Biocontrol Potential of Endophytic Actinobacteria against Fusarium solani, the Causal Agent of Sudden Decline Syndrome on Date Palm in the UAE

Abstract

Thirty-one endophytic streptomycete and non-streptomycete actinobacteria were isolated from healthy date palm root tissues. In vitro screening revealed that the antifungal action of isolate #16 was associated with the production of cell-wall degrading enzymes, whereas with diffusible antifungal metabolites in isolate #28, albeit their production of volatile antifungal compounds. According to the 16S rRNA gene sequencing, isolates #16 and #28 were identified as Streptomyces polychromogenes UAE2 (Sp; GenBank Accession #: OK560620) and Streptomyces coeruleoprunus UAE1 (Sc; OK560621), respectively. The two antagonists were recovered from root tissues until 12 weeks after inoculation, efficiently colonized root cortex and xylem vessels, indicating that the date palm roots are a suitable habitat for these endophytic isolates. At the end of the greenhouse experiments, the development of sudden decline syndrome (SDS) was markedly suppressed by 53% with the application of Sp and 86% with Sc, confirming their potential in disease management. Results showed that the estimated disease severity indices in diseased seedlings were significantly (p < 0.05) reduced from 4.75 (scale of 5) to 2.25 or 0.67 by either Sp or Sc, respectively. In addition, conidial numbers of the pathogen significantly (p < 0.05) dropped by 38% and 76% with Sp and Sc, respectively, compared to infected seedlings with F. solani (control). Thus, the suppression of disease symptoms was superior in seedlings pre-inoculated with S. coeruleoprunus, indicating that the diffusible antifungal metabolites were responsible for F. solani retardation in these plants. This is the first report of actinobacteria naturally existing in date palm tissues acting as microbial antagonists against SDS on date palm.

Keywords: Fusarium solani; Streptomyces; biocontrol; date palm; endophytic actinobacteria; sudden death syndrome.

Figure 1

Total population of potential BCAs in date palm roots. The tested endophytic actinobacterial populations in root tissues of date palm grown under greenhouse conditions were sampled at different time points. Values are means of eight replicates ± SE for each sampling from two independent experiments. Mean values followed by different letters are significantly (p < 0.05) different according to Duncan’s multiple range test. Isolates #16 and #28 represent Streptomyces polychromogenes UAE2 (BCA1) and Streptomyces coeruleoprunus UAE1 (BCA2), respectively. BCA, biological control agent; wpt, weeks post treatment.

Figure 2

Colonization of endophytic BCAs in root tissues of date palm. Light micrograph of semi-thin sections of date palm root tissues (A) non-inoculated control (C; left), or inoculated with either isolate #16 (BCA1; middle) or #28 (BCA2; right) (1000X); (B) spore germination within the root cortex cells inoculated with either isolate #16 (left) or #28 (right) (1000X) and (C) spores of isolate #28 in the root xylem of date palm (1000X). In (A–C), all sections were stained with 0.1% toluidine blue showing the distribution of spores in root tissues (red arrows) and germinating spores within the root cells (yellow arrows) of date palm. Bars: 20 µm. Isolates #16 and #28 represent Streptomyces polychromogenes UAE2 and Streptomyces coeruleoprunus UAE1, respectively. BCA, biological control agent; P, pith, N, nucleus; C, cortex; X, xylem.

Figure 3

Intercellular colonization of date palm root tissues by the endophytic BCAs. Transmission electron micrograph of ultra-thin sections of date palm root tissues of non-inoculated control (C; left; 6000X) and inoculated with isolate #16 (BCA1; middle; 20,500X) or #28 (BCA2; right; 11,500X). All sections were stained with uranyl acetate and lead citrate showing the distribution of spore chains (red arrows). Bars: 2 µm (left); 500 nm (middle); 1 µm (right). Isolates #16 and #28 represent Streptomyces polychromogenes UAE2 and Streptomyces coeruleoprunus UAE1, respectively. BCA, biological control agent; N, nucleus.

Figure 4

Effect of the BCAs on hyphae and cytoplasm of Fusarium solani. Abnormalities observed in the hyphal morphology including septum formation and cytoplasmic contents of F. solani, following exposure to (A) isolate #16 on CCB; or (B) filter-sterilized crude culture filtrate of isolate #28 on FMEB, compared to their corresponding control (C). White arrow indicates cytoplasmic lysis. Yellow and red arrows indicate hyphal non-septate formation and cytoplasmic coagulation, respectively. Light microscopy images were taken at 1000X magnification. Isolates #16 and #28 represent Streptomyces polychromogenes UAE2 (BCA1) and Streptomyces coeruleoprunus UAE1 (BCA2), respectively. BCA, biological control agent; CCB, colloidal chitin broth; FMEB, fish meal extract broth.

Figure 5

Molecular, cultural, and morphological characteristics of Streptomyces coeruleoprunus UAE1. Identification of S. coeruleoprunus UAE1 (BCA2) according to (A) the dendrogram that showing the phylogenetic relationships between S. coeruleoprunus UAE1 (NCBI accession #: OK560621) and other members of Streptomyces spp. based on the 16S rRNA sequences prepared by the neighbor-joining method; (B) light blue aerial mycelium (left) and pink to gray-violet substrate mycelium (right) growing on (ISP medium 3) supplemented with yeast extract, and (C) scanning electron micrograph (5500X) of the straight spore chains (section rectiflexibiles) and smooth-surfaced spores of S. coeruleoprunus UAE1. In (A), numbers at nodes indicate percentage levels of bootstrap support based on a neighbor-joining analysis of 500 resampled datasets. GenBank accession numbers are given in parentheses. BCA, biological control agent.

Figure 6

Preventive application of BCAs on Fusarium solani-inoculated date palm seedlings in the greenhouse. (A) Suppression of Fusarium wilt disease on date palm seedlings cv. Barhi using BCA1 Streptomyces polychromogenes UAE2 (isolate #16) and BCA2 Streptomyces coeruleoprunus UAE1 (isolate #28) at 15 (top) and 35 (bottom) dpt; (B) and recovery of root tissues infected with F. solani and previously inoculated with BCA at 35 dpt. (C) Number of conidia (×10⁴ spores/mL); and (D) disease severity index (DSI) after recovery of the pathogen from affected date palm root tissues (n = 16) treated with BCAs at 35 dpt. In (A–D), seedlings inoculated with F. solani at 5 days after BCA treatments. In (C,D), values (means ± SE of 16 replicates from two independent experiments) with different letters are significantly different from each other at p = 0.05. In (D), DSI was based on a scale of 5:0 = no infection, 1 = 1–10%, 2 = 11–25%, 3 = 26–50%, 4 = 51–75% and 5 = 76–100% damage including necrosis, white area in leaves or rotting in roots. Isolates #16 and #28 represent Streptomyces polychromogenes UAE2 (BCA1) and Streptomyces coeruleoprunus UAE1 (BCA2), respectively. BCA, biological control agent; C, non-inoculated seedling (control); Fs, infected seedlings with F. solani only; Sp or Sc, seedlings inoculated with only S. polychromogenes UAE2 or S. coeruleoprunus UAE1, respectively; Sp + Fs or Sc + Fs, seedlings inoculated with the individual BCA (either S. polychromogenes UAE2 or S. coeruleoprunus UAE1, respectively) 5 days prior to F. solani inoculation; dpt, days post treatment.