Diversity and spoilage potential of microbial communities associated with grape sour rot in eastern coastal areas of China

Samples of sour rot-affected grapes

Sour rot-affected grapes infested with fruit flies were collected from vineyards in Yantai (N36°27′, E117°10′), Shandong Province, China. Approximately 1.0 g of rotten tissue was sliced from each sour rot-affected grape (Muscat), and the tissues from 100 sour rot-affected grapes were collected together into one 50-mL sterile centrifuge tube. Three replicates from a total of 300 sour rot-affected grapes were stored at −80 °C for 16S rDNA and ITS high-throughput sequencing. Another three replicates were used for the separation and identification of culturable microorganisms in sour rot-affected grapes.

16S rDNA and ITS high-throughput sequencing analysis

Diversity of culturable microorganisms in sour rot-affected grapes

The above samples with three replicates were suspended in phosphate-buffered saline (PBS, 0.2 M, pH 7.2) at ratios of 1:10³, 1:10⁴ and 1:10⁵. The suspension (200 µL; different concentrations) was spread on nutrient agar medium and potato dextrose agar medium with three replicates each. After culturing at 25 °C for 48 h on nutrient agar and 7 d on potato dextrose agar a total of 80 colonies were picked and restreaked for purity using the primary media.

Spoilage potential assay of cultivated bacteria and fungi

Isolated bacteria and fungi were tested for spoilage potential on grape berries. Healthy grape berries of Midnight Beauty, a susceptible variety, were collected and surface sterilized with 1% sodium hypochlorite (NaClO) solution for one minute. Excess NaClO was removed by washing the berries twice in sterile distilled water. The experimental berries were pricked 2–3 mm deep using a dissecting needle to simulate the wounds made by fruit flies during egg laying or other mechanical damage. The bacterial suspension and fungal spore suspension were prepared with a concentration of approximately 1 × 10⁶ cfu/ml or conidia/ml in the suspension. This suspension (5 µl per berry) was used to inoculate the wounds of healthy grape berries. Sterile water was used instead of the suspension as a negative control. Two methods were used to observe the spoilage potential of bacteria and fungi. In the merged placement method, 10 grape berries were placed in a single Petri dish (10 cm in diameter and three cm in height) to simulate grape clusters in the field. In the separate placement method, each of 10 grape berries was placed in a single culture bottle (2.5 cm in diameter and three cm in height). Three replicates were established for both methods and each bacterium and fungus treatment. Subsequently, the inoculated grape berries were kept in a moisture chamber at 27 °C/25 °C (day/night) and 80% humidity, and symptoms were recorded on the 5th day. The bacterial and fungal species were reisolated from these artificially inoculated grape berries using NA medium and PDA medium, respectively. The resulting culture was compared with the original culture (Hyun et al., 2001).

Based on the ratio of the infected area to the total area, grading was performed as follows (Rouxel et al., 2013; Zhou et al., 2014): 0, no disease spot; 1, less than 5% of the total area infected; 3, 5% to 25% of the total area infected; 5, 25% to 50% of the total area infected; 7, 50% to 75% of the total area infected; 9, 75% to 100% of the total area infected. The incidence (%) and the McKinney index of bacteria and fungi were calculated according to the following formulas:

(1)${\displaystyle \mathrm{The}\text{percentage}\mathrm{of}\text{incidence}\left(\text{\%}\right)=100\ast \frac{\mathrm{the}\text{number}\mathrm{of}\text{diseased}\text{berries}}{\mathrm{the}\text{number}\mathrm{of}\mathrm{all}\text{berries}}}$ (2)${\displaystyle \mathrm{The}\text{McKinney}\text{index}=100\ast \sum _{k=0}^{\mathrm{n}}\frac{kx}{9N}}$

where, x is the value for each grade; n is the number of diseased berries at each level; and N is the total number of fruits investigated.

Sequencing and alpha diversity analyses

The mean lengths of 16S rDNA of bacteria and ITS gene of fungi generated from metagenomic DNA extracted from rotting grapes were 413 ± 3 bp and 279 ± 5 bp, respectively. The OTU numbers for bacteria and fungi were 1,343 ± 283 and 1,039 ± 387 respectively (Table 1). Rarefaction curves of three samples for 16S rDNA and ITS sequences reached asymptotes (Fig. S1), which suggests coverage was sufficient. The number of sequences for each OTU decreased rapidly by the OTU rank of 18 (Fig. S1). The flat curve indicated a high degree of sequencing uniformity.

In a phylogenetic tree of the top 50 bacterial OTUs, 15 OTUs were classified as phylum Firmicutes, class Bacilli. Among the other 35 Proteobacteria, 21 OTUs belonged to the class Alphaproteobacteria, three to Betaproteobacteria, and 11 to Gammaproteobacteria (Fig. S2). In a phylogenetic tree of the top 50 fungal OTUs, one belonged to Basidiomycota and eight were not identified based on searches against the ITS database. Among the other 41 Ascomycota OTUs, 29 belonged to the class Saccharomycetes, nine to Sordariomycetes, and two to Dothideomycetes (Fig. S3).

