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Historical advancements in understanding bacteriocins produced by rhizobacteria for their application in agriculture 了解根瘤菌产生的细菌素在农业中应用的历史性进展
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-31 DOI: 10.1016/j.rhisph.2024.100908
Sonia Fischer , Viviana López-Ramírez , Jorge Asconapé

Abiotic stress, such as high temperatures, droughts and soil salinity, as well as biotic stress, such as pythopathogenic bacteria, are causing serious damage to crops and they result in significant economic losses. In addition, excessive application of agrochemicals has deteriorated productive agricultural land, contributed to spreading antimicrobial resistance genes among pathogenic microorganisms and caused damage to human health. Developing alternative strategies to the use of chemicals is an ongoing challenge for achieving a sustainable agriculture. In this context, biological products, including bacteriocins, enhance crop growth and health without harming the environment. Bacteriocins are proteinaceous compounds that exhibit high specificity and kill competitors closely related to the producing bacteria. They are secreted by both Gram-negative bacteria and Gram-positive bacteria, and they have been used to treat bacterial infections in humans and animals, and to preserve food. In recent years, studies that projected the use of bacteriocins in agriculture have increased due to their high biotechnological potential. These bacteriocins have been explored as plant biostimulants or as biocontrol agents, and provide an innovative solution to the problems in agriculture. In particular, tailocins have a great potential as antimicrobials because they are very stable, extremely specific, and efficient as killers; in fact, a single particle is enough to kill a susceptible cell.In this review, we examine the bacteriocins produced by rhizobacteria and their application for a sustainable agriculture, a topic that has not been addressed extensively yet. In addition, we discuss bacteriocin expression in plants and the study of bacteriocins through omics.

高温、干旱和土壤盐碱化等非生物胁迫以及热病原菌等生物胁迫对农作物造成了严重损害,并导致重大经济损失。此外,过量施用农用化学品使农业生产用地恶化,导致病原微生物的抗菌素抗性基因扩散,并对人类健康造成损害。要实现农业的可持续发展,就必须制定替代化学品使用的战略。在这种情况下,包括细菌素在内的生物制品可以在不损害环境的情况下促进作物生长和健康。细菌素是一种蛋白质化合物,具有高度特异性,能杀死与产生细菌密切相关的竞争者。它们由革兰氏阴性菌和革兰氏阳性菌分泌,已被用于治疗人类和动物的细菌感染以及保存食物。近年来,由于细菌素具有很高的生物技术潜力,预测细菌素在农业中的应用的研究越来越多。这些细菌素已被探索用作植物生物刺激剂或生物控制剂,为解决农业问题提供了创新方案。在这篇综述中,我们研究了根瘤菌产生的细菌素及其在可持续农业中的应用,这是一个尚未得到广泛研究的课题。此外,我们还讨论了细菌素在植物中的表达以及通过全息图学对细菌素的研究。
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引用次数: 0
Phytotoxicity of Dactylonectria spp. exudates on kiwifruit vine and profile of secondary metabolites for understanding their relationship with the host plant Dactylonectria spp.渗出物对猕猴桃藤的植物毒性以及次生代谢物概况,以了解它们与寄主植物的关系
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-27 DOI: 10.1016/j.rhisph.2024.100906
Luisa M. Manici , Francesco Caputo , Simona Luccioli , Alessandro Frattarelli , Emilia Caboni

Kiwifruit vine decline syndrome (KVDS), which is responsible for a progressive decline in yields in Italy, is linked to a non-specific reduction in root development, the biotic or abiotic causes of which are still unclear. Dactylonectria is one of the most common soil-borne fungi associated with kiwifruit vines, However, its role in KVDS has not yet been clarified; therefore, Dactylonectria functional relationship with kiwifruit vine at rhizosphere level was investigated focusing solely on its exudates. Six isolates, four from kiwifruit-bearing soils belonging to D. torresensis. D. ecuadoriensis, D. novozelandica and D. pauciseptata, and two D. torresensis from a virgin soil, was tested for phytotoxicity of exudates by growing kiwifruit in vitro plants on MS media amended at 50% with sterile fungal filtrates. Parallelly, low molecular weight (LMW) metabolic profile of fungal filtrates was evaluated with untargeted metabolomics approach using LC-MS/TOF. The fungal isolates from kiwifruit bearing soil were much richer in LMW metabolites than those from virgin soil. Non host specific mycotoxins such as Aflaxoxin B2, Alternariol and Alterlactone were found in these isolates along with a number of metabolites most of which were characterised by antimicrobial properties. D. torresensis isolates were clearly discriminated from the other three species based on metabolic profile. A negative Pearson correlation between the growth index of kiwifruit vitro-plants and LMW metabolite count per isolate (richness) showed that the latter were associated with phytotoxicity, but the degree of correlation (r = −0.639) indicated that they were not the only component responsible. The results suggest that kiwifruit vines may alter the composition of the Dactylonectria spp. community in the root zone, which in turn may directly or indirectly contribute to kiwifruit vine decline syndrome.

