Pythium Damping-Off and Root Rot of Capsicum annuum L.: Impacts, Diagnosis, and Management

Capsicum annuum L. is a significant horticulture crop known for its pungent varieties and used as a spice. The pungent character in the plant, known as capsaicinoid, has been discovered to have various health benefits. However, its production has been affected due to various exogenous stresses, including diseases caused by a soil-borne pathogen, Pythium spp. predominantly affecting the Capsicum plant in younger stages and causing damping-off, this pathogen can incite root rot in later plant growth stages. Due to the involvement of multiple Pythium spp. and their capability to disperse through various routes, their detection and diagnosis have become crucial. However, the quest for a point-of-care technology is still far from over. The use of an integrated approach with cultural and biological techniques for the management of Pythium spp. can be the best and most sustainable alternative to the traditionally used and hazardous chemical approach. The lack of race-specific resistance genes against Pythium spp. can be compensated with the candidate quantitative trait loci (QTL) genes in C. annuum L. This review will focus on the epidemiological factors playing a major role in disease spread, the currently available diagnostics in species identification, and the management strategies with a special emphasis on Pythium spp. causing damping-off and root rot in different cultivars of C. annuum L.


Introduction
Cultivars of Capsicum annuum L. (Solanaceae) are an essential part of many cuisines worldwide as a spice. C. annuum L. is a semi-perennial herbaceous plant that is usually grown as an annual crop worldwide, including in India [1]. The genus Capsicum is split into three gene complexes based on the crossing ability between species [2], of which the species of C. annuum complex (C. annuum, C. frutescens, and C. chinense) is commercially most important [3]. In 2018, worldwide production of dry chilies and peppers was estimated to be up to almost 4.2 million tons over an area of 1.8 million hectares. India is the biggest producer of dry chilies and pepper grown worldwide. Capsaicinoids are distinctive of the Capsicum genus and are produced entirely inside the fruit placenta [4], which causes the characteristics of fruit pungency in this genus [5]. Capsaicinoids are a mixture of at least 23 alkaloids (vanillylamines), a major representative of which are dihydrocapsaicin and control Pythium spp.-related diseases in Capsicum since 2010, and resistant breeding against Pythium spp.

Pythium Species: Causal Agent of Damping-Off and Root Rot
The genus Pythium is a part of the Peronosporales order of the class Oomycetes or the phylum Oomycota, a member of the kingdom Chromista (Stramenopiles) [36]. The Pythium spp. are no longer considered to be true fungi; instead, they were found to be more closely related to algae [37]. The class Oomycetes differ from true fungi in that they have coenocytic hyphae, a diploid vegetative stage, cell wall content of β-glucan and cellulose, biflagellate zoospores with tinsel, and whiplash flagella [37,38]. The Pythium genus contains species that are pathogenic to plants [39][40][41], animals [42], algae [43], and other fungi [44]. More than 330 species of Pythium have been identified to date (www.mycobank.org), of which many are pathogenic to plants. These pseudo-fungi cause many plant diseases, including damping-off, root rot, collar rot, soft rot, and stem rot in different production systems such as nurseries, greenhouses, and agricultural fields [45][46][47]. In C. annuum plants, many Pythium spp. have been observed to cause diseases. Some of these species have been identified using the morphological keys available and gene sequences ( Table 1). The next few sections discuss the ecology of the pathogen involved and the critical factors involved in disease epidemics on C. annuum cultivars caused by various Pythium spp.

