The spread of C. annuum wilt and the high incidence of damage, together with the few studies of the associated microorganisms that inhabit the rhizosphere, result in great concern for all personnel involved in the cultivation and management of this agriculturally important crop. In the present study, we analyzed the diversity and structure of bacterial and fungal communities through 16S rRNA and ITS region amplicon-based metagenomic sequencing in rhizosphere soil of healthy as well as wilt-diseased plants under organic farming conditions. From the results, we identified a diverse community of microorganisms, of which several bacterial and fungal members were either shared by, or exclusive to, the rhizospheres of C. annuum plants depending on their health condition.
The taxonomic assignment confirms that rhizospheric soil of C. annuum is composed mainly of the Proteobacteria, Gemmatimonadetes, Actinobacteria, Bacteroidetes, and Acidobacteria phyla, the first being the most abundant. The phylum Proteobacteria has been reported in many studies for its association with the rhizosphere of plants, particularly in some studies on soil growing C. annuum [4, 34, 60, 75]. In general, at the genus level, microbial diversity was very high and homogeneous, independent of the health conditions of the plants, although genera such as Kaistobacter, Bacillus, Rubrobacter, Streptomyces, Balneimonas, Nitrospira, and Salinimicrobium were slightly more abundant than others in the rhizosphere. These genera have been reported as the most frequent in soil [21, 71]. Little information is available about the genus Kaistobacter; however, some studies have reported species of this genus as being associated with active disease suppression in the rhizosphere of tobacco plants [21, 37]. Moreover, genera including Bacillus, Rubrobacter, and Streptomyces have been reported as suppressors of rhizosphere fungi that are pathogenic to other plant species, such as Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahliae [9, 12, 30, 56, 73]. These species were also found in this study, so future analyses should be carried out to evaluate the biological control capabilities of these bacterial members.
The Ascomycota, Mucoromycota, Mortierellomycota, and Basidiomycota were the most abundant fungal phyla, of which the first had the highest abundance. Although it is reported as a widely found phylum in the rhizosphere, only few studies have described the fungal communities associated with C. annuum as well as other plants belonging to the Solanaceae family [46, 58]. The genus Aspergillus, the most abundant in this study, has been reported for its antifungal activity against Phytophthora capsici ; however, as that study was conducted on in vitro bioassays, caution must be exercised in drawing conclusions on biological interactions that occurunder in vivo conditions.
Studies that have evaluated the α-diversity of rhizosphere communities in agricultural crops have reported variations in the diversity, presumably because of the different factors to be considered at the time of the study (e.g., temperature, plant age, sampling season, crop rotation). Similar to the results reported in this study on bacterial richness and diversity, in plants of the Solanaceae and Piperaceae families a high bacterial diversity has been reported regardless of whether the plants were healthy or diseased [28, 35], but this was not so for evenness, which significantly declined when the crop became diseased . In fungal communities, similar to what occurred in this study, a higher αdiversity has been reported in healthy plants than in diseased plants[35, 62, 72]. It is worth mentioning that the agronomic practices in the plots sampled in this study were organic, so soil microorganisms had not been exposed to agrochemicals.
In terms of community structure, several studies showed changes in the β-diversity of bacterial and fungal rhizosphere communities, caused by plant pathogen infestation ; our results also demonstrated that the β-diversity was segregated according to plant health condition. These differences in microbial communities may have occurred for several reasons, such as modification of soil properties due to attack and colonization of C. annuum plants by pathogens (e.g., pH, nutrient solubility, O2, CO2, moisture), which triggered a modification of the ecological niche and resulted in the recruitment of microorganisms that exert either deleterious or beneficial effects on the plants .
