSY21 to Colonize Maize Leaves
DZSY21 在玉米叶片上定植的能力

To investigate colonization ability of the endophytic strain DZSY21 in maize leaves, DZSY21 was tagged with kanamycin and chloramphenicol. The double-resistance strain DZSY21^Kan,chl was selected on LB agar with kanamycin (200 μg/mL) and chloramphenicol (15 μg/mL). After approximately 20 generations of growth in the antibiotic medium with kanamycin (200 μg/mL) and chloramphenicol (15 μg/mL), the stability of the DZSY21^Kan,chl were evaluated by comparing the number of CFUs after the last subculture, and analyzing the characteristics of colony of DZSY21^Kan,chl and DZSY21. The results indicated the colonies of the DZSY21^Kan,chl (Figure 2B) and the strain DZSY21 (Figure 2A) were all smooth, moist and milky white, and the number of CFUs of the DZSY21^Kan,chl in different culture generation had no obvious difference. Additionally, the diameter of colony of B. maydis treated with the strain DZSY21^Kan,chl (Figure 2D) was similar to that of the DZSY21 (Figure 1B), while the colony of B. maydis treated with water was basically full of Petri dish(Figure 2C). The antifungal activity of DZSY21^Kan,chl was stable with 67.00% inhibition, compared to 61.85% for the wild-type strain DZSY21. And the DZSY21^Kan,chl was also identified as Bacillus subtilis. Therefore, the double-resistance strain DZSY21^Kan,chl was regarded as the mutant of DZSY21, the colonization ability of the DZSY21 in maize leaves was clarified through utilizing strain DZSY21^Kan,chl.
为了研究内生菌株 DZSY21 在玉米叶片中的定殖能力，用卡那霉素和氯霉素标记了 DZSY21。在含有卡那霉素（200 μg/mL）和氯霉素（15 μg/mL）的 LB 琼脂上筛选出了双重抗性菌株 DZSY21 ^Kan,chl 。在含有卡那霉素（200 μg/mL）和氯霉素（15 μg/mL）的抗生素培养基中生长约 20 代后，通过比较最后一次亚培养后的 CFU 数量，并分析 DZSY21 ^Kan,chl 和 DZSY21 的菌落特征，评估了 DZSY21 ^Kan,chl 的稳定性。结果表明，DZSY21 ^Kan,chl （图 2B）和菌株 DZSY21（图 2A）的菌落均为光滑、湿润的乳白色，DZSY21 ^Kan,chl 在不同培养代的 CFU 数量无明显差异。此外，用菌株DZSY21 ^Kan,chl 处理的麦地那龙线虫菌落直径（图2D）与DZSY21（图1B）相似，而用水处理的麦地那龙线虫菌落基本布满培养皿（图2C）。DZSY21 ^Kan,chl 的抗真菌活性稳定，抑制率为 67.00%，而野生型菌株 DZSY21 的抑制率为 61.85%。而 DZSY21 ^Kan,chl 也被鉴定为枯草芽孢杆菌。因此，双抗菌株 DZSY21 ^Kan,chl 被认为是 DZSY21 的突变体，通过利用菌株 DZSY21 ^Kan,chl 明确了 DZSY21 在玉米叶片中的定殖能力。

FIGURE 2 图 2

FIGURE 2. Morphology and antifungal activity of the strain DZSY21^Kan,chl: (A) the morphology of the strain DZSY21; (B) the morphology of the strain DZSY21^Kan,chl; (C) the colony of B. maydis treated with water incubated for 7 days; (D) the colony of B. maydis treated with strain DZSY21^Kan,chl incubated for 7 days.
图 2.菌株 DZSY21 ^Kan,chl 的形态和抗真菌活性：(A) 菌株 DZSY21 的形态；(B) 菌株 DZSY21 ^Kan,chl 的形态；(C) 用清水培养 7 天的 B. maydis 菌落；(D) 用菌株 DZSY21 ^Kan,chl 培养 7 天的 B. maydis 菌落。

