Engineered Phomopsis liquidambaris with Fhb1 and Fhb7 Enhances Resistance to Fusarium graminearum in Wheat.

Fusarium head blight is one of the most serious diseases caused by Fusarium graminearum in wheat. Here, we developed a new way to prevent and control Fusarium head blight by introducing the resistance genes Fhb1 and Fhb7 into the endophytic fungus Phomopsis liquidambaris, named PL-Fhb1 and PL-Fhb7, respectively, which could colonize wheat. The wheat seedlings were preinoculated with PL-Fhb1 and PL-Fhb7 to enhance the resistance against deoxynivalenol (DON) and PL-Fhb1 and PL-Fhb7 inhibited the growth of F. graminearum by 73% and 49%, respectively. The incidence rate of diseased spikes decreased to 35.2% and 45.4%, and the corresponding DON levels for wheat grains decreased from 13.2 to 1.79 µg/g and from 13.2 µg/g to 0.39 µg/g when the leaves were preinoculated with PL-Fhb1 and PL-Fhb7 after overwintering, respectively. The incidence rates of diseased spikes decreased to 25.7% and 34.7%, and the DON levels for wheat grains decreased from 17.48 µg/g to 1.23 µg/g and from 17.48 µg/g to 0 µg/g when the wheat flowers were inoculated with PL-Fhb1 and PL-Fhb7, and the wheat flowers were subsequently infected with F. graminearum, respectively. It was confirmed that DON was transformed into DON-glutathione (GSH) by PL-Fhb7 using high-performance liquid chromatography-mass spectrometry (HPLC-MS). However, PL-Fhb1 may have increased plant immunity and enhanced the resistance to F. graminearum. This study indicates that engineered endophytes can improve the resistance to Fusarium head blight and presents a new method for the biological control of Fusarium head blight.

Activation of Naringenin and Kaempferol through Pathway Refactoring in the Endophyte Phomopsis Liquidambaris.

Abundant gene clusters of natural products are observed in the endophytic fungus Phomopsis liquidambaris; however, most of them are silent. Herein, a plug-and-play DNA assembly tool has been applied for flavonoid synthesis in P. liquidambaris. A shuttle plasmid was constructed based on S. cerevisiae, E. coli, and P. liquidambaris with screening markers URA, Amp, and hygR, respectively. Each fragment or cassette was successively assembled by overlap extension PCR with at least 40-50 bp homologous arms in S. cerevisiae for generating a new vector. Seven native promoters were screened by the DNA assembly based on the fluorescence intensity of the mCherry reporter gene in P. liquidambaris, and two of them were new promoters. The key enzyme chalcone synthase was the limiting step of the pathway. The naringenin and kaempferol pathways were refactored and activated with the titers of naringenin and kaempferol of 121.53 mg/L and 75.38 mg/L in P. liquidambaris using fed-batch fermentation, respectively. This study will be efficient and helpful for the biosynthesis of secondary metabolites.

The prevalence of heifer mastitis and its associated risk factors in Huanggang, Central China.

The purposes of this study were to determine the prevalence of heifer clinical mastitis (HCM) and heifer subclinical mastitis (HSCM), isolate HCM-causing bacteria, and assess the association of some risk factors in Huanggang, Central China. A total of 1,374 lactating heifers from three dairy farms were examined in the present study; 22.64% of heifers were positive for mastitis, out of which, 3.86 and 18.78% were with HCM and HSCM, respectively. Of the 67 HCM samples, 91.05% were single growth, 7.46% mixed growth, and 1.49% no bacterial growth. Coagulase-negative staphylococci (CNS) accounted for 30.98% of the isolates followed by Escherichia coli (29.58%), Staphylococcus aureus (16.9%), and Streptococcus uberis (11.27%). Logistic regression analyses showed that heifer milk yield lower than 3 L had the highest prevalence of HCM compared to heifer milk yield of more than 3 L (P < 0.01), and prevalence of HCM was significantly high in heifers with the presence of teat lesions (P < 0.01). Moreover, the heifer milk yield lower than 3 L had the highest prevalence of HSCM compared to heifer milk yield of more than 3 L (P < 0.01). These two factors were significantly associated with the occurrence of heifer mastitis, which needs to be considered in the prevention and control strategies of the disease.