Antagonism and biocontrol of walnut blight by sweet osmanthus endophytic bacterium OFE17

Walnut blight caused by Xanthomonas arboricola pv. juglandis (Xaj) is an important bacterial disease for walnut production worldwide. The objective of the present study was to characterize one endophytic bacterium, namely OFE17 from Osmanthus fragrans leaves, evaluate its potential biocontrol efficiency against the disease, and identify the underlying probable mechanisms of its function. Based on morphology, biochemical and physiological characteristics, 16S-rDNA and gyrB sequences, and antibiotic production genes, the endophyte OFE17 was tentatively identified as Bacillus sp. A disease control efficiency of up to 68.69% was observed through a biocontrol test on detached immature walnut fruits under controlled conditions. OFE17 can produce protease, cellulase, amylase, siderophores, and demonstrates phosphate dissolving ability. However, the OFE17 is unable to produce extracellular lipase, IAA (indoleacetic acid), and has no nitrogen fixation capability. The active compounds of OFE17 were primarily non-protein compounds, and the optimum organic extraction solvent was chloroform. Through specific PCR detection, it contains the genes ituA and ituD which play a key role in active compound synthesis of iturin A synthetase. This study added a promising biocontrol agent candidate for the disease control and laid a foundation for further exploration.


Introduction
Walnut blight is the most serious bacterial disease in walnut production. Its causal agent is Xanthomonas arboricola pv. juglandis (Xaj) (Vauterin et al. 1995). It was initially recognized in 1901 in California, United States (Pierce 1901). Since then, the disease has been recorded worldwide (Frutos 2010;Moragrega and Özaktan 2010;Burokiene and Pulawska 2012;Kaluzna et al. 2014;Lamichhane 2014). Xaj overwinters on infected dormant buds, cankers and can survive epiphytically, then invades into host plants through stomata, leaf scars, or wounds. The pathogen may even penetrate through the husk and into the nutmeat in severe infections (Scotton et al. 2015). The disease symptoms include black blight on leaves, wilt, and necrosis of branches and petioles. The typical symptoms on walnut fruitlets are oily, black sunken patches that are centrally located (Lamichhane 2014). The pathogen also serves as a major contributor of brown apical necrosis (BAN) (Belisario et al. 2002;Moragrega and Özaktan 2010;Moragrega et al. 2011) and walnut trunk vertical oozing canker (VOC) (Hajri et al. 2010;Palacio-Bielsa et al. 2012). The disease outbreaks caused by Xaj on Juglans trees have sparked international concern, suggesting the possibility of future epidemics (Lamichhane 2014) and leading to the recent recruitment of Xaj into the CABI (Centre for Agriculture and Bioscience International) invasive species compendium list (https:// www. cabi. org/ isc/ datas heet/ 56946).
Chemical control has been a major counter-measure and a standard routine in walnut blight management for a long time (Buchner et al. 2001;Ginibre et al. 2001;Ninot et al. 2002). However, its overuse has adverse consequences on orchards and their surroundings, particularly the emergence of copper-resistant Xaj strains (Lee et al. 1994;Pereira et al. 2015;Giovanardi et al. 2016). Therefore, the development and utilization of biocontrol agents, especially those originating from plant endophytes and their derivatives, is highly appealing.
Endophytes are facultative or obligate symbiotic microorganisms that live in healthy plant tissues (Schulz and Boyle 2007). The main mechanisms of endophytes acting as plant biocontrol agents usually rely on attacking the pathogen and boosting the host plant resistance. Their anti-pathogen mechanisms include antimicrobial compounds, antagonistic effects, competition, and hyper-parasitic bacteriolysis (Kamber et al. 2012;Eljounaidi et al. 2016;Romero et al. 2019). The mechanisms for protecting the plant include triggering induced systemic resistance, which produces molecules useful for plant defense and structural support (Ek-Ramos et al. 2019), and growth promotion based on hormones or nutrition (Eljounaidi et al. 2016;Hossain and Chung 2019;Romero et al. 2019).