Understand the shape of the DZSY21^Kan,chl by transmission electron microscopy was in favor of estimating whether the strain could colonize plant leaves. The DZSY21^Kan,chl was observed by transmission electron microscopy and presented rhabditiform and globosity (Figure 3B), the shape of the strain DZSY21^Kan,chl was the same as that of the wild-type DZSY21 (Figure 3A). Then maize leaves were inoculated with the suspensions (1 × 10⁸ CFU/mL) of the double-resistance strain DZSY21^Kan,chl (50 mL per plant), 24 h after inoculation with the DZSY21^Kan,chl, bacterial cells were found to be localized to the intercellular spaces of leaf tissues. Furthermore, colonization of the DZSY21^Kan,chl in maize leaves did not have any significant effects on the cell tissues of leaves, chloroplast and starch grains were normal, and the bacterial strain and maize leaves formed a harmonious endophytic relationship (Figures 3D,E). No bacteria were observed in the control plants (Figure 3C). Using the dilution plating method, the population density of the DZSY21^Kan,chl in maize leaves was detected, the population density reached 6.35 × 10³ CFU/g leaf tissue at 15 days post-inoculation, and remained above 2.17 × 10³ CFU/g leaf tissue until 30 days post-inoculation. No labeled strains could be isolated from the control plants during the course of the experiments (Figure 3F). Quantification studies revealed the maize leaves were successfully colonized by DZSY21.
通过透射电子显微镜了解 DZSY21 ^Kan,chl 的形状有利于估计该菌株是否能在植物叶片上定植。通过透射电镜观察，DZSY21 ^Kan,chl 呈横纹状和球状（图 3B），菌株 DZSY21 ^Kan,chl 的形状与野生型 DZSY21 相同（图 3A）。然后用双抗菌株 DZSY21 ^Kan,chl 的悬浮液（1 × 10 ⁸ CFU/mL）接种玉米叶片（每株 50 mL），接种 DZSY21 ^Kan,chl 24 h 后，发现细菌细胞定位于叶组织的细胞间隙。此外，DZSY21 ^Kan,chl 在玉米叶片中的定殖对叶片细胞组织没有明显影响，叶绿体和淀粉粒正常，细菌菌株与玉米叶片形成了和谐的内生关系（图 3D、E）。对照植株中未观察到细菌（图 3C）。使用稀释平板法检测了玉米叶片中 DZSY21 ^Kan,chl 的种群密度，接种后 15 天，种群密度达到 6.35 × 10 ³ CFU/克叶片组织，直到接种后 30 天，种群密度一直保持在 2.17 × 10 ³ CFU/克叶片组织以上。在实验过程中，无法从对照植株中分离到标记菌株（图 3F）。定量研究表明，玉米叶片被 DZSY21 成功定殖。

FIGURE 3 图 3

FIGURE 3. Development of the strain DZSY21^Kan,chl in the maize leaves 24 h post-inoculation: (A) the different shapes of the wild-type DZSY21 observed by transmission electron microscopy (a, the spherical DZSY21; b, the rod-shaped DZSY21); (B) the different shapes of DZSY21^Kan,chl observed by transmission electron microscopy(c, the spherical DZSY21^Kan,chl; d, the rod-shaped DZSY21^Kan,chl); (C) TEM photographs of maize leaf tissue treated with water; (D,E) TEM photographs of maize leaf tissue treated with the DZSY21^Kan,chl (Ch, chloroplast; N, Nuclear; Sg, Starch grain; e and f, the DZSY21^Kan,chl), and; (F) population dynamics of the DZSY21 mutant on maize leaf tissue.
图 3.菌株 DZSY21 ^Kan,chl 在接种后 24 小时在玉米叶片中的发育情况：(A) 透射电子显微镜观察到的野生型 DZSY21 的不同形状（a，球形 DZSY21；b，杆状 DZSY21）；(B) 透射电子显微镜观察到的 DZSY21 ^Kan,chl 的不同形状（c，球形 DZSY21 ^Kan,chl ；d，杆状 DZSY21 ^Kan,chl ）；(C）用水处理的玉米叶片组织的 TEM 照片；（D,E）用 DZSY21 ^Kan,chl 处理的玉米叶片组织的 TEM 照片（Ch，叶绿体；N，核；Sg，淀粉粒；e 和 f，DZSY21 ^Kan,chl ），以及；（F）DZSY21 突变体在玉米叶片组织上的种群动态。

Biocontrol on Southern Corn Leaf Blight with DZSY21
用 DZSY21 对南方玉米叶枯病进行生物防治

To verify if Bacillus subtilis DZSY21 was able to induce resistance in maize, which might be an indirect factor promoting maize growth, we sprayed the lipopeptides (1 mg/mL) and DZSY21 suspensions (1 × 10⁸ CFU/mL) on the leaves of the maize plants (50 mL per plant), and subsequently challenged leaves with the pathogen B. maydis, from the fourth day after the challenge, the symptoms of southern corn leaf blight appeared in all groups, with the prolonging of time of growth, there were obvious differences in the symptoms of maize leaves in different groups. Compared with the negative control (Figure 4A), the plants inoculated with lipopeptides (Figure 4B), DZSY21 suspensions (Figure 4C) and carbendazim wettable powder (Figure 4D) respectively could produce resistance phenotype characterized by the appearance of few small tan necrotic spots on the leaves.
为了验证枯草芽孢杆菌DZSY21是否能诱导玉米产生抗性，这可能是促进玉米生长的间接因素，我们将脂肽（1 mg/mL）和DZSY21悬浮液（1×10 ⁸ CFU/mL）喷洒在玉米植株叶片上（每株50 mL），然后用病原菌B.随着生长时间的延长，各组玉米叶片症状有明显差异。与阴性对照（图 4A）相比，分别接种脂肽（图 4B）、DZSY21 悬浮液（图 4C）和多菌灵可湿性粉剂（图 4D）的植株能产生抗性表型，表现为叶片上出现少量棕褐色坏死小斑点。

