Strawberry production worldwide is strained by Colletotrichum and Phytophthora species. Strawberry anthracnose by Colletotrichum spp. has been serious threat and various controls including chemical fungicides were suggested. C. fructicola is a part of C. gloeosporioides species complex, and DNA markers were developed to differentiate the fungal species from other Colletotrichum spp. causing strawberry anthracnose (Gan et al., 2017). Many Korean C. gloeosporioides isolates causing strawberry anthracnose was re-classified as C. fructicola (Nam et al., 2013). Fungicides azoxystrobin and prochloraz-Mn protected strawberry crown anthracnose by dipping the seedlings in fungicides before planting in fruit production fields (Kim et al., 2002; Nam et al., 2014). Control efficacies of azoxystrobin, pyraclostrobin and thiophanate-methyl as protectants were found in strawberry plant fields over 3 years against C. gloeosporioides‒causing crown rot (MacKenzie et al., 2009). Phytophthora cactorum infection led to leather rot and crown rot of strawberry (Grove et al., 1985; Lim et al., 1998; Madden et al., 1991). Azoxystrobin, mefenoxam, and pyraclostrobin showed protectively controlled strawberry leather rot, whilst postinfection treatment with mefenoxam only decreased the leather rot (Rebollar-Alviter et al., 2007). Metalaxyl and fosetyl-Al reduced the leather rot by foliar-spraying and soil-drenching in strawberry fruit production fields (Ellis et al., 1998). In South Korea, two and six fungicides were registered to control Phytophthora rot in strawberry nursery and fruit production fields, respectively, in 2020. However, pyraclostrobin and iminoctadine tris (albesilate) were not investigated for the strawberry Phytophthora rot yet.

Lasiodiplodia theobromae infects many plants such as grapevine, mango, and peach trees (Burruano et al., 2008; Li et al., 2014; Saeed et al., 2017; Úrbez-Torres et al., 2008). Strawberry dieback by L. theobromae was reported in nursery of South Korea and fruit production fields in Turkey (Nam et al., 2016; Yildiz et al., 2014). Strawberry cv. ‘Seolhyang’, planted over 80% of production fields in South Korea, was highly susceptible to the dieback, which can be a great concern (Nam et al., 2016). Unfortunately, appropriate controls have not been demonstrated for the strawberry dieback.

Pyraclostrobin controlled anthracnose on strawberry, bean, grape, and pepper plants (Conner et al., 2004; Gao et al., 2017; Samuelian et al., 2014; Turechek et al., 2006). However, recent occurrences of pyraclostrobin-resistant C. acutatum isolates in pepper and strawberry fields warned of frequently using pyraclostrobin (Forcelini et al., 2016; Kim et al., 2019). Iminoctadine tris (albesilate) has shown in vitro antifungal activity against Alternaria dauci causing carrot Alternaria leaf spot and Aspergillus tubingensis causing Shine Muscat bunch rot (Do et al., 2020; Kim et al., 2020), and protectively and curatively reduced cucumber leaf spots by Corynespora cassiicola (Zhu et al., 2019). Iminoctadine tris (albesilate) was registered for strawberry anthracnose and grey mould in South Korea, but hardly demonstrated for other strawberry diseases.

Antagonistic microbes have controlled strawberry diseases. Endophytic bacterium Azospirillum brasilense REC3 in strawberry roots promoted plant growth as well as controlled anthracnose via enhanced plant immunity including augmented phenolics and pathogenesis-related gene expressions in plants (Tortora et al., 2012). Decreased anthracnose by Bacillus amyloliquefaciens S13-3 derived from soil was closely related with the bacterium-produced antimicrobial lipopeptides (Mochizuki et al., 2012; Yamamoto et al., 2015). Dipping strawberry roots in Trichoderma harzianum and T. viride conidial suspensions decreased leather rot (Porras et al., 2007). Pseudomonas fluorescence F113 and Serratia plymuthica HRO-C48 from rhizosphere of sugar beet and oilseed rape plants, respectively, reduced the strawberry root rot (Barahona et al., 2011; Kurze et al., 2001).