The endophytes from the Bacillus genus are an important biocontrol resource, as they can often produce lipopeptides, plant hormones, polysaccharides, and several enzymes linked to phenylpropanoid metabolism (Ek-Ramos et al. 2019). For example, the citrus root endophyte B. licheniformis has a significant effect on Huanglongbing (Trivedi et al. 2011). The endophytic Bacillus from wheat plays a significant role in the control of wheat tan spot disease (Drechslera tritici-repentis) (Larran et al. 2016). Maize seed endophytic Bacillus spp. have been found to be effective against the fungal pathogen Sclerotinia sclerotiorum (Massawe et al. 2018). With greenhouse experiments, B. subtilis E1R-j reduced take-all disease caused by Gaeumannomyces graminis var. tritici in wheat seedlings by more than 60% (Liu et al. 2009). B. subtilis 7PJ-16 (endophyte from mulberry) exhibits strong antifungal activity and can promote plant growth in the field (Xu et al. 2019). B. thuringiensis TbL-22 can effectively and sustainably control streptomycin-resistant citrus canker caused by X. citri subsp. citri (Islam et al. 2019).
Phyllospheric fungi and bacteria are abundant in sweet osmanthus (Osmanthus fragrans) plant (Li et al. 2014;Sun et al. 2018). About 70% of the obtained endophytic fungi from sweet osmanthus could produce antifungal metabolites against at least one plant pathogenic fungi. Additionally, the ethyl acetate extracts of seven species of endophytes from sweet osmanthus showed broad inhibition against six phytopathogens with inhibition rates from 20 to 80% (Liu et al. 2010). In the case of O. fragrans fruit endophyte B. safensis strain B21, its culture filtrate showed antifungal activity against rice blast pathogen Magnaporthe oryzae on plates and in the field whose results exceeded even those of carbendazim pesticide application (Rong et al. 2020).
For walnut blight biocontrol, there have been a few exploratory studies. For instance, essential oils and aqueous extracts from dozens of medicinal plants such as Ziziphora persica, Mentha piperita, and Allium sativum showed antibacterial effects on Xaj (Soltani and Aliabadi 2013). Bacteriophages isolated from walnut orchard soils (phyllosphere and rhizosphere) in the south island of New Zealand have been proposed as potential walnut blight biocontrol agents (McNeil et al. 2001). Recently, three lytic bacteriophages (f20-Xaj, f29-Xaj, and f30-Xaj) with effectiveness against Xaj were isolated from walnut trees in Chile (Retamales et al. 2016). Besides bacteriophages, there have been reports about biocontrol agents from bacteria. Thirty-five antagonistic bacteria effective against Xaj were discovered, 29 of which were isolated from healthy walnut leaves (Ozaktan et al. 2012). Amongst them, four Pseudomonas fluorescens strains have been identified and shown to significantly reduce symptoms on walnut leaves (41% to 82%) caused by Xaj (Ozaktan et al. 2012).
The goal of this study is to seek antagonistic bacteria against the pathogen Xaj, and provide more candidates for environmentally friendly control of the pathogen. In this paper, we obtained a promising endophytic bacterium OFE17 from healthy O. fragrans leaves, and identified it as Bacillus sp. Its biocontrol effect on detached immature walnut fruits was also evaluated. Furthermore, the possible active substances and the related genes, and plant growth-promoting factors were investigated.

Endophyte isolation and Xaj strains
Healthy sweet osmanthus (O. fragrans) leaves were collected from Xiaogan city, Hubei province, China. Leaves were washed with tap water followed by sterilized water three times. They were then cut into around 1 cm 2 patches, rinsed with ddH 2 O thrice, and surface disinfected for 3 min in 75% ethanol, 0.1% HgCl 2 for 1 min. This was followed by a final five rinses with ddH 2 O. The disinfected patches were pulverized thoroughly and suspended in ddH 2 O for 10 min. The leaf debris suspension was spread onto YPGA plates (yeast powder 5 g, peptone 5 g, D-glucose 10 g, deionized water 1000 mL, agar 15 g, pH 7.2) (Hajri et al. 2010) plate, and incubated at 28 ℃ till the endophyte colony developed. The antagonistic effect was observed via the dual culture method (Lin et al. 2014). Briefly, Xaj (200 µl, OD 600 = 0.5) was streaked onto a YPGA plate, the centers of which were then immediately inoculated with fresh endophyte. After incubating for 2-3 d at 28 ℃, the plates with a zone of inhibition were identified as those with a potential antagonistic effect on Xaj. The endophytes were picked for purification and subsequently stored in 30% glycerol at -80 ℃.
Forty-two Xaj strains were isolated from different counties in Hubei province used in this study listed in Table 1. The Xaj strains were grown on YPGA medium at 28 ℃ (Hajri et al. 2010), and stored at -80℃ with 30% glycerol in our laboratory.

