Isolation and screening of antifungal endophytic actinomycetes
In this study, 122 endophytic actinomycete strains were isolated from healthy and diseased rice segments. Among them, 65.57% strains showed antagonistic activity against M. oryzae, and the frequency of antagonistic endophytes was associated with the tissue source, culture medium, and temperature by statistical analysis (Table 1). More antagonistic actinomycetes appeared from healthy rice stems, and on the TWYE and MS media at 37°C. One isolate, Ahn75 (CCTCC No. M 2019890), which was obtained from a healthy stem on TWYE medium at 37°C, showed an inhibition rate of 53.49% ± 3.38% against M. oryzae by dual culture assay.
Identification of strain Ahn75
Strain Ahn75 grew on different agar media. However, it grew better on ISP2 medium than PDA medium. When it was grown on ISP2 agar medium, the colonies were round with white aerial mycelium and gray-green spores after 2–3 d. The substrate mycelium was white at early cultivation and then turned brown gradually (Fig. 1a).
From 16S rRNA gene sequence analysis using BLAST at NCBI, the strain was classified as belonging to the genus Streptomyces. The phylogenetic tree based on the neighbor-joining method showed that strain Ahn75 had the closest sequence similarity with type strain S. griseobrunneus ATCC 4.1838 (99.48%) (Fig. 1b). To improve taxonomic identification, MLSA of five housekeeping genes (atpD, gyrB, recA, rpoB, and trpB) was used to analyze strain Ahn75. A neighbor-joining tree based on five housekeeping genes suggested that strain Ahn75 was most similar to S. griseobrunneus ATCC 4.1838 and S. bacillaris CGMCC 4.1584 (Fig. 1c), which are known as heterotypic synonyms of the same species according to Rong and Huang (2010). All loci obtained from strain Ahn75 showed 98.93% and 99.05% sequence similarity to S. griseobrunneus ATCC 4.1838 and S. bacillaris CGMCC 4.1584, respectively, and the evolutionary distance calculated with Kimura 2 parameters was below the species definitive MLSA distance of 0.007 , which suggested that Ahn75 is the same species as S. griseobrunneus or S. bacillaris.
Activity of strain Ahn75 against M. oryzae
The mycelial growth of M. oryzae on ISP2 agar medium containing culture filtrate of Ahn75 was inhibited, and the inhibition rate was dependent on the concentration of the culture filtrate (Fig. 2, Fig. 3a). When the concentration of the culture filtrate ranged from 1% to 10%, the inhibition rate of mycelial growth gradually increased from 25.05% to 80.88% with an IC50 value of 2.21% (95% confidence interval, 0.522% to 4.645%).
Meanwhile, the spore germination of M. oryzae was also be inhibited by the culture filtrate of Ahn75 (Fig. 3b). When 50% culture filtrate by volume was used, the inhibition rate of spore germination reached 79.15% ± 7.18%.
Using a phase-contrast microscope, it was observed that the mycelia of M. oryzae became shorter when treated with 2% and 5% culture filtrate of Ahn75, as did the single cell, and part of the cells appeared swollen, distorted, or formed vesicle structures (Fig. 4).
These results indicated that the culture filtrate of Ahn75 can significantly inhibit mycelial growth and spore germination of M. oryzae with concentration dependence, and cause mycelia to be shorter, swollen, or distorted.
Plant growth-promoting features of strain Ahn75
Strain Ahn75 developed small hydrolysis zones around the colonies grown on Pikovskaya’s and Aleksandrov medium plates, indicating phosphate and potassium solubilizing ability. Ahn75 also showed a positive result in nitrogen fixation by growing well on nitrogen-free medium for more than 5 generations. Ahn75 decolored the blue-colored ferric CAS complex to orange with a clear zone surrounding the colony on the CAS plate, which indicated the presence of siderophores (data not shown). These results suggests that Ahn75 can decomposite mineral phosphate and potassium, fix atmospheric nitrogen, and produce siderophores.
Genomic features of strain Ahn75
The draft genome of strain Ahn75, which was deposited at GenBank under accession number JAJQWZ000000000, had a consensus length of 7,550,402 bp assembled by 115 scaffolds. Gene predictions resulted in 6980 open reading frames (ORFs). The function of 6435 ORFs was annotated and 3556 ORFs were classified into 21 COG categories by their function. Among the 21 COG categories, the cluster for “amino acid transport and metabolism” represented the largest group (368, 9.97%), followed by “transcription” (351, 9.51%) and “general function prediction” (314, 8.51%) (Fig. 5).
