Friend or Foe? The Endophytic Fungus Alternaria tenuissima Might Be a Major Latent Pathogen Involved in Ginkgo Leaf Blight

Abstract

Ginkgo leaf blight, one of the most economically important ginkgo diseases, has become very prevalent in many places in China. Flavonoids and endophytes are both considered important in ginkgo plant functioning. However, little is known about the potential relationships among ginkgo leaf blight pathogens, flavonoid accumulation profiles in infected leaves, and ginkgo leaf endophytes. In this study, the flavonoid accumulation profiles in infected leaves, pathogens of ginkgo leaf blight, and the endophytes of healthy ginkgo leaves were characterized. The levels of total flavonoids in the healthy parts of the infected leaves were significantly higher than those in the healthy leaves. Furthermore, Alternaria tenuissima, Botryosphaeria dothidea, and Dothiorella gregaria were identified as pathogens of ginkgo leaf blight; among them, A. tenuissima was the major pathogen. The in vitro experiments showed that flavonoids (apigenin, luteolin, and kaempferol) could significantly inhibit the growth of one or more pathogens at a concentration of 10 mg/L. Furthermore, fifty-six ginkgo leaf endophytic fungi (GLEF) from healthy ginkgo plants were isolated and characterized. Among them, Alternaria spp. were the most abundant, and GLEF55 shared the same ITS sequence with the pathogen Alternaria tenuissima. Thereafter, four flavonoid-producing endophytes were selected and their effects on the growth of pathogens were evaluated. The extracts of GLEF55 could significantly inhibit the growth of the pathogens B. dothidea and D. gregaria simultaneously in vitro, but not the growth of the pathogen A. tenuissima. Furthermore, the dual cultures of the candidate GLEF and ginkgo leaf blight pathogens revealed that GLEF55 had a similar growth rate to that of A. tenuissima and D. gregaria, but its growth rate was significantly slower than that of B. dothidea. Finally, the GLEF exhibited variable roles when facing pathogens in ginkgo leaves. Among them, GLEF55 showed similar pathogenicity as the pathogen A. tenuissima when they were dually cultured in ginkgo leaves. By contrast, GLEF17 (an uncultured soil fungus) could significantly counteract the pathogenic effects of A. tenuissima and D. gregaria, but it dramatically exacerbated the pathogenic effects of B. dothidea. Larger lesion areas were observed on the side of ginkgo leaves where GLEF39 (Alternaria sp.) or GLEF54 (Aspergillus ruber) and pathogens were simultaneously inoculated, which suggested that the pathogenicity of specific endophytic fungi occurred when plants were wounded. Overall, A. tenuissima, a major pathogen of ginkgo leaf blight, might lurk inside the plants as a friendly endogenous fungus and convert into a hostilely pathogenic mode at a particular time. This study proposed a possible cause of ginkgo leaf blight and provided potential theoretical guidance for its prevention.