**2. Results**

## *2.1. Disease Development in A. philoxeroides Was Delayed Compared with A. sessilis*

The pathogenicity of *R. solani* was tested on invasive *A. philoxiroides* and native *A. sessilis*. Disease symptoms and necrotic lesions developed on leaves of both plant species (Figure 1a). However, *A. philoxiroides* showed slower disease progression compared to *A. sessilis* (Figure 1a,b). *Alternanthera sessilis* exhibited prominent disease symptoms at 24 hpi, whereas *A. philoxiroides* had no visible symptoms and mycelium was only observed at the site of inoculation under the microscope, indicating minimal fungal colonization (Figure 1c). Necrotic lesions were noticeable only at 48 hpi in *A. philoxiroides* (Figure 1a).

**Figure 1.** Disease symptoms caused by *R. solani* on invasive *A. philoxiroides* and native *A. sessilis* showing (**a**) in vitro detached leaf assay; (**b**) in planta pathogenicity test; and (**c**) microscopic observation of the infected area on leaves (at 24 hpi). Four-week-old plant leaves were inoculated with mycelium plugs of *R. solani* and disease symptoms were observed at various time intervals (24, 48, 72 and 96 hpi). For in planta tests, plastic bags were placed on the leaves after inoculation to avoid drying of plugs and moisture development for better infections. In (**c**), the inoculated area is circled with a black marker. After removing plugs, threads of fungal mycelium can be seen on the leaf surface in *A. philoxiroides*, indicating fungal colonization. This is in contrast to the clear necrotic disease lesions in *A. sessilis*.

Infected leaves stained with trypan blue clearly highlighted the region of cell death on the leaves (Figure 2a). *Alternanthera philoxiroides* had a significantly lower cell death area compared to *A. sessilis* across all time intervals post-inoculation with *R. solani* (Figure 2b). Furthermore, the amount of ion leakage was significantly higher at earlier time periods (<24 hpi) in *A. sessilis* compared to *A. philoxiroides*, which showed higher leakages at a later time interval (>48 hpi; Figure 2c). Overall, these results suggest that, although both plants are susceptible to *R. solani*, the invasive *A. philoxiroides* showed slower disease progression compared to the native *A. sessilis*.

**Figure 2.** Trypan blue staining and ion leakage tests of *R. solani* infected leaves from invasive *A. philoxiroides* (AP) and native *A. sessilis* (AS) at different time intervals after inoculation. (**a**) Before and after staining with trypan blue for visualizing dead plant leave tissues. Circled areas on leaves are at 0 h (un-inoculated control) and 24 h (inoculated) before staining. (**b**) Cell death areas represented by means ± standard errors (SE) from three biological replicates with different letters representing the groups that were significantly different from other groups as determined by a one-way analysis of variance (ANOVA), followed by a multiple comparison using Duncan's method (*p* < 0.05). (**c**) Electrolyte leakage of leaf discs infected by *R. solani* from 2 h to 96 h measured using an electrolytes' conductivity meter. Electrolytic conductivity increased in native AS during earlier time intervals; however, it increased in invasive AP at later time intervals compared to the un-inoculated control (0 h). Error bars indicate means ± SE (number of disks = 12 for each species) and two discs from each plant representing a total of six biological replicates for each species. Asterisks indicate significantly different from native plants using Duncan's method (*p* < 0.01).

#### *2.2. Expression Divergence of Defense Hormone Genes between A. philoxiroides and A. sessilis*

Expressions of all six genes (*PAL*, *PR3*, *LOX*, *JAR1*, *PR6,* and *EIN3*) were quantified in both the local and systemic leaves of the two species against *R. solani* (the fold–change ratio of defense hormones and their responsive genes are presented in Table S1). Expression differences were observed in both infected local (Figure 3) and systemic leaves (Figure 4), as well as the hormone content (Figure S1) between *A. philoxiroides* and *A. sessilis* during disease development caused by *R. solani* (Table 1 and Table S1). Furthermore, expression differences were also observed in the hormone pre-treatment group between the two species (Figure 5). The key difference was that JA and ET-signaling was differentially regulated in *A. sessilis*. JA was partially suppressed in *A. sessilis*, whereas there was a consistent reduction in expression in *A. philoxiroides* (Figure 3a,b). ET-*EIN3* expression was reduced in *A. sessilis* but was induced in *A. philoxiroides* (Figure 3e). SA level was induced in both species (Figure 3c,d). In addition, weaker or no antagonistic cross-talk was observed between SA (Figure 3c,d) and JA-signaling (Figure 3a,b) in *A. sessilis*. In contrast, there was strong cross-talk in *A. philoxiroides* (Figure 3 and Table 1).

**Figure 3.** qPCR analysis of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)- responsive gene expressions between invasive *A. philoxiroides* (AP) and native *A. sessilis* (AS). Four-week-old plants were infected with *R. solani* and the samples were harvested for RNA extractions at the indicated time intervals (0 to 96 hours) after inoculations. Un-inoculated leaves were used as a control (0 h). Specific primers were used for *JAR1* (**a**), *PR6* (**b**), *PAL* (**c**), *PR3* (**d**) and *EIN3* (**e**) with *Actin* (control) as shown in Supplementary Table S2. Values represent means ± SE from three biological replicates. The asterisks (\*) represent significantly different levels at each time period and were determined using one-way ANOVA, followed by a multiple comparison using Duncan's method (*p* < 0.05).

**Figure 4.** Expression analysis of SA, JA and ET-responsive genes for systemic acquired resistance tests between *Alternanthera philoxiroides* (AP) and *A*. *sessilis* (AS). Four-week-old plants were infected with *R. solani* and samples of healthy un-inoculated leaves were sampled for RNA extractions at the indicated time intervals (0 to 96 hours). 0 h is from the samples of plant completely un-infected for control. Relative expression of *JAR1* (**a**), *LOX* (**b**), *PR6* (**c**), *PAL* (**d**), *PR3* (**e**), *EIN3* (**f**) and *Actin* (control) were tested using gene specific primers at the indicated time intervals. Error bars show ± SE from three biological replicates and the asterisks (\*) represent significantly different levels, which were determined via a one-way ANOVA, followed by a multiple comparison using Duncan's method (*p* < 0.05).

**Figure 5.** Expression analysis of SA, JA and ET-responsive genes for hormone pre-treatment tests between *Alternanthera philoxiroides* (AP) and *A*. *sessilis* (AS). Four-week-old plants were sprayed with MeJA pretreatment (**a**,**b**) SA pretreatment (**c**,**d**), and ET pretreatment (**e**) before being inoculated with *R. solani*. Samples were harvested for RNA extractions at the indicated time intervals after inoculations. Un-inoculated leaves were used as a control (0 h). Specific primers were used for *JAR1* (**a**), *PR6* (**b**), *PAL* (**c**), *PR3* (**d**) and *EIN3* (**e**) with *Actin* (control) being tested using gene specific primers at the indicated time intervals. Values represent means ± SE from three biological replicates. Asterisk (\*) represent significantly different levels at each time period and was determined via a one-way ANOVA, followed by a multiple comparison using Duncan's method (*p* < 0.05).


**Table 1.** Expression differences in defense hormones and their responsive genes in the invasive *A. philoxeroides* and native *A. sessilis* during *R. solani* pathogenesis. See Table S1 for fold–change ratios of each gene for each treatment in both the invasive and native species.