Pythium Species: Causal Agent of Damping-Off and Root Rot
The genus Pythium is a part of the Peronosporales order of the class Oomycetes or the phylum Oomycota, a member of the kingdom Chromista (Stramenopiles) [36]. The Pythium spp. are no longer considered to be true fungi; instead, they were found to be more closely related to algae [37]. The class Oomycetes differ from true fungi in that they have coenocytic hyphae, a diploid vegetative stage, cell wall content of β-glucan and cellulose, biflagellate zoospores with tinsel, and whiplash flagella [37,38]. The Pythium genus contains species that are pathogenic to plants [39][40][41], animals [42], algae [43], and other fungi [44]. More than 330 species of Pythium have been identified to date (www.mycobank.org), of which many are pathogenic to plants. These pseudo-fungi cause many plant diseases, including damping-off, root rot, collar rot, soft rot, and stem rot in different production systems such as nurseries, greenhouses, and agricultural fields [45][46][47]. In C. annuum plants, many Pythium spp. have been observed to cause diseases. Some of these species have been identified using the morphological keys available and gene sequences ( Table 1). The next few sections discuss the ecology of the pathogen involved and the critical factors involved in disease epidemics on C. annuum cultivars caused by various Pythium spp.

Ecology
Pythium spp. are soil-borne fungi. Indeed, members of this genus are no longer considered to be fungi but still share a striking similarity with true fungi in having vegetative (mycelium), asexual (sporangia and zoospores), and sexual (oospores) stages in their life cycle [54]. Figure 2 provides a standard overview of the disease cycle of Pythium species in pepper plants. Pythium spp.inhabit the soil either as a propagule (Zoospore, oospore, or sporangia) or in the mycelial form. The oospores are the primary survival structure and infecting unit of the many species of the Pythium genus in the soil, which can stay viable in the soil for long durations [37,55]. However, sporangia of some Pythium species have also been reported to be survival propagules in soil [56,57]. The survivability of propagules of Pythium in soil follow two critical mechanisms: (1) the constitutive dormancy of oosporesi.e., their germination requires an internal stimulation other than the external stimulus, because of which all the propagules present in the soil do not germinate simultaneously; and (2) the formation of secondary resting propagules in the case of a shortfall of ample nutrients [55,58]. The propagules can germinate and penetrate the host plant very rapidly in the presence of exudates from roots and other supporting exogenous stimuli, which makes their control very difficult [39]. Sporangia can infect the plant either directly through hyphal tube germination or by producing zoospores in the presence of high moisture conditions [55]. Besides the propagules, Pythium species also survive saprophytically as mycelium in the soil and usually colonize the fresh organic substrate. However, this stage of their life cycle is very prone to competition by other soil colonizers [59]. In the seeds, seedlings, and younger roots of mature plants, after penetration, Pythium usually survives necrotrophically, thus eventually leading to mortality.

Epidemiology
Pythium spp.are naturally attracted to the exudates released from the germinating seeds, which are stimulatory to the growth of their propagules. While germinating, the seeds imbibe water and release exudates, making them the primary target of infection by a Pythium propagule [55]. Exogenous environmental factors have a critical role in the predisposal of plants to infection and disease spread in the host after infection by Pythium. However, the favorable conditions for disease spread by Pythium tend to vary with the species [60]. Damages ranging from small outbreaks to an epidemic-level scale depend mostly on variables such as moisture, temperature, and organic matter content; however, factors such as pH and soil type also play a crucial role.
Temperature is undeniably the critical factor in the spread of disease by Pythium. An array of temperature ranges represent thriving conditions for various Pythium spp. such as P. graminicola have been observed to cause more damage in Capsicum at high cardinal temperatures [21]. Temperature is undeniably the critical factor in the spread of disease by Pythium. An array of temperature ranges represent thriving conditions for various Pythium spp. such as P. graminicola have been observed to cause more damage in Capsicum at high cardinal temperatures [21]. Besides supporting the growth of Pythium, high-temperature conditions, in some cases, place additional abiotic stress on the plants, which eventually enhances the severity of root rot [61]. A high moisture content in the soil supports the motility of zoospores and increases the size of the spermosphere-factors that play a crucial role in infections by Pythium propagules [55]. Hyder et al. [62] observed high disease incidences and an increased severity of damping-off in chili pepper crops in areas with canal irrigation systems, and Majeed et al. [30] also reported that spaces with high natural moisture were highly infested. A greenhouse environment with damp conditions and high temperatures provides thriving conditions for Pythium [63]. Muthukumar et al. [64] observed the highest incidences of damping-off in chili in clay soils, which was attributed to their high moisture-holding capacity. Organic matter content in the soil sometimes supports the saprophytic growth of Pythium in the absence of antagonistic microbial communities. Hansen and Keinath [65] observed organic amendments of Brassica species that, rather than being suppressive, supported the increase of Pythium populations.