FIGURE 4 图 4

FIGURE 4. Induction of the systemic resistance in maize plants by Bacillus subtilis DZSY21: (A) plants drenched with a conidial suspension of B. maydis; (B) plants drenched with lipopeptides and then challenged with a conidial suspension of B. maydis; (C) plants drenched with suspensions of DZSY21 and then challenged with a conidial suspension of B. maydis; (D) plants drenched with carbendazim wettable powder and then challenged with a conidial suspension of B. maydis; (E) the graph of disease index of different groups, leaves incubated for 24 h with (a) water control, (b) lipopeptides, (c) suspension of endophytic strain DZSY21, and (d) 50% carbendazim wettable powder and then challenged with B. maydis. Disease index was calculated at different days after challenge with B. maydis. Data are expressed as the average of three replicates ± standard deviation.
图 4.枯草芽孢杆菌 DZSY21 对玉米植株系统抗性的诱导：（A）用 maydis 菌的分生孢子悬浮液浸泡植株；（B）用脂肽浸泡植株，然后用 maydis 菌的分生孢子悬浮液对植株进行侵染；（C）用 DZSY21 悬浮液浸泡植株，然后用 maydis 菌的分生孢子悬浮液对植株进行侵染；（D）用多菌灵可湿性粉剂浸泡植株，然后用 maydis 菌的分生孢子悬浮液对植株进行侵染；（E）用枯草芽孢杆菌 DZSY21 诱导玉米植株系统抗性的曲线图。(E)不同组的病害指数图，叶片经(a) 水对照、(b) 脂肽、(c) 内生菌株 DZSY21 悬浮液和 (d) 50%多菌灵可湿性粉剂培养 24 小时后，再受麦地那龙线虫分生孢子悬浮液的侵染。在麦地那龙线虫侵染后的不同天数计算病害指数。数据以三个重复的平均值 ± 标准偏差表示。

The disease index of the maize leaves was calculated 4, 6, 8, 10, and 15 days after challenge with the pathogen, and was used to detect the resistant response of different groups. The results showed the disease index of the leaves was reduced in all inoculated plants pretreated with the DZSY21, lipopeptides and carbendazim wettable powder as compared with the negative control, and the resistant responses of DZSY21 and its lipopeptides were always better at different period (Figure 4E). At 8 days after challenge with the pathogen B. maydis, the disease index of DZSY21 and its lipopeptides were 15.60 and 19.30, respectively (Figure 4E), at the same time, the disease index of 50% carbendazim wettable powder was 32.80 (Figure 4E). Pre-treatment with DZSY21 and its lipopeptides also retarded disease development. At the 15 day time point, the disease index of DZSY21 and its lipopeptides were 33.50 and 36.42, respectively, compared to 40.78 for 50% carbendazim wettable powder (Figure 4E).
在病原体侵染后 4、6、8、10 和 15 天计算玉米叶片的病害指数，用于检测不同组的抗性反应。结果表明，与阴性对照相比，所有用 DZSY21、脂肽和多菌灵可湿性粉剂预处理过的接种植株的叶片病害指数都有所降低，而且在不同时期，DZSY21 及其脂肽的抗性反应都较好（图 4E）。在病原菌 B. maydis 侵染后 8 天，DZSY21 及其脂肽的疾病指数分别为 15.60 和 19.30（图 4E），同时 50%多菌灵可湿性粉剂的疾病指数为 32.80（图 4E）。DZSY21 及其脂肽的预处理也能延缓病害的发展。在 15 天的时间点，DZSY21 及其脂肽的疾病指数分别为 33.50 和 36.42，而 50%多菌灵可湿性粉剂的疾病指数为 40.78（图 4E）。

