Inuences of Culture Media, Temperature and Light/Dark Conditions on Growth and Antifungal Activity of Streptomyces spp. Against Botrytis cinerea, in vitro and on Tomato Leaf

The inuences of culture media, temperature, and light/dark conditions on growth and antifungal activity of the three strains Streptomyces spp. against Botrytis cinerea was studied. Results of in vitro study indicated that the GYM agar and incubated at 28°C exhibited good mycelial growth and a spore mass production of the three strains of Streptomyces spp. On the other hand, the PDA and incubation at 21°C were suitable for the mycelial growth of B. cinerea . Moreover, light/dark conditions had an effect on the growth of the two strains of S. philanthi . The strains RL-1-178 and RM-1-138 of S. philanthi grown in all media and incubation temperatures tested possessed antifungal activity against B. cinerea (100% inhibition) while S. mycarofaciens showed different results on PDA (83% inhibition) and GYM (88% inhibition) with the optimum incubation temperature at 21°C. Then, the antifungal compounds in culture ltrates produced by the three antagonistic strains against B. cinerea were tested on tomato leaf. They showed a signicantly higher inhibitory effect on the symptoms of blight disease on tomato leaf compared with the control. The better protection ecacy against B. cinerea on tomato leaf was observed with the culture ltrates of S. philanthi RM-1-138 (82.89% and 0.33 cm 2 lesion areas symptoms). Moreover, the antifungal compounds in the culture ltrate of S. philanthi RM-1-138, identied by GC-MS, were greatly altered relative to concentration components under different temperatures and light/dark conditions tested. Our results clearly demonstrated that the environmental factors have an inuence on antifungal activity of the three strains of Streptomyces spp.


Introduction
The necrotrophic fungi Botrytis cinerea Pers. [teleomorph Botryotiniafuckeliana (de Bary) Whetzel] is among the world's most dangerous fungal pathogens that caused economically important diseases in numerous fruits and vegetables (Kamensky et al. 2003;Elad et al. 2015). Biocontrol is an environmentally friendly and e cient alternative to chemical fungicide management of these pathogens (Chen et al. 2016;Kamil et al. 2018). Species of Streptomyces are potential biocontrol agents since they are ubiquitous in the environment and many of them produce secondary metabolites such as enzyme inhibitors and antibiotics with diverse biological activities, including the ability to inhibit plant pathogenic fungi (VazJauri et al. 2016). Several species of Streptomyces have been isolated and used to control plant pathogens in various crops, such as B. cinerea (Ge et al. 2015;Kim et al. 2015;Bi and Yu 2016;Boukaew et al. 2017;. The in uence of culture media and temperatures on the growth of antagonistic Streptomyces sp. and microbial plant pathogens are important parameters. The temperature is known to have profound in uences on the growth and production of bioactive compounds in Streptomyces species (Srinivasan et al. 1991) and disease development of pathogen B. cinerea. Several studies have shown that the growth of Streptomyces species is dependent on the culture medium and temperature (Kontro et al. 2005;Rashada et al. 2015). The medium containing 3 % starch, 0.75% peptone, 0.025% yeast extract, 1% (w/v) soybean meal and minor/trace elements, initial pH of 6.5, and incubation at 28°C was the best condition for growth and antifungal activity of S. platensis 3-10 against Plasmodiophora brassicae (Shakeel et al. 2015). GYM, containing 0.4% glucose, 0.4% yeast extract, and 1.0% malt extract, with an initial pH of 7.5 and a temperature of 30°C were found to be optimum for both cell growth and antifungal activity of S. philanthi RM-1-138 against Rhizoctonia solani (Boukaew and Prasertsan 2014). The medium consists of 3.0% glucose, 3.5% corn starch, 2.5% soybean meal, 1.2 mM MgCl 2 , and 5.9 mM glutamate, and a temperature of 28°C was found to be the best condition for growth and antifungal activity of S. rimosus AG-P1441 against Phytophthora capsici (Ju et al. 2015). Soyabean meal medium has in uenced the production of antibacterial compounds of Streptomyces sp. (Khattab et al. 2016).
