ANTAGONIST ACTIVITY OF Streptomyces sp. Y20 AGAINST FUNGI CAUSING DISEASES IN PLANTS AND FRUITS †

Background : Crop microbial pathogens reduce the production and quality of agricultural products. They cause substantial increase costs for producers of fruits, vegetables, and ornamental plants with negative consequences on economy and food security at household, national and global levels. Annually, the losses represent around 40% to 50% for root crops, vegetables, and fruits. Chemical control with fungicides can prevent, kill, mitigate, or inhibit the growth of plant pathogenic fungi. Nevertheless, biological control with microorganisms and natural molecules is an increasingly popular alternative to protect crops. Objective: Here, the antagonist activity of soil Streptomyces sp. Y20 against the pathogenic fungi causing diseases in plants and fruits was evaluated. Methodology : Streptomycetes bacteria was isolated from soils collected at open field cultures of local farms with tomato. The antagonism was evaluated in vitro via a dual confrontation experiment against fungal species of Fusarium , Lasiodiplodia , Colletotrichum , Aspergillus , Botrytis, and Sclerotium . Streptomyces sp. Y20 was characterized phenotypically and molecularly identified by the 16S rDNA gene. The biosynthetic gene clusters for polyketide synthases (PKS Type I) and non-ribosomal peptide synthase (NSPS) were detected. Results : Preliminary, the isolate Y20 was selected by the higher antagonism against F. oxysporum f sp. lycopersici . Taxonomic characterization of the isolate Y20 by the analysis of the 16S rDNA sequence led to its identification as member of Streptomyces genus. Spore surface morphology by Scanning Electronic Microscopy (SEM) showed barrel-like spores. Antagonistic activity of Streptomyces sp Y20 was comparable to the commercial strain S. lydicus WYEC108 (P > 0.5). However, there was a superior antagonism of Y20 strain versus the commercial strain WYEC108 against F. oxysporum f sp. lycopersici , Fusarium sp. CDBB1172, F. oxysporum , Lasiodiplodia sp., and Aspergillus sp. (P < 0.05). Implications: Soil streptomycetes with in vitro antagonistic activity on plant pathogenic fungi could be a natural alternative to the use of chemical fungicides to control plant diseases. Conclusion : This study presented a novel soil Streptomyces specie which showed in vitro antagonism against a diversity of plant pathogenic fungal species. Streptomyces strain Y20 could be used as a biocontrol agent.


INTRODUCTION
To satisfy the world demand for food in a sustainable way, it is essential to ensure agricultural production without risks to the environment and people. The pests and diseases that affect crops are a factor that puts food security at risk because they damage crops and reduce the availability and access to food (FAO, 2017). Phytopathogenic fungi are microorganisms that every year put agricultural production at risk. They can destroy up to a third of the annual production (Almeida et al., 2019). Approximately, $220 billion per year is the global economic cost of plant diseases, with 20-40% of crop production lost to pests (FAO, 2019). Synthetic fungicides have long been used to control fungi, but these compounds also cause environmental pollution, damage human and animal health, and suffer from the development of resistant strains (Moshi and Matoju, 2017;Akram et al., 2018).
Antagonistic bacteria and their metabolites can be an alternative to chemical fungicides in the manage of fungal diseases in the control of postharvest decay (Syed-Ab-Rahman et al., 2018). Several mechanisms are involved in how microorganisms can act against phytopathogens such as parasitism, cross protection, antibiosis, and competition (Shoda, 2000). Bacteria of the genus Streptomyces have been widely recognized for their known ability to produce fungicides, antibiotics, extracellular hydrolytic enzymes (lipases, amylases, proteases, chitinases, glucanases, xylanases), and other bioactive compounds that inhibit the growth of phytopathogenic microorganisms in a natural and safe way (Evangelista-Martínez et al., 2017;Chen et al., 2018). The biopesticide market has commercial products based on Streptomyces species such as Streptomyces lydicus WYEC108 and Streptomyces griseoviridis K61 (Lahdenperä, 1987;Yuan and Crawford, 1995). Streptomyces lydicus WYEC108 on fungal species of the genera Fusarium, Rhizoctonia, Pythium, Phytophthora, Botrytis, and Sclerotinia. Streptomyces griseoviridis K61 inhibits the growth of Fusarium, Phytophthora, Alternaria, Pythium, Rhizoctonia, and Botrytis.
Sustainable agriculture is a useful strategy for biological management of plant diseases and provides fruits and vegetables free of synthetic pesticides (Di Francesco et al., 2016). Therefore, the objective of the study was to evaluate the antagonistic activity of soil Streptomycetes against phytopathogenic fungi that affect plants and fruits in their post-harvest stage.