猕猴桃藤蔓衰退综合症(KVDS)是造成意大利猕猴桃产量逐渐下降的原因,它与非特异性的根系发育减少有关,其生物或非生物原因尚不清楚。Dactylonectria 是与猕猴桃藤相关的最常见土传真菌之一,但它在猕猴桃根瘤蚜中的作用尚未明确;因此,研究人员仅从其渗出物入手,调查了 Dactylonectria 与猕猴桃藤在根瘤层的功能关系。六种分离物中,四种来自猕猴桃种植土壤,分别属于 D. torresensis、D. ecuadoriensis、D.通过在用无菌真菌滤液添加 50% 的 MS 培养基上培养猕猴桃离体植株,测试了渗出物的植物毒性。同时,利用 LC-MS/TOF 非靶向代谢组学方法对真菌滤液的低分子量(LMW)代谢谱进行了评估。与原始土壤中的真菌相比,从猕猴桃生长土壤中分离出的真菌富含更多的低分子量代谢物。在这些分离物中发现了非宿主特异性真菌毒素,如 Aflaxoxin B2、Alternariol 和 Alterlactone,以及许多代谢物,其中大多数具有抗菌特性。根据代谢特征,D. torresensis 分离物明显有别于其他三个物种。猕猴桃试管植物的生长指数与每个分离物的低分子量代谢物数量(丰富度)之间的负皮尔逊相关性表明,后者与植物毒性有关,但相关程度(r = -0.639)表明,它们不是唯一的致病成分。结果表明,猕猴桃藤蔓可能会改变根区 Dactylonectria 属群落的组成,进而直接或间接导致猕猴桃藤蔓衰退综合症。
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引用次数: 0
Growth regulators on shooting and adventitious rooting of Mimosa caesalpiniifolia adult stem cuttings 生长调节剂对含羞草成茎扦插枝条生长和不定根的影响
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-24 DOI: 10.1016/j.rhisph.2024.100901
Deyvid Luis da Silva Sousa, Paulo César da Silva Santos, Moema Barbosa de Sousa, Erika Rayra Lima Nonato, Eliane Cristina Sampaio de Freitas, Ricardo Gallo

Mimosa caesalpiniifolia, crucial for fencing and firewood in Brazil's Caatinga dry forest, presents a promising avenue for commercial propagation. The challenges faced in seed propagation, such as the scarcity of inputs and the difficulties in overcoming dormancy, highlight the advantages of vegetative methods. These methods emerge as a viable alternative, enabling uniform and large-scale production of clonal plantlets. This technique ensures consistent plant quality, which is crucial given the wide variety of uses for this species. Additionally, the use of plant growth regulators is critical to enhance the success of vegetative propagation, promoting the formation of robust roots and overall development of plants from cuttings of adult trees. Recognizing the significance of creating clonal forests of M. caesalpiniifolia and the necessity for understanding methods to expedite this endeavor, this study was designed to assess the shooting and adventitious rooting of stem cuttings sourced from adult M. caesalpiniifolia trees, examining the effects of auxinic agents. M. caesalpiniifolia cuttings were harvested through vegetative rescue from six randomly selected adult trees, treated with indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) at concentrations of 0, 1,000, 2,000, and 4000 mg L−1, using a completely randomized design in a 2 × 4 factorial arrangement. The evaluations included assessing root presence, number of shoots, vigor of clonal plantlets and plant survival under greenhouse conditions, shade house conditions, and full sun exposure. The study did not reveal any significant interaction between growth regulators and their concentrations under these conditions. IBA at intermediate concentrations enhanced root growth, while cuttings without growth regulators exhibited superior aerial development. A decrease in survival percentages with increasing concentrations of regulators was observed, while cuttings without regulators demonstrated better morphological development. The study suggests for large-scale nurseries, natural rooting and shoot formation in M. caesalpiniifolia cuttings may suffice for successful propagation without IBA or IAA.