Identification of endophyte OFE17
One strain with a strong antagonistic effect against Xaj DW3F3 on the YPGA plates was chosen for further investigation, and it was denoted as OFE17 (represent O. fragrans endophyte isolated in 2017). The endophyte OFE17 was characterized by cellular and colony morphology, Gram stain, endospore stain, flagellar stain, motility, methyl red, urease, catalase, and salt tolerance tests according to Dong and Cao (2001) methods. Meanwhile, the bacterium was identified using fatty acid analysis (Microbial Identification Inc, MIDI). This method is based on Fatty Acid Methyl Ester (FAME) profiles combined with gas chromatography (Agilent 6850). Chem Station software was used according to the manufacturer's instructions. Furthermore, OFE17 genomic DNA was extracted by EZ-10 Spin Column Bacterial Genomic DNA Mini-Prep Kit (Sangon Biotech Shanghai Co., Ltd.) for subsequent molecular identification. The PCR procedure and primers of 16S rDNA and gyrB sequences referred to Fu et al. (2016) and Wang et al. (2007). The 16S rDNA and gyrB amplicons were sequenced by Sangon Biotech Shanghai Co., Ltd., then subjected to Blastn against NCBI database.

Antagonism of OFE17 on multiple Xaj isolates
We obtained OFE17 by screening plant endophytes against the strain Xaj DW3F3. Subsequently, we tested its effects on 42 Xaj isolates from walnut leaves, buds, and fruits between 2015 to 2017 in our laboratory. The antagonistic assay was performed as described in Sect. 2.1. We used ddH 2 O (2 µl) and Kanamycin (50 mg/mL) (2 µl) as the negative and positive control, respectively. The inhibitory zone and endophyte colony size were measured after a 3-day incubation at 28 ℃. The inhibitory rate was calculated by dividing the inhibitory zone diameter (mm) by the colony diameter (mm). Each treatment was conducted in triplicate, and the experiment was repeated three times.

Biocontrol assay on detached walnut fruit
For inoculum preparation, OFE17 and Xaj DW3F3 cell suspensions were adjusted to 10 8 CFU/mL (OD 600 = 0.5) separately and confirmed by plating on appropriate media. The Xaj DW3F3 strain isolated from immature fruit was chosen as the target pathogen due to its high pathogenicity and readily accessible genome (Fu et al. 2018). Symptomless immature walnut fruits with similar sizes were collected before shell hardening, 30-40 days after setting, from 5-8-year-old walnut (Juglans regia, cv. Qingxiang) trees. Detached nuts were thoroughly surface disinfected with 75% ethanol and carefully rinsed with sterile distilled water. The pathogen and endophyte inoculation was carried out by a stabbing and infiltration method adapted from previous studies (Moretti and Buonaurio 2010;Frutos and López 2012;Ozaktan et al. 2012). Each fruit was punctured with nine pores in a 3 × 3 alignment with a sterilized needle. The pathogen DW3F3 was applied by infiltrating 100 µl cell suspension into the stabbed sites, and 100 µl ddH 2 O was used as a control. Endophyte OFE17 was inoculated at five different time points: 2 days before, 1 day before, the same day, 1 day post, and 2 day post pathogen inoculation. Fruits without endophyte treatment were used as the negative control. Each treatment was applied to 10 detached fruits, and the experiments were conducted independently twice in parallel. Fruits were then placed on wet filter paper in closed plastic boxes, which were kept for 14-21 days in a chamber at 26 °C and 80% relative humidity (Moretti and Buonaurio 2010;Frutos and López 2012). The size of the diseased area in mm 2 was measured 15 days post pathogen inoculation. We used the following formula to calculate the biocontrol efficiency: biocontrol efficiency = (negative control area [mm 2 ]-treatment area [mm 2 ]) / negative control area [mm 2 ] × 100%.