The disease reduction of the DZSY21 was evaluated in suppressing southern corn leaf blight development through the disease index assessment measure. The disease reduction was shown in all inoculated plants as compared with the negative control (Table 2). The disease reduction by strains DZSY21 and lipopeptides were 60.41% and 51.02% in 8 days after challenged-inoculation with the pathogen (Table 2). Pre-treatment with DZSY21, lipopeptides and carbendazim wettable powder also resulted in a slower progression of disease development. By day 15, the disease reduction of DZSY21, lipopeptides and carbendazim wettable were 42. 24, 37.24, and 31.03%, respectively (Table 2). Apparently the best disease reduction was achieved in the treatment with DZSY21, which showed significantly greater disease suppression than the chemical control. The results suggest that the inhibitory effect of lipopeptides is not significantly different than that of DZSY21, indicating that lipopeptides produced by DZSY21 could be the primary mechanism of disease suppression.
通过病害指数评估指标，评价了 DZSY21 在抑制南方玉米叶枯病发展方面的病害减轻效果。与阴性对照相比，所有接种植株的病害都有所减轻（表 2）。在接种病原体 8 天后，菌株 DZSY21 和脂肽对病害的抑制率分别为 60.41% 和 51.02%（表 2）。使用 DZSY21、脂肽和多菌灵可湿性粉剂进行预处理后，病害发展速度也有所减缓。到第 15 天，DZSY21、脂肽和多菌灵可湿性粉剂对病害的抑制率分别为 42.24%、37.24%和 31.03%（表 2）。显然，用 DZSY21 处理的病害减少效果最好，其病害抑制效果明显高于化学对照。结果表明，脂肽的抑病效果与 DZSY21 的抑病效果无明显差异，说明 DZSY21 产生的脂肽可能是抑病的主要机制。

TABLE 2 表 2

TABLE 2. The disease reduction of southern corn leaf blight after leaves treatment with strain DZSY21.
表 2.用菌株 DZSY21 处理叶片后，南方玉米叶枯病的发病率降低情况。

Molecular Mass of DZSY21 Lipopeptides
DZSY21 脂肽的分子质量

Bacillus species could produce a structurally diverse group of antimicrobial lipopeptides, including surfactin, iturin, and fengycin families (Wang et al., 2010b; Cai et al., 2013). The lipopeptides of DZSY21 was further characterized by MALDI-TOF-MS analysis for molecular mass and determination of lipopeptide groups. The DZSY21 lipopeptides contain members of the antifungal surfactin A, surfactin B, and fengycin families. The molecular mass of fengycin in the range m/z 1449.8–1491.8 was similar to previous published molecular mass (Pathak et al., 2012) (Figure 5A), mass spectra of fengycin including m/z 1449.8, 1463.8, 1477.8, and 1491.8 represented lipopeptide groups with different numbers of carbon atoms (m/z 14) in their fatty acid chains, and the compound at m/z 1449.8 represented a H adduct of fengycin (Figure 5A). The molecular mass of surfactin B was in the range of m/z 994.6–1032.6. These represented H, Na, and K adducts of surfactin B, respectively (Figure 5B), and were similar to previous published molecular masses (Mikkola et al., 2004). The other surfactin A was also confirmed by mass spectra of m/z 1022.6–1060.6, the mass spectra of m/z 1022.6–1060.6 represented H, Na, and K adducts of surfactin A, respectively (Figure 5C), the mass agrees with previous studies (Luo et al., 2015; Jiang et al., 2016). And the MALDI-TOF-MS characterization did not show any peaks corresponding to iturin lipopeptide.
芽孢杆菌可产生结构多样的抗菌脂肽，包括表面活性素、伊曲肽和芬吉肽家族（Wang 等，2010b；Cai 等，2013）。通过 MALDI-TOF-MS 分析，对 DZSY21 脂肽的分子质量和脂肽基团进行了进一步表征。DZSY21 脂肽含有抗真菌的表面活性素 A、表面活性素 B 和芬吉星家族成员。在 m/z 1449.8-1491.8 范围内的芬吉肽的分子质量与之前公布的分子质量相似（Pathak 等人，2012 年）（图 5A），包括 m/z1449.8、1463.8、1477.8 和 1491.8 在内的芬吉肽质谱代表了其脂肪酸链中具有不同碳原子数（m/z 14）的脂肽基团，而 m/z 1449.8 处的化合物代表了芬吉肽的 H 加合物（图 5A）。表面活性剂 B 的分子质量在 m/z 994.6-1032.6 之间。这分别代表表面活性素 B 的 H、Na 和 K 加合物（图 5B），与之前公布的分子质量相似（Mikkola 等人，2004 年）。另一种表面活性素 A 也得到了 m/z 1022.6-1060.6 的质谱证实，m/z 1022.6-1060.6 的质谱分别代表了表面活性素 A 的 H、Na 和 K 加合物（图 5C），其质量与之前的研究一致（Luo 等，2015；Jiang 等，2016）。而MALDI-TOF-MS表征未显示任何与伊图灵脂肽相对应的峰。