含羞草是巴西卡廷加旱林中重要的栅栏和木柴用材,是一种前景广阔的商业繁殖途径。种子繁殖面临的挑战,如投入不足和克服休眠困难,凸显了无性繁殖方法的优势。无性繁殖法是一种可行的替代方法,可实现克隆小苗的统一和大规模生产。这种技术可确保植物质量的一致性,鉴于该物种用途广泛,这一点至关重要。此外,植物生长调节剂的使用对于提高无性繁殖的成功率至关重要,它能促进从成年树木扦插的植株形成健壮的根系并促进植株的整体发育。本研究认识到创建 M. caesalpiniifolia 克隆林的重要意义,以及了解加快这一努力的方法的必要性,因此旨在评估从成年 M. caesalpiniifolia 树上扦插的茎的生根和不定根情况,并检查辅助剂的效果。从随机挑选的六棵成年M. caesalpiniifolia树上通过无性系拯救收获插条,用浓度为0、1,000、2,000和4,000 mg L-1的吲哚-3-丁酸(IBA)和吲哚-3-乙酸(IAA)处理,采用2 × 4因子排列的完全随机设计。评估包括在温室条件、荫棚条件和全日照条件下评估根的存在、芽的数量、克隆小植株的活力和植株存活率。研究结果表明,在这些条件下,生长调节剂及其浓度之间没有明显的相互作用。中等浓度的 IBA 能促进根系生长,而不含生长调节剂的插条则表现出较好的气生发育。随着调节剂浓度的增加,成活率有所下降,而不含调节剂的插条则表现出更好的形态发育。该研究表明,对于大规模苗圃来说,M. caesalpiniifolia 插条的自然生根和嫩枝形成可能足以在不使用 IBA 或 IAA 的情况下成功繁殖。
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引用次数: 0
PGPg_finder: A comprehensive and user-friendly pipeline for identifying plant growth-promoting genes in genomic and metagenomic data PGPg_finder:用于从基因组和元基因组数据中识别植物生长促进基因的全面且用户友好的管道
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-23 DOI: 10.1016/j.rhisph.2024.100905
Thierry Alexandre Pellegrinetti , Gabriel Gustavo Tavares Nunes Monteiro , Leandro Nascimento Lemos , Renato Augusto Corrêa dos Santos , Artur Gomes Barros , Lucas William Mendes

Identifying and comparing plant growth-promoting traits (PGPT) within whole-genome and metagenomic sequencing data can significantly advance agricultural research and promote sustainable crop production. This study introduces PGPg_finder, a comprehensive pipeline designed to annotate and compare PGPT from both whole-genome and metagenome sequencing datasets. This pipeline utilizes direct sequence annotation alongside de novo assembly methods to accurately detect PGPT. By cross-referencing sequences from the PLaBAse database, it identifies and quantifies the presence of these genes within the original datasets, facilitating an intuitive comparison of the abundance and distribution of PGPT across various samples. We evaluated the performance of PGPg_finder by analyzing genomes from five rhizobacterial strains: Paenibacillus vini, Paenibacillus polymyxa, Fictibacillus sp., Brevibacillus agri, and Bacillus cereus, and also metagenomic samples from bulk soils subjected to forest-to-pasture conversion in the Amazon rainforest. The genomic workflow revealed several genes associated with substrate utilization, abiotic stress neutralization, phosphate solubilization, and iron acquisition. It also identified genes unique to specific lineages, including those associated with colonization and plant-derived substrate usage in P. polymyxa, quorum sensing response and biofilm formation in P. vini, heavy metal detoxification and nitrogen acquisition in B. agri, and spore production and neutralizing biotic stress in B. cereus. The strain Fictibacillus sp. presented several unique genes related to surface attachment, stress response, xenobiotic degradation, phosphate solubilization, and phytohormone production. The use of PGPg_finder highlights its potential to uncover novel inoculants and strains. The metagenomic workflow distinguished plant-growth promotion gene profiles between soils from the Amazon rainforest and pasture, with the latter showing a profile more aligned with simple carbohydrate consumption, abiotic stress tolerance, motility and chemotaxis, and phosphorus mineralization. Native forests exhibited a profile associated with the degradation of complex organic matter, oxidative stress tolerance, xenobiotic degradation, bactericidal activity, iron acquisition, and volatile pathways. These findings underscore the effectiveness and sensitivity of PGPg_finder in accurately identifying and comparing PGPT genes, highlighting both commonalities and variations across samples. The application of this pipeline has the potential to significantly facilitate the identification of plant growth-promoting microbes.