Detection and Diagnosis of Pythium Species
Pythium spp. pathogenic to C. annuum plants have been identified using morphological features and sequencing marker regions of the DNA of the isolated pathogen. Levesque and Cock [66] found the sporangial morphology to be the most reliable morphological feature in species identification, which was very much consistent with major clades formed in molecular characterization. However, the correct identification of the Pythium spp. is often hindered by the inability of heterothallic species to form reproductive structures in cultures, overlapping some characters, and the inability to distinguish intraspecific variation [21,66,67]. Nevertheless, the identification based on morphological features is neither very time-efficient nor very reliable. Besides supporting the growth of Pythium, high-temperature conditions, in some cases, place additional abiotic stress on the plants, which eventually enhances the severity of root rot [61]. A high moisture content in the soil supports the motility of zoospores and increases the size of the spermosphere-factors that play a crucial role in infections by Pythium propagules [55]. Hyder et al. [62] observed high disease incidences and an increased severity of damping-off in chili pepper crops in areas with canal irrigation systems, and Majeed et al. [30] also reported that spaces with high natural moisture were highly infested. A greenhouse environment with damp conditions and high temperatures provides thriving conditions for Pythium [63]. Muthukumar et al. [64] observed the highest incidences of damping-off in chili in clay soils, which was attributed to their high moistureholding capacity. Organic matter content in the soil sometimes supports the saprophytic growth of Pythium in the absence of antagonistic microbial communities. Hansen and Keinath [65] observed organic amendments of Brassica species that, rather than being suppressive, supported the increase of Pythium populations.

Detection and Diagnosis of Pythium Species
Pythium spp. pathogenic to C. annuum plants have been identified using morphological features and sequencing marker regions of the DNA of the isolated pathogen. Levesque and Cock [66] found the sporangial morphology to be the most reliable morphological feature in species identification, which was very much consistent with major clades formed in molecular characterization. However, the correct identification of the Pythium spp. is often hindered by the inability of heterothallic species to form reproductive structures in cultures, overlapping some characters, and the inability to distinguish intraspecific variation [21,66,67]. Nevertheless, the identification based on morphological features is neither very time-efficient nor very reliable.
Pathogen detection and diagnosis have a vital role in correlating epidemiological factors with the species involved in the disease spread. Damping-off and root rot diseases sometimes involve a complex of pathogens, including species other than Pythium spp. [68]. The early diagnosis of Pythium can help in managing the further spread of the disease. The accurate detection of the relevant species can prevent the wasteful usage of pesticides and other resources because different species differ in their susceptibility to control strate-Microorganisms 2021, 9, 823 6 of 17 gies [38]. Thus, the potential diagnostic techniques have the prerequisites of being rapid, accurate, easy to use, and cost-effective. The detection and diagnostic methods currently used for plant pathogens can be broadly divided into two categories; i.e., immunodiagnostic methods and nucleotide-based methods [69]. In the context of Pythium spp., recent studies have been primarily focused on advanced PCR-based molecular techniques such as real-time PCR, multiplex PCR, and loop-mediated isothermal amplification (LAMP). The Internal Transcribed Spacer (ITS) region, including ITS 1, 5.8 s, and ITS 2 sequences of nuclear rDNA, is the most frequently used sequence for identifying Pythium spp. [20,21,49,70]. The primers developed by White et al. [71], based on some conserved sequences in eukaryotic DNA, as well as the primers based on the ITS sequence of the specific species, are used most frequently in the PCR amplification of the ITS region of the specific species.