Bacillus siamensis H30-3 promoted Chinese cabbage plant growth under normal ambient and adverse environments like high temperature- and high temperaturedrought stresses (Lee et al., 2018; Shin et al., 2019). B. siamensis H30-3 was antagonistic to Alternaria brassicicola and Colletotrichum higginsianum causing black spot and anthracnose diseases in Chinese cabbage, respectively, but diseases by A. brassicicola and C. higginsianum were reduced in cv. Ryeokgwang, not cv. Buram-3-ho (Lee et al., 2018). Chinese cabbage soft rot by Pectobacterium carotovorum subsp. carotovorum was alleviated by B. siamensis H30-3 (Shin et al., 2019). These results indicate that B. siamensis H30-3 has broad antimicrobial activities against fungi and bacteria.

In this study, in vitro antifungal activities of two fungicides pyraclostrobin and iminoctadine tris (albesilate) were demonstrated against C. fructicola, P. cactorum, and L. theobromae by singly or simultaneously. B. siamensis H30-3 was applied to the three pathogens to investigate whether the bacterial strain arrests the pathogen growths through diffusible and volatile antifungal machineries. Tolerance of B. siamensis H30-3 to the two fungicides was evaluated for simultaneous usage potentials with chemical controls for integrated strawberry disease management. Simultaneous treatment with two fungicides with or without B. siamensis H30-3 volatiles on the three pathogen growths was also investigated.

In vitro antimicrobial activities of pyraclostrobin and iminoctadine tris (albesilate) were evaluated against C. fructicola, P. cactorum, and L. theobromae (Fig. 1). Colony formation (Fig. 1A) and mycelial growths (Fig. 1B) of C. fructicola were gradually decreased by increasing pyraclostrobin (0, 0.00625, 0.0125, 0.025, 0.05, and 0.1 mg/ml) and iminoctadine tris (albesilate) (0, 0.00625, 0.0125, 0.025, 0.05, and 1 μg/ml). Linear repression analyses demonstrated strong correlation of fungicides concentrations with reduced growths of C. fructicola, shown by increasing concentrations of pyraclostrobin (Y = ‒14.79X + 100.59, R² = 0.8943) and iminoctadine tris (albesilate) (Y = ‒17.308X + 123.71, R² = 0.9689).

Increasing pyraclostrobin concentrations at 0.00625, 0.0125, 0.025, 0.05, and 0.1 mg/ml resulted in significant decreases in mycelial growth of C. fructicola to ca. 56.9%, 50.2%, 42.5%, 28.3%, and 15.2% compared to mock-treated control. Same dosage range of pyraclostrobin showed more drastic suppressions in mycelial growths of P. cactorum and L. theobromae. Minimal concentration of 0.00625 mg/ml of pyraclostrobin reduced growth of P. cactorum and L. theobromae by ca. 18.9% and 17.5%, respectively. More doses of pyraclostrobin to 0.1 mg/ml did not change P. cactorum suppression efficacy, whilst increasing doses to more than 0.025 to 0.1 mg/ml gradually more suppressed the growths of L. theobromae. Increasing iminoctadine tris (albesilate) concentrations at 0.00625, 0.0125, 0.025, 0.05, and 0.1 μg/ml led to significant decreases in growth of C. fructicola to ca. 89.2%, 78.0%, 62.0%, 34.9%, and 14.6% compared to mock-treated control. Same dosage ranges of iminoctadine tris (albesilate) has no antimicrobial activity against P. cactorum, and only slightly reduced growth inhibition was found against L. theobromae by 0.05-0.1 μg/ml. These results suggest that pyraclostrobin can be applied to control both Phytophthora rot and dieback caused by P. cactorum and L. theobromae, respectively. Antimicrobial activities of iminoctadine tris (albesilate) by more than 0.1 μg/ml remains investigated.