在全基因组和元基因组测序数据中识别和比较植物生长促进性状(PGPT)能极大地推动农业研究,促进作物的可持续生产。本研究介绍了 PGPg_finder,这是一个综合管道,旨在注释和比较全基因组和元基因组测序数据集中的 PGPT。该管道利用直接序列注释和从头组装方法来准确检测 PGPT。通过交叉引用 PLaBAse 数据库中的序列,它能识别并量化原始数据集中这些基因的存在,从而有助于直观地比较 PGPT 在不同样本中的丰度和分布。我们通过分析五种根瘤菌株的基因组评估了 PGPg_finder 的性能:我们评估了 PGPg_finder 的性能,我们分析了五株根瘤菌的基因组:Paenibacillus vini、Paenibacillus polymyxa、Fictibacillus sp.、Brevibacillus agri 和 Bacillus cereus,还分析了亚马逊雨林中从森林到牧场转换过程中大块土壤的元基因组样本。基因组工作流程揭示了与底物利用、非生物压力中和、磷酸盐溶解和铁获取相关的若干基因。它还确定了特定菌系的独特基因,包括与多粘菌属(P. polymyxa)的定殖和植物源底物利用、维尼菌属(P. vini)的法定量感应反应和生物膜形成、农杆菌属(B. agri)的重金属解毒和氮获取以及蜡样芽孢杆菌属(B. cereus)的孢子生产和生物压力中和有关的基因。Fictibacillus sp.菌株在表面附着、应激反应、异生物降解、磷酸盐溶解和植物激素产生方面有几个独特的基因。PGPg_finder 的使用凸显了其发现新型接种剂和菌株的潜力。元基因组工作流程区分了亚马逊雨林土壤和牧场土壤中促进植物生长的基因图谱,后者的图谱更符合单碳水化合物消耗、非生物胁迫耐受性、运动性和趋化性以及磷矿化。原生林显示出与复杂有机物降解、氧化应激耐受性、异生物降解、杀菌活性、铁获取和挥发性途径相关的特征。这些发现强调了 PGPg_finder 在准确识别和比较 PGPT 基因方面的有效性和灵敏度,突出了不同样本之间的共性和差异。该管道的应用有可能极大地促进植物生长促进微生物的鉴定。
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引用次数: 0
Close linkage between available and microbial biomass phosphorus in the rhizosphere of alpine coniferous forests along an altitudinal gradient 沿海拔梯度高山针叶林根瘤层中可利用磷与微生物生物量磷之间的密切联系
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-22 DOI: 10.1016/j.rhisph.2024.100904
Min Li , Xi He , Peipei Zhang , Ruihong Wang , Jipeng Wang , Xinjun Zhang , Huajun Yin

The rhizosphere is a hotspot of soil phosphorus (P) transformation, which profoundly influences the P status of plants. Although P is projected to limit the ability of forests to serve as a carbon sink, it remains unclear how rhizosphere P availability responds to changing environments in alpine forests. Here, we investigated changes in rhizosphere available P across a series of altitudinal bands (2850 m, 2950 m, 3060 m and 3200 m) in alpine forests and examined the potential regulators of rhizosphere P availability, including temperature and soil biotic and abiotic properties. The results showed that rhizosphere P availability decreased up to the 3060 m site but then increased at the 3200 m site. A structural equation model showed that temperature and soil properties (pH and organic carbon content) indirectly affected rhizosphere available P through amorphous iron/aluminum oxides and microbial biomass P, which had negative and positive effects on rhizosphere available P, respectively. Thus, sorption by soil minerals and turnover of microbial biomass P may be key processes regulating P availability. In contrast, soil organic acids and acid phosphatase, which may promote the release of P by ligand exchange and mineralization, respectively, did not show a positive relationship with rhizosphere available P. Overall, our findings highlight the potential role of microbial biomass as a labile P pool that provides readily available P by turnover and protects P from sorption by soil minerals, which could help in elucidating the mechanisms by which plants maintain their P nutrient supply in alpine ecosystems under environmental changes.