FIGURE 5 图 5

FIGURE 5. MALDI-TOF mass spectra of lipopeptides: (A) MALDI-TOF mass spectra of lipopeptides for the presence of antibiotic groups fengycin; (B) MALDI-TOF mass spectra of lipopeptides for the presence of antibiotic groups surfactin B; (C) MALDI-TOF mass spectra of lipopeptides for the presence of antibiotic groups surfactin A.
图 5.脂肽的 MALDI-TOF 质谱：（A）抗生素基团芬吉星存在时脂肽的 MALDI-TOF 质谱；（B）抗生素基团表面活性素 B 存在时脂肽的 MALDI-TOF 质谱；（C）抗生素基团表面活性素 A 存在时脂肽的 MALDI-TOF 质谱。

DZSY21 Mediates the Defense Response in Maize Plants
DZSY21 介导玉米植株的防御反应

To determine the signaling pathways mediated by DZSY21, the expression of target plant genes known to function in the SA or JA/ET pathways were analyzed. Namely PR1 (an SA-responsive marker gene), defensin (PDF1.2) (JA/ET response marker gene), lipoxygenase (LOX) (a JA-responsive marker gene) and an ERF that could be expressed in the plant defense mechanism (Van Loon and Bakker, 2006; Pieterse et al., 2009) were used in this study. Maize leaves were harvested in the 12, 24, 36, 48, and 60 h separately after inoculation with DZSY21, and the changes in gene expression were analyzed by qRT-PCR.
为了确定 DZSY21 介导的信号通路，分析了已知在 SA 或 JA/ET 通路中起作用的目标植物基因的表达。本研究使用了 PR1（SA 响应标记基因）、防御素（PDF1.2）（JA/ET 响应标记基因）、脂氧合酶（LOX）（JA 响应标记基因）和可在植物防御机制中表达的 ERF（Van Loon 和 Bakker，2006 年；Pieterse 等人，2009 年）。分别在接种 DZSY21 后的 12、24、36、48 和 60 小时收获玉米叶片，并通过 qRT-PCR 分析基因表达的变化。

We observed that the expression of PR-1 was strongly induced in DZSY21- treated plants at levels 4.41- times in 24 h, compared to the control plants treated with water, then expression levels of PR-1 were gradually reduced in 36, 48, and 60 h and the PR-1 transcripts were induced to 2.74-, 2.25-, and 2.01-fold, respectively (Figure 6A). The LOX was gradually increased from 12 to 48 h after inoculation with DZSY21, and the expressions of LOX was strongly induced in DZSY21- treated plants with 4.49- times in 48 h, compared to the control plants (Figure 6C). The dynamic of PDF1.2 expression in DZSY21 – treated plants were similar in the expressions of LOX, the PDF1.2 transcripts were slightly induced to 1.72-, 1.38-, and 1.41-fold in 36, 48, and 60 h in DZSY21-treated plants, respectively (Figure 6B). Meanwhile, the expression of ERF in DZSY21-treated plants was lower than in the non-bacterized controls, and the higher expression of ERF was only 0.73 in 48 h after inoculation with DZSY21 (Figure 6D), indicating the expression was not enhanced in the presence of the DZSY21. These defense-related genes PR1, LOX, and PDF1.2 were concurrently expressed in the leaves of DZSY21-treated plants, suggesting simultaneous activation of the salicylic acid (SA) -and the jasmonic acid (JA) -dependent signaling pathways by DZSY21. There was no evidence of any necrotic lesions in treated plants.
我们观察到，与用水处理的对照植株相比，DZSY21-处理植株的 PR-1 表达在 24 h 内被强烈诱导了 4.41 倍，然后 PR-1 的表达水平在 36、48 和 60 h 内逐渐降低，PR-1 转录物被诱导的水平分别为 2.74、2.25 和 2.01 倍（图 6A）。接种 DZSY21 后 12~48 h，LOX 的表达量逐渐增加，48 h 内 DZSY21 处理植株的 LOX 表达量是对照植株的 4.49 倍（图 6C）。DZSY21处理植株中PDF1.2的表达动态与LOX的表达动态相似，DZSY21处理植株的PDF1.2转录本在36、48和60 h内分别被小幅诱导至1.72倍、1.38倍和1.41倍（图6B）。同时，ERF 在 DZSY21 处理植株中的表达量低于未分化对照，接种 DZSY21 后 48 h ERF 的高表达量仅为 0.73（图 6D），表明在 DZSY21 存在下ERF 的表达量并未增强。这些防御相关基因 PR1、LOX 和 PDF1.2 在 DZSY21 处理的植株叶片中同时表达，表明 DZSY21 同时激活了依赖于水杨酸（SA）和茉莉酸（JA）的信号通路。处理过的植株没有任何坏死病变的迹象。

