连作障碍导致作物产量和品质下降，制约作物集约化生产。我国是甘薯生产大国，有限的耕地限制了甘薯换地耕作，长期连作导致的甘薯产量下降、病害高发等连作障碍现象日益严重，但目前针对甘薯连作障碍的研究仍处于起步阶段。厘清甘薯连作障碍因子，并针对性地研发甘薯连作障碍的消减技术，有助于推动甘薯产业高质量发展。本研究以典型甘薯连作障碍农田为研究对象，从微生态角度解析甘薯连作的障碍因子，进而以导致连作障碍的致病菌为研究材料筛选出高效生防菌，并结合全基因组和多种生化分析明确生防菌的作用机制与抑菌物质，最后通过田间试验分析其应用潜力。主要研究结果如下：
1. 以典型甘薯连作障碍农田和非连作农田为研究对象，通过比较不同处理下根际微生物组、代谢组和土壤性质等的差异，联合分析连作甘薯根际微生态特征。发现连作甘薯根际微生物多样性下降，周刺座霉（Volutella）和Neoidriella等致病性真菌显著富集，芽孢杆菌（Bacillus）等有益菌丰度显著降低。真菌网络受连作影响程度较高，网络复杂度下降，关键微生物为周刺座霉属。微生物降解芳香族化合物功能下降，苯甲酸、生物素等潜在自毒物质在甘薯根际积累。连作后甘薯块根产量、土壤pH和速效钾（AK）含量显著降低，土壤微生物群落与pH、速效钾和铵态氮（NH4+-N）的相关性最高。其中，土壤pH和速效钾与芽孢杆菌均呈正相关，与部分植物病原真菌（周刺座霉和Neoidriella）呈负相关，而铵态氮与它们呈相反趋势。通过偏最小二乘路径模型（PLS-PM）发现，连作年限主要通过土壤真菌群落间接影响块根产量。以出现典型连作障碍的甘薯农田中发病甘薯植株及其根际土壤为研究材料，从中分离潜在致病真菌，结合致病性分析明确尖孢镰刀菌（Fusarium oxysporum）和腐皮镰孢菌（F. solani）是导致甘薯连作障碍的主要致病菌，为后续甘薯连作障碍的生物防治提供材料。
2. 以甘薯连作障碍主要致病菌为靶标病原菌，从抗根腐病的“济薯26”不同组织分离的内生菌中筛选生防菌，测试其抑菌谱，并基于基因组学探究其防病促生相关功能基因（簇）。发现贝莱斯芽孢杆菌（B. velezensis）S14对尖孢镰刀菌和腐皮镰孢菌的抑制率分别为76.9%和70.3%，而且对甘薯黑斑病、基腐病和炭腐病等常见病害致病菌的抑制率均在80.0%以上，对小麦纹枯病和赤霉病致病菌的抑制率分别为93.7%和81.0%，表明生防菌S14具有广谱抑菌效果。基于基因组学预测到生防菌S14具有Surfactin、Macrolactin H、Bacillaene、Fengycin、Difficidin、Bacillibactin和Bacilysin等脂肽和聚酮类抑菌物质的生物合成基因簇，还存在efp、cheC-D、flgB-G、fliA-T、epsA-O、kinA-E、srfAA-AD等与细菌定殖相关的基因，dhbF、phzF、treP、treRtrpA-E、aroB-C、aroH等与IAA和其他促进植物生长的物质合成相关的基因，以及与养分循环相关（pstA-C、narG-J）和诱导系统抗性的基因（alsD）。
3. 以生防菌S14为研究材料，分析其对病原菌菌丝形态和细胞活性的影响以明确其抑菌机制，结合质谱检测主要抑菌物质。发现生防菌S14产生的挥发性有机化合物（VOCs）和脂肽作用于病原菌菌丝细胞膜，使细胞膜完整性丧失，引起菌丝形态畸变，诱导菌丝细胞内产生大量活性氧，引起氧化应激，最终导致菌丝细胞死亡。结合顶空固相微萃取法和气相色谱仪-质谱仪（HS-SPME/GC-MS）分析出VOCs中存在具有抑菌活性的N-亚硝基甲乙胺和3-叔丁基-2,2,4,4-四甲基戊-3-醇。经液相色谱-串联质谱联用仪（LC-MS/MS）鉴定发现生防菌S14的抑菌脂肽中主要包括常见的丰原素（Fengycin）、伊枯草素（Iturin）和表面活性素（Surfactin）等环状脂肽，同时鉴定出线性Fengycin，其中m/z为1453的线性C14 Fengycin A此前未见报道。此外，无病原菌处理下，生防菌S14处理的甘薯生物量增加45.7%；镰刀菌处理下，生防菌S14的应用能够使甘薯生物量增加65.4%。生防菌还能够提高甘薯苯丙氨酸解氨酶（PAL）、多酚氧化酶（PPO）和过氧化物酶（POD）等防御酶活性，诱导甘薯产生抗病性。此外，以生防菌S14为试验材料，通过田间试验验证其应用潜力。发现生防菌能够促进甘薯生长，使单株块根产量提高41.9%，降低病害发生率。生防菌S14处理的甘薯根际富集芽孢杆菌等有益菌，降低周刺座霉和镰刀菌的丰度，致使真菌网络中周刺座霉不再成为关键微生物。
综上所述，本研究通过对典型甘薯连作障碍农田采样，分析周刺座霉属和尖孢镰刀菌是导致甘薯连作障碍的主要因子。从抗性品种中筛选的贝莱斯芽孢杆菌S14具有广谱抑菌活性，该菌株能够通过产生抑菌脂肽和VOCs导致病原真菌菌丝细胞死亡、抑制菌丝生长，通过溶解养分和产生生长素促进植物生长，通过诱导甘薯防御酶活性增强抗病能力。生防菌S14的田间应用能够有效提高甘薯块根产量，富集有益微生物、抑制周刺座霉和镰刀菌等致病菌，降低甘薯病害发生率，进而缓解甘薯连作障碍。研究结果为贝莱斯芽孢杆菌S14消减甘薯连作障碍提供了理论和实践依据。

