**1. Introduction**

Numerous plant species have been directly or indirectly introduced to new habitats as ornamentals, or as sources of food and fiber. However, many of these species have become invasive and pose a

serious threat to agriculture, biodiversity and ecosystem function [1]. Following initial introduction, the spread of an invasive plant species could be further enhanced by global climate change (e.g., increasing CO<sup>2</sup> emissions) [2,3]. Much of the previous research in invasion biology has focused on the ecological and evolutionary factors that contribute to plant invasions [4,5]. Due to the unavailability of genomic resources [6], the genetic factors underlying invasion success are still not well understood.

Invasive plants may encounter novel abiotic and biotic stresses across the introduction– naturalization–invasion continuum. These stresses can affect their survival and reproduction, and can act as barriers to plant invasion. Successful invasive plant species possess various attributes (such as rapid adaptation, fast growth and spread, or high fecundity), and have effective defenses against natural enemies, which allow them to overcome barriers to invasion [7,8]. Higher resistance against generalist herbivores and pathogens may benefit invasive plants more than non-invasive species in new regions [8,9]. The enemy release hypothesis (ERH) and the biotic resistance hypothesis (BRH) have been proposed to explain the success and limitations that invasive species experience based on studies of novel plant–natural enemy interactions [10,11]. The ERH proposes that exotic plants will be less impacted by natural enemies compared to their native range because they have escaped their specialist herbivores or pathogens [12]. Therefore, the success of invasive plants in new environments can be attributed to the allocation of resources from defense to growth to outcompete other plants (evolution of increased competitive ability hypothesis or EICA) [13]. There has been a lot of support for the ERH, including reduced impacts by herbivores on invasive plants [14–16], as well as reduced attacks by above ground fungal and soil pathogens [14,15,17–20]. In contrast, the BRH states that native enemies should limit the growth of exotic plants in new ranges [21]. Many studies support the BRH [11,22–25], while some studies support both the ERH and BRH [26,27].

The improved performance of invasive plants in a competitive environment to enemy attack may be due to genetic changes acquired during the invasion process [6,28]. The use of genomic and transcriptomic technologies could identify the genetic architecture underlying the success of invasive plant species. A recent transcriptomic study was used to compare the gene expression profiles of introduced (North American) and native (European) populations of the Canada thistle, *Cirsium arvense*, in response to nutrient deficiency and shading [29]. This study identified significant differences in R-protein mediated defense and expression pattern between introduced and native populations of *C*. *arvense* [29]. Similarly, in the common ragweed (*Ambrosia artemisiifolia*), an invasive species to Europe, candidate genes were identified using oligonucleotide microarrays under light and nutrient stress conditions that were thought to contribute to invasiveness [30]. In addition, weedy sunflower genotypes of *Helianthus annuus* naturalized in the USA were tested for variations in gene expression compared to wild non-weedy species [31]. This study found extensive genetic differentiation between the two species [31]. However, until now, only a few investigations have been undertaken to elucidate the genomic mechanisms responsible for the adaptation of invasive plants to biotic stresses.

Generally, a multitude of plant defense pathways are activated in response to microbial pathogens [32]. The first line of active defense occurs at the plant cell surface, when generalist microbe elicitors (the microbe or pathogen-associated molecular patterns, i.e., MAMP or PAMPs) are produced by pathogens such as, flagellin (from bacteria) [33], chitin (from fungal pathogens) [34], β-glucan (from oomycete pathogens) [35], or effectors (from specialized pathogens) [32]. Plants detect these elicitors by pattern-recognition receptors (PRRs) within the cell membrane, which leads to PAMP-triggered immunity (i.e., PTI) [36]. In the case of effectors, receptors with nucleotide-binding domains and leucine-rich repeats (NLRs or *R* genes) [37] are used, which lead to effector-triggered immunity (i.e., ETI) [38,39]. The evolutionary development of the plant immune system is represented as a Zig-zag model [40], where specialist pathogens often co-evolve with their host (see Han [41] for evolutionary dynamics between plants and pathogens). Invasive plants generally lack the need for defense against specialist pathogens in their new ranges (due to lack of co-evolution) [42] and therefore can invest more energy into growth or reproduction [12,13].

