鸡舍环境中tri5基因阳性镰孢菌的分离及其产毒特性

Isolation and toxogenic characterization of tri5 positive Fusarium from poultry houses

摘要:

摘要：【目的】探讨利用tri5-PCR鉴定产毒镰孢菌的可行性以及tri5阳性菌株的产毒特性和产毒条件。评估鸡舍空气和固体基质中镰孢菌分离株中产单端孢霉烯族毒素潜力的发生率。【方法】利用编码单端孢霉烯族毒素合成酶的起始基因（tri5基因）对139株镰孢菌分离株进行PCR检测，并对tri5阳性菌株进行产毒培养，通过免疫亲和柱-高效液相色谱方法来检测培养产物中的T-2毒素和HT-2毒素含量。【结果】共筛选到42株tri5阳性菌株，其中分离自鸡舍空气中的10株tri5阳性菌株经产毒培养后7株菌株产生T-2毒素（1.36ng/mL~5ng/mL）或HT-2毒素（6.1ng/mL~17.1ng/mL）。在5℃-20℃间隔24h变温、光照与黑暗间隔24h交替、前期振荡后期静止的培养条件下培养9d镰孢菌的产毒量最高；镰孢菌的产毒量与温度和时间显著相关，而与菌丝体干重无显著相关性。【结论】与传统鉴定产毒镰孢菌的方法比较，tri5-PCR更适用于快速、准确地检测大量鸡舍环境样本中的产毒镰孢菌。本研究为养殖环境的产毒镰孢菌的危害预警和控制提供技术支撑和理论依据。

Abstract:

Abstract: [Objective] We used tri5-PCR technique to identify the toxin-producing Fusarium strains, the toxogenic characteristics and toxicogenic condition of tri5 positive strains. We also evaluated the potential trichothecene-producing level of the Fusarium strains from the air and solid materials in poultry houses. [Methods] Tri5 gene, the start gene encoding trichothecene synthase, was taken to detect 139 Fusarium isolates by PCR. Toxogenic culture was carried out for tri5 positive Fusarium strains, and the quantities of T-2 and HT-2 toxins in toxogenic cultures were measured by high performance liquid chromatography (HPLC) after immune-affinity column clear-up. [Results] Among the 42 tri5 positive strains determined by tri5-PCR, all 10 tri5 positive strains from the air in poultry houses were found to produce T-2 toxin (1.36ng/mL~5ng/mL) or HT-2 toxin (6.1ng/mL~17.1ng/mL) after culture. The optimal conditions for toxogenic culture were 9 days of culture with 5℃-20℃ temperature fluctuation and light-dark alternating at 24h intervals, shaking at early stage and non-shaking at later stage. The toxin production of tri5 positive strains was significantly affected by temperature and time, but it has no correlation with the dry mass of mycelium. [Conclusion] In comparison with the traditional method, tri5-PCR is a rapid method for accurate detection of toxogenic Fusarium isolates in large amounts of environmental samples from poultry houses. The results provide a technical support and theoretical basis for early hazard warning and control of toxogenic Fusarium strains in animal raising environments.