Soil sampling and streptomycetes isolation
Soil samples were collected from open field cultures of local farms with tomato var. Pony Express located at Santo Domingo, Oxkutzcab,México (20°11'12.444'' N 89°31'28.415'' W). All samples were collected with an auger by drilling down to a 10 cm depth. They were subsequently placed in presterilized plastic bags and were processed after 36 h.
The Streptomycete isolation and colony selection was based on the typical morphological features and was performed as described previously (Evangelista-Martínez, 2014b). Repeated streaking of a spore sample used a toothpick from single colonies onto fresh international Streptomyces Project media 2 (ISP 2) agar plates; this step produced pure isolates. A suspension of spores or mycelium cells stored at -20 °C in 20% (w/v) glycerol was used to prepare a working general inoculum (GI) with a turbidity of 0.5 on the McFarland standard.

Molecular identification
The genomic DNA was purified from a spore suspension using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich). The 16S rRNA gene amplification and sequencing analysis were performed using the universal oligonucleotides fD1 (5′-CCGAATTCGTCGACAACAGAGTTT GATCCTGGCTCAG-3′) and rD1 (5′-CCCGGGATCCAAGCTTAAGGAGGTGATCCAG CC-3′) (Weisburg et al., 1991). The PCR fragments were amplified as established by Evangelista-Martínez (2014a) using the GoTaq Hot Start Polymerase (Promega). Direct sequencing of both DNA strands was determined at Macrogen (Seoul, Korea). Sequences were assembled and trimmed using CLC Main Workbench 6 (CLC Bio). The sequencing data were BLAST analyzed using the non-redundant GeneBank database (http://www.ncbi.nlm.nih.gov/). Phylogenetic analysis was performed at Phylogeny.fr (http://www.phylogeny.fr). Multiple alignments were generated using the ClustalW (v 2.1); poorly aligned positions and divergent regions were removed with Gblocks (v 0.91b). A phylogenetic tree based on the neighbor-joining method was constructed under Kimura's two-parameter model. Bootstrap confidence analysis was carried out with 1000 replications. Partial sequences of 16S rDNA gene of Streptomyces sp. strain Y20 was deposited in the GenBank database under accession MW485004.

Characterization of Streptomyces sp. Y20
The phenotypic features of the Y20 isolate were evaluated based on Shirling and Gottlieb (1966) with slight modifications. To evaluate the phenotypic characteristics of Y20 isolate, 2 μl of GI were inoculated in different media: ISP 2 for colony differentiation and color, ISP 7 for melanin production, and IPS 9 for hydrolysis test of complex substrates. The RAL color chart was used for coloring description. To evaluate the growth characteristics of Y20 isolate, 2 μl of GI suspension were inoculated in Petri plates containing different culture agar media: ISP 2, ISP2 added with 0.5% (w/v) pancreatic digest casein, ISP 9, ISP 9 with 0.5% (w/v) pancreatic digest casein, nutrient agar (NA), tryptone yeast extract agar (TYA), Czapek-Dox agar (CDA), King B agar (KB), and PDA. All media were incubated for 14 days at 29 °C; substrate and aerial mycelium growth as well as spore production were determined.
Antibiotic susceptibility by the disk diffusion method was performed as stated in Evangelista-Martínez et al. (2020). An antibiotic multidisc for Gram-positive bacteria II (Bio-Rad®, Hercule, CA, USA) was used by triplicate. The inhibitory halo diameter was measured with a caliper.

Scanning electron microscopy
An agar block of 10 × 10 mm with an active mycelium growth of a 14-days culture of Y20 isolate was placed into an empty Petri plate. It was then sealed with 3M Micropore surgical tape and kept at 4 °C. After eight days, the agar pieces were analyzed using a scanning electronic microscope EVO-50 (Carl Zeiss) at the Facultad de Ciencias de la Universidad Autónoma de Querétaro, México.

Antagonistic evaluation
A dual confrontation assay was used to evaluate the antagonistic activity of the Streptomycete isolates on the growth of phytopathogenic fungi (Bredholdt et al., 2007). An initial selection of antagonistic Streptomycetes on the fungal pathogen F. oxysporum f. sp. lycopersici was implemented. An inoculum of Streptomycetes spores after 15 days of growth was performed with a toothpick and dispersed into a square area of 7 × 14 mm, 1.0 cm from the edge of the ISP 2 agar plates. Spores of the reference strain S. lydicus WYEC108 were inoculated at the opposite side of the plate. Thereafter, an agar plug (9 mm diameter) covered with mycelium of a 10-12 days culture of the fungus was placed at the center of the plate and maintained in an incubator at 29 °C. The growth controls consisted of fungus disk in ISP 2 in the absence of Streptomycete isolates. All experiments were performed in triplicate. Measurements were made with a caliper when the radial growth of the fungi colonies grow near the edge in the growth control Petri plates. The percentage of inhibition (PI) was calculated with the formula: PI (%) = (FR − AR)/FR × 100, as described in Evangelista-Martínez et al. (2020); FR, represents the radial growth of the fungus (mm) of a control culture, and AR represents the radial growth (mm) in the direction of the Streptomycete.
Subsequent in vitro assays to determine the potential biocontrol activity of the selected Streptomyces isolates against diverse plant fungal pathogens were performed. All measurements were conducted in triplicate.

