Plant Invasion by Chromolaena Odorata Alters Soil Microbiome and Provides Insight Into the Role of the Fcb Group of Bacteria


 Purpose Plant invasion provides an excellent working model for understanding the effects of soil microbial communities associated with natural plant populations. In this study, we investigated the diversity and abundance of soil microbial communities of Chromolaena odorata, one of the most invasive weeds, and Tridax procumbent, a native plant in the same family. Methods Elemental analysis of carbon, hydrogen and nitrogen in soil samples was performed using a CHN analyzer. Meta-genome analyses were performed using Oxford Nanopore sequencing. The data assembly, analysis, and classifications of all functional categories were performed using the SqueezeMeta v1.0.0 Pipeline.ResultsThere is an increase in pH along with an increase in C, H, and N in the soil of the invasive plant C. odorata. Copiotrophic microbiome along with the gene families responsible for transport, metabolism of carbohydrates and cellular processes are more abundant in C. odorata soil. In contrast, oligotrophic microbial communities along with gene families involved in energy production and cellular maintenance are more abundant in the T. procumbens soil.ConclusionOur studies show the association of plant invasion with an increase in nutrient cycling and foster the development of copiotrophic organisms, including Bacteroides. This is associated with an increase in carbohydrate metabolism and substrate utilization, as well as activation of pathways for stress adaptation and resistance.


Introduction
Chromolaena odorata (L.) King and Robinson (C. odorata) are some of the most widely distributed tropical shrubs. As one of the most invasive weeds in the world, it is a serious problem in Central and West Africa, India, Australia, Paci c Islands, and Southeast Asia (McFadyen 2003). It copes with a broad range of climates and has spread to major continents like Asia. When established, it can create pure stands in disturbed areas, grasslands, fallows, and forest plantations (Kriticos et al. 2005). A few studies have been conducted on the invasion of C. odorata. Humid climates are preferable to the establishment and growth of C. odorata, and that tropical humid regions with latitudes between 9°30' and 10°N are likely to be suitable habitats for C. odorata under future climate conditions (Fandohan et al. 2015). Invasion among natural communities by these introduced species poses a major threat to biodiversity (Panetta and Gooden 2017). Worldwide Diverse native plant communities are di cult to re-establish on soils that were once harboured by invasive plants ( Van der Putten et al. 2013). In India, 1,599 alien plant species belong to 841 genera and 161 families, making alien ora 8.5% of India's vascular ora (Reshi and Khuroo 2012). There are several hypotheses to explain alien species invasions, the prominent of which focus on the role of habitat disturbance, competitors and consumers, soil nutrient cyclin (Chabrerie et al. 2019). Many studies have focused on identifying traits that promote plant invasiveness. The in uence of soil microbial communities on invasion by these plants needs investigation. Identifying the potential mechanisms that allow invasive species to thrive is essential for controlling existing and future invasions. The soil microbiota changes over time and plays an important role in determining plant productivity and species diversity. In recent years, the interaction between soil microbes and invaded plants has gained increasing attention (Chernov and Zhelezova 2020). Species introduced into an ecosystem can alter edaphic factors, altering the structure and functioning of the microbial community (Bell et al. 2020). However, it remains unclear how microbes contribute to the invasive process.
With the rapid and continuous development of cultivation-independent metagenomic approaches, our knowledge of soil microbes has increased (Neelakanta and Sultana 2013). Microbial communities in native soils consist of a few dominant species and many other rare taxa (Jousset et al. 2017). Deep metagenomic sequencing enables understanding genomic insights into a low abundance population and may reveal a wide range of plant-microbe interactions in soil. By comparing the soil microbiome of invasive and native species, the difference between the microbial composition of these two communities may enable better explanation, which would contribute to understanding the role of microbial communities in the mechanisms underlying C. odorata invasion. The extensive spread of C. odorata species and its suitability for climate change requires immediate attention to elucidate the mechanism of invasion and the contributing factors. In this work, the soil properties of C. odorata and its microbiota were studied. Tridax procumbens, a native plant of the Asteraceae family, was used as a control.