FIGURE 6 图 6

FIGURE 6. Expression of the key genes involved in the salicylic acid (SA) or jasmonic acid (JA) and ethylene dependent defense signaling pathways in maize in different periods after inoculation. (A) Expression of pathogenesis related protein (PR1); (B) Expression of plant defensing factor (PDF 1.2); (C) Expression of lipoxigenase (LOX); (D) Expression of epidermal repair factor (ERF). The graph shows expression levels of defense marker genes after normalization to the control gene actin. Data are expressed as the average of three replicates ± standard deviation.
图 6 玉米接种后不同时期参与水杨酸（SA）或茉莉酸（JA）和乙烯依赖性防御信号通路的关键基因的表达。(A）致病相关蛋白（PR1）的表达；（B）植物防御因子（PDF 1.2）的表达；（C）脂氧化酶（LOX）的表达；（D）表皮修复因子（ERF）的表达。图中显示的是与对照基因肌动蛋白归一化后的防御标记基因表达水平。数据以三个重复的平均值±标准偏差表示。

Discussion 讨论

Eucommia ulmoides is a rare and precious plant, and it is not easily infected by plant diseases and insect pests, has a longlife time, and is used in Chinese traditional medicine. The endophytes isolated from E. ulmoides tissue could have useful functions, such as biocontrol and plant-growth-promoting activities and producing the same bioactive compounds like host plant (Chen et al., 2010; Liu et al., 2016). In this study, the biocontrol mechanisms of DZSY21 against southern corn leaf blight were evaluated. Our results could establish a framework for screening biocontrol strains and inform the design of appropriate protocols for using biocontrol strains.
杜仲是一种稀有的珍贵植物，不易受植物病虫害侵染，寿命长，是中药材。从杜仲组织中分离出的内生菌可能具有生物防治和促进植物生长等有用功能，并能产生与寄主植物相同的生物活性化合物（Chen 等，2010；Liu 等，2016）。本研究评估了 DZSY21 对南方玉米叶枯病的生物防治机制。我们的研究结果可为筛选生物防治菌株建立一个框架，并为设计使用生物防治菌株的适当方案提供参考。

The efficacy of endophytes as BCAs is dependent on many factors, including: host specificity, population dynamics, pattern of host colonization, and ability to move within host tissues (Backman et al., 1997). Endophytic bacteria need be grown robustly and a considerable population needs to be established in the internal plant tissues. DZSY21 isolated from E. ulmoides leaves were able to enter and colonize the internal leaves of maize, and were able to persist for 30 days. DZSY21 is also capable of colonizing other plants (i.e., lack of host specificity). It has been reported that colonization ability might be linked to certain factors, such as lipopolysaccharides, flagellas, and pili (Compant et al., 2010); further studies will be required to elucidate the mechanisms of colonization in DZSY21.
内生菌作为生物活性成分的功效取决于许多因素，包括：宿主特异性、种群动态、宿主定殖模式以及在宿主组织内移动的能力（Backman 等人，1997 年）。内生细菌需要生长旺盛，并且需要在植物内部组织中建立相当大的种群。从 E. ulmoides 叶片中分离出的 DZSY21 能够进入玉米内部叶片并定植，而且能够持续 30 天。DZSY21 还能在其他植物上定植（即缺乏宿主特异性）。据报道，定殖能力可能与某些因素有关，如脂多糖、鞭毛和纤毛（Compant 等人，2010 年）；要阐明 DZSY21 的定殖机制，还需要进一步的研究。

One of the most important modes of action for endophytic bacteria is antagonism mediated by different compounds with antifungal properties, especially the genus Bacillus. Lipopeptides, such as surfactin, bacillomycin, and fengycin, are major antimicrobial compounds secreted by Bacillus spp., and possess antifungal, antibacterial, immunosuppressive, antitumor, or other physiologically relevant bioactivities (Chen et al., 2008). In this study, the lipopeptides of DZSY21 belonging to Bacillus spp. showed antifungal activity and was highly effective in reducing disease index. The mycelium protoplasm of pathogen exposed to DZSY21 lipopeptides were deformed and contorted. The lipopeptides were characterized by MALDI-TOF analysis, and were found to contain members of the antifungal surfactin A, surfactin B, and fengycin families. Fengycin and surfactin are strong antifungal compounds secreted by Bacillus spp., which inhibit filamentous fungi by antagonizing sterols, phospholipids, and oleicacid in fungal membranes (Romero et al., 2007; Alvarez et al., 2012). Our results demonstrate that direct antifungal activity was the most dominant method of action of DZSY21 against southern corn leaf blight.
内生细菌最重要的作用方式之一是由具有抗真菌特性的不同化合物（尤其是芽孢杆菌属）介导的拮抗作用。脂肽，如表面活性素、杆菌霉素和芬吉星，是芽孢杆菌属分泌的主要抗菌化合物，具有抗真菌、抗细菌、免疫抑制、抗肿瘤或其他生理相关的生物活性（Chen 等，2008 年）。在这项研究中，属于芽孢杆菌属的 DZSY21 的脂肽显示出抗真菌活性，对降低疾病指数非常有效。接触到 DZSY21 脂肽的病原体菌丝原生质体发生变形和扭曲。通过 MALDI-TOF 分析对这些脂肽进行了表征，发现其中含有抗真菌的表面活性素 A、表面活性素 B 和芬吉星家族成员。芬奇霉素和表面活性素是芽孢杆菌属分泌的强抗真菌化合物，它们通过拮抗真菌膜上的固醇、磷脂和油酸来抑制丝状真菌（Romero 等人，2007 年；Alvarez 等人，2012 年）。我们的研究结果表明，直接抗真菌活性是 DZSY21 对南方玉米叶枯病最主要的作用方式。