Immunological methods rely on antigen-antibody specificity. Although immunological methods for Pythium have been minimal because of the difficulty in producing species-specific antibodies [37], they have a better potential for use in point of care test systems operated by non-specialists [72]. Ray et al. [73,74] used the polypeptides released during fungal infections as immunogens to develop polyclonal antibodies to detect early P. aphanidermatum infections in turmeric and ginger rhizomes, respectively. In an ELISA analysis, the developed antibodies showed a good sensitivity at very low concentrations. Although the antigen load in the early stages of infection in rhizome was remarkably low compared to more severely infected rhizomes, the infection was still detectable.
Similar to the rapid detection of the pathogen, inoculum quantification is also important. In real-time PCR amplification, the PCR product is monitored as it is amplified after each cycle, providing an estimate of the pathogen present in the environmental sample [75]. Based on the inoculum present in the soil and plant, the amount of pesticide application, crop area selection, non-host species, and seasonal variation in the pathogen spread can be determined. Furthermore, the resistant varieties and genetically modified resistant crops can be studied by observing the pathogen count in asymptomatic plant parts [76]. Li et al. [77] estimated the population densities of P. intermedium in forest soils using a real-time PCR technique. The primers Pf002 and Pr002b, designed using the ITS region of the P. intermedium, were found to be highly species-specific. For the sensitive and quantitative observation of Pythium spp., real-time PCR assays were also developed by Li et al. [77], Li et al. [78], and Van der Heyden et al. [79].
Very often, multiple Pythium spp.are involved simultaneously in plant infections. Multiplex PCR facilitates the simultaneous identification of the species present within a sample. The multiple primers used in a single reaction are designed to hybridize specifically to target DNA and amplify the target regions with different amplicon sizes that can easily be separated by electrophoresis [38,80]. Nine primers-18S-69F, 18S-1118R, AsARRR, AsAPH2B, AsTOR6, AsVANF, AsPyF, AsGRAF, and AsPyR-for identifying five species of Pythium, P. arrhenomanes, P. graminicola, P. aphanidermatum, P. torulosum, and P. vanterpooli that cause diseases in turfgrass were designed by Asano et al. [80]. In the DNA extracted from plant samples showing symptoms, multiplex PCR was more efficient in detecting the Pythium spp. compared to pathogen isolation using the selective medium. Based on the isolation of multiple Pythium spp. from one plant sample, it was postulated that Pythium spp. would be involved in latent infections, and subsequently, visible symptoms were observed only when the conditions optimal for disease induction occurred. Ishiguro et al. [81] developed a multiplex PCR detection method to identify high-temperature-growing Pythium spp., P. aphanidermatum, P. helicoides, and P. myriotylum in soil samples. Species-specific primers-kkMYRR for P. myriotylum and kkhel F1mods2/kkhel R2 for P. helicoides-were newly designed. Other pre-designed primers-AsPyF for P. aphanidermatum and P. myriotylum, AsAPH2B for P. aphanidermatum -and two universal primers-18S69F and 18S1118R-were also used. Their analysis concluded that the primers were highly species-specific and could efficiently detect specific species from environmental samples.
Loop-mediated isothermal amplification (LAMP), unlike other PCR techniques, does not involve a thermal cycler; instead, nucleic acids are amplified under isothermal conditions [82]. The use of four different primers explicitly designed for six different target gene regions makes it highly specific [83]. Two loop primers were also used in an attempt to reduce the amplification time. The amplification of nucleic acids relies on Bst DNA polymerase, which uses autocycling strand displacement for DNA synthesis [84]. Fukuta et al. [82] developed a LAMP assay to detect P. myriotylum detection in hydroponic solution samples. The five primers-F3, B3, FIP, BIP, and B-Loop-designed using the ITS sequence of P. myriotylum and related species from the B1 phylogenetic clade of the Pythium efficiently amplified the target genes at 60 • C. The DNA extracted from the hydroponic nutrient solution showed the presence or absence of P. myriotylum accurately, which was confirmed by the plate culture method. The presence of double-stranded DNA amplified by primers and Bst DNA polymerase was observed by the fluorescence produced due to double-strand binding intercalating dye. As the number of double-stranded DNA increases, fluorescence increases. Additionally, LAMP assays were also developed for P. irregulare [85], P. aphanidermatum [85], P. ultimum [86], and P. spinosum [87]. Cao et al. [88] used a modification of LAMP called RealAmp, where LAMP products were quantified in real-time. The quantitative detection of the pathogen was done by measuring the increase in turbidity derived from magnesium pyrophosphate to determine the amplified DNA product. The only limitation associated with LAMP is the false-positive results produced due to the presence of multiple primers, which enhances the sensitivity of the assay manifolds [82]. The LAMP method has a high potential for use in field conditions because of the low requirement for sophisticated instruments; additionally, the environmental samples can be analyzed directly without the need for DNA extraction [83,86].