Sample collection and analysis
We collected soil samples from C. odorata and T. procumbens plants growing in the local areas of Nalanchira (8.5392 N 76.94 E), Thiruvananthapuram Kerala, India. Soil samples beneath ve plants at a depth of 0-10 cm and a radius of 20 cm were collected and immediately stored at -80 °C until analysis.
Elemental analysis of carbon, hydrogen, and nitrogen was performed using a CHN analyzer (2400 CHN elemental analyzer from Perkin Elmer) according to the manufacturer's instructions.

DNA extraction
We have performed the extraction of DNA from soil samples using a commercial kit (Purelink microbiome, Thermo Fischer) designed to extract DNA from soil samples. Following the manufacturer's instructions, DNA was extracted by bead beating and puri ed by column chromatography. The extracted DNA was quanti ed using the Qubit instrument (Thermo Fischer). After staining with Ethidium Bromide, resolution on a 0.8% agarose gel was used to analyze the quality of the extracted DNA. DNA was stored at -20°C until further processing. To avoid contamination, all procedures were performed aseptically.

DNA sequencing and bioinformatics analysis
Raw sequence data generated from the nanopore platform were ltered and assessed quality with the nanoQC software used for nanopore sequence data quality checking. Soil metagenome analysis of C. odorata and T. procumbens was performed using SqueezeMeta v1.0.0 Pipeline (Tamames and Puente-Sánchez 2019), which can be run in three different modes. The rst mode is the sequential mode, where the sequenced metagenome data are treated individually and analyzed. The second mode is the coassembly mode, where the sequenced reads are pooled to perform a single assembly, and the third is the merged mode, where a single set of contigs is generated from the assembled data. The sequenced samples were assembled to maximize the robustness of contig construction using MEGAHIT assembler (Li et al. 2015) with default parameters and the resulting contigs were merged into a single co-assembly. MEGAHIT assembler assembles the whole data with no pre-processing steps, like partitioning and normalization. It produces longer contig N50 and average contig length with large reads aligned to the assembly. Contig statistics were done using prinseq (Schmieder and Edwards 2011). Redundant contigs were removed using CD-HIT (Schmieder and Edwards 2011). Contigs were merged using Minimus2 (Treangen et al. 2011).
The contigs were annotated to identify the function and taxonomic a liations. Here, contigs corresponding to the same organism are identi ed using different properties of their sequences, such as composition or abundance, and clustered into genome bins. Binning was done by the software MaxBin2 inside the SqueezeMeta Pipeline. After this step, samples were mapped against assembled contigs and were used to calculate taxonomic and functional statistics. Gene prediction from the contigs was performed with Prodigal (Hyatt et al. 2010) and redundant contigs were removed using CD-HIT

Soil physicochemical properties
The soil sample of C. odorata is more acidic than that of T. procumbens. C. odorata soil had a pH of 5.4 and T. procumbens soil had a pH of 6.8. CHN analyses were conducted to determine the composition of carbon (C), hydrogen (H), and nitrogen (N). The results showed that C, H, and N were abundant in the soil samples of C. odorata compared to T. procumbens soil (Table 1). Compared to the T. procumbens (0.90%), the soil of C. odorata contains the highest percentage of C (9.78%). The H content is 0.28% less than T. procumbens soil (0.48%). The N content of the C. odorata soil is 0.74% and that of the T. procumbens soil is 0.18%.

Bioinformatics analysis
Based on microbial genome sequencing using nanopore sequencing, 2923 raw reads were generated for the C. odorata soil sample and 2350 raw reads for T. procumbens soil. The quality of the raw data was checked using the nanoQC software program for quality checking of nanopore sequencing data. In both cases, the quality of the raw data was satisfactory. Figures 1a and 1b illustrate the quality plots based on the Phred scores. The average Phred score of the sequencing reads for the samples was calculated as ≥20. Megahit was used to assemble samples individually, followed by the merging of the resulting contigs into a single co-assembly. The total assembly statistics for the co-assembled genome comprise 684256 bases and 312 reads shown in supplementary Table 1.