根瘤层是土壤磷(P)转化的热点,它对植物的磷状况影响深远。虽然预计磷会限制森林作为碳汇的能力,但根圈磷的可用性如何应对高山森林中不断变化的环境仍不清楚。在这里,我们研究了高山森林中一系列海拔带(2850 米、2950 米、3060 米和 3200 米)根瘤层可利用钾的变化,并考察了根瘤层可利用钾的潜在调节因素,包括温度、土壤生物和非生物特性。结果表明,在海拔 3060 米以下的地点,根圈钾的可利用性降低,而在海拔 3200 米的地点,根圈钾的可利用性提高。结构方程模型显示,温度和土壤特性(pH 值和有机碳含量)通过无定形铁/铝氧化物和微生物生物量 P 间接影响根瘤层可利用钾,而无定形铁/铝氧化物和微生物生物量 P 分别对根瘤层可利用钾有负面和正面影响。因此,土壤矿物质的吸附作用和微生物生物量钾的周转可能是调节钾可用性的关键过程。总之,我们的研究结果凸显了微生物生物质作为一个可变型钾库的潜在作用,它通过周转提供随时可用的钾,并保护钾不被土壤矿物质吸附,这有助于阐明高寒生态系统中植物在环境变化下维持钾养分供应的机制。
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引用次数: 0
Climate–resilient strategy for shortening the crop cycle in rice and the host influenced rhizosphere microbiome 缩短水稻作物周期的气候适应性战略以及受宿主影响的根瘤微生物组
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-22 DOI: 10.1016/j.rhisph.2024.100903
P. Umadevi , S. Gopala Krishnan , M. Nagarajan , S. Manivannan , K.K. Vinod , A.K. Singh

We present the field rapid generation advancement procedure for shortening the crop cycle in rice. The practical protocol developed by us using different maturity groups of rice showed promising early flower induction. We observed a shortening of crop cycle to about 35–40 days depending on the variety. The spacing between the plants was the major influencer for flower induction compared to other interventions tested viz., clipping, potassium di-hydrogen phosphate and paclobutrazol spray. Our raised bed direct seeding strategy completely avoided the transplantation. The work flow for flower induction can be a reference to increase generations of rice breeding. The rhizosphere bacterial dynamics using 16srRNA gene amplicon sequencing showed the Acinetobacter population abundance as a key mediator and marker for flowering time. The alpha diversity at flowered and unflowered stage showed the species richness as Control < Pusa Sugandh 5 < BPT-5204. The rice rhizosphere of this ecosystem had an abundance of Methylotrophs that utilize methane as a carbon source suggesting that the developed method is a green technique suitable for generation advancement that can be replaced for flooded condition assisted breeding in rice. Selective enrichment of functional abundance between varieties suggested the host - influenced microbiome for early flowering in rice. This protocol is expected to greatly accelerate the process of new variety breeding and the construction of mapping populations.

我们介绍了用于缩短水稻作物周期的田间快速育种程序。我们使用不同成熟度的水稻组开发的实用方案显示出了良好的早花诱导效果。我们观察到,根据品种的不同,作物周期缩短至约 35-40 天。与其他干预措施(如剪枝、磷酸二氢钾和喷洒 paclobutrazol)相比,株距是诱导开花的主要影响因素。我们的高床直接播种策略完全避免了移植。诱导开花的工作流程可为水稻育种的增代提供参考。利用 16srRNA 基因扩增片段测序法对根瘤菌动态进行了研究,结果表明,Acinetobacter 种群丰度是开花时间的关键介质和标记。开花期和未开花期的α-多样性显示物种丰富度为对照组< Pusa Sugandh 5 < BPT-5204。该生态系统的水稻根瘤层中有大量利用甲烷作为碳源的养甲菌,这表明所开发的方法是一种绿色技术,适用于水稻的世代交替,可替代水淹条件下的辅助育种。品种间功能丰度的选择性富集表明,受宿主影响的微生物组可促进水稻早花。该方案有望大大加快新品种培育和制图群体构建的进程。
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引用次数: 0
Serratia marcescens AB1: A rhizosphere bacterium mitigating the acetochlor stress on the soil environment Serratia marcescens AB1:一种减轻土壤环境中乙酰氯压力的根瘤菌
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-16 DOI: 10.1016/j.rhisph.2024.100898
Meiqi Dong , Yufeng Xiao , Bingbing Yang , Siya Wang , Liangpeng Sun , Zhe Han , Hao Zhang , Xian Wu