B. cinerea requires several speci c growth conditions for growth and reproduction. It is well known that growth and sporulation are important phases during the life of fungi, which is considerably in uenced by external growth factors (Mishra and Tripathi 2015). Among the external growth factors, nutrition was one of the determinants that were previously proved by several workers in many fungal pathogens using different culture media sources (Kasar et al. 2004;Rani and Murthy 2004;Younis et al. 2004;Kim et al. 2005;Zhao et al. 2010;Lazarotto et al. 2013). Potato dextrose agar and incubation, ranging from 18 to 22°C were the best conditions for the growth of B. cinerea (Whipps 1987;Aqueveque et al. 2017;Boukaew et al. 2017;Meng et al. 2020).
Light conditions play a signi cant role in the growth of Streptomyces spp. and pigment production (Koyama et al. 1976;Takano et al. 2015;Khattab et al. 2016) and during disease development and pathogenicity of B. cinerea (Fillinger and Elad 2016;Zhang et al. 2016;Cheng et al. 2019;Meng et al. 2020). In Streptomyces, carotenoid production is a widespread metabolic activity, which occurs in a constitutive, light-dependent, or cryptic manner (Koyama et al. 1976). Koyama et al. (1975) found that light could enhance pigment production in the growing mycelia of S. phaeopurpureus 5125, S. salmonicida 5472, and S. fulvissimus more than cultivated under dark conditions. The pigmented antibiotics of S. coelicolor A3 are produced under light induction (Takano et al. 2005). Light is known to affect the population of several fungi and the circadian clock of one fungal phytopathogen has been linked to the pathogen's virulence programme (Hevia et al. 2015). B. cinerea is a light-responsive strain that actively senses light conditions to ne-tune its development and pathogenicity (Zhang et al. 2016). Light is an essential developmental signal for the B. cinerea as it triggers the exclusive formation of conidia, whereas constant darkness initiates the sole formation of sclerotia (Fillinger and Elad 2016). Zhang et al. (2016) reported that B. cinerea could produce conidia production in dark conditions more than in light conditions. Under these circumstances, the present study aimed to study the growth conditions of antagonistic bacteria and pathogenic fungi in different growing conditions. The objectives of this study are (i) to study the in uences of cultures media, temperature, and light/dark conditions on the growth of the three strains of antagonistic bacteria Streptomyces spp. and pathogenic fungi of B. cinerea, (ii) to evaluate the inhibitory ability of the three strains of Streptomyces spp. on the growth of B. cinerea in vitro and in tomato leaf and (iii) to identify the chemical composition of the antifungal compounds of the selected strain of Streptomyces spp. grown under different temperatures and light/dark conditions by GC-MS.

Microorganisms
The antagonistic strain SS-2-243 of Streptomyces mycarofaciens and the strains RM-1-138 and RL-1-178 of S. philanthi were isolated from the rhizosphere of chili peppers in southern Thailand (Boukaew et al. 2011) and kept on 20% glycerol at -20°C as a stock culture. They were sub-cultured freshly on glucose yeast-malt agar (GYM) and incubated at 28°C for 10 days before use in this study. Spore suspensions of each strain of Streptomyces spp. were prepared by removing the spores from a 10-day-old culture and suspending them in sterile distilled water, then adjusting the spore concentration with sterile distilled water to 10 7 spores per milliliter using a hemocytometer.
The pathogen strain Botrytis cinerea was isolated from infected tomatoes, showing typical gray mold symptoms. The fungal strains were grown on potato dextrose agar (PDA) slants and kept at 4°C. The fungal pathogen was sub-cultured freshly on PDA and incubated at 21°C for 3 days before use in this study.
In uence of culture media, temperature, and light/dark conditions on the growth of Streptomyces spp., and B. cinerea In a previous study, Boukaew et al. (2011) have shown that GYM medium and incubation at 28°C were the best conditions for the growth of the three strains of Streptomyces spp. However, the growth conditions on PDA medium and 21℃ are not yet known. A 10 7 spores mL -1 of each strain of Streptomyces spp. were streaked on GYM agar and PDA and incubated at 21 and 28°C. To evaluate their ability to grow at different mediums and temperatures, the growth and pigment of spore mass were qualitatively determined after 14 days of incubation. Each treatment had three replicates.
