Pepper Mild Mottle Virus: An Infectious Pathogen in Pepper Production and a Potential Indicator of Domestic Water Quality

Pepper (Capsicum spp.; Family: Solanaceae; 2n = 24) is an important crop cultivated worldwide for the consumption of its fresh and dried processed fruits. Pepper fruits are used as raw materials in a wide variety of industrial processes. As a multipurpose vegetable crop, there is a need to increase the yield. However, yield productivity of pepper is severely constrained by infectious plant pathogens, including viruses, bacteria, fungi, and oomycetes. The pepper mild mottle virus (PMMoV) is currently one of the most damaging pathogens associated with yield losses in pepper production worldwide. In addition to impacts on pepper productivity, PMMoV has been detected in domestic and aquatic water resources, as well as in the excreta of animals, including humans. Therefore, PMMoV has been suggested as a potential indicator of domestic water quality. These findings present additional concerns and trigger the need to control the infectious pathogen in crop production. This review provides an overview of the distribution, economic impacts, management, and genome sequence variation of some isolates of PMMoV. We also describe genetic resources available for crop breeding against PMMoV.


Introduction
Vegetable crops are rich sources of basic food nutrients, including vitamins, minerals, and dietary fiber, as well as several antioxidant compounds required for human health [1]. Pepper (Capsicum spp.; family: Solanaceae) is one of the most important vegetable crops globally, and is widely cultivated for the consumption of its fruits, either fresh, dehydrated, or processed in spicy condiments [2]. As a versatile crop, pepper is widely cultivated in diverse climatic conditions in both fields and protected environments. At the global and regional scales, fresh pepper fruits and processed products are highly traded, making cultivation of the crop an important source of employment for many people, especially smallholder farmers around the world [3]. The crop is in demand for industrial uses, especially for the extraction of useful volatile molecules or compounds, including capsaicin, carotenoids, and tocopherol compounds, and for use as an ingredient in agrofoods, cosmetics, food preservatives, additives, antimicrobial preparations, and pharmaceuticals [2]. Therefore, it is important to increase the pepper fruit yield. However, the success of pepper cultivation in relation to productivity of the fruits is influenced by environmental conditions, which can place stress on the crop, leading to fruit yield loss and quality reduction [4]. Abiotic and biotic conditions are major environmental threats that limit the yield of vegetable crop species, and efforts to control the associated impacts usually require an investment of resources leading to increased production costs. Fruit yield loss in pepper continues to be recorded in many growing areas, which is largely attributed to a wide array of phytopathogens [5]. Pepper plants are susceptible to different plant pathogens,

The Genus Tobamovirus
Among the various genera, the tobamoviruses include several well-characterized plant viruses (Table 1) containing a positive-sense single-stranded RNA (ssRNA+) genome [9]. The genome of tobamoviruses has a 5 -capped RNA containing four open reading frames that encode a replication protein (~130 kDa), a read-through product (~180 kDa), nonstructural cell-to-cell movement proteins (30 kDa), and a coat protein (CP; 17.5 kDa) [10,12]. The encoded proteins vary across different strains andisolates ( Table 1). The replication protein has a methyltransferase-like domain responsible for the 5 capping of progeny RNAs and an RNA helicase-like domain, whereas the read-through protein contains an RNA-dependent RNA polymerase-like (RdRp) domain [13,14]. The genus Tobamovirus is evolutionarily diverse and comprises several highly damaging plant viruses, such as PMMoV, cucumber green mottle mosaic virus, and tomato brown rugose fruit virus (Table 1, Figure 1).

Genome Sequence Variationof PMMoVIsolates
PMMoV has a monopartite genome [51], consisting of a single RNA molecule that is protected in a shell or capsid. The capsid is composed of proteins that form a the rodshaped virion of the virus [7]. The virion contains a nonenvelopedssRNA+ genome [52,53]. The first complete genome sequence of PMMoV (Isolate: PMMoV-S) was reported in 1991 [54]. Several experiments have since been conducted to provide information about the complete genome sequencing of PMMoV isolates (Table 2, Figure 2). Complete genome sequencing projects of PMMoV have revealed the presence of variation among different isolates in terms of nucleotide sequence length ( Table 2). The genome of PMMoV encodes four different types of proteins: replication-associated proteins (126 kDa and 183 kDa), a movement protein (30 kDa), and a CP (17 kDa) [10,52,55]. Given the challenges regarding the mechanisms of controllingplant viruses, their dynamic and evolvability nature, it is vital to understand the evolutionary relationships among different isolates of the PMMoV pathogen. This knowledge will provide important information for understanding the genetic or mutations associated with resistance-breaking ability of PMMoV strains or isolates, and efficiency of diagnostic tools to use. In addition, knowledge on evolutionary relationships among different pathogen isolates is significant for disease management, germplasm evaluation and crop breeding. Phylogenetic trees based on sequences of coat proteins of different PMMoV isolates whose genomes have been completely sequenced are shown in Figure 2.

