Effects of ferment fertilizer from waste grape berry on soil bacterial diversity in vineyards

Abstract:【Objective】Due to diseases and pests, climate conditions and management practices, a cer- tain amount of waste grape is produced in the vineyards every year. The disposal of the waste berry has always been one of the problems besetting the farmers, which seriously restricted the clean and efficient production of large-scale vineyards. Producing ferment fertilizer with waste grape is an effective way to solve the problem. Many studies have shown that the application of ferment fertilizer can promote the decomposition and utilization of soil nutrients and improve yield and quality of crops. However, the ef- fects of the application of ferment fertilizer on soil microbial diversity remain unclear. The objective of this study is to evaluate the effects of application of ferment fertilizer from waste grape berry on soil mi- crobial diversity and community structure characteristics in vineyards【. Methods】Illumina MiSeq high- throughput sequencing technology was adopted to evaluate the impact of the application of ferment fer- tilizer made from waste grape on soil microbial diversity and community structure characteristics. CK indicates the grapevines that were applied with traditional fertilizer, while F treatment indicates the grapevines that were applied with ferment fertilizer. Total bacterial genomic DNA from soil samples of CK and F were extracted using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA,USA) and stored at −20 °C prior to further analysis. PCR amplification of V3–V4 region of the bacteri- al 16S rRNA genes was performed using the forward primer 338F (5’-ACTCCTACGGGAGGCAGCA- 3’) and the reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). Thermal cycles consisted of initial denaturation at 98 °C for 2 min, followed by 25 cycles consisting of denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension of 5 min at 72 °C. PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA), and then sequencing was performed using the Illlumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China)【. Results】A total of 568 608 sequences were ob- tained by Illumina MiSeq sequencing, among which 99 757 sequences were obtained in CK and 89 779 sequences were obtained in F. Alpha diversity index showed that the Chao1 and ACE index were signifi- cantly lower in F than in CK, which indicated that the richness of soil bacteria community was signifi- cantly decreased by application of grape waste ferment fertilizer. The Venn analysis showed that the CK and F shared 4 235 OTUs, while CK had 1 271 unique OTUs and F had 1 034 unique OTUs. The com- munity structure composition on phylum level showed that the top 5 phyla in abundance in CK wereProteobacteria (24.67%), Chloroflexi (22.10%), Actinobacteria (16.23%), Acidobacteria (14.98%) andGemmatimonadetes (4.29% ), while the most abundant 5 phyla in F were Proteobacteria (29.73% ),Chloroflexi (17.87% ), Actinobacteria (16.62% ), Acidobacteria (13.52% ) and Gemmatimonadetes(4.54%). The phylum community structure composition was similar between CK and F. However, the abundance distribution was significantly different. The community structure composition on genus level indicated that the top 5 abundant genus in CK were Haliangium (1.96%), RB41 (1.46%), Roseiflexus(1.41%), Streptomyces (1.14%) and 11-24 (1.07%), while the 5 most abundant genus in F were Azoto- bacter (4.03%), Haliangium (1.81%), RB41 (1.87%), Roseiflexus (1.62%) and Streptomyces (1.31%). Significant differences were observed in both community structure composition and abundance distribu- tion between CK and F on genus level. The heatmap analysis on the top 50 genus showed that CK1, CK2 and CK3 samples had high community structure similarity and clustered into one group. F2 and F3 samples clustered into on group with high similarity, and then clustered together with the CK group. F1 samples showed significant difference with other samples. NMDS and PLS-DA analyses were used to group the experimental samples and evaluate the similarity of community structure among different samples. The results indicated that the CK and F groups can be better distinguished according to NMDS2 and PLS-DA1 factors. Metastats analysis was adopted to identify the dominant species in dif- ferent groups. The results showed that 3 phyla and 63 genera were different between CK and F. PhylaBRC1 and Planctomycetes were enriched in CK, while phylum Ignavibacteriae was enriched in F. Among the top 20 different genera, 15 genera were enriched in CK and 5 genera enriched in F.【Conclu- sion】Application of the ferment fertilizer from waste grape showed clear effects on soil microbial diver- sity and community structure characteristics. Significant differences on phylum and genus levels were observed between CK and F groups. The Chao1 and ACE index indicated that application of this fertiliz- er is effective to reduce the soil microbial diversity in vineyard. It also promotes some microbial popula- tions, such as Nitrobacter and Mesorhizobium, which plays a very important role in nitrogen cycle.