The in uence of light conditions on the growth of Streptomyces spp. was also studied. A 10 7 spores mL -1 of each strain of Streptomyces spp. were streaked on the selected medium and incubated in the light (photo period of light-dark (L:D) at 14:10 hours) and dark (24 hours of dark) growth chamber at 21°C for 14 days. Samples were taken at a time interval to evaluate the ability of Streptomyces spp. to grow in the light/dark condition.
Most researchers have shown that the PDA and incubation, ranging from 18 to 22°C were the best conditions for the growth of B. cinerea (Whipps 1987;Aqueveque et al. 2017;Meng et al. 2020). However, the growth conditions on GYM medium and 28℃ are not yet known. A 5-mm-diameter mycelial plug, excised from a three-day-old B. cinerea colony, was transferred to the center of GYM and PDA plate media and incubated at 21 and 28°C. The ability of pathogenic fungi to grow at different mediums and temperatures was determined after three days of incubation. Growth was measured based on the radius of mycelium produced (in cm). Each treatment had three replicates.
In uences of culture media and temperature on antifungal activity of Streptomyces spp. against B. cinerea The two strains of S. philanthi RM-1-138 and RL-1-178 and S. mycarofaciens SS-2-243 were evaluated for their antagonistic properties against B. cinerea using a dual culture technique (Boukaew et al. 2011). For each strain of Streptomyces spp., a streak of spore suspension at 10 7 spores mL -1 was deposited on one side of a GYM or PDA media in Petri dishes. Plates were then incubated in a growth chamber for seven days at 28°C.
A 5-mm-diameter mycelial plug, excised from a three-day-old sample of the pathogenic fungi colony, was transferred to the center of each plate. As a control, a mycelial plug of the pathogenic fungi was placed on a GYM and PDA plate without any Streptomyces strain. The dual culture plates were incubated in a growth chamber at 21°C and 28°C for three days, after which the radial mycelial growth of the pathogenic fungi was measured and compared to that of the control. Three replicates were conducted for each Streptomycesthe pathogenic fungal strain combination. The colony size in each treatment was recorded and the percentage inhibition of hyphal growth was calculated by the following formula: In vivo antagonistic activity of culture ltrates produced by Streptomyces spp. against B. cinerea on tomato leaf The culture ltrates of S. mycarofaciens SS-2-243 and the strains RL-1-178 and RM-1-138 of S. philanthi were prepared in a 250 mL ask containing 100 mL GYM medium (pH adjusted at 7.0 before autoclaving) and incubated in the light (photoperiod L:D 14:10 hours) growth chamber at 28°C. After three days of incubation, 5 mL aliquots of this culture was transferred into 100 mL fresh GYM medium and incubated for ten days under the same condition. The culture broth was centrifuged (8880 ×g for 20 min), then ltered through a 0.45 mm Millipore membrane to recover the culture ltrate. These culture ltrates were sprayed on tomato leaf Streptomyces spp. (10 mL/leaf). When the leaves were dried (1 h), they were removed, and three lea ets were placed in a transparent plastic box with humid absorbent paper to maintain high relative humidity (close to 100 %). Mycelial plugs (5-mm in diameter) of B. cinerea excised from the growing margin of three-day-old PDA cultures were deposited onto the center of the lea ets of tomato. Leaves were treated with GYM medium as a control. After 2 days of growth with 12 h of light and 12 h of darkness alternately at 28 ℃, the lesion areas were measured (Zhang et al. 2020).
Each treatment included three replicates and each replicate consisted of three lea ets (three lea ets/plastic boxes). The strain of Streptomyces spp. possessing the highest in vivo antagonistic activity was selected for the next study.