Microbial remediation, a significant research focus in bioremediation, shows promise in addressing pollution. In this study, the optimal medium for acetochlor-degrading bacteria AB1 was determined by the response surface method as 29.94 g L−1 sucrose, 10.06 g L−1 yeast extract, and 20.32 g L−1 NaCl. The single-factor method identified optimum degradation conditions, including a temperature of 30 °C, pH of 7.0, inoculation with 3% AB1, and an initial acetochlor concentration of 10 mg L−1. The strain reached a maximum degradation rate of 79.87% within 5 days. AB1 performed nitrogen fixation, phosphorus dissolution, potassium hydrolysis, siderophore production, and biofilm formation. In the presence of acetochlor, it also induced the upregulation of genes, wza and luxS. Utilizing a green fluorescent protein and rifampicin-resistant strain LAB1-gfp, it demonstrated stable colonization in maize rhizospheres and soils, enhancing growth and degradation. This reduced the acetochlor half-life to 12.77 days and increased soil enzyme activity, providing a theoretical foundation for acetochlor bioremediation.

微生物修复是生物修复领域的一个重要研究重点,在解决污染问题方面前景广阔。本研究采用响应面法确定了乙酰氯降解菌 AB1 的最佳培养基为 29.94 g L-1 蔗糖、10.06 g L-1 酵母提取物和 20.32 g L-1 氯化钠。单因素法确定了最佳降解条件,包括温度 30 °C、pH 值 7.0、接种 3% AB1 和初始乙草胺浓度 10 mg L-1。该菌株在 5 天内达到 79.87% 的最大降解率。AB1 具有固氮、溶磷、水解钾、产生苷元和形成生物膜的功能。在乙酰氯存在的情况下,它还能诱导 wza 和 luxS 基因的上调。利用绿色荧光蛋白和利福平抗性菌株 LAB1-gfp,它在玉米根瘤和土壤中实现了稳定定殖,增强了生长和降解能力。这将乙草胺的半衰期缩短至 12.77 天,并提高了土壤酶的活性,为乙草胺的生物修复提供了理论基础。
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引用次数: 0
Novel rapid screening assay to incorporate complexity and increase throughput in early-stage plant biological testing 新型快速筛选测定法可在早期植物生物测试中考虑复杂性并提高通量
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-16 DOI: 10.1016/j.rhisph.2024.100897
Sabrina M. Pittroff, Alexander R. Brems, Rune J. Brunshøj, Johan V. Christiansen, Emma Melgaard, Morten Lindqvist Hansen, David Llorente Corcoles, Jonathan Funk, Vilhelm K. Møller, Søren D. Petersen, Rasmus J.N. Frandsen, Niels B. Jensen, Lars Jelsbak
<div><p>The search for new biological products with a positive impact on crop performance is typically initiated by laboratory based <em>in vitro</em> assays. However, live plants and their associated microbes are often removed from <em>in vitro</em> testing assays as a way to reduce biological complexity (variation) and facilitate molecular techniques in the pursuit of uncovering mode-of-action (MoA) mechanisms. Nevertheless, when studying biological candidates intended for use in agriculture, it is essential to incorporate this complexity and validate mechanisms under conditions as close to <em>in situ</em> as possible in order to understand the capacities and MoA of the biologicals in the intended application environments. To address this paradox, we have developed a high-capacity early-stage plant assay that incorporates a live non-sterile plant while also enabling molecular MoA investigations, and that can be conducted in laboratories without greenhouse facilities. The high-capacity design features plants grown in 8-chamber transparent boxes to allow for multiplex imaging and increased biological replicates for greater statistical power. The transparent box design allows the visualization of shoots, roots, tagged-microbes, or visible substrates, and further non-destructive access to shoots or roots for sampling. The boxes are held in racks that hold eight plant boxes during growth in a 19 by 17 cm space, further increasing the throughput to >670 plants per m<sup>2</sup> and easing the logistical challenges of plant assays. Furthermore, the box can support various levels of microbial complexity with the option to select the plant growth medium that meets experimental objectives, as well as using sterile or non-sterile seeds. A script-based post-imaging quantification was developed to automate image processing and allow for individual plant readings, further enabling increased statistical confidence. As proof of concept, we use the high-capacity plant system to evaluate the biocontrol potential of <em>Pseudomonas protegens</em> and the biostimulation potential of <em>Pseudomonas koreensis</em>, and are in both cases able to show statistically significant differing plant biomass between treatments under these closer-to-nature conditions. We further demonstrate that the high-capacity plant system is suitable for paired molecular investigations by performing metabolomics and qPCR DNA quantification directly from the plant box to explore <em>in situ</em> chemical MoA, as well as confirm the survival of the <em>P. protegens</em> strains to validate their role in the improved plant phenotype. In conclusion, the study presents a modular high-capacity plant assay system that enables increased throughput functional testing of microbial biocontrol and biostimulant candidates <em>in planta</em>. This novel assaying system saves time, reduces human error, provides quantitative and non-destructive <em>in planta</em> data, and can be used in laboratories
寻找对作物生长有积极影响的新型生物产品通常是通过实验室体外试验来进行的。然而,活体植物及其相关微生物往往被排除在体外试验之外,以降低生物复杂性(变异)并促进分子技术的发展,从而揭示作用模式(MoA)机制。然而,在研究拟用于农业的候选生物时,必须将这种复杂性纳入其中,并在尽可能接近原位的条件下验证机制,以了解生物在预期应用环境中的能力和作用模式。为了解决这一矛盾,我们开发了一种高容量的早期植物检测方法,该方法结合了非无菌活体植物,同时还能进行分子分子行为学研究,并且可以在没有温室设施的实验室中进行。高容量设计的特点是植物生长在 8 室透明箱中,可进行多重成像,并增加生物重复数以提高统计能力。透明箱的设计允许对芽、根、标记微生物或可见基质进行可视化,并可进一步对芽或根进行非破坏性取样。在 19 x 17 厘米的空间内,植物盒可放置在可容纳 8 个植物盒的架子上,从而将吞吐量进一步提高到每平方米 670 个植物,并减轻了植物检测的物流挑战。此外,植物箱还可以支持各种复杂程度的微生物,选择符合实验目标的植物生长培养基,以及使用无菌或非无菌种子。我们还开发了基于脚本的成像后量化功能,以实现图像处理的自动化,并允许对单个植物进行读数,从而进一步提高统计置信度。作为概念验证,我们使用大容量植物系统评估了蛋白假单胞菌的生物防治潜力和韩国假单胞菌的生物刺激潜力,在这两种情况下,都能在更接近自然的条件下显示出不同处理之间在统计学上显著不同的植物生物量。我们还进一步证明,大容量植物系统适用于配对分子研究,可直接从植物箱中进行代谢组学和 qPCR DNA 定量,以探索原位化学摩尔效应,并确认 P. protegens 菌株的存活,以验证它们在改善植物表型中的作用。总之,该研究提出了一种模块化高容量植物检测系统,可提高植物体内微生物生物控制和生物刺激候选菌功能测试的通量。这种新型检测系统可节省时间、减少人为错误、提供定量和非破坏性的植物体数据,并可在没有温室设施的实验室中使用。因此,我们相信它提供了一种有效的早期测试选择,在体外测试和温室测试之间架起了一座桥梁,并将加快发现优质的下一代农业生物产品。
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引用次数: 0
A simple, cost-effective and optimized protocol for collecting root exudates from soil grown plants 收集土壤种植植物根部渗出物的简单、经济、优化方案
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-14 DOI: 10.1016/j.rhisph.2024.100899
Stefanie Döll , Hannah Koller , Nicole M. van Dam