Bacterial suspension (10⁸ cfu/ml) of B. siamensis H30-3 and the three pathogens were co-inoculated on 1/2-potato dextrose agar (PDA) media as described previously (Lee et al., 2018). B. siamensis H30-3 showed diffusible antimicrobial activities against C. fructicola, P. cactorum, and L. theobromae during dual cultures (Fig. 2). Compared to each mock-treated control at 8, 16, and 4 days after the cultures, mycelial growths of C. fructicola, P. cactorum, and L. theobromae were markedly limited as cultured with B. siamensis H30-3 simultaneously (Fig. 2A). Significantly reduced colony radius of the three phytopathogens were shown in Fig. 2B. Volatiles from B. siamensis H30-3 cultured on different media exhibited in vitro antimicrobial activities against C. fructicola, P. cactorum and L. theobromae (Fig. 3). Volatiles from B. amyloliquefaciens, B. pumilus and B. velezensis strains showed antifungal activities against diverse fungal species (Asari et al. 2016; Lim et al., 2017; Morita et al., 2019). Reduced colonies of the three pathogens were found on 1/2-PDA media covered different culture media tryptic soy agar, nutrient agar, and Luria-Bertani agar media drop-inoculated by B. siamensis H30-3 (Fig. 3A). Particularly, aerial mycelia of P. cactorum was drastically reduced by B. siamensis H30-3 volatiles. Distinct difference in growth inhibition by the different bacterial media was not found (Fig. 3B). B. siamensis H30-3 has several antimicrobial lipopeptide-encoding genes such as bacD, bmyA, ituA, and srfA (Lee et al., 2018), which may be involved in the suppressed C. fructicola, P. cactorum, and L. theobromae.

In vitro bactericidal activities of the two fungicides against B. siamensis H30-3 were investigated (Fig. 4). B. siamensis H30-3 (105 cfu/ml) were initially cultured in 4-ml of nutrient broth with or without increasing concentrations of the two fungicides at 30°C for 24 h and the bacterial growths were evaluated spectrophotometrically at OD₆₀₀ (Hong et al., 2016). Bacterial growth was declined by more than 0.025 mg/ml of pyraclostrobin and no change was found by increased doses (0.05-0.1 mg/ml). Iminoctadine tris (albesilate) doses from 0.00625 to 0.1 μg/ml has no antibacterial effect on B. siamensis H30-3. Bacillus spp. as biological control agents were suggested to be integrated into disease management with chemical fungicides (Jacobsen et al., 2004; Korsten et al., 1997; Lee et al., 2012). B. siamensis H30-3 can be considered for strawberry disease control with the two fungicides. However, use of less than 0.0125 mg/ml pyraclostrobin will be recommended to avoid its negative effect on the growth of B. siamensis H30-3 during their simultaneous application.

Antimicrobial effects of the two chemical fungicides with B. siamensis H30-3 volatiles were investigated (Fig. 5). Co-treatment with pyraclostrobin (0.00625 mg/ml) and iminoctadine tris (albesilate) (0.00625 μg/ml) without B. siamensis H30-3 volatiles resulted in more significantly suppressed growths of C. fructicola compared to the one treated with single fungicide. However, P. cactorum and L. theobromae was not synergistically suppressed by the co-treatment with the two fungicides. Together with B. siamensis H30-3 volatiles, mycelial growths of C. fructicola were more efficiently decreased by pyraclostrobin and/or iminoctadine tris (albesilate). The fungicide-suppressed P. cactorum was rather relieved by B. siamensis H30-3 volatiles. No significant difference in growths of L. theobromae treated with the fungicides by B. siamensis H30-3 volatiles. It should be solved how B. siamensis H30-3 volatiles will apply to the strawberry growing fields. Identification of B. siamensis H30-3 volatile compounds and improved production of the antimicrobial volatile compounds may pave a way for more efficient and reliable biological control of strawberry diseases.

Taken together, fungicides pyraclostrobin and iminoctadine tris (albesilate) and B. siamensis H30-3 showed their antimicrobial activities against the C. fructicola, P. cactorum, and L. theobromae singly or in combination. Use of B. siamensis H30-3 volatiles with pyraclostrobin and/or iminoctadine tris (albesilate) can be a more useful for controlling strawberry anthracnose by C. fructicola. In planta disease control efficacies of the chemical and biological agents will provide more sustainable production of strawberry fruits.