Identi cation of antifungal compounds produced by the selected strain of Streptomyces spp. grown in different light/dark conditions and temperatures by GC-MS A 5 mL aliquots of the selected strain of Streptomyces spp. was transferred into a 200 mL GYM medium, incubated in the light (photoperiod L:D 14:10 hours) and dark (24 hours) growth chamber at 21°C and 28°C. After 10 days of incubation, the culture broth was centrifuged (8880 ×g for 20 min), then ltered through a 0.45 mm Millipore membrane to recover the culture ltrate. The chemical composition of the antifungal compounds in all culture ltrates was determined. Each culture ltrate was acetylated (Sangmanee and Hongpattarakere 2014), then analyzed by a gas chromatograph-mass spectrometer (GC-MS) (7890 B GC-7000 MS, Agilent, USA) equipped with a VF-WAXms column (30 m, lm thickness 0.25 μm, I.D. 0.25 mm). Mass spectra were obtained using the scan modus (total ion count, 35-500 m/z). The retention time and fragmentation pattern in the mass spectra were used to identify each compound by comparing them to those available standards from the Library of the National Institute of Standards and Technology (NIST).

Statistical analysis
The data were submitted to analyses of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) version 26 (IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp). A Pvalue < 0.05 was considered signi cant.

Results
In uences of culture media, temperature, and light conditions on the growth of Streptomyces spp. Table 1A represents the growth level of S. mycarofaciens SS-2-243 and the two strains of S. philanthi (RM-1-138 and RL-1-178) under different media and temperatures. The growth of these three strains depended on the nutrient composition of the media and incubation temperature. Streptomyces spp. had an excellent spore mass production depending on the temperature and medium composition. The two strains of S. philanthi showed higher growth on GYM medium and incubation at 28°C than on PDA medium incubated at 21°C. GYM media contained higher nutrients (glucose and yeast-malt extract) for microbial growth than PDA (potato and dextrose) 21℃. Unlike S. philanthi, culture media had no in uence on the growth of S. mycarofacien but the strain was more affected by incubation temperature.
The strain grew better at 28 o C than at 21 o C. Therefore, the optimum temperature for growth of all three strains of Streptomyces spp. was 28℃. While the two strains of S. philanthi preferred GYM to PDA, the medium composition did not affect S. mycarofacien.
Light/dark conditions in uenced the growth of S. mycarofaciens SS-2-243 but did not affect the two strains of S. philanthi (RM-1-138 and RL-1-178) (Table 1B). S. mycarofaciens SS-2-243 grew better under dark conditions than the light conditions In the case of B. cinerea, the culture media (PDA and GYM) and temperatures (21 and 28°C) had a profound effect on the mycelial growth (colony diameter) after three days of incubation (Fig. 1). The PDA medium and temperature at 21℃ gave signi cantly higher mycelial growth of B. cinerea with an average colony diameter of 8.41 cm compared with the GYM (6.86 cm).
In uence of culture media and temperature on antifungal activity of Streptomyces spp. against B. cinerea In uence of culture media and temperature on antifungal activity of S. mycarofaciens SS-2-243 and the two strains of S. philanthi against B. cinerea were investigated (Fig. 2). Figure 2A shows that the three strains of Streptomyces spp. exhibited 83-100% inhibition on the mycelial growth of the pathogenic fungi on PDA or GYM medium after three days incubation at 21 or 28°C, compared with the control treatment without Streptomyces spp. Signi cant differences in antifungal activity of S. mycarofaciens SS-2-243 were observed between medium and temperature (the same as the result of growth). Notably, the strains RM-1-138 and RL-1-178 of S. philanthi showed the strongest antagonistic activity (100% inhibition) against the mycelial growth of B. cinerea tested on both PDA and GYM medium after incubation at 21 or 28°C. The growth appearance of each Streptomyces against the pathogenic fungi on PDA and GYM plates after incubation at 21℃ and 28°C is illustrated ( Fig. 2B and C).
