*3.7. Biocontrol Potential of Liquid Formulation and Methanol Extract of S. tuirus AR26*

Healthy chilli fruits inoculated with *C. scovillei*, *C. truncatum*, *F. oxysporum* and the coinoculation of three pathogens produced typical fruit rot symptoms in the form of lesions of up to 2.5, 2.2, 2.6 and 2.9 cm, respectively, seven days after inoculation with the pathogens. Fruits that were not inoculated with the pathogens (healthy control) did not develop fruit rot symptoms, indicating that *C. scovillei*, *C. truncatum* and *F. oxysporum* were the causative agent of the anthracnose disease. Chilli fruits inoculated with the liquid formulation of *S. tuirus* AR26 caused no symptoms or damage to the fruits, indicating its non-pathogenic nature and biocontrol ability (Table 3). The liquid formulation of *S. tuirus* AR26 at both the concentrations 5 mL/L and 10 mL/L caused significant reductions in disease symptom when compared to the pathogen-inoculated control. Application of the liquid formulation of *S. tuirus* AR26 at 10 mL/L completely (100%) suppressed the fruit rot lesions caused by *C. truncatum*, *F. oxysporum* and *Cscovillei + C. truncatum + F. oxysporum. C. scovillei* inoculated fruits recorded 87.9% disease reduction with a corresponding lesion size of 0.30 cm when compared to the *C. scovillei* inoculated control (2.48 cm) (Figure 8). The liquid formulation at 5 mL/L concentration reduced the lesion size by 70.85%, 82.68%, 67.32% and 77.08%, respectively for *C. scovillei*, *C. truncatum*, *F. oxysporum* and the co-inoculation of all the three pathogens with corresponding lesion size of 0.73 cm, 0.38 cm, 0.85 cm and 0.63 cm. Irrespective of pathogens, the metabolites in the methanol extract of antagonist also had significant inhibitory effect on the suppression of fruit rot lesions on chilli fruits compared to the pathogen-inoculated control (Table 4). However, the percentage inhibition of the fruit rot lesion by the antagonist metabolites was significantly lower than the active culture formulation of *S. tuirus* AR26. Antagonist metabolites reduced the lesions up to 70.10%, 62.45%, 53.08% and 44.85% caused by *C. truncatum*, *C. scoviellei*, *F. oxysporum* and co-infection of three pathogens, respectively.

**Figure 7.** Phylogenetic tree representing the evolutionary relationships of five potent antagonistic actinobacterial isolates isolated from chilli plants. Neighbour joining (NJ) phylogenetic tree constructed from 16S rRNA sequences shows the position of five potent actinobacterial isolates (highlighted in red) and all isolates belong to the genera *Streptomyces*. Bootstrap values (expressed as percentages of 1000 replications) are shown at the nodes. *Pseudomonas fluorescens* 1-42 (MK88064) was used as an outgroup. GenBank accession numbers are given in parenthesis.


**Table 3.** Antifungal efficacy of liquid formulation of *Streptomyces tuirus* AR26 against chilli fruit rot pathogens.


**Table 3.** *Cont.*

The values are the mean of three replications. The means in a column followed by the same superscript letters are not significantly different at *p* = 0.05. Values in parenthesis are arc sine transformed.