Data analysis
The PI is expressed as means ± standard deviation (SD). The means were compared using an one-way analysis of variance (ANOVA) followed by the Tukey test (P = 0.05). The statistical analyses were performed with the MiniTab v18 program (Minitab, LLC).

Isolation and preliminary selection of antagonistic streptomycetes
A total of 46 Streptomycete-like strains were isolated and preserved at the Actinomycetes Germplasm Bank at CIATEJ. All strains were evaluated for their ability to inhibit the growth of Fusarium oxysporum f. sp. lycopersici. The results showed that isolates Y20 and Y44 antagonized the fungal pathogen at a PI of 49.2% and 27.1%, respectively. Further antagonistic evaluations over other fungal phytopathogens were conducted for the Y20 isolate.

Molecular characterization
The analysis of the partial 16S rRNA gene sequence (1463 bp) from the isolate Y20 revealed a close relation to other sequences belonged to the Streptomyces genus. The phylogenetic tree showed that strain Streptomyces sp. Y20 is related to species with antifungal activity and that produce antimicrobial metabolites (Figure 1). The endophytic bacteria S. cavourensis produce antifungal metabolites such as flavensomycin, humidin, and bafilomycin; bafilomycin B1 and C1 inhibited the mycelial growth of Fusarium spp, Rhizoctonia solani, and Botrytis cinerea (Skarbek and Brady, 1978;Pan et al., 2015). Streptomyces californicus produces borrelidin-an antibacterial and antifungal metabolite that inhibited F. oxysporum and Aspergillus species (Saisivam et al., 2008). A cell-free ferment filtrate produced by S. pratensis inhibited the mycelia growth of Botrytis cinerea and diminished the lesion expansion of the mold infection on detached leaves and postharvest fruits (Lian et al., 2017). Moreover, there was antagonism and significant inhibition effects on the wheat scab pathogen F. graminearum by Streptomyces pratensis S10. S. pratensis had control effects on fungal pathogens in the plot experiments (Zhang et al., 2020). Molecular identification was confirmed by its phenotypic features.

Morphological and physiological characterization
Morphologically, the colonies of Streptomyces sp. Y20 have a distinctly dusty appearance and a light-ivory substrate mycelium when grown on ISP 2 agar media. There is white to cream-colored aerial mycelia and a cream-colored spore mass. Some physiological features were also observed (Table 1).
Microscopic observation by SEM showed that aerial hyphae morphology was smooth and flexous with a rectiflexible spore chain type with segmented barrellike spore chains of the hyphae (Li et al., 2016). (Figure 2).

Antagonistic activity of Streptomyces sp. Y20
The antagonistic activities of Streptomyces sp Y20 against fungal phytopathogens are shown in Table 2.

Detection of biosynthetic gene clusters of secondary metabolites
PCR detection of the biosynthetic clusters of genes involved in the production of specialized secondary metabolites showed DNA fragments corresponding to ~1400 bp for PKS-I and two amplified fragments of ~ 700 bp and ~ 800 bp for NRPS. No biosynthetic genes for PKS type II were detected for Y20. Streptomycetes are a group of bacterium widely recognized as bioactive metabolites producers; some species contain in their genome more than 20 biosynthetic gene clusters for secondary metabolites (Challis and Hopwood, 2003); for instance, 22 secondary metabolite-producing gene clusters in S. yeochonensis CN732 have been identified (Malik et al., 2020). Several approaches have suggested that Streptomyces genus might produce over 100,000 antimicrobial metabolites. This is a high number of compounds relative to the small percentage that has been identified (Watve et al., 2001). These results suggest that Streptomyces sp. Y20 could produce antifungal compounds.

CONCLUSION
Biological control of fungal pathogens with microorganisms is a natural alternative to the use of chemical fungicides in the crop fields. Streptomyces sp. Y20 has a wide antagonistic and inhibitory capacity and can inhibit the phytopathogenic fungi that affect horticultural crops. This strain is antagonistic to the growth of Fusarium and Colletotrichum species that