In addition to competition and direct antagonism, endophytic bacteria could control disease through indirect mechanisms. This includes ISR in the host plant, which involves a enhanced capacity to mobilize cellular defense responses before or upon pathogen challenge (Maryline et al., 2007; Verhagen et al., 2010) and induction of stress-related genes expression (Verhagen et al., 2004). ISR has been observed in some PGPB (Pieterse et al., 2001; Pahm et al., 2007; van Loon et al., 2009; Liu et al., 2010). Bacterial-mediated ISR involves elicitation of the ISR pathway, generation and translocation of the ISR signal, and ISR signal transduction leading to ISR-related gene expression and resistance (Pieterse et al., 2001; Liu et al., 2010).
除了竞争和直接拮抗之外，内生细菌还可以通过间接机制控制疾病。这包括宿主植物的 ISR，其中包括在病原体挑战之前或之后增强调动细胞防御反应的能力（Maryline 等人，2007 年；Verhagen 等人，2010 年）以及诱导压力相关基因的表达（Verhagen 等人，2004 年）。在一些 PGPB 中观察到了 ISR（Pieterse 等人，2001 年；Pahm 等人，2007 年；van Loon 等人，2009 年；Liu 等人，2010 年）。细菌介导的 ISR 包括 ISR 途径的激发、ISR 信号的产生和转运，以及 ISR 信号转导导致 ISR 相关基因的表达和抗性（Pieterse 等人，2001 年；Liu 等人，2010 年）。