Host Range and Symptoms Associated with PMMoV
Members of the genus Tobamovirus are capable of infecting different Solanaceae species, including tomato, tobacco, and eggplant. Although PMMoV infects species of pepper and tobacco, other susceptible hosts, such as Dracaenabraunii [73], tomato [1], and Parispolyphylla var. yunnanensis [72], have been reported. Symptomsof infection by PMMoV occurmainly on leaves and fruits (53). At the early stage of infection, plants generally show mild foliar mosaicism. Infected plant leaves subsequently develop mottled, deformed, and chlorotic features [74] ( Figure 3A-C). In severe infections, especially those occurring at the early stage of growth, affected plants become stunted, resulting in reduced yields [55]. Fruits of affected plants are decreased in size and appear deformed, necrotic, mosaic, and lumpy or blistered ( Figure 3D), thus reducing their market value. The symptoms of PMMoV-affected plants are usually more prominent when infection occurs at the early stages of plant growth. Severe yield losses are likely to occur if an infection is not detected early [59].
PMMoV occurmainly on leaves and fruits (53). At the early stage of infection, plants generally show mild foliar mosaicism. Infected plant leaves subsequently develop mottled, deformed, and chlorotic features [74] (Figure 3A-C). In severe infections, especially those occurring at the early stage of growth, affected plants become stunted, resulting in reduced yields [55]. Fruits of affected plants are decreased in size and appear deformed, necrotic, mosaic, and lumpy or blistered ( Figure 3D), thus reducing their market value. The symptoms of PMMoV-affected plants are usually more prominent when infection occurs at the early stages of plant growth. Severe yield losses are likely to occur if an infection is not detected early [59].

Mode of PMMoV Transmission in Plants
PMMoV can be transmitted mechanically via contact with contaminated sources, including working equipment such as gloves and workers' clothing, during normal crop management. The pathogen is both seed-and soil-borne and can be transmitted through the use of contaminated seeds in planting as well as planting in infected soils. On seeds, PMMoV occurs on the outer coat of the seed and is transmitted nonembryonically [69]. As the pathogen is seed-borne, it can be easily introduced across different environments, which is likely the reason for the spread of the pathogen in many pepper-growing areas. Wounds and microscopic abrasions on seeds or plant parts facilitate viral entry into the host. PMMoV can remain stable and serve as inocula when adsorbed on plant debris (leaves, stems, and roots), soil, humus, greenhouse structures, and working tools for prolonged periods.

Global Distribution and Economic Importance of PMMoV
Overall, plant viral diseases account for considerable yield losses and the severity of their impacts on crop production can be accelerated by changing patterns of climate, international trade, and pathogen adaptation for rapid evolution [75]. Globally, annual crop yield losses resulting from plant viruses in the form of small-scale yield reductions to total crop failure havebeen estimated to have a value of more than USD 30 billion [76]. In the early half of the 1950s, PMMoV was described for the first time as a latent strain of tobacco mosaic virus in the USA [77]. In 1984, PMMoV was first described in the literature by an Italian group as a distinct virus [78]. Currently, disease incidences associated with the pathogen are spread across different parts of the world ( Table 2). PMMoV is

Mode of PMMoV Transmission in Plants
PMMoV can be transmitted mechanically via contact with contaminated sources, including working equipment such as gloves and workers' clothing, during normal crop management. The pathogen is both seed-and soil-borne and can be transmitted through the use of contaminated seeds in planting as well as planting in infected soils. On seeds, PMMoV occurs on the outer coat of the seed and is transmitted nonembryonically [69]. As the pathogen is seed-borne, it can be easily introduced across different environments, which is likely the reason for the spread of the pathogen in many pepper-growing areas. Wounds and microscopic abrasions on seeds or plant parts facilitate viral entry into the host. PMMoV can remain stable and serve as inocula when adsorbed on plant debris (leaves, stems, and roots), soil, humus, greenhouse structures, and working tools for prolonged periods.

Global Distribution and Economic Importance of PMMoV
Overall, plant viral diseases account for considerable yield losses and the severity of their impacts on crop production can be accelerated by changing patterns of climate, international trade, and pathogen adaptation for rapid evolution [75]. Globally, annual crop yield losses resulting from plant viruses in the form of small-scale yield reductions to total crop failure havebeen estimated to have a value of more than USD 30 billion [76]. In the early half of the 1950s, PMMoV was described for the first time as a latent strain of tobacco mosaic virus in the USA [77]. In 1984, PMMoV was first described in the literature by an Italian group as a distinct virus [78]. Currently, disease incidences associated with the pathogen are spread across different parts of the world ( Table 2). PMMoV is particularly adapted to survive under extreme conditions, such as warm, hot, and humid climates. The fast-spreading nature of the pathogen poses a serious threat to pepper cultivation and food security. In addition, the broad scope of PMMoV isolates is indicative of the pathogen's exceptional adaptation, which may enable some strains to easily overcome known resistance genes and even expand their host range. Meanwhile, the severity of impacts resulting from PMMoV infection in pepper differs according to the isolