Effectiveness of carboxylic acids from Pichia membranifaciens against coffee rust

ABSTRACT Coffee rust is a fungal disease that has affected every coffee-producing region in the world. Given that the effectivity of the protectant and systemic fungicides applied routinely to control the spread of the causative agent of the disease (Hemileia vastatrix) has gradually diminished, besides are harmful to mammals and ecosystems, the objective of this work was to search for a mixture of harmless natural compounds with the potential to be applied in the field. So, a yeast strain producing a battery of long-chain carboxylic acids (CA) with fungicide properties was isolated from soil of coffee crop and identified as Pichia membranifaciens by ITS sequencing. Culture conditions of the yeast were optimized and the CA in the solution were characterized by Gas Chromatography-Mass Spectrometry (GC-MS) as ethyl formate (55.5 g L-1), octadecenoic acid (3.5 g L-1), propionic acid (7.2 g L-1), 3-(octadecanoyl)-propionic acid (7.2 g L-1) and methyl acetate (8.4 g L-1). Randomized field studies were conducted in three different locations in Chiapas, México. Five treatments were tested including three concentrations of the CA solution (389, 584 and 778 ppm) and copper oxychloride (5 000 ppm) as conventional control. The initial coffee rust incidence averages varied between sites: Maravillas (3-9%), Santo Domingo (10-16%) and Búcaro (16-22%). The treatments of CA solution proved to be effective at slowing down the progress of the rust disease even for the sites where initial incidence was high. Likewise, the CA solution reduced the viability of H. vastatrix spores, as assessed by fluorescence microscopy.摘要：咖啡锈病（Coffee rust）是一种侵染全球所有咖啡产区的真菌病害。当前，常规用于防控该病害病原菌（咖啡锈菌Hemileia vastatrix）的保护性和内吸性杀菌剂不仅防控效果逐渐减弱，且对哺乳动物及生态系统均具有危害性。为此，本研究旨在筛选出一组具备田间应用潜力的无害天然化合物混合物。研究从咖啡种植园土壤中分离得到一株可产生一系列具有杀菌活性的长链羧酸（CA）的酵母菌株，通过内部转录间隔区（ITS）测序将其鉴定为膜毕赤酵母（Pichia membranifaciens）。本研究优化了该酵母的培养条件，并通过气相色谱-质谱联用仪（Gas Chromatography-Mass Spectrometry, GC-MS）对发酵液中的羧酸组分进行定性定量分析，确定其主要成分及对应含量为：甲酸乙酯（55.5 g·L⁻¹）、十八烯酸（3.5 g·L⁻¹）、丙酸（7.2 g·L⁻¹）、3-（十八烷酰基）-丙酸（7.2 g·L⁻¹）及乙酸甲酯（8.4 g·L⁻¹）。在墨西哥恰帕斯州的三个不同试验点开展了随机区组田间试验，共设置5组处理，分别为3种不同浓度的羧酸溶液（389、584和778 ppm），以及浓度为5000 ppm的氧氯化铜作为常规对照药剂。各试验点的初始咖啡锈病发病率存在差异：马拉维利亚斯（Maravillas）为3%~9%、圣多明各（Santo Domingo）为10%~16%、布卡洛（Búcaro）为16%~22%。结果表明，羧酸溶液处理可有效延缓锈病的病情进展，即使在初始发病率较高的试验点亦表现出良好防控效果。此外，通过荧光显微镜检测证实，羧酸溶液可显著降低咖啡锈菌孢子的活力。