The timely recognition of the invading pathogen and a rapid, effective induction of defense responses are required for resistance to disease in plants [43]. Plant hormones play a key regulatory role in inducing defense responses shortly after the perception of a pathogen, through an extensive transcriptional reprogramming of genes involved in hormonal signaling [44]. In addition, the plant defense hormones play a critical role in response to adverse environmental conditions [45–48]. Abscisic acid (ABA) plays a central role in plant defense to abiotic stresses, such as salt and drought stress [49]. Salicylic acid (SA) is a major plant defense hormone induced by infections from biotrophic and hemi-biotrophic pathogens [50,51]. SA, jasmonic acid (JA), and ethylene (ET) also play key roles under biotic stresses [52]. These induced defenses and hormone signaling networks have been well characterized in model organisms and crop plants, such as *Arabidopsis* [53,54], tobacco [55] and tomato [56], but are not well known in invasive plants. For example, pyrosequencing was used to identify molecular signaling networks linked with paradormancy in underground vegetative buds of invasive *Cirsium arvense*. Interestingly, the plant hormone auxin and ABA-signaling was found to regulate paradormancy, allowing plants to resist weed control methods (e.g., chemical and biological controls), thereby enhancing their invasiveness [57]. Currently, there is a broad interest among invasion biologists to unravel the genetic mechanisms of resistance and defense responses of invasive plants. In this study, we hypothesize that invasive plant species have higher or enhanced resistance to microbial pathogens compared to native congeners. We also predict that the endogenous defense mechanism and signaling (especially the defense hormones SA, JA, and ET) play a crucial role in resistance, thereby benefiting the invasion success of invasive plants.

The alligator weed, *Alternanthera philoxeroides* (Martius) Grisebach, is an amphibious stoloniferous perennial herb [58]. It is native to South America [59] and was first introduced to China in the late 1930s as a forage crop from Japan [60]. It is the most noxious invasive plant in China [61,62] where it is a significant weed in rice farms, causing an estimated agricultural loss of \$75 million per year [63]. Populations of *A. philoxeroides* growing in China have extremely low genetic diversity, which is attributed to the predominance of a single genotype (likely due to a single recent introduction) and extensive vegetative propagation by cuttings since being introduced [60,64].

*Alternanthera philoxeroides* is highly vulnerable to insect herbivore attack. For example, more than 15 generalist insects were found to feed on *A. philoxeroides* in China [11]. A specialist beetle, *Agasicles hygrophila* (alligator weed flea beetle) from South America was introduced to the USA [59] and China [11] to control *A. philoxeroides*. However, only a few disease incidences in *A. philoxeroides* have been reported. For example, species of *Nimbya* have been found to cause leaf and stem spot on *A. philoxeroides* in Australia [65]. *Nimbya alternantherae* has been identified as a biocontrol agent in Brazil [66], while species of *Fusarium* have been used as a biocontrol for alligator weed in China [67]. *Rhizoctonia solani* has been shown to be pathogenic to *A. philoxeroides*, and has also been found to infect a related species, *Alternanthera sessilis*, in the USA [68] and India [69]. Earlier preliminary pathogenicity screening tests showed that *R. solani* (ACCC 30374) is more virulent on *A. philoxeroides* compared to *Fusarium oxysporum* f.sp *cubense* (ACCC 36369) (SS Qi, unpublished data). Therefore, *A. philoxeroides* represents a good model to study pathogen resistance and defenses in an invasive plant species following invasion.

In this study, we aimed to isolate the defense hormones (SA, JA, and ET) and associated genes in invasive *A. philoxeroides* and its native congener *A. sessilis* to test for differences in gene expression between the species against a generalist necrotrophic fungus, *Rhizoctonia solani*. Although signaling of these defense hormones and their cross-talk in response to pathogens are well documented in *Arabidopsis* and other model plant species [53–56], our study is the first to examine these phenomena in wild populations of a co-occurring invasive and native congener pair. Furthermore, divergence in gene expression between the species may allow us to identify patterns of defense signaling that may be responsible for enhanced resistance in invasive *A. philoxeroides*. Specifically, we ask the following questions: (1) Is invasive *A. philoxeroides* less susceptible to the pathogen (*R. solani*) compared to its native congener *A. sessilis*? (2) Are there differences in gene expression between the native and invasive

species after inoculation with *R. solani*? (3) Are there differences in resistance between infected and un-infected neighboring leaves? To address these questions, we performed in vitro and in planta leaf inoculations using *R. solani*. Six hormones and their responsive genes (three JA, two SA, and one ET) were successfully isolated from both invasive *A. philoxeroides* and native *A. sessilis* for expression analysis using RT-qPCR in response to three treatments. The first included *R. solani* inoculation for susceptibility tests, the second comprised un-infected samples for systemic resistance tests, and the third included hormone pretreatments using SA, JA, and ET (prior to inoculation with *R. solani*).