PCR-based diagnosis provides advantages over conventional diagnostic procedures of rapid detection, sensitivity, and the ability to avoid culturing a target pathogen [75]. However, using PCR technology to detect pathogens in environmental samples has its own shortcomings. The extraction of Pythium DNA from environmental samples is hindered by the presence of PCR inhibitors, the thick walls of dormant structures, and the strong binding of microbes to soils or plant tissue [37,89,90]. The purity of extracted DNA, the state of infection in plant tissue, and pathogen distribution in environmental samples can also drastically affect the results [38]. PCR amplification cannot provide a delimitation between viable and non-viable propagules because DNA can persist for more extended periods even after cell death, thus hindering the assessment of a control strategy [91]. Using real-time PCR with environmental samples can result in the incorrect quantification of the pathogen in the test sample because of the presence of fluorescent molecules, the difference in the copy number of nuclear rDNA between isolates, and the ambiguity of extracted DNA from living and dead propagules [38,77,92]. Other than the technical limitations, the skill requirements, high-end instruments, and high costs limit the use of PCR-based diagnosis in actual field-based studies.

Cultural Control
Cultural techniques such as organic amendments, soil solarization, and cover cropping have been observed to control Pythium-related diseases in C. annuum crops. The solarization of soil has been a prevalent practice to manage various soil-borne diseases in greenhouses and nursery beds. Soil solarization is a pre-sowing management approach that generally reduces colony-forming units (CFUs) or propagules of various soil-inhabiting pathogens as well as other microbes, as observed by Akhtar et al. [93], where CFUs of Pythium spp.were reduced to 1.67 × 10 4 after 8 weeks of solarization as compared to 5.00 × 10 4 in presolarized soils at a depth of 15 cm.
The use of isothiocyanates (ITCs) producing Brassicaceae cover crops for soil biofumigation has acquired increasing interest as an alternative to restricted chemical fumigants such as methyl bromide for soil-borne disease control [94]. Although isolates of Pythium spp. were effectively inhibited by ITCs in in-vitro conditions [95], field experiments using Brassica cover for biofumigation were not very efficient in reducing Pythium populations in pepper crops [65]. However, high ITC levels were observed in field soils after the pulverization and incorporation of Brassica cover crops in soils [65]. In contrast, Handiseni et al. [96] saw an improvement in pepper seedling emergence of 40%-60% when the ITC-producing intact seed meals of Brassica napus and Brassica juncea were incorporated into P. ultimum-infested soils. The difference in observations by Hansen and Keinath [65] and Handiseni et al. [96] can be attributed to the variability in sensitivity to ITC of different species of Pythium.