Taxonomy and distribution
Taxonomic analysis of the C. odorata soil revealed that 99 reads mapped to bacteria, 7 reads mapped to Eukaryota, and 65 reads mapped to an unknown taxon. For the T. procumbens soil sample, 78 reads mapped to bacteria, 24 reads mapped to Eukaryota, and 40 reads mapped to unknown taxa. In the interactive taxonomic distribution of total microbial diversity based on contig taxonomy, the Krona data shows that the C. odorata soil contains 58% bacteria, 4% eukaryotes, and 38% unknown species. Among the bacterial species, 28% are Terrabacteria, 26% are Proteobacteria, 14% are FCB bacteria and 30% are other bacteria. Actinobacteria make up 71% of the Terra bacteria. Bacteroidetes make up 79% of the FCB group bacteria. In the T. procumbens soil samples, Proteobacteria accounted for 24% of the total bacterial population, 38% of Terrabacteria and 4% of Acidobacteria. Among the Proteobacteria, 32% were Beta Proteobacteria, 16% were Alpha Proteobacteria, and 11% were Delta Proteobacteria. The Terrabacteria comprised 53% Actinobacteria, 13% Chloro exia and 10% Tenericutes ( Figure. 2a and 2b).
From the taxonomy assigned to the contigs of the co-assembled whole-genome metagenome, the distribution of the top Phyla and Class are shown in Figure 3 and Figure 4. The results show that Proteobacteria and Actinobacteria are more abundant in C. odorata soils compared to T. procumbens soils. In addition, the Bacteroidetes were found in large numbers in C. odorata soil. The phylum Chlorofelxi, Planctomycetes, Acidobacteria, Arthropoda, and Tenericutes, and Tenericutes are abundant in T. procumbens soils. Among Proteobacteria, Alpha Proteobacteria, and Gamma Proteobacteria classes were more abundant in C. odorata soils, while Beta Proteobacteria and Delta Proteobacteria were more abundant in T. procumbens soils. Species of Ignavibacteria were common within the class of Saprospiria.
Chordates were observed among eukaryotes in C. odorata soils. However, Cloro exi and insects were abundant in the T. procumbens soil. According to KEGG annotation, metagenomic ORFs were aligned with the genes associated with 145 metabolic pathways. Among the functional categories in KEGG, genes involved in metabolism are the most abundant in C. odorata soil and T. procumbens soil. Interestingly, carbohydrate metabolism was the most abundant metabolic pathway (31%) in C. odorata soils. With C. odorata plants, the second abundant category was involved in cellular processes. It is noteworthy that in the cellular process; the genes associated with amino acid transporters were abundant in C. odorata soils. There was an increase in pathways associated with genetic information and processing in Chromolaena species (Fig.5a) Besides this, the most abundant functional categories of the gene were Branched-chain amino acids, ABC transporter-periplasmic leucine binding subunit livK (K0199) and its orthologues genes in C. odorata soil. Glutathione S transferase (K00799), Fe-S assembly proteins (K09014) were also abundant in the C. odorata soil sample (Fig.5b). In T. procumbens sample Elongation factor G (K02355), the large subunit of ribosomal protein L13 (K02871), Monovalent cation H+ antiporter (K03316) was the prominent category.
In COG category glycosyl transferase (COG 0435), ABC-Type transporter system involved in Fe-S cluster assembly permease component (COG 0719) ABC transporter substrate-binding protein (ENOG410XNWR), Glusamylase and related glycosyl hydrolases (COG3387) were the abundant categories. With T.