Assay of enzymes and plant growth-promoting substances
Four enzymes related to biocontrol potential were tested by qualitative methods as follows. Protease production was assayed on non-fat milk medium (non-fat milk 12 g/L, nutrient agar 20 g/L, pH 7.0) (Zhao et al. 2010). Lipase production was assayed on olive oil medium (olive oil 25 ml/L, peptone 10 g/L, beef extract 5 g/L, glucose 3 g/L, PVA 10 g/L, NaCl 0.5 g, Tween 80 0.5 mL, bromocresol purple 0.016%, agar 15 g/L, pH 7.5) (Ouyang et al. 2012). Cellulase production was assayed on Congo red medium (Congo red 0.2 g/L, yeast extract 10 g/L, peptone 20 g/L, K 2 HPO 4 0.5 g/L, MgSO 4· 7H 2 O 0.25 g/L, Carboxymethyl Cellulose 10 g/L, agar 15 g/L, pH 7.0) (Shi et al. 2017). Amylase production was assayed on starch medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, dissolvable starch 20 g/L, agar 15 g/L) (Wang et al. 2014). Plates were cultured at 28 ℃ for 24 h to 48 h. Amylase detection plates were read by adding iodine solution (0.1 mol/L) stain and letting sit for 20 min. Plates with a transparent halo were identified as positive, and those without were considered negative. Four plant growth-promoting factors were also assayed. The CAS (chrome azurol S) agar plate (10 ml/L 20% sucrose, 10 mL 1 mM MgSO 4 , 10 mL 10% casein acids hydrolysate, 100 mL Phosphate Buffered Saline, 100 mL chrome azurol S) assay was used to test the ability to produce siderophore (Silva-Stenico et al. 2005). IAA detection was performed using Salkowski's reagent in liquid culture (Rahman et al. 2010). The culture supernatant was combined with Salkowski's reagent (2% of 0.5 M FeCl 3 in 35% HClO 4 solution) in the dark for 30 min. A color change to red meant plates were IAA positive, otherwise they were interpreted as negative. Phosphate solubilizing capacity was tested on NBRIP (National Botanical Research Institute Phosphate) medium (Nautiyal 1999). Nitrogen fixing capacity was assayed by streaking the strain onto nitrogendeficient Ashby's agar medium according to Muangthong et al.'s methods (Muangthong et al. 2015), with growth being indicative of nitrogen fixation ability. All experiments were additionally performed without endophyte inoculation as a negative control. All assays were done three times.

Temperature and pH effect on OFE17 cell-free filtrate
OFE17 was cultured in LB for 24 h at 28 ℃ on a shaker (180 r/min). The supernatant was harvested by centrifuging twice at 13,000 r/min for 20 min each, then filtering through a 0.22 µm filter thrice. The cell-free filtrate was subjected to temperature stability tests at 40 ℃, 60 ℃, 80 ℃, 100 ℃, and 120 ℃ for 30 min, with room temperature (RT) as a control. The cell-free filtrate was adjusted to pH values of 2, 4, 6, 8, 10, and 12 to assay its stability, with non-adjusted pH cellfree filtrate as a control. The antagonistic effect was tested by adding 20 µL cell-free filtrate to the medium central well of Xaj DW3F3 on YPGA. Each treatment contained three plates, with the experiments performed twice.

Active substance extracts
Protein-like active substances were salted out by gradual addition of ammonium sulfate (up to 70%) to OFE17 cell-free filtrate, followed by dialysis in 0.02 mol/L phosphate-buffered saline. The HCl-methanol method was used to extract non-protein substances from the cell-free filtrate (Chen et al. 2008). Three polarity organic solvents (petroleum ether, ethyl acetate, and chloroform) were further used to extract nonprotein active substance from the cell-free filtrate and the extracts were dissolved in methanol (Chen et al. 2008). These extracts were tested for inhibitory effect against Xaj DW3F3 via the plate confrontation culture method described above, with methanol used as control. Each treatment had three replicates, and the experiment was done twice.