Continuous cropping obstacles lead to a decline in crop yield and quality, restricting intensive crop production. China is a large sweetpotato producer, limited arable land restrictions on sweetpotato farming, long-term continuous cropping led to a decline in sweetpotato yield, high incidence of diseases and other continuous cropping obstacles phenomenon is becoming more and more obvious, but at present for sweetpotato continuous cropping obstacles is still insufficient research. To clarify the factors of sweetpotato continuous cropping factors, and to target the development of sweetpotato continuous cropping obstacles mitigation technology can help to promote the high-quality development of the sweetpotato industry. In this study, we took a typical sweetpotato continuous cropping obstacle farmland as the research object, analyzed the obstacle factors of continuous sweetpotato crop from the perspective of microecology, and then took the pathogenic microbe leading to continuous cropping obstacle as the research material, screened the biocontrol bacteria, and analyzed the mechanism of the action of the biocontrol bacteria and its inhibitory substances with the combination of the whole genome and a variety of equipments, and analyzed the potential of its application through the field trial of the biocontrol bacteria. The main research results are as follows:

1. Taking typical sweetpotato continuous cropping obstacle farmland and non-continuous cropping farmland as research objects, the microecological characteristics of continuous cropping sweetpotato rhizosphere were analyzed by comparing the differences in rhizosphere microbiomes, metabolomes and soil properties under different treatments. It was found that the rhizosphere microbial diversity declined after continuously sweetpotato cropped, with significant enrichment of pathogenic fungi such as Volutella and Neoidriella, and significant reduction in the abundance of beneficial bacteria such as Bacillus. The fungal network was affected by the higher degree of continuous cropping, and the network complexity was reduced, and the key microorganisms was Volutella. The function of microorganisms in degrading aromatic compounds decreased, and potential autotoxic substances such as benzoic acid and biotin accumulated in the rhizosphere of sweetpotato. Sweetpotato tuber yield, soil pH and available potassium (AK) content significantly decreased after continuously cropped, and soil microbial communities showed the highest correlation with pH, quick-acting potassium and ammonium nitrogen (NH₄⁺-N). Among them, soil pH and AK were positively correlated with Bacillus and negatively correlated with some phytopathogenic fungi (Volutella and Neoidriella), while NH₄⁺-N showed the opposite trend. Partial least squares path modeling (PLS-PM) revealed that the number of years of continuous cropping affected tuber yield indirectly mainly through the soil fungal community. Taking the morbid sweetpotato plants and their rhizosphere soils in typical sweetpotato continuous cropping obstacle farmland as research materials, we isolated potentially pathogenic fungi from them, and combined with the pathogenicity analysis, we clarified that Fusarium oxysporum and F. solani are the main pathogens leading to the continuous cropping obstacle of sweetpotato, so as to provide the materials for the subsequent biocontrol of the continuous cropping of sweetpotato.

