Fungal community and diversity in rhizospheric soil with root rot in an apple orchard

Abstract:【Objective】The relative abundance, community composition and diversity of fungi were in- vestigated to reveal the effects of root rot disease on rhizospheric soil microbe. Meanwhile, the reported pathogens associated with root rot disease were examined and the potential biocontrol agents were pre- dicted.【Methods】Rhizospheric soil from healthy and root rot trees in the same orchard were collected, which were denoted as Healthy and Diseased. By digging up the top soil, the roots were exposed. A part of the roots was cut and shaked gently to remove the loosely adhering soil, and then the tightly adhering soil was collected. Then, the soil samples were put into liquid nitrogen and stored in an ultralow temper- ature freeze at - 80 °C for DNA extraction. The total DNA was extracted from each sample using the Fast DNA Spin Kit for soil. With ITS5-1737F and ITS2-2043R as the primer pair, the fungal internal transcribed spacer (ITS) region of rDNA was amplified and then sequenced by means of Illumina HiSeq high- throughput sequencing technology. The high quality paired- end reads were merged by FLASH. The operational taxonomic units (OTUs) were obtained with 97% similarity cutoff using  UPARSE. Based on the rarefying OTU relative abundance for taxa, the taxonomy of each ITS sequence was analyzed by RDP Classifier algorithm against the UNITE 7.2 using a confidence threshold of 70%. The significant difference in alpha-diversity were calculated using student’s T test with Mothur v.1.30.【Results】Approximately 0.58 million high quality sequence tags were obtained. The rarefaction curves showed that as the numbers of reads increased, the sobs index tended to be gentle, so the sequenced re- sults were reasonable and could reflect the actual situation of collected samples. Finally, Healthy and diseased rhizospheric soil samples got 708 and 1 006 OTUs, respectively, mainly affiliated to 11 phy- lum, 33 classes, 148 families, and 263 genera. The result showed that 611 OTUs were detected both in healthy and diseased samples, while 97 OTUs and 395 OTUs were respectively observed only in healthy and diseased soil. The alpha- diversity index (Sobs, Shannon, Simpson, ACE, Chao) analysis showed that Sobs, Shannon, ACE, and Chao index in diseased soil were significantly higher than in healthy soil, while Simpson index was lower than that with healthy soil. Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Mortierellomycota and Rozellomycota were observed in all sam- ples. The dominant phylum included Ascomycota, Basidiomycota and Chytridiomycota. Especially As- comycota possessed absolute advantage. In addition, there was a large amount of unclassified fungi in all samples. Ascomycetes fungi in diseased soil were significantly lower (p=0.045) than those in healthy soil. At class level, communities with relative abundance above 1% were arranged as Dothideomycetes, Sordariomycetesa, Leotiomycetes, Tremellomycetes, Eurotiomycetes and Mortierellomycetes. The rela- tive abundance of Dothideomycetes in healthy soil was significantly higher than that in diseased soil. Moreover, Leotiomycetes and Eurotiomycetes were lower than those in diseased soil. At genus level, healthy soil was enriched with more unclassified Pleosporales, Plectosphaerella and Cladosporium, and unclassified Nectriaceae, Ilionectria, Guehomyces, Trichoderma, Phoma and Lophiostoma. While diseased soil was enriched with more unclassified Ascomycota, Pseudogymnoascus, Fusarium, Nagan- ishia, Mortierella, Trimmatostroma and Knufia. In order to analyze the potential pathogens that caused apple root rot disease, the relatively abundance of reported pathogens associated with fruit root rot were calculated. The result showed that seven genera(Alternaria, Cylindrocarpon, Cylindrocladiella, Fusari- um, Ilyonectria, Pestalotiopsis and Rosellinia )were observed both in healthy and diseased soil. Among those genera，Fusarium and Rosellinia that had been reported causing apple root rot were enriched in the root rot soil, but the difference between health and disease soil was not significant. At the same time，the potential biocontrol agents such as Trichoderma，Penicillium and Paecilomyces existed in both healthy and diseased soil. Trichoderma was enriched in healthy soil, and Penicillium was enriched in diseased soil.【Conclusion】The OTUs numbers, observed species, fungal diversity in the rhizospheric soil of root rot were significantly higher than those in healthy soil. Infected by the root rot disease, more fungi would appear in rhizospheric soil. The dominant fungal communities at phylum level were Ascomycota, Basidiomycota and Mortierellomycota. Dominant classes included Dothideomycetes, Sordario- mycetes, Leotiomycetes, Tremellomycetes, Eurotiomycetes and Mortierellomycetes. The pathogens Fusarium and Rosellinia associated with apple root rot had higher relative abundance in the diseased soil than in the healthy soil, so we speculated that Fusarium and Rosellinia maybe were the mainly patho- gens causing apple root rot in this orchard. However, more laboratory separation experiment and patho- genicity detection should be undertaken to validate the speculation. Meanwhile, potential biocontrol agent Trichoderma, Penicillium and Paecilomyces were observed both in healthy and diseased soil. Although we could not clearly judge which agent played a crucial role by high-throughput sequencing, some beneficial clues can be gotten to rapidly find out possible biocontrol fungi.