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从连作条件下的平邑甜茶健康根系分离得到1株内生真菌A1,通过形态学特征、培养特性观察鉴定该菌属于青霉属,将其ITS r DNA序列和β-tubulin序列与Gen Bank中已知标准菌株的ITS r DNA序列和β-tubulin序列进行比对,并利用MEGA5.05软件构建青霉菌A1菌株进化树。结果表明,青霉菌A1与草酸青霉Penicillium oxalicum(KF152942.1,KC344990.1)序列同源性最高,为100%,最终将青霉菌A1鉴定为草酸青霉A1。在马铃薯培养基(PDA)和玉米粉培养基上分别将内生真菌A1与尖孢镰孢菌、串珠镰孢菌、层出镰孢菌和腐皮镰孢菌进行对峙试验,结果在PDA培养基上对菌丝生长抑制率分别为42.0%、74.0%、47.0%和54.5%,拮抗系数达到Ⅱ级或Ⅰ级,在玉米粉培养基上菌丝生长抑制率分别为60.5%、82.2%、68.4%和53.4%,拮抗系数达到Ⅱ级或Ⅰ级,说明草酸青霉A1对4种苹果连作障碍病原镰孢属真菌具有较好的抑制作用。平邑甜茶盆栽试验结果表明,与连作土对照相比,A1菌肥显著促进了连作平邑甜茶幼苗鲜样质量、干样质量的增加,分别增加2.53倍和2.35倍,说明草酸青霉A1菌肥能在一定程度上减轻苹果连作障碍。使用实时荧光定量技术对土壤中的尖孢镰孢菌基因拷贝数进行检测,发现A1菌肥处理的土壤中尖孢镰孢菌的基因拷贝数量大幅下降,说明草酸青霉菌A1可以抑制其生长。A1菌株可作为防控苹果连作障碍的拮抗菌进一步研究。
An endophytic fungus A1 was isolated from the healthy roots of Malus hupehensis under continuous cropping. The morphological characteristics and culture characteristics of the strain were identified as belonging to the genus Penicillium. ITS ITS DNA sequences and β-tubulin sequences were compared with Gen Bank It is known that ITS r DNA sequence and β-tubulin sequence of standard strains are aligned, and the phylogenetic tree of Penicillium sp. A1 strain is constructed by using MEGA5.05 software. The results showed that Penicillium Al had the highest homology of 100% with Penicillium oxalicum (KF152942.1, KC344990.1), and Penicillium sp. A1 was identified as Penicillium oxalicum A1. Endophytic fungi A1 were confronted with Fusarium oxysporum, Fusarium moniliforme, Fusarium graminearum and Fusarium solani on potato culture medium (PDA) and cornmeal medium, respectively. The mycelial growth inhibition rates were 42.0%, 74.0%, 47.0% and 54.5%, respectively, and the antagonistic coefficients reached Grade Ⅱ or Ⅰ. The mycelial growth inhibition rates were 60.5% and 82.2% on the maize meal medium, respectively. 68.4% and 53.4%, respectively, indicating that the antagonistic coefficient reached Grade Ⅱ or Grade Ⅰ, indicating that Penicillium oxalicum A1 has a good inhibitory effect on four pathogenic Fusarium pathogens of continuous cropping obstacle. The results of pot experiment of PingYi sweet tea showed that compared with continuous cropping soil control, A1 fertilizer significantly increased the fresh quality and the dry weight of the continuous cropping sweet potato seedlings by 2.53-fold and 2.35-fold, respectively, indicating that A1 oxalate strain A1 Fertilizer to some extent reduce the obstacles to continuous cropping. Using real-time fluorescence quantitative detection of Fusarium oxysporum gene copy number in soil, it was found that the number of Fusarium oxysporum gene copies significantly decreased in A1 soil, indicating that Penicillium oxalicum A1 can inhibit its growth. The A1 strain can be used as antagonistic bacteria to prevent and control apple continuous cropping obstacle.