Chemical Control
Despite their well-known detrimental effects, such as environmental toxicity and accumulation at different trophic levels [97], the use of chemical pesticides is still relevant because of their effective results. Fungicides used as a soil drenching, fumigation, and seed treatment have been used to control soil-borne disease. Saha et al. [98], in a four-year-long study, studied the effect of the treatment of chili seeds with the fungicidal formulations Thiram 75 WS and Captan 50 WP on incidences of damping-off caused by P. aphanidermatum. Treatment of seeds using Thiram 75 WS at the dose of 2.5 kg/g of seeds reduced the preemergence damping-off losses from 29.06% in untreated control to 6.52% in treatment and post-emergence damping-off losses from 59.12% in the untreated control to 16.15% in treatment conditions. It was even attempted to integrate metalaxyl with biocontrol agent Trichoderma harzianum as a seed treatment, which also produced good results as the percentage of seed rot from 93.75% in untreated seeds was reduced to 52.08% in treated seeds [28]. However, using a single active ingredient has resulted in the development of resistant varieties, as can be seen in metalaxyl-resistant Pythium spp. [99,100]. A combination of different fungicides, creating a cocktail of alternative chemistries, has provided the solution to overcome the resistance developed by Pythium spp. [99,100]. The two or more active compounds used in this pesticide cocktail mostly act additively, but synergism has also been observed in some cases [101]. It is suggested to use Previcur 840 SL containing propamocarb (47.3%) and fosetyl (27.7%) as a combination of active ingredients, which is recommended for the effective control damping-off disease caused by Pythium spp. in chili [102]. The efficacy of mixed fungicides in vitro on isolates of Pythium isolated from diseased parts of chili plants was studied by Dubey et al. [21], who observed Vitavax (Carboxin 37.5% + Thiram 37.5%) to be highly effective, inhibiting the growth of mycelia by 93.3% at a concentration of 100 ppm.
As discussed in the previous sections, high moisture content in the soil is conducive to the development of Pythium spp.; thus, events of extreme rainfall can lead to severe disease outbreaks. Saha et al. [103] observed high mortality in the Capsicum crop after Hurricane Frances, which was further aggravated due to the biological vacuum created by pre-plant metalaxyl fumigation. The fumigation resulted in a severe reduction in beneficial soil microbiota, which later supported the outbreak by Pythium spp. Kokalis-Burelle et al. [104] also observed Pythium epidemics in methyl bromide-treated plots of pepper plants in subsequent years, which were attributed to the biological vacuum created by methyl bromide fumigation. Due to the fewer soil microorganisms present in the soil, no competition was present to Pythium. Thus, disease forecasting based on variables such as the number of pathogen propagules, the susceptible host, and the conducive environment becomes crucial in deciding the timing and dose of fungicides to be used [105].

Biological Control
The antagonistic ability of many fungal, bacterial, and algal isolates has been investigated directly and indirectly against phytopathogens, as shown in Table 2. The activity and efficacy of biocontrol agents on damping-off in seeds and seedlings depends very much on the texture of seeds, the associated microflora in the soil, and the physiological characters of the plant. Therefore, it can be concluded that the efficacy of biocontrol tends to vary from species to species and crop to crop [68]. The antagonism exhibited by microbial biocontrol agents can follow different modes of actions such asc ompetition, induced systemic resistance, antibiosis, and mycoparasitism ( Figure 3).