Through genome mining by the antiSMASH tool, 40 candidate secondary metabolite clusters were predicted in the Ahn75 genome, including three lactones, seven non-ribosomal peptide synthetases (NRPS) (valinomycin, salinomycin, griseoviridin, malacidin, phosphonoglycans and daptomycin), two Nrps-like, three Nrps-pks (diisonitrile antibiotic SF2768, cosmomycin D, and SGR PTMs.), four siderophores (desferrioxamine B, griseobactin, coelichelin, and ficellomycin), eight polyketide antibiotics (bafilomycins, salinomycin, brasilinolide, nonactin, auricin, alkylresorcinol, and herboxidiene), five terpenes (geosmin, hopene, isorenieratene, and stambomycin), six peptides, and two others (Table S1). The total lengths of these gene clusters were estimated to be about 1196 kb, which suggested that 15.84% of the genome may be occupied by genes concerned with the biosynthesis of secondary metabolites, a far higher proportion than that found in other sequenced genomes. Among these secondary metabolites, valinomycin inhibited the mycelia growth of M. oryzae with an inhibition rate of 52.27% ± 4.55% at a concentration of 15 µg·mL−1 in our inhibition assay. Siderophore and bafilomycin have been reported to be active against M. oryzae (Zeng et al. 2018; Zhang et al. 2011), and lactones, Nrps, and polyketide antibiotics were reported to possess excellent antibacterial and antifungal activities (Katz and Baltz 2016; Pimentel-Elardo et al. 2010; Sansinenea and Ortiz 2011). The exhibition of multiple antimicrobial metabolite gene clusters provide a good genetic basis for Ahn75 as a biocontrol resource.
Furthermore, the Ahn75 genome also contains a series of genes/gene clusters associated with plant growth promotion, including siderophore biosynthesis, nitrogen fixation, phosphate and potassium transport, growth-promoting hormones (indole acetic acid (IAA), phytase, trehalose), and spermidine (Table S2). Siderophore can not only inhibit plant pathogens but also promote plant growth by providing iron to the plant. In addition, five nitrogen utilization related genes, nifU coding for the nitrogen fixation protein, glnB coding for nitrogen regulatory protein II, moeA and moaD coding for molybdenum cofactor biosynthesis protein, nir coding for nitrite reductase, four phosphate transporter genes pstA, pstB, pstC, and pstS, and three potassium transporter genes trkA, ktrB, and kdpFABC were found in the Ahn75 genome. IAA synthesis related genes ysnE coding for N-acetyltransferase, dhaS coding for indol 3-acet-aldehyde dehydrogenase, trpC coding for indole-3-glycerol-phosphate synthase, yhcX coding for nitrilase, the phytase synthesis gene phy, trehalose synthesis-related genes, tpsA coding for alpha, alpha-trehalose-phosphate synthase, trePP coding for trehalose 6-phosphate phosphorylase, and treZ coding for malto-oligosyltrehalose trehalohydrolase were also identified in the Ahn75 genome. The genome also contains spermidine synthase gene speE, and agmatinase gene speB, which are also involved in plant growth and shoot differentiation (Chen et al. 2019).
This genetic basis suggests that strain Ahn75 has great potential as a biocontrol and growth-promotion agent in rice cultivation.
Biocontrol and growth-promoting efficacy of strain Ahn75
The biocontrol and growth-promoting efficacy of strain Ahn75 was assessed in pot experiments. The leaf blast disease symptoms detected at the tillering stage showed that leaves treated with Ahn75 developed significantly less lesions than the control leaves treated with water. The majority (64.17%) of plants developed disease lesions after pathogen infection, but only small parts (26.04%) of plants treated with Ahn75 before pathogen infection developed disease lesions, which suggests that Ahn75 can protect rice plants against leaf blast by reducing disease incidence by 59.76% (Table 2). Meanwhile, rice plants treated with Ahn75 before pathogen infection showed the increase in wet and dry weight of grains by 56.33% and 50.65%, respectively, compared to the control plants only infected with fungal pathogens (Table 2).