In vivo antagonistic activity of culture ltrate produced by Streptomyces spp. against B. cinerea on tomato leaf Because control e cacy in vitro is not always consistent with the results in vivo, the disease controls of the e cacy of culture ltrate produced by three strains of Streptomyces sp. against gray mold was evaluated on tomato leaf as a screening procedure for predicting the actual control e cacy. The development and expansion of disease symptoms induced by B. cinerea were inhibited effectively by culture ltrate produced by the strain SS-2-243 of S. mycarofaciens and the strains RM-1-138 and RL-1-178 of S. philanthi and in in vivo leaf test. As shown in Figure 3A, the lesion areas on tomato leaves inoculated by B. cinerea were signi cantly reduced (P < 0.05) after being treated with culture ltrate produced by Streptomyces spp. using detached leaf assay. For leaves in the control group with no treatment of culture ltrate, the lesion areas extended to 3.12 cm 2 after 2 days incubation at 28℃, whereas for the leaves treated with the culture ltrate produced by S. mycarofaciens SS-2-243 and the strains RL-1-178 and RM-1-138 of S. philanthi, the lesion areas were limited to 1.78, 0.98 and 0.33 cm 2 , respectively, (Fig. 3B). The cultural ltrates of S. philanthi RM-1-138 showed a strong e cacy protection on tomato leaf caused by B. cinerea (82.89%) (Fig. 3C). Therefore, the culture ltrates of this strain RM-1-138 were selected to identify the chemical composition.

Discussion
This paper provides useful information related to the in uence of culture media, temperatures, and light conditions on growth and antifungal activity by the antagonistic strains of Streptomyces mycarofacien SS-2-243 and S. philanthi RL-1-178 and RM-1-138 against Botrytis cinerea in vitro and tomato leaf. Besides, the antifungal compounds produced by the promising strain grew at different lights, and temperatures were identi ed by GC-MS. The results revealed that culture media, temperatures, and light conditions had a profound effect on the growth of the three antagonistic strains and fungal pathogenic strains. Moreover, temperatures and light conditions in uenced metabolic compounds produced by the selected strain RM-1-138 of S. philanthi.
The culture media and environmental factors were found to be the most in uential factors related to the growth and production of secondary metabolites of microorganisms (Saxena et al. 2001;Kim et al. 2005;Saha et al. 2008). Kathiresan et al. (2005) reported that the ability of bacteria to produce antimicrobial agents can be increased or lost under different culture conditions. In our study, the growth medium, temperature, and light/dark conditions had a signi cant effect on the three strains of Streptomyces spp. The strains RL-1-178 and RM-1-138 of S. philanthi were grown on GYM medium and incubated at 28°C and grew and produced a spore mass higher than on PDA medium incubated at 21°C. GYM medium containing glucose and yeast-malt extract was more enriched with nutrients for microbial growth than PDA containing only potato and dextrose. Nitrogen and carbon sources were reported to have a profound in uence on the growth and production of secondary metabolites by Streptomyces species (Narayana and Vijayalakshmi 2008). The study indicated that the nutrient source has an important on bacterial and fungal growth.
The light/dark cycle in uenced the growth of the three antagonistic strains of Streptomyces spp. There were no signi cant differences in the growth of S. philanthi RL-1-178 and RM-1-138 between light and dark conditions. On the other hand, S. mycarofaciens SS-2-243 had a signi cantly higher growth rate under dark conditions than light conditions. Different previous studies have shown that S. phaeopurpureus 5125, S. salmonicida 5472, and S. fulvissimus could grow well in light conditions (Koyama et al. 1975). Some strains of Streptomyces species such as S. viridosporus, S. lividans and S. coelicolor were reported to be sensitive to light, while others remained unaffected (S. ambofaciens and S. pilosus) (Imbert and Blondeau 1999). It has been demonstrated that light conditions in uenced the growth of Streptomyces sp.
B. cinerea is able to deploy a wide range of strategies to complete its infection cycle. The induction of these strategies must be mainly in response to environmental conditions. Culture media (PDA and GYM) and temperature (21 and 28℃) in uenced the mycelial growth of B. cinerea. The PDA medium and temperature at 21℃ gave a rapid mycelial growth of B. cinerea while GYM medium was not signi cantly different after incubation at 21 or 28 ℃. This nding agreed with that of Ciliberti et al. (2016) who reported that the highest mycelial growth and conidia production of B. cinerea was observed on PDA and temperature at 20℃. Several other researches also stated that PDA was the best media for mycelial growth (Xu et al. 1984;Maheshwari et al. 1999).