Identification of endophyte OFE17
The OFE17 cells are rod-shaped and Gram-positive, the colonies were cream-colored with surface wrinkles and irregularly edges on YPGA medium (Fig. 1). It has an endospore, peritrichous flagella, and motility ability on a semi-solid medium. The methyl red and urease tests were negative, the catalase reaction was positive, and there was no salt tolerance at 15%. OFE17 shared the highest score of 93.31% with B. subtilis (Library RTSBA6 6.21, Sim Index 0.756) in the fatty acid analysis using the MIDI bacteria identification system. The 16S rDNA (accession No. MT921614) was 1398 bp in length and had the greatest similarity with B. subtilis with a score of 100% by Blastn in the NCBI database. The gyrB (MT957084) sequence was 1131 bp in length, with a 98% similarity to B. subtilis. Based on the morphology, biochemical characteristics, fatty acid analysis, and conserved gene sequences, the endophyte OFE17 was tentatively identified as Bacillus sp.

Antagonism to different Xaj strains
We found endophyte OFE17 was antagonistic to all 42 tested Xaj isolates, although the effect on the representative strain DW3F3 was not the strongest of those tested ( Table 1). The highest inhibition ratio was against the strains BKA2 and BKA2-1 with an inhibition ratio of 1.94 ± 0.43 and 1.58 ± 0.38, respectively. These two isolates were isolated from Baokang county, Hubei Province, and have typical bacterial blight symptoms on immature walnut fruits.

Biocontrol effect on detached fruitlets
Through the stabbing and infiltration method, we tested OFE17's biocontrol effect on DW3F3. The results showed OFE17 had an obvious control effect on walnut blight caused by Xaj DW3F3, regardless of the time of treatment (before, at the same time as, or post pathogen inoculation) (Fig. 2). Based on the diseased area on the fruits, the biocontrol efficiency was calculated. The biocontrol efficiency ranged from 20.3% to 68.69% among the five treatment time points (Fig. 2). The best biocontrol efficiency (68.9%) achieved was the inoculation of the biocontrol agent OFE17 48 h post pathogen inoculation.

Enzyme and plant growth-promoting factors assay
The OFE17 grown on a non-fat milk medium produced a transparent halo after 2 days of incubation (Fig. 3). This means it was capable of degrading casein by producing protease. However, for lipase detection on olive oil medium, there was no transparent halo, suggesting it does not produce lipase. After it was rinsed by phosphate saline buffer, orange halos around colonies were readily observed on the cellulase detection medium. Therefore, OFE17 is capable of cellulase production. After staining with the iodine solution, the dissolvable starch degraded by the OFE17 colony could be observed with halos (Fig. 3).
On the CAS agar plate siderophore test, a dark blue edge formed around the colonies, revealing that OFE17 is capable of producing siderophores. On NBRIP medium, opaque orange halos were produced by phosphate solubilization (Fig. 3). However, the fact that the endophyte can't grow on nitrogendeficient Ashby's agar medium implied it cannot fix nitrogen. The OFE17 is also incapable of producing IAA, as evidenced by the lack of color change in the IAA-production test.
The crude protein was obtained through salting out by exposing the filtrate to 70% saturated ammonia sulfate. The crude protein displayed antibacterial activity, but the bacteriostatic activity was not as strong as the cell-free filtrate before salting-out (Fig. 5). Based on these results, the dominant active substance is likely not limited to proteins. This is reinforced by the observation that the non-protein products showed high antibacterial activity. Therefore, we speculated that the dominant active substances are non-protein compounds. Furthermore, we used three organic solvents to extract the active substances from the cell-free filtrate. The extracts in chloroform had the strongest activity against Xaj DW3F3 on YPGA plates (Fig. 6). Therefore, we speculate chloroform is an optimum solvent for the active substance extraction from the cell-free filtrate.

Active substance related genes in OFE17
We amplified related genes by PCR with specific primers targeting ituD and ituA. They are the key enzyme genes in synthesizing active substances Iturin A synthetase D and Iturin A synthetase A, respectively. These results confirmed that OFE17 is a strain with the genetic capability to produce antagonistic compounds. The full length of OFE17 ituD (MT957087) sequence is 653 bp, and the homology was the highest with Bacillus sp. CY22 (AF534917.1) (similarity 98%). The ituA (MT957085) sequence is 823 bp in length, and the homology was the highest with B. subtilis strain MH25 (EU263005.1) (similarity 99%). The ituA (MT957086) sequence is 488 bp in length, and the homology was highest with B. subtilis Bacillomycin D (AY137375.1) (similarity 98%). Accordingly, the strain OFE17 has the potential to synthesize Iturin A and Iturin, which might be one of the crucial determinants in OFE17 antagonistic effect on walnut blight pathogen Xaj.