Root exudates play a pivotal role in belowground interactions in both ecological and agricultural contexts. The metabolic composition of exudates profoundly influences the dynamics of these interactions, thereby shaping the intricate relationships between plants, microbes, and soil environments. Recent advances in mass-spectrometry have facilitated the analysis of root exudate metabolic composition to a greater depth. Previously used methods primarily analyze root exudates in hydroponic systems, or employ hybrid methodologies, which cultivate plants in soil and transitioning them briefly to hydroponic systems for exudate collection. Modern day ecological studies demand that exudates are collected in their natural habitats, because this will provide a more ecologically meaningful exudate metabolic profile. However, collecting exudates from soil grown plants poses several challenges with regard to the collection procedures, amongst others, the need for recovery after excavation of the roots, the collection period, and the solution in which to collect. Here, we present an optimized, cost-effective protocol for root exudate collection from potted plants, which is readily adaptable to field-grown specimens. Using tomato plants grown in pots, we examined and optimized various parameters: the collection medium (water versus nutrient solution), the use of wetted glass beads versus roots submerged in water, the recovery phase post-substrate removal, and the duration of exudation. Employing liquid chromatography-mass spectrometry (LC-MS), we assessed total amount of exudate, the number of features and background noise. Subsequent to data processing and statistical analyses, we assessed the chemical classes within exudates and variations in key metabolites among the different methods. Our results showed that each of the tested parameters can influence the outcome in different ways. Omitting the recovery phase increased the numbers of features and exudate amounts, likely due to adding metabolites from damaged roots, whereas the exudation medium and the duration of exudation had fewer effects. Based on our results, we propose to collect exudates in beakers containing ultrapure water, and to collect exudates for 4 h after a 24 h recovery phase. This is a straightforward and economical approach for collecting root exudates from soil-grown plants which is suitable for LC-MS analysis.