2. Taking the main pathogens of sweetpotato continuous cropping obstacle as target pathogens, Bacillus velezensis S14 was screened from endophytes isolated from different tissues of root-rot-resistant sweetpotato, tested for its inhibitory spectrum, and investigated its disease-preventing and promoting related functional genes (clusters) based on genomics. It was found that B. velezensis S14 inhibited F. oxysporum and F. solani by 76.9% and 70.34%, respectively, and inhibited the pathogenic fungi of common diseases such as black rot, foot rot and charcoal rot of sweetpotato by more than 80.0%, and the inhibition of the pathogenic fungi of wheat blight and scab by 93.7% and 81.0%, respectively. This indicates that strain S14 has a broad-spectrum inhibitory effect. It was predicted that the biosynthetic gene clusters of Surfactin, Macrolactin H, Bacillaene, Fengycin, Difficidin, Bacillibactin and Bacilysin, and some genes associated with bacterial colonization, such as efp, cheC-D, flgB-G, fliA-T, epsA-O, kinA-E and srfAA-AD. Besides, there also have some genes associated with synthesis of IAA and other substance that promotes plant growth (dhbF, phzF, treP, treR, trpA-E, aroB-C, aroH), as well as genes associated with nutrient cycling (pstA-C, narG-J) and induction of systemic resistance (alsD).

3. Using biocontrol bacteria S14 as the research material, the mechanism of bacterial inhibition was clarified through the morphology and cellular activity of the pathogenic bacterial hyphae, and the main bacterial inhibitory substances were detected by mass spectrometry. It was found that the volatile organic compounds (VOCs) and lipopeptides produced by S14 act on the cell membrane of the pathogenic fungal mycelium, causing the loss of cell membrane integrity, resulting in the aberration of mycelium morphology, inducing the production of a large amount of reactive oxygen species in the mycelium cells, causing oxidative stress, and ultimately leading to the death of the mycelium cells. Ethanamine,N-methyl-N-nitroso and 3-Pentanol,3-(1,1-dimethylethyl)-2,2,4,4-tetramethyl with antifungal activity were analyzed in VOCs by combining headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that the inhibitory lipopeptides of strain S14 mainly included common cyclic lipopeptides such as Fengycin, Iturin, and Surfactin, and linear Fengycin was also identified, of which the linear C14 Fengycin A with an m/z of 1453 was found, which has not been previously reported. In addition, strain S14 treatment increased sweetpotato biomass by 45.7% under pathogen-free treatment, and increase sweetpotato biomass by 65.4% under Fusarium spp. treatment. The biocontrol bacteria were also able to increase the activities of sweetpotato defense enzymes such as PAL, PPO and POD, and induced sweetpotato to develop disease resistance. Furthermore, biocontrol bacteria S14 was used as experimental material, and its application potential was verified through field trials. It was found that the strain S14 could promote sweet potato growth, increase tuber yield by 41.9%, and reduce the incidence of diseases. The sweetpotato roots treated with strain S14 were enriched with beneficial bacteria, such as Bacillus, and the abundance of Volutella and Fusarium were reduced, so that Volutella was no longer a key microorganism in the fungal network.

In summary, in this study, through sampling typical sweetpotato cropping obstacle farmland, we analyzed that Volutella and Fusarium are the main factors leading to sweetpotato cropping obstacle, and the screening of B. velezensis from resistant varieties has broad-spectrum inhibitory activity, which can lead to the death of mycelial cells of the pathogenic fungi and inhibit the growth of mycelium through the production of bacterial inhibitory lipopeptides such as Surfactin, Iturin, and Fengycin, and VOCs, and the linear C14 Fengycin A at m/z 1453 was also identified among the bacterial lipopeptides. In addition to its antifungal effects, S14 was able to promote plant growth by solubilizing nutrients and producing growth hormone, as well as inducing the activity of sweetpotato defense enzymes. The field application of strain S14 could effectively increase the yield of sweetpotato tubers, enrich beneficial microbe, inhibit the pathogenic fungi such as Volutella and Fusarium, reduce the incidence of sweetpotato diseases, and then mitigation the sweetpotato continuous cropping obstacle. This study provides a theoretical and practical basis for the mitigation of sweetpotato continuous cropping obstacle by B. velezensis S14.

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