Biological Control
The antagonistic ability of many fungal, bacterial, and algal isolates has been investigated directly and indirectly against phytopathogens, as shown in Table 2. The activity and efficacy of biocontrol agents on damping-off in seeds and seedlings depends very much on the texture of seeds, the associated microflora in the soil, and the physiological characters of the plant. Therefore, it can be concluded that the efficacy of biocontrol tends to vary from species to species and crop to crop [68]. The antagonism exhibited by microbial biocontrol agents can follow different modes of actions such asc ompetition, induced systemic resistance, antibiosis, and mycoparasitism ( Figure 3).  Table 2 shows that Bacillus spp. and Pseudomonas spp. are the most frequently used and most successful bacterial biocontrol agents in pot trials and field applications. However, both of these have diverse modes of action. Endophytic Pseudomonas fluorescens EBS20 isolated from chili plants produced an antibiotic phenazine, which reduced the in vitro growth of Pythium spp. [51]. The production of antimicrobial substances by Pseudomonas spp. and Bacillus spp. was observed by Muthukumar et al. [106] and Amaresan et al. [107], respectively. The disease-suppressing activity of plant growth-promoting rhizobacteria has also been correlated with its efficiency in promoting plant growth [106]. Many species of Pythium that are pathogenic to plants have a very high optimum temperature for growth at which some biocontrol agents are not very effective [108]. Mehetre and Kale [109] attempted to assess a thermophile Bacillus licheniformis NR1005 for its antagonism against P. aphanidermatum, and promising results were obtained in suppressing the disease.  Table 2 shows that Bacillus spp. and Pseudomonas spp. are the most frequently used and most successful bacterial biocontrol agents in pot trials and field applications. However, both of these have diverse modes of action. Endophytic Pseudomonas fluorescens EBS20 isolated from chili plants produced an antibiotic phenazine, which reduced the in vitro growth of Pythium spp. [51]. The production of antimicrobial substances by Pseudomonas spp. and Bacillus spp. was observed by Muthukumar et al. [106] and Amaresan et al. [107], respectively. The disease-suppresupssing activity of plant growth-promoting rhizobacteria has also been correlated with its efficiency in promoting plant growth [106]. Many species of Pythium that are pathogenic to plants have a very high optimum temperature for growth at which some biocontrol agents are not very effective [108]. Mehetre and Kale [109] attempted to assess a thermophile Bacillus licheniformis NR1005 for its antagonism against P. aphanidermatum, and promising results were obtained in suppressing the disease.  68.88% reduction in the growth of mycelia.
9.10% and 12.33% incidences of pre and post-emergence damping-off when EBC 5 and EBC 7 were combined in pot culture using seed coating, as opposed to 30.66% and 34% in control.
Production of antifungal metabolites reduced mycelial growth in-vitro. [106] Pseudomonas fluorescens EBC 7 65.93% reduction in the growth of mycelia.
2% incidences of damping-off as opposed to 14.67% in control in field conditions.
81.18% reduction in damping-off incidence over control in pot culture using seed treatment.
75%-100% reduction in disease index over control in pot culture using root inoculation.
75%-100% reduction in disease index over control in pot culture using root inoculation.
100% reduction in disease index over control in pot culture using root inoculation.  Seed treatment with ethyl acetate extract of culture filtrate reduced mortality to 10%-20% as opposed to 60%-70% in untreated controls in pot culture.
Trichoderma spp. was found to be the most frequently studied fungal biocontrol agent for the control of Pythium spp.-related diseases in Capsicum plants. Trichoderma spp. has been observed to compete with phytopathogens directly by showing a competitive saprophytic ability through the production of cellulase enzymes, thus competing with Pythium spp. directly in their ecological niche [112]. The production of antimicrobial substances by Trichoderma was also observed to be one of the mechanisms in the control of Pythium spp. [112,113].
Compatible strains of bacteria and fungi were also observed to inhibit the activity of Pythium spp. in vitro and in vivo; using a different mechanism of disease suppression, exhibiting synergistic effects, proved more effective than individual antagonists [52,114]. In the combined treatment of Pseudomonas fluorescens EBL 20-PF and T. viride TVA on chili plants, Muthukumar et al. [52] observed induced resistance in the plant itself. They noticed increased activities of resistance-inducing enzymes and the accumulation of phenolic compounds, resulting in a significant decrease in disease incidences.
Non-native antagonist isolates generally face resistance by pre-established and acclimatized microflora and often fail to establish themselves [107]. Antagonists isolated from the plant rhizosphere and from the plant itself have shown convincing results in reducing incidences of damping-off [51,53,113]. However, as previously discussed, propagules of Pythium spp. can germinate and infect very rapidly, sometimes even with a shortage of nutrients and water, thus escaping natural antagonism [58]. An antagonist can only be efficient against Pythium spp. if provided with an advantage in colonizing the roots and rhizosphere of the plant. Early inoculation of the biocontrol agent before seeding or inoculation close to the seed or seedling can increase the efficacy of an antagonist [115]. The rapid colonization of an antagonist can also provide an extra advantage for its success [109,110].