在生态和农业环境中,根系渗出物在地下相互作用中发挥着关键作用。渗出物的代谢组成深刻影响着这些相互作用的动态,从而塑造了植物、微生物和土壤环境之间错综复杂的关系。质谱分析法的最新进展有助于更深入地分析根系渗出物的代谢组成。以前使用的方法主要分析水培系统中的根系渗出物,或采用混合方法,即在土壤中培育植物,然后将其短暂过渡到水培系统中收集渗出物。现代生态研究要求在自然栖息地收集渗出物,因为这将提供更具生态意义的渗出物代谢概况。然而,从土壤生长的植物中采集渗出液在采集程序上面临着一些挑战,其中包括根部挖掘后的恢复需要、采集时间以及采集溶液。在此,我们提出了一种优化的、具有成本效益的盆栽植物根部渗出物采集方案,该方案可随时适用于田间种植的标本。利用盆栽番茄植物,我们研究并优化了各种参数:收集介质(水与营养液)、使用浸湿的玻璃珠与浸没在水中的根、移除基质后的恢复阶段以及渗出的持续时间。我们采用液相色谱-质谱法(LC-MS)评估了渗出物的总量、特征数量和背景噪声。在数据处理和统计分析之后,我们评估了渗出物中的化学类别以及不同方法中主要代谢物的差异。我们的结果表明,每个测试参数都会以不同的方式影响结果。省略恢复阶段会增加特征的数量和渗出物的数量,这可能是由于增加了来自受损根系的代谢物,而渗出介质和渗出持续时间的影响较小。根据我们的研究结果,我们建议在装有超纯水的烧杯中收集渗出液,并在 24 小时恢复阶段后收集 4 小时的渗出液。这是收集土壤栽培植物根部渗出物的一种直接而经济的方法,适用于 LC-MS 分析。
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引用次数: 0
Lettuce seedlings rapidly assemble their microbiome from the environment through deterministic processes 莴苣幼苗通过确定性过程从环境中快速组合微生物群
IF 3.7 3区 生物学 Q1 PLANT SCIENCES Pub Date : 2024-05-11 DOI: 10.1016/j.rhisph.2024.100896
Nesma Zakaria Mohamed , Leonardo Schena , Antonino Malacrinò

Plant-associated microorganisms have significant impacts on plant biology, ecology, and evolution. Although several studies have examined the factors driving variations in plant microbiomes, the mechanisms underlying the assembly of the plant microbiome are still poorly understood. In this study, we used gnotobiotic plants to test (i) whether seedlings create a selective environment and drive the assembly of root and leaf microbiomes through deterministic or stochastic processes, and (ii) whether seedlings structure the microbiome that is transferred through seeds using deterministic processes and whether this pattern changes when seedlings are exposed to the environmental microbiome. Our results show that the microbiome of gnotobiotic plants (i.e., inherited through seeds) is not under the selective influence of the host plant but changes quickly when plants are exposed to soil microbiomes. Within one week, plants were able to select microorganisms from the inocula, assemble the root microbiome, and assemble the shoot microbiome. This study supports the hypothesis that plants at early developmental stages might exert strong selective activity on their microbiomes and contribute to clarifying the mechanisms of plant microbiome assembly.

植物相关微生物对植物生物学、生态学和进化有重大影响。尽管已有多项研究探讨了植物微生物组变异的驱动因素,但人们对植物微生物组的组装机制仍然知之甚少。在这项研究中,我们利用无性繁殖植物来测试:(i) 幼苗是否创造了一个选择性环境,并通过确定性过程或随机过程来驱动根和叶微生物组的组装;(ii) 幼苗是否利用确定性过程来构建通过种子转移的微生物组,以及当幼苗暴露于环境微生物组时,这种模式是否会发生变化。我们的研究结果表明,非生物植物(即通过种子遗传的植物)的微生物组不受宿主植物的选择性影响,但当植物暴露于土壤微生物组时,微生物组会迅速发生变化。一周之内,植物就能从接种菌中选择微生物,组建根部微生物群,并组建芽部微生物群。这项研究支持了植物在早期发育阶段可能对其微生物组产生强烈选择活动的假设,并有助于阐明植物微生物组的组装机制。
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