Discussion
Biological invasions have been identi ed as a growing threat to global sustainability, and their impact on soil microbial communities has attracted much attention in recent years. C. odorata has a worldwide distribution, in neotropical areas affected by human activities. Once introduced, C. odorata can become dominant within a short period (McFadyen and Skarratt 1996). C. odorata can increase soil nutrient levels (Wei et al. 2017) and inhibit native plants (Mangla et al. 2008). Given the wide distribution of this C. odorata species and its ability to adapt to climate change, it is imperative to understand the factors that in uence its invasion mechanisms.
Plant growth is promoted by the interplay of soil microbes and the physiological environment. In our study, we analyzed soil physicochemical factors and microbial diversity in soil samples of C. odorata and compared them with the soil of the native plant T. procumbens. We collected both soil samples from the same location. Soil pH can be considered a key variable due to its in uence on microbial activity, nutrient availability, and plant growth. Our results show that soil samples of C. odorata are more acidic than those of T. procumbens, which may be due to the acidic extrudates generated by the invasive plants (Ikhajiagbe 2016). There is growing evidence that soil nutrient availability is a major abiotic factor that can in uence the success of alien invasive plants. An alien invasive plant usually shows higher e ciency in nutrient utilization and greater exibility in nutrient management compared to its native counterpart (Osborne and Gioria 2018). Analysis of CHN shows that the soil of C. odorata contains a higher concentration of C, H and N than that of T. procumbens. This may lead to nutrient-rich conditions in C. odorata soils, which promote nutrient cycling and accelerate the growth of invasive plants. This is consistent with previous reports showing that the amount of C and N in soil appears to increase with the severity of C. Our results support the hypothesis that copiotrophic groups (e.g., Proteobacteria and Actinobacteria) with fast growth rates increase under nutrient-rich conditions, whereas oligotrophic groups (e.g., Acidobacteria and Chloro exi), which have slower growth rates, decrease (Fierer et al. 2005). This shift in microbial community structure associated with a nutrient-rich environment may play an important role in plant invasion. In soils treated with arti cial water rich in organic and mineral loads, the abundance of Gamma Proteobacteria, Cytophagia, Saprospiria and Sphingobacteria increased. Similarly, we have observed the abundance of these bacterial genera in soil samples with invasive plant C. odorata.
We also observed a signi cant decrease in arthropod communities in C. odorata soils. This correlates with earlier reports that show a reduction in the arthropod community during invasion (van Hengstum et al. 2014). In many ecosystems, arthropods are the most important primary consumers and necessary for the pollination and dispersal of a variety of plant species (Chapman and Chapman 1998).
Evidence suggests that invaders negatively affect the visitation and reproductive success of native cooccurring plants (Morales and Traveset 2009), and invaders may even disrupt the mutualistic relationships between native species and insects (Traveset et al. 2013). The response of arthropods to plant invasions should be of particular concern given their abundance, diversity and indispensable roles as herbivores, pollinators, predators and prey.

Importance of FCB group of bacteria in plant invasion
We detected an abundance of bacteria belonging to the FCB group in the soils of C. odorata. The FCB group comprises Fibrobacteres, Chlorobi and Bacteroidetes. Bacteroidetes occur in many ecosystems and play an important role in the degradation of polymeric organic matter (Zheng et al. 2021). Because of their high abundance, their ability to degrade polysaccharides, and their diverse enzyme systems, Bacteriodates play an important role in the carbon and nutrient cycles (Gupta 2004). They have an effective genomic organization in which most of their CAZymes are localized in the polysaccharide utilization loci. Carbohydrate active enzymes (CAZymes) are responsible for the synthesis, modi cation, and degradation of carbohydrate biopolymers in plants. According to our results, the genes involved in carbohydrate metabolism were abundant in the soil of C. odorata. This indicates that copiotroph organism like Bacteroidetes which produce a variety of carbohydrate-active enzymes (Larsbrink and McKee 2020) plays a major role utilization of plant-derived polysaccharides and thereby accelerating the growth of invasive plants. Ignavibacteria, a member of the Bacteroidetes, have been isolated from hot water sediments from Yellowstone National Park and have been reported to metabolize nitrogen and aromatic compounds (Tian et al. 2015). Cytophaga Flavobacterium (CF), are bacteria that may cause soil degradation. Several of these organisms exist all over the world and are known for their role in degrading organic compounds. Studies have shown that they are present in the soil, especially in the root zone. In addition, these bacteria produce a wide range of hydrolytic enzymes that can degrade complex carbohydrates. This suggests that the FCB group play a crucial role in plant invasion. Because of their adaptability to changing conditions and substrate utilization, they threaten the ecological balance in changing climatic conditions that favour plant invasions. Ether-linked lipids in their membrane can help Bacteroidetes adapt to the different stress conditions they are exposed to during their development (Siliakus et al. 2017;Villanueva et al. 2021).

