Characterization and bioefficacy of grapevine bacterial endophytes against Colletotrichum gloeosporioides causing anthracnose disease

Abstract

Introduction: Grapevine (Vitis vinifera L.), one of the economically important fruit crops cultivated worldwide, harbours diverse endophytic bacteria (EBs) responsible for managing various fungal diseases. Anthracnose (Colletotrichum gloeosporioides) (Penz.) is one of the major constraints in quality grape production and therefore its management is a major concern among the grape growers.

Materials and methods: Among the 50 EBs isolated from healthy leaf segments from the eight grapevine genotypes, biologically potential 20 EBs were purified and identified based on morphological, and biological characteristics and sequence analysis of 16S rRNA region. The antagonistic activities of EBs against Colletotrichum gloeosporioides were studied in vitro conditions.

Results: The colony morphologies of EBs are white and yellow-coloured colonies, circular to irregular in shape, and entire, and flat margins. Among the 20 purified EBs, 19 isolates were found to be Gram-positive except one i.e., MS2 isolate. The 12 isolates reduced nitrate and 14 isolates produced urease enzyme. The in vitro assay revealed that two isolates, SB4 and RF1, inhibited 56.1% and 55.6% mycelial growth of C. gloeosporioides, respectively. Further, the identity of EBs was confirmed through PCR amplification of the 16S rRNA region resulting in ~1400 bp size amplicons. The sequence analysis of representative 15 isolates revealed that 5 EB isolates viz., SB5, CS2, RG1, RF1, C1 were identified as Bacillus subtilis with >99% sequence identity, two EBs viz., SB3, and CS1 were identified as B. subtilis subsp. subtilis, two EBs viz., SB1, and CS4 were identified as B. licheniformis. The SB2 isolate was identified as Bacillus sp., whereas SB4 as Brevibacillus borstelensis, TH1 as B. velezensis, TH2 as B. tequilensis, CS3 as B. pumilus and MS1 as Micrococcus luteus were identified.

Conclusion: The phylogenetic analysis of 16S rRNA sequence revealed eight distinct clades and showed the close clustering of identified species with the reference species retrieved from NCBI GenBank. The current investigation provides the scope for further field evaluations of these endophytic microbes for managing anthracnose disease.

Keywords: Bacillus subtilis; Brevibacillus borstelensis; Micrococcus luteus; biological control; endosymbionts; grapes; molecular characterization.

Figure 1

Colony color and morphological characteristics of 20 bacterial endophytes isolated from leaf segments of eight grapevine genotypes (Vitis vinifera and Vitis rotundifolia) cultivated at experimental fields of the Indian Council of Agricultural Research (ICAR)-National Research Centre for Grapes, Pune, Maharashtra, India. All the bacterial strains were cultivated on nutrient agar (NA) medium for 2 days at 28°C. The details regarding the morphological characterization of all isolates are mentioned in Supplementary Table S1. CS, Cabernet Sauvignon; C, Crimson Seedless; MS, Manjari Shyama; RG, Red Globe; SB, Sauvignon Blanc; S, Shiraz; TH, Thompson Seedless; RF, Vitis rotundifolia.

Figure 2

Antibiotic sensitivity assay of 16 bacterial endophytes isolated from the leaf segments of eight grapevine genotypes cultivated at the Indian Council of Agricultural Research (ICAR)-National Research Centre’s experimental farm.

Figure 3

Antagonistic activity of 15 bacterial endophytes isolated from the leaves of eight grapevine genotypes evaluated against Colletotrichum gloeosporioides causing anthracnose disease in grapevine in India. The antagonistic activity of these fungal endophytes against test pathogens was evaluated and categorized. On the right-hand side is the bacterial colony streaked, and on the left-hand side is the Colletotrichum gloeosporioides test pathogen inoculated. The high antagonist bacterial endophytes showed the restricted growth of the test pathogen and vice versa.

Figure 4

Phylogenetic dendrogram of 15 bacterial endophyte species characterized based on the 16S rRNA gene sequence information indicated in bold letters and reference sequences of respective species retried from the National Center for Biotechnology Information (NCBI) GenBank was inferred using the Neighbor-Joining method. The original tree with the sum of branch length (SBL) = 1.65455554 was observed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. This analysis involved 28 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1,585 positions in the final dataset. The evolutionary analyses were conducted in MEGA11.