Discussion
In this study, we obtained a promising biocontrol candidate for walnut blight disease control. We found the biocontrol effects differed in efficacy depending on when the OFE17 was applied. The highest biocontrol effect was 68.89%, the lowest biocontrol effect was 20.3%. This is interesting given that the relevant studies have not necessarily examined different timepoints of biocontrol agent application. For instance, epiphytes from the phylloplane of healthy walnut trees significantly reduced immature nut bacterial blight by 41% to 77%, with only one biocontrol treatment time which occurred 24 h before Xaj inoculation (Ozaktan et al. 2012).
We found that when inoculating OFE17 after pathogen inoculation by one or two days, the biocontrol effects were better than applying the endophyte before the pathogen inoculation. This might suggest the antagonistic strain OFE17 is not capable of disease prevention. Actually, in practice, there are still many ways we need to improve methods of biocontrol agent application (Anyasi and Atagana 2019).
While walnut blight is caused primarily by Xaj, there are some other bacteria or fungi reported to cause the disease (Fang et al. 2020;Kim et al. 2020). Consistent with this observation, only 1/6 yellow-pigmented Gram-negative bacterial isolates that were recovered from symptomatic walnut were identified as Xaj (Zarei et al. 2019). Therefore, in the disease management practice relying on just one biocontrol agent will make it difficult to control the disease completely.
The OFE17 cell-free filtrate has a strong antagonistic effect on Xaj DW3F3. Interestingly, it is very stable at various temperatures and pH strengths, even at extremes like 120 ℃ and pH values of 2 and 12. These properties of OFE17 are very similar to endophyte B. safensis strain B21 which was isolated from O. fragrans fruits. The antifungal activity (rice blast pathogen M. oryzae) of its methanol-extracted compounds were stable at a wide range of pH values (1-9) and temperatures (40-100 °C) (Rong et al. 2020). Given these properties, the active substances could not be proteins. This speculation was verified by the fact that the salted-out extract only showed partial activity.
To further investigate the active substances in the cellfree filtrate, different extraction methods and solvents were employed. The active substances demonstrated bacteriostatic activity with three kinds of organic solvents, and chloroform was the best among them. Many of the studied Bacillus in the literature showed the presence of antimicrobial compounds in the culture supernatant. These were either peptides, bacteriocins, or secondary metabolites (de Almeida Lopes et al. 2018). Two antifungal compounds were identified as iturin A2 and iturin A6 (Rong et al. 2020). This is mirrored in our study, where PCR detection revealed that the strain OFE17 has the potential to synthesize Iturin A and Iturin. It is also possible that Bacillus, as endophytes isolated from different plants, might act as biocontrol agents through the production of volatile organic compounds (VOC). These have been shown to reduce sclerotial production and inhibit the mycelial growth of S. sclerotiorum (Massawe et al. 2018). However, exactly what the active substances in this study are needs to be investigated further using Liquid Chromatography (LC) and Mass Spectrometry (MS) techniques.
The literature presents several reports on the control of walnut blight, including extracts from medicinal plants (Soltani and Aliabadi 2013), bacteriophage screening (McNeil et al. 2001;Dömötör et al. 2016), and antagonistic bacteria screening (Ozaktan et al. 2012). The biocontrol effect identified in this study is comparable to those, based on our assay on detached walnut fruits. We tried testing detached leaves, but the leaves were quick to discolor which led to difficulty recognizing the symptoms and calculating the biocontrol effect. So, using young walnut trees as test material in a greenhouse may be more reliable and closer to field practices. Further, it is possible that applying biocontrol agents in a mixture, such as a cocktail of bacteriophages, could improve the performance above that of a single biocontrol agent.
In conclusion, the endophyte OFE17 from O. fragrans is a valuable candidate for walnut blight biocontrol. It showed a strong antagonistic effect on all 42 Xaj strains of walnut blight pathogen in vitro. Based on the analysis of its secreted proteins and related genes, it may achieve this result in multiple ways.