Functional category
Functional annotation shows that one of the most abundant genes expressed in the soil microbiome of C. odorata was the Branched-chain amino acids ABC transporter-periplasmic leucine binding subunit livK and its orthologous genes. Studies suggest that catabolism of Branched-chain amino acids may contribute to plant resistance to pathogens by modulating the defence pathways (Zeier 2013). The second most abundant category was the Fe-S cluster assembly protein SufB FE -S. It is necessary for the proper functioning of several metabolic pathways and resistance to genotoxic and abiotic stresses. In addition, they are involved in resistance to non-host plants and plant immunity (Fonseca et al. 2020). Iron and sulfur (Fe-S) clusters are essential for several biochemical processes, including respiration, photosynthesis and nitrogen xation (Gao 2020) Bacterial glutathione transferase (GST) is another functional category that is over-expressed. Bacterial GSTs mediate several processes, including degradation of xenobiotics, defence against chemical and oxidative stress, and resistance to antimicrobial agents. In addition, GSTs play a role in many metabolic processes including, the degradation of lignin, the biotransformation of dichloromethane, and the reductive dechlorination of pentachlorophenol (Allocati et al. 2009). Glutathione transferases (GSTs) are ancient, ubiquitous, multifunctional antioxidant enzymes that play important roles in plant growth and development. Recent studies suggest the involvement of GSTs in cell signalling kinases, ion channel formation and modulation, oxidation-reduction reactions, etc. GSTs can also act as ligands and are involved in the transport of auxins within cells (Kumar and Trivedi 2018). This indicates the involvement of genes in pathogen resistance and stress tolerance are involved more in invasive soil. Several oligopeptide importers, 3-isopropyl malate dehydrogenase, enzymes of amino acid biosynthesis and Elongation factor G are expressed in the soil of T. procumbens. These genes are involved in the transport of nutrients, cell signalling, amino acid biosynthesis and protein synthesis. A recent study using genome analyses also shows that gene families involved in the transport and metabolism of carbohydrates and amino acids often predominate in copiotrophic microorganisms. Oligotrophs exhibited a higher prevalence of gene families involved in energy production and conversion. Translation e ciency through codon optimization was an important factor in controlling the growth pattern of oligotrophs and copiotrophs. This is correlating with our results which show that the gene family responsible for transport, metabolism of carbohydrates and cellular processes are more prevalent in invasive soil microbial communities. However, gene families involved in energy production and environmental information and processing are more prevalent in the microbial community of T. procumbens.
These results and our ndings suggest that copiotrophic organisms such as Bacteroides play an important role in plant cell invasion. Our studies show the association of plant invasion with an increase in nutrient cycling fosters the development of copiotrophic organisms. This is also associated with an increase in carbohydrate metabolism and substrate utilization, as well as activation of pathways for stress adaptation and resistance.

Conclusion
We found an increase in pH along with an increase in the availability of C, H, and N in the soil of C. odorata, which could enhance a shift from a copiotrophic to an oligotrophic microbial community in the microbiome. The invasive plants might be able to alter soil conditions and re-establish soil microbial communities once they have invaded, thereby gaining access to soil nutrients more readily than their associated competitors. This could be accompanied by an increase in gene families involved in substrate utilization, adaptation to stress, and resistance to pathogens. Plant invasion can alter soil biochemical properties, nutrient cycling, and microbial diversity, creating an ecological imbalance. The development and application of a microbial consortium in conjunction with altering the nutrient-rich environment is an alternative strategy to promote the growth of native plant species. We present here a preliminary analysis of the meta-genome as a basis for further study. Further studies are needed to fully understand the mechanisms of invasive plants and their impact on environmental restoration.

Declarations Data Availability Statement
The datasets presented in this study can be found in the online repository, https://www.ncbi.nlm.nih.gov/ with an accession number PRJNA746476 49. Zhou Z, Tran PQ, Kieft K, Anantharaman K (2020) Genome diversi cation in globally distributed novel marine Proteobacteria is linked to environmental adaptation. ISME J 14:2060-2077 Figure 1 Phred quality score values for (a) C. odorata soils sample and (b) T. procumbens soil sample. The raw data quality was checked with the nanoQC software used for nano sequence data quality checking. The

Figures
x-axis displays the base position in the read, and the y-axis shows quality scores.