**Figure 8.** *Cont*.

**Figure 8.** Antifungal efficacy of liquid formulation and methanol extract of *S. tuirus* AR26 against chilli fruit rot pathogens. (**A**) Fruits inoculated with sterile distilled water; (**B**) fruits inoculated with methanol alone; (**C**) fruits inoculated with liquid formulation of *S. tuirus* AR26 at 10 mL/L; (**D**) fruits inoculated with *C. scovillei* and methanol extract of *S. tuirus* AR26; (**E**) fruits inoculated with *C. scovillei* alone; (**F**) fruits inoculated with *C. scovillei* and liquid formulation of *S. tuirus* AR26 at 10 mL/L; (**G**) fruits inoculated with *C. truncatum* and methanol extract of *S. tuirus* AR26; (**H**) fruits inoculated with *C. truncatum* alone; (**I**) fruits inoculated with *C. truncatum* and liquid formulation of *S. tuirus* AR26 at 10 mL/L; (**J**) fruits inoculated with *F. oxysporum* and methanol extract of *S. tuirus* AR26; (**K**) fruits inoculated with *F.oxysporum* alone; (**L**) fruits inoculated with *F. oxysporum* and liquid formulation of *S. tuirus* AR26 at 10 mL/L AR26; (**M**) fruits inoculated with *C. scovillei*, *C. truncatum*, *F. oxysporum* and methanol extract of *S. tuirus* AR26; (**N**) fruits inoculated with *C. scovillei*, *C. truncatum*, *F. oxysporum* alone; (**O**) fruits inoculated with *C. scovillei*, *C. truncatum*, *F. oxysporum* and liquid formulation of *S. tuirus* AR26 at 10 mL/L.

**Table 4.** In vivo antifungal efficacy of methanol extract of *S. tuirus* AR26 against chilli fruit rot pathogens.



**Table 4.** *Cont.*

The values are the mean of three replications. The means in a column followed by same superscript letters are not significantly different at *p* = 0.05. Values in parenthesis are arc sine transformed.

#### **4. Discussion**

The use of synthetic fungicides is a common practice among farmers for many decades for the management of chilli fruit rot disease; however, this can cause several ill effects to the environment and living creatures. The most urgent and necessary activity of human society is to eliminate the use of fungicides in food crops [66] like chilli which are directly consumed by people. Hence, protecting crops with safe biocontrol agents will not only address concerns about fungicide residues in fresh and processed products, but also increase the export value of fungicide-free food products in domestic as well as world markets. Furthermore, antagonistic microbe–plant interactions reduce the dependence on chemical pesticides by upto 20% [67]. Native actinobacteria adopting a dual role as a biocontrol agent and biofertilizer is a more sustainable, promising and versatile candidate towards the eco-friendly management of plant disease with multiple benefits to society and the ecosystem as a whole.

Hence, in the present study, around 52 actinobacterial isolates were screened for their antagonistic activity against the chilli fruit rot pathogens. Among which, six isolates AR1, AR10, AR26, AL5, AL7 and AFE2 exerted strong antifungal activity against all three pathogens with an inhibition zone of >2 cm and belonged to the genus *Streptomyces*. It was evidenced from previous literature that several species of *Streptomyces* have emerged as biocontrol agents that are safe alternatives to synthetic fungicides for the management of phytopathogens [68,69]. There is ample scientific evidence indicating the successful interaction of various *Streptomyces* spp. with chilli plants to curtail the infection of fruit rot pathogens both at pre- and post-harvest levels. Shahbazi et al. [70] reported that *Streptomyces rochei* strain P42 displayed the highest inhibitory activity against *C. acutatum*, *C. capsici* and *C. gloeosporioides. S. griseocarneus* R132 inhibited the development of anthracnose symptom in chilli fruits [47], and likewise the application of *S. violaceoruber* fermentation broth reduced the incidence of the chilli anthracnose under greenhouse conditions [30].

The results of the present study also revealed that a higher proportion of native rhizospheric actinobacteria exert strong antagonistic activity against *C. scovillei* and *C. truncatum* compared to phyllospheric and endophytic isolates. Similar results were also highlighted by Shahbazi et al. [70], who reported that out of 66 native rhizosphere strains of streptomycetes, 16 strains showed very strong to moderate inhibition against *C. acutatum*, *C. capsici* and *C. gloeosporioides.* Many researchers have reported that diverse species of actinobacteria are recognized to play a crucial function in the rhizosphere by suppressing pathogenic species, as well as promoting the growth and multiplication of beneficial microbes. *Streptomyces* is one of the most dominant and promising biocontrol bacterial genera of plant diseases which efficiently colonise the plant rhizosphere and are known to produce over two-third of antibiotics with the ability to inhibit a wide range of phytopathogens [71,72]. Hyder et al. [73] stated that eight native rhizospheric bacterial isolates obtained from chilli plants were

found to exert antifungal activity against damping pathogen *Phytophthora capsici* in vitro and in vivo.

Based on the dual culture and paired antibiosis assay, the actinobacterial isolate AR26 obtained from chilli rhizosphere, which was subsequently identified as *Streptomyces tuirus*, was found to be the most effective isolate in inhibiting the mycelial growth of all the three tested pathogens. This finding is in accordance with the results of Chaudhry [74] who reported that *S. tuirus* strongly inhibited carrot cavity spot pathogen *Pythium violae* including various other pathogens such as *Phytophthora spinosum*, *Phytopythium helicoides*, *Fusarium oxysporum*, *Fusarium falciforme*, *Fusarium solani*, *Sclerotium rolfsii*, and *Sclerotinia sclerotiorum*. Scanning electron micrographs of the interaction of *S. tuirus* with fruit rot pathogens in the dual culture plate revealed mycelial deformities like shrinkage, distortion and aggregation of *C. scovillei*, *C. truncatum* and *F. oxysporum* hyphae, in contrast to dense, smooth and regular mycelium in the control plate. Xu et al. [75] also observed severe morphological and internal abnormalities such as the shrinkage and aggregation of *Magnaporthe oryzae* hyphae when treated with the culture filtrate of rice endophyte *Streptomyces hygroscopicus* OsiSh-2.

*S. tuirus* also exhibited positive results for most of the antifungal bioassays under study. It is the most potent antagonist to produce almost all the tested extracellular hydrolytic enzymes, most prominently cellulase and chitinase which are reported to be the important hydrolytic enzymes responsible for the biocontrol ability of an antagonist. High chitinase-producing strains are more antagonistic to fruit-rotting pathogens compared to low-chitinase-producing strains [76]. Jha and Modi [77] and Bhattacharyya et al. [78] pointed out that the genus *Streptomyces* is an efficient producer of various lytic enzymes, which plays an important role in the biological control of plant diseases by degrading the cell wall of phytopathogenic fungi made up of chitins and glucans. It is also evident from the earlier reports that *Streptomyces* spp. are significantly responsible for the suppression of plant diseases through the production of chitinase, glucanase [40] and protease [79]. Shahbazi et al. [70] stated that the production of hydrolytic enzymes, especially chitinases, can be considered as a potential antagonistic mechanism against chilli anthracnose pathogens. Therefore, the production of these enzymes will help to select potential actinobacterial isolates for the biological control of the tested pathogens.

*S. tuirus* AR26 is also a highly efficient synthesizer of siderophore which is considered to be one of the most important mechanisms for the biocontrol of plant pathogens [80], in which the antagonist inhibits pathogen growth by depriving it of the available iron in the environment [81]. Hence, it is possible that the siderophore-producing ability of *S. tuirus* AR26 might also have contributed to the suppression of mycelial growth of all the tested pathogens. It is similar to the finding of Liotti et al. [47] who reported the possible role of siderophore of *S. griseocarneus* R132 in the biocontrol of *F. oxysporum* in chilli.

Volatile, non-volatile and thermostable compounds of the *S. tuirus* isolate AR26 also reported significant antifungal activity, particularly volatile organic compounds, which recorded the maximum antifungal activity against the fruit rot pathogens. Many *Streptomyces* spp. were reported to produce various volatile compounds that were effective against the anthracnose disease in various crops [82]. The volatile compounds from *Streptomyces philanthi* RM-1-138 and *Streptomyces* spp. are highly potent for the biocontrol of chili anthracnose caused by *C. gloeosporioides* PSU-NY8 [14] and cucumber anthracnose caused by *C. orbiculare* [83] respectively in the post- harvest pathosystem. Metabolites produced by *Streptomyces* include bioactive compounds such as macrolide, benzoquinones, aminoglycosides, polyenes, and nucleoside antibiotics that are involved in the suppression of various phytopathogens [84,85].

The results of detached fruit assay revealed that application of active antagonists in the form of a liquid bio-formulation was found to be most effective against all the three pathogens compared to methanol extracts. The active culture of the antagonist *S. tuirus* AR26 in the liquid bio formulation caused a significant reduction in the expression of fruit rot symptom, ranging from 87.9% to as high as 100%. It completely suppressed the expression of symptoms caused by *C. truncatum*, *F. oxysporum* and *C. scovillei* + *C. truncatum*, *F. oxysporum* in chilli fruits, which is approximately 30%, 50% and 55% higher than the suppression by the methanol extract. Our finding is in line with the research findings of Sadeghian et al. [50] who also reported that active antagonists as practical formulations seem more effective compared to crude extracts against the bitter rot of apple fruits caused by *C. gloeosporioides*.

Therefore, the inhibition of fruit rot pathogens observed in this study might be due to the antagonistic potential of *S. tuirus* AR26 through the production of antifungal compounds, siderophores, chitinase or through the synergistic action of all these mechanisms. It has been documented in earlier findings that the antifungal ability of actinobacteria might be due to the synergistic activity of two or more antagonistic mechanisms. Furthermore, Evangelista-Martínez [86] also reported that the *Streptomyces* sp. CACIA-1.46HGO strain inhibited the hyphal growth of many fungal plant pathogens by the production of secondary metabolites, extracellular enzymes and probably by the combined effect of these mechanisms. It is well understood from the findings of Yasmin et al. [87] who reported that the antagonistic activity of *Pseudomonas* spp. E227, E233, Rh323, *Serratia* sp. Rh269 and *Bacillus* sp. might be due to the production of siderophores, lytic enzymes and HCN or the synergistic interaction of these two or with other metabolites.

#### **5. Conclusions**

The management of chilli fruit rot disease still continues to be the focus of intensive research. Though there are several ways of managing this disease, none of the methods were found to be completely successful when applied alone. Hence a preliminary attempt was made to screen the antifungal activity of native actinobacteria against fruit rot pathogens under in vitro conditions. Current results confirmed the potentiality of native actinobacterial isolate *S.tuirus* AR26 to be exploited as a biointensive component under an integrated disease management strategy. The actinobacteria *S. tuirus* AR26 exhibited multifarious biocontrol mechanisms such as the production of volatile, non-volatile and thermostable compounds, competition for iron through the synthesis of siderophores, and production of extracellular lytic enzymes such as chitinase and cellulases. Hence, *S. tuirus* AR26 has a great scope for evaluating its biocontrol potential against chilli fruit rot disease under field conditions as well against a broad spectrum of post-harvest plant pathogens. Larger investigations in the future will demonstrate such possibilities. As farmers become increasingly aware of the concept of sustainable agriculture and organic farming, use of this actinobacteria based bio-formulation will definitely address concerns about ecologically sustainable and socially acceptable long-term solutions to tackle notorious fruit rot pathogens.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/life13020426/s1, Figure S1: Paired antibiosis assay for antifungal activity of actinobacterial isolates against chilli fruit rot pathogens; Figure S2: Relative ability of actinobacterial isolates to produce siderophore and extracellular hydrolytic enzymes; Figure S3: Screening of actinobacterial isolates for amylase activity; Figure S4: Screening of actinobacterial isolates for cellulase activity; Figure S5: Screening of actinobacterial isolates for chitinase activity; Figure S6: Screening of actinobacterial isolates for protease activity; Figure S7: Screening of actinobacterial isolates for siderophore production; Table S1: In vitro antifungal activity of actinobacterial isolates against chilli fruit rot pathogens.

**Author Contributions:** Conceptualization, R.R., K.P., L.R., R.A., L.P., T.R. and G.K.; methodology, R.R., L.R., K.P., R.A. and L.P.; validation, R.R., K.P., L.R. and R.A.; formal analysis, R.R.; investigation, R.R.; K.P. and L.R.; resources, K.P., L.R., R.A., L.P., T.R. and G.K.; data curation, R.R.; writing—original draft preparation, R.R.; writing—review and editing, R.R., K.P. and R.A.; visualization, R.R., K.P. and L.R.; supervision, K.P.; project administration, K.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The dataset supporting the conclusions of this article are included within the article and its additional files.

**Conflicts of Interest:** The authors declare no conflict of interest.