In maize, there is little information regarding induction of the ISR pathway using endophytic microbes. Only a few studies have been reported showing inducing expression of defense-related genes in maize elicited by beneficial bacteria or exogenous JA (Van Loon and Bakker, 2006). Here, we identified some characteristic genes in maize that are similar to known or deduced functions involved in the SA and JA/ET pathways (Klosterman et al., 2011); these genes are suspected to play a role in generating signals for the activation of certain defense responses and protecting plants from damage associated with defense response. We found that defense-related genes PR1 and LOX were highly expressed in the leaves of DZSY21-treated plants. However, the expression of ERF did not increase. Thus, LOX and PR1 are likely responsible for conferring resistance against B. maydis infection in maize. Previously, LOX expression was shown to be stimulated by JA. The application of SA has been shown to trigger the expression of the PR1 gene. ISR is generally independent of the SA signaling pathway and is not associated with major alterations in the expression of defense-related genes, but is rather associated with the priming of defenses (Verhagen et al., 2004; van Loon et al., 2009). It has been well-established that there is cross-talk between the SA- and JA/ET-dependent signaling pathways (Koornneef and Pieterse, 2008), the SA and JA/ET signaling pathways interact antagonistically stimulating either one leads to the suppression of the other (Koornneef and Pieterse, 2008). However, the results in this study indicate that LOX and PR1 are the defense-related genes responsible for conferring resistance on maize plants by both SA and JA pathways. Some studies have previously reported the induction of genes from both the SA and JA/ET pathways with endophytic microbes (Van Loon and Bakker, 2006), and Niu and associates found the Bacillus cereus AR156, a plant growth-promoting rhizobacterium, mediated ISR to P. syringae DC3000 in Arabidopsis through parallel activation of the SA- and JA/ET-signaling pathways, which leads to an additive effect on the level of induced disease resistance (Niu et al., 2011). Additionally, it has been reported that the Bacillus spp. elicit ISR in several plant species through enhanced peroxidise activity, increased production of chitinase isozymes and glucanase, and accumulation of SA (Kloepper et al., 2004). B. thuringiensis induced resistance to R. solanacearum in tomato plants through activation of the SA-dependent signaling pathway and suppression of the JA-dependent signaling pathway (Takahashi et al., 2014). Thus, some researchers have suggested that the specific ISR signal transduction pathway promoted by beneficial microbes is dependent on the strain, the host plant, and/or the pathogen.
在玉米中，有关利用内生微生物诱导 ISR 途径的信息很少。只有少数研究报告显示有益细菌或外源 JA 诱导了玉米防御相关基因的表达（Van Loon 和 Bakker，2006 年）。在此，我们在玉米中发现了一些特征基因，它们与已知或推断的参与 SA 和 JA/ET 途径的功能相似（Klosterman 等人，2011 年）；这些基因被怀疑在产生激活某些防御反应的信号以及保护植物免受与防御反应相关的损害方面发挥作用。我们发现，防御相关基因 PR1 和 LOX 在 DZSY21 处理过的植物叶片中高表达。然而，ERF 的表达量并没有增加。因此，LOX 和 PR1 可能是赋予玉米对 B. maydis 感染的抗性的原因。在此之前，LOX 的表达已被证明受到 JA 的刺激。研究表明，施用 SA 会触发 PR1 基因的表达。ISR 通常独立于 SA 信号通路，与防御相关基因表达的重大改变无关，而是与防御的启动有关（Verhagen 等人，2004 年；van Loon 等人，2009 年）。已经证实，依赖 SA 和 JA/ET 的信号通路之间存在交叉作用（Koornneef 和 Pieterse，2008 年），SA 和 JA/ET 信号通路之间存在拮抗作用，刺激其中一个会导致抑制另一个（Koornneef 和 Pieterse，2008 年）。然而，本研究的结果表明，LOX 和 PR1 是通过 SA 和 JA 途径赋予玉米植株抗性的防御相关基因。之前有研究报告称，内生微生物可诱导 SA 和 JA/ET 途径中的基因（Van Loon 和 Bakker，2006 年），Niu 及其同事发现，促进植物生长的根瘤菌枯草芽孢杆菌 AR156 通过平行激活 SA 和 JA/ET 信号途径，介导拟南芥对 P. syringae DC3000 的 ISR，从而对诱导的抗病性水平产生叠加效应（Niu et al、2011).此外，据报道，枯草芽孢杆菌通过增强过氧化物酶活性、增加几丁质酶同工酶和葡聚糖酶的产量以及积累 SA，诱导多种植物产生 ISR（Kloepper 等人，2004 年）。苏云金杆菌通过激活依赖 SA 的信号通路和抑制依赖 JA 的信号通路，诱导番茄植株对 R. solanacearum 产生抗性（Takahashi 等人，2014 年）。 因此，一些研究人员认为，有益微生物促进的特定 ISR 信号转导途径取决于菌株、寄主植物和/或病原体。

In summary, we show that the suppression of southern corn leaf blight in maize by DZSY21 is a result of direct antagonism of antifungal lipopeptides produced by the DZSY21, as well as indirect inhibition through ISR. SA- and JA-mediated signal pathways are involved in the induction of ISR. Identifying the mechanisms of different biocontrol agents is important because helps to establish a theoretical basis for the design and appropriate use of biocontrol strains.
总之，我们的研究表明，DZSY21 对玉米南方玉米叶枯病的抑制是 DZSY21 产生的抗真菌脂肽直接拮抗以及通过 ISR 间接抑制的结果。SA 和 JA 介导的信号途径参与了 ISR 的诱导。确定不同生物防治剂的机制非常重要，因为这有助于为设计和适当使用生物防治菌株奠定理论基础。

Author Contributions 作者供稿

These studies were designed by TD, HJ. BS and XC carried out the major experimental analyses and prepared all figures and tables. SG, QW, and DH complete experiment of plant disease culture inoculation. TD analyzed the data and drafted the manuscript. SX contributed to revisions of the manuscript. HJ assisted in explaining the results and revised the final version of the manuscript. All authors have read and approved the final manuscript.
这些研究由 TD 和 HJ 设计。BS 和 XC 进行了主要的实验分析，并绘制了所有图表。SG、QW和DH完成了植物病害培养接种实验。TD 分析数据并起草手稿。SX 参与了手稿的修改。HJ 协助解释结果并修改了手稿的最终版本。所有作者均已阅读并认可最终稿件。

Conflict of Interest Statement
利益冲突声明

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
作者声明，本研究在进行过程中不存在任何可能被视为潜在利益冲突的商业或经济关系。

Acknowledgment 鸣谢

This work was supported by the National Natural Science Foundation of China (31371980, 31301324).
这项工作得到了国家自然科学基金（31371980、31301324）的支持。

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