Virulence Mechanism of Pythium spp. and Challenges in Resistant Breeding against Pythium spp.
Disease development by Pythium spp. involves an extensive repertoire of carbohydrateactive enzymes (CAZymes), including glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, proteases, etc., which help in plant cell wall penetration and further colonization [116,117]. Lévesque et al. [117], in their study of the P. ultimum genome, observed high sequence similarity and synteny with another phytopathogenic oomycete, Phytophthora, sharing genes and encoding enzymes involved in the metabolism of carbohy-drates. However, the absence or underrepresentation of many notable genes in Pythium encoding for cutinases, xylanases, pectinases, etc., that are present in the Phytophthora genome suggests some difference in the methods of virulence between these oomycetes [118]. Even the expression of cellulases and pectinases in Pythium spp. are limited; they are sufficient for hyphal penetration but not for complete saccharification. Furthermore, the absence of these complex carbohydrate-degrading enzymes and the presence of α-glucosidase, α-amylases, α-glucoamylases, and invertases, degrading the plant starch and sucrose, establishes the affirmation of phytopathogenic Pythium towards young plant tissues with no secondary growth [116]. The presence of genes encoding proteases families such as subtilisin-related proteases, metalloproteases, and E3 ligases, inducing necrosis and cellwall degradation in the core Pythium spp. genes and the absence or lack of RXLR effectors in Pythium support its non-host specificity and necrotrophic lifestyle [117][118][119]. Unlike Phytophthora, Pythium spp. have been observed to lack RXLR effectors with avirulence activities [118,119]. The absence of these avirulence factors has been correlated with the lack of gene-for-gene resistance against Pythium spp. Thus, the absence of a virulence factor producing RXLR effectors makes the identification of race-specific resistance genes against Pythium very difficult to explore [120]. However, Ai et al. [121] have recently identified RXLR effectors in nine Pythium spp. that share a common ancestor with Phytophthora. RXLR effectors in Pythium spp. exhibited necrosis-inducing activities resulting in plant cell death. The difficulties in identifying race-specific resistance genes against Pythium spp. make quantitative gene expression a more comfortable approach for developing resistant varieties. Several major and minor quantitative trait loci (QTL) associated with root rot resistance have been identified in Pythium spp.-affected crops such as snap bean [122] and soybean [120,[123][124][125]. The partial (horizontal) resistance achieved through the involvement of multiple QTLs in the soybean plants leading to transgressive segregation has been observed to confer a common response pattern against multiple Pythium spp. [120,125]. As Pythium spp.-affected crops are often inhabited by several Pythium spp., the development of a variety with resistance against multiple species would be more efficient.

Conclusions
As one of the significant horticulture crops in India and worldwide, improvements in Capsicum production yield are a primary target that needs to be achieved. Biotic stress due to different soil-borne pathogens poses a significant threat to that target. Different Pythium spp. have been reported to cause infections in Capsicum cultivars with a disparate reproductive behavior and growth environment. These factors make the detection of the pathogen and correct identification of the species of paramount importance. The technology for the detection and diagnosis of pathogens has evolved from polyclonal antibodies to the use of multiple species-specific primers for a single species. The use of LAMP technology has provided a possible escape from the use of sophisticated instruments and pure DNA isolation. However, the search for a point-of-care technology that does not require a specific skill set and can provide results in a shorter time frame is still far from over.
Although biological control has provided a viable alternative to chemical management, in a natural setup, the comparative disease control of Pythium spp. has not been achieved yet. Additionally, the successful expression of antagonism by the microbes partially depends upon the cultural and environmental conditions. An integrated system of cultural control and microbial control can provide the desired targets in Pythium spp. disease control. The use of disease-resistant varieties is another sustainable alternative for disease control. However, in this case, no research work could be found involving Pythium-resistance development in C. annuum cultivars. The candidate QTL genes for the resistance against multiple Pythium spp. could prove to be groundbreaking in resistance breeding in C. annuum L.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All the data is present in the manuscript.