The differential abundance test showed no significant difference in the bacterial rhizosphere regardless of plant health condition, whereas the abundance in the fungal rhizosphere differed significantly between plant conditions. In the rhizosphere of healthy plants, a significant difference was observed in the genus Mortierella, members of which have been reported as plant growth promoters, as well as antibiotic and phytohormone producers, thereby improving resistance to phytopathogens in plants of agricultural importance [42, 48, 74]. Moreover, in the rhizosphere of wilted plants, significant differences were shown in several fungal taxa, particularly in some genera that have been reported as phytopathogenic agents in various crops. This has led us to hypothesize that this increase in abundance of Thanatephorus and Fusarium species is responsible for triggering the symptomatology that affects the plants, and which subsequently allows other opportunistic pathogens (e.g., Rhizopus, Curvularia, Cladosporium, and Alternaria) to jointly infect the plant until its decay.
The genus Rhizopus was found with a higher abundance in the rhizosphere of the wilted plants, and this pathogen has been previously associated with the deterioration of crops in chili pepper [1, 19] and other plants, such as soybean , apple , and sugar beet . Similarly, Thanatephorus, Fusarium, and Alternaria have been found to be dominant fungal genera in rhizosphere soil of chili pepper [5, 18, 50, 64, 69], soybean , mung beans , and maize  as causal agents of wilt, root rot, leaf spot, and/or fruit and seed rot. Also, Curvularia is a plant pathogen ; C. lunata has been reported as the causal agent of maize leaf spot , leaf spot disease of Clerodendrum indicum , and root rot of strawberry . Finally, Cladosporium species have been reported as pathogenic fungi of members of the Solanaceae family such as tomato  and chili pepper , in which they cause foliar damage. Surprisingly, Phytophthora was not found in the rhizosphere of wilted or healthy chili pepper plants, while other studies carried out even in the same region have reported it as the causal organism of pepper wilt .
Diseases caused by soil-borne plant pathogens can be difficult to control for a variety of reasons, as many soil-borne pathogens produce persistent resistance structures that can survive in the soil for many years. Even in the absence of a susceptible host, the agricultural practices focused on reducing pathogens are either unsuitable or insufficient, as well as the selective pressure from the other microorganisms in the rhizosphere, because of competition for nutrients and essential elements . However, as many other groups of microorganisms affect plant health, their pathogenic action could occur in conjunction. For example, it has been demonstrated that pathogens have the ability to secrete effector molecules that can affect the communication between plants and beneficial microorganisms, and that they can also recruit other microorganisms that compete against native microorganisms and help in the colonization of the host [6, 59].
In summary, our findings provide evidence that wilt-disease in C. annuum has an impact on reducing diversity and changes in the structure of bacterial and fungal rhizosphere communities. We found a complex and diverse microbial community, composed of bacterial members with a homogeneous abundance. In contrast, the abundance of some members of the fungal community was a little more heterogeneous. Several of the fungal genera we found have been reported as phytopathogens in chili pepper and other plants where a change in their individual abundance was observed, increasing significantly as the chili pepper plants wilted. In addition, further experiments should be done that isolate the pathogens found as well as the potentially beneficial microorganisms from the rhizosphere soil, to study their importance and focus on the interactions within soil microbial communities in an effort to elucidate possible biocontrol strategies. Finally, it would be interesting to explore, through metatranscriptomic analysis, the metabolic capabilities that are triggered in the rhizosphere when a plant enters a state of disease, in order to review the mechanisms of action exerted by both potentially pathogenic microorganisms and those that confer resistance to the plants.
Availability of data and materials
The datasets generated and analyzed during the current study are available in the NCBI Sequence Read Archive (SRA) repository under the BioProject with accession code PRJNA728362.
RG-E, LNM-C, and GDA-Q designed the research; LNM-C conducted the project administration; GDA-Q conducted the rhizosphere soil sampling; RG-E performed the processing and preparation of the samples; ZYM-R performed bioinformatic analysis and prepared the figures; RG-E wrote the original draft; RG-E, LNM-C, ZYM-R, CGL, and GDA-Q participated in the analysis of data, reviewed and edited the manuscript. All authors read and approved the final manuscript.