Plants and microbes used in this study are listed in Table 1.

Surfactin sodium salt was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Iturin A was purified from solid-state cultures of B. subtilis ATCC21556 by organic solvent extraction followed by column chromatography, as previously described [9]. Briefly, solid-state culture of B. subtilis ATCC 21556 was extracted by ethyl acetate (EtOAc): 1-butanol (BuOH) = 7:3 (v/v), and then, organic phase was dried up by a rotary evaporator. The resulting residue was dissolved in EtOAc: methanol (MeOH) = 90:10 (v/v) and applied to a silica gel column (SI60µm SIZE:60., Shoko Science, Kanagawa, Japan). Iturin A was eluted by a linear gradient of EtOAc - MeOH (90:10 to 50:50 within 30 min). The elution containing iturin A were combined and dried up by a rotary evaporator. The resulting residue was dissolved in 50% MeOH, and applied to an ODS column (Octa Decyl Silyl) resin (Wakogel 50C18, Wako Pure Chemical Industries, Osaka, Japan). Iturin A was eluted by a linear gradient of MeOH - DDW (50:50 to 75:25 within 60 min, followed by 75:25 for 30 min). Purified Iturin A was >98% pure measured at 210 nm by HPLC.

Disease suppression assay was conducted in an air-controlled greenhouse at 24˚C. Host plants were propagated by a hydroponic culture with Hoagland solution [11] unless otherwise described. Population of bacterial suspensions for inoculation on host plants is listed in Table 1.

We previously reported on disease suppression by soil amendments of purified surfactin or iturin A against Fusarium yellows of tatsoi, Brassica rapa var. rosularis [9]. We found that both surfactin and iturin A significantly suppress disease caused by this soil-borne pathogen. However, the disease suppression by surfactin and iturin A can be negated by applying excess amounts of either cLP in soil.

Surfactin is a strong haemolytic, antibacterial and antiviral molecule [14]. By contrast, members of iturin family present limited antibacterial and no antiviral activity but they have a strong in vitro antifungal action [15]. Beyond that, these molecules have been reported as inducers of defense response in host plant by stimulation of the induced systemic resistance phenomenon [7] [16].

Significant disease suppression against bacterial cabbage leaf spot was observed after root-treatment of host plants with both surfactin and iturin A at a range of 0.125 to 1 µM (Figure 1(a) and Figure 1(b)). Moreover, disease suppression was negated after application of more than 2 µM of root-treatments for both of surfactin and iturin A, respectively, whereas no disease symptoms were observed with 4 µM of surfactin or iturin A root-treatments without pathogen-inoculation (Figure 1(a) and Figure 1(b)).

This data more clearly demonstrates that the disease suppression is due to the effects of induced disease resistance by surfactin and iturin A, instead to the antimicrobial activities of surfactin and iturin A directly against soil-borne pathogens. Furthermore, it is notable that the loss of disease suppressive activities of surfactin and iturin A at high concentrations occurred via lack of induced disease resistance, with no visible plant disease symptoms observed unless a pathogen is applied.

On tomato, significant disease suppression against bacterial speck was observed by root-treatment of surfactin at a range of 1 to 4 µM (Figure 1(c) and Figure 1(d)). On the other hand, no significant disease suppression was observed at a range of 0.25 to 32 µM of iturin A treatments.

In tobacco (Nicotiana tabacum), which belongs to the same family (Solanaceae) as tomato, surfactin treatment of tobacco cells elicits induced disease resistance related events, whereas no responses were observed following iturin A treatments [17]. Our data suggests that a specificity between surfactin and Solanaceae plants may exist for eliciting induced disease resistance.

Similar to cabbage, high surfactin treatments over 8 µM negated disease

suppression activities whereas no disease symptoms were observed with up to 32 µM of surfactin treatment without pathogen inoculation.

On rice, both surfactin and iturin A showed disease suppression activity against bacterial leaf blight of rice after root treatments. However, the range of concentrations that elicited disease suppression differed between surfactin and iturin A (Figure 1(e) and Figure 1(f)). The range of concentration that showed significant disease suppression by surfactin was 0.06 to 0.5 µM in hydroponic culture. This disease suppression range was lower than that observed for iturin A treatments, which was 0.5 to 2 µM in hydroponic culture. Moreover, similar to the above plants, 1 µM of surfactin or 4 µM of iturin A treatments negated the observed disease suppression, whereas no disease symptoms were observed at 1 µM of surfactin- and 4 µM of iturin A-treatments, respectively, in pathogen-free plants.

On disease suppression through treatment of purified surfactin or mycosubtilin, a member of iturin family, against rice sheath blight on rice, and followed the rice plant response through gene expression analysis for induced disease resistance related genes [6]. With respect to their findings, both surfactin and mycosubtilin elicited expression of induced disease resistance related genes on rice. However, in that study, only surfactin showed disease suppressive activity at the 35 µM treatment used against the fungal disease on rice, whereas no disease suppression was observed following mycosubtilin treatment at the same concentration. Based on our findings, we propose that mycosubtilin might also show disease suppression against rice fungal disease at a different range of mycosubtilin concentration.

On soybean, both purified surfactin and iturin A treatments showed significant disease suppression against bacterial blight of soybean (Figure 1(g) and Figure 1(h)). However, the concentration of surfactin that showed significant disease suppression was 1 µM in hydroponic culture, which was higher than the significantly effective range of iturin A treatment (0.13 and 0.25 µM in hydroponic culture). Moreover, 2 µM of surfactin or 0.5 µM of iturin A treatments negated the disease suppression, whereas no disease symptoms were observed by both surfactin- and iturin A-treatments at 2 µM in pathogen-free soybean plants.

The treatment of cucumber seeds with a mixture of 1 µM of semi-purified of iturin, surfactin and fengycin, respectively, showed no disease suppression against cucurbit anthracnose on cucumber leaf caused by Colletotrichum lagenarium [18].

In this study, iturin A treatments of cucumber roots showed significant disease suppression against cucurbit angular leaf spot at a range of 0.25 to 1.0 µM in hydroponic culture, whereas the negation of disease suppression was observed at 2 µM and higher of iturin A in hydroponic culture (Figure 1(i) and Figure 1(j)), similar to the other plants above. On the other hand, surprisingly, significant disease enhancement was observed following surfactin treatments at a range of 0.25 to 4 µM at 3 days post inoculation. With respect to the previous report [15], we deduce that by including surfactin in the mixture of cLPs, it might have acted antagonistically against the disease suppression by iturin A.