The microbial diversity, as determined by the Shannon index and Simpson index, was higher for bacteria than fungi, and richness, as determined by the Chao1 index and ACE index, was higher for fungi than bacteria (Table 2).

Microbial taxonomic analysis

Proteobacteria (72%) and Firmicutes (27%) were dominant among the 19 bacteria phyla identified (Fig. 1A). The proportion of other bacteria was less than 1%. The dominant genera in sour rot-affected grapes were Acetobacter (38%), Gluconobacter (24%), Bacillus (12%), and Lactococcus (Fig. 1B).

Ascomycota (94%) was the dominant phylum in the identified fungal community (Fig. 2A). The dominant species identified in sour rot-affected grapes were Issatchenkia terricola (18%), Colletotrichum viniferum (13%), Hanseniaspora vineae (13%), Saprochaete gigas (4%), and Candida diversa (4%), and 32% of isolates were not taxonomically identified (Incertae sedis sp.) (Fig. 2B).

Diversity of culturable microorganisms in sour rot-affected grapes

We cultured 15 bacterial strains from sour rot-affected grapes infested by fruit flies (Table 3). We identified Firmicutes as the dominant phylum (60%), with nine Gram-positive bacteria species (i.e., Staphylococcus saprophyticus, Lactococcus garvieae, Lactobacillus plantarum, two Lysinibacillus species, and four Bacillus species). We also isolated six Gram-negative bacteria species assigned to the phylum Proteobacteria. All bacterial taxa presented positive results in catalase reaction, gelatin, H₂S, and ammonia production assays whereas they presented negative results for fermentation with the methyl red. Strains classified as Cronobacter malonaticus, Cronobacter sakazakii, and Klebsiella pneumoniae presented negative results for these biochemical tests (Table 4).

We cultured ten fungi from sour rot-affected grapes. Five were classified as Deuteromycotina, including Cladosporium oxysporum, Alternaria tenuissima, Geotrichum gigas, Fusarium proliferatum, and Nigrospora sp. (Table 3). C. oxysporum, with bottle-green colonies, developed into conidia by asexual reproduction. A. tenuissima colonies, with a white front side and brown reverse side, developed into conidia in the form of a chain lattice. The hyphae of Saprochaete gigas or Geotrichum gigas, with white colonies, developed into arthrospores by asexual reproduction. F. proliferatum, with red colonies, had branched conidiophores and sickle or long column-shaped conidia. Nigrospora sp. had irregular colonies, branched conidiophores, and ball-shaped conidia. Five species (i.e., Penicillium citrinum, P. georgiense, Aspergillus niger, A. aculeatus, and A. oryzae) belonged to Ascomycotina. The sporophores of P. citrinum and P. georgiense grew from hyphae and developed into brush-like structures. These two Penicillium species differed in colony color. The conidia of A. niger, A. aculeatus, and A. oryzae were black, green, and yellow, respectively (Fig. 3).

Spoilage potential of culturable bacteria and fungi for grape sour rot

All 15 bacterial species and 10 fungal species demonstrated the potential to spoil grapes. In the merged placement method, all of the microorganisms except for B. amyloliquefaciens, caused cracking and infection in grapes (Fig. 4A). In the separate placement method, all of fungi except for Saprochaete gigas, could cause infection in grapes. Obvious symptoms were not detected in the grapes treated by bacterium (Fig. 4B). The bacterial species and the fungal species reisolated from these spoiled grapes using NA medium and PDA medium were confirmed as the original microorganisms summarized in Table 3.

The incidence and McKinney index of 15 bacterial species and 10 fungal species were significantly different from those of the control (sterile water and LB medium) using the merged placement method (incidence: F = 10.44, P < 0.01; McKinney index: F = 43.28, P < 0.01; Fig. 5A and Fig. 5B). Fungal isolates demonstrated stronger spoilage potential in the grape berries with an incidence of more than 75%. Except for C. oxysporum and P. citrinum, the McKinney index of all other fungi exceeded 50%, which was greater than that of bacteria. Three Aspergillus species and P. georgiense showed 100% spoilage on the McKinney index. Healthy grapes were also highly sensitive to A. tenuissima and F. proliferatum, which high McKinney index values of 52 ± 1 and 50 ± 2, respectively. Among the bacteria, the incidence and McKinney index of two Cronobacter species, Serratia marcescens and Lysinibacillus fusiformis, were higher than those of the other bacteria. B. amyloliquefaciens and B. cereus led to less serious spoilage than other bacteria (Fig. 4, then Fig. 5). The percentages of incidence and McKinney index of microorganism were lower using separated infection method than merged method, while those of fungi were also higher than bacteria (Percentage of incidence: F = 90.52, P < 0.01; McKinney index: F = 50.49, P < 0.01; Fig. 5C and Fig. 5D). The C. oxysporum had the highest spoilage potential among microbial taxa, which was different from the results obtained under merged infection.