The PDA medium, with a temperature of 21°C not only had a profound positive effect on growth but also on the antifungal activity of S. mycarofaciens SS-2-243 against gray mold B. cinerea. The two strains of S. philanthi (RM-1-138 and RL-1-178) showed the strongest antagonistic activity (100% inhibition) against the mycelial growth of B. cinerea tested on both media at an incubation temperature of 21 and 28°C in vitro test. The three antagonistic strains of Streptomyces spp. produced antifungal substances and secreted them into the agar medium in dual culture plates.
In addition to the in vitro trials, the three antagonistic strains of Streptomyces spp. also exhibited positive results against B. cinerea during the in vivo trials on tomato leaves with remarkable protection e ciency after 2 days of cultivation at 28℃. Blight symptoms and lesion diameter caused by the fungal pathogen in tomato leaves were all signi cantly reduced by the strains of S. mycarofaciens SS-2-243 as well as S. philanthi RM-1-138 and RL-1-178 when compared with the control. Among the three strains tested, the antifungal compounds in the culture ltrates produced by S. philanthi RM-1-138 (82.89%) showed the highest inhibitory effect on the protection of tomato leaf caused by B. cinerea when compared with S. philanthi RL-1-178 (68.45%) and S. mycarofaciens SS-2-243 (42.78%), respectively. These results con rmed the potential of an antifungal compound in the secondary metabolites produced by antagonistic microorganisms to control gray mold B. cinerea in this study or other plant pathogenic fungi such as Streptomyces sp. The antimicrobial substances produced by Streptomyces were variable and diversi ed depending on multiple factors. Therefore, the in uence of temperatures (21 and 28℃) and light/dark conditions on antifungal production of the selected strain S. philanthi RM-1-138 was studied. Interestingly, the metabolic compounds produced by this particular strain were greatly altered by relative concentration components under the conditions tested. In light/dark conditions tested, the anti-B. cinerea of S. philanthi RM-1-138 was established at a higher level in several metabolic compounds in the dark condition (11 and 32 antifungal compounds after incubation at 21 and 28℃, respectively) than in the light condition (11 and 19 antifungal compounds after incubation at 21 and 28℃, respectively). Moreover, we found that the dominant antifungal compounds showed signi cant differences between dark and light conditions.
Temperature conditions tested showed that incubation temperature plays a signi cant role in the number of antifungal compounds and dominance compound of S. philanthi RM-1-138. Incubation was at 21℃ and, the dominant component was acetic acid (68.77%, dark condition and 67.41%, light condition) while at 28℃ the dominant components were propanamide (20.68 %, dark condition) and benzeneacetamide (43.58%, light condition). Besides, we found that acetic acid, 2-propanone, and 2-furanmethanol were detected in all conditions tested. Moreover, some of the antifungal compounds such as lactic anhydride, 2,5-pyrrolidinedione, 1-methyl, propanamide, benzeneacetamide, butyl aldoxime, 2-methyl-, syn-were found only under incubation at 28℃. According to this study, different environmental factors may release similar or different types of antifungal substances, with a variety of effects. All researchers proposed that Streptomyces spp. was commonly reported for its broad spectrum of antimicrobial activity (Ouhdouch et al. 2001;Prabavathy et al. 2006;Prapagdee et al. 2008). Our results clearly demonstrated that the light/dark condition have an in uence on a rich source of secondary metabolites of S. philanthi RM-138. Tables   Table 1 Influences     In uences of culture media (potato dextrose agar; PDA and glucose yeast-malt agar; GYM) and temperature (21 and 28℃) on mycelial growth of B. cinerea were determined after three days incubation.

Declarations
Values are the meaning of three replications (±SD).  . The data presented are the mean ± SD (n = 6). The same letter on the bars for each column indicates no signi cant difference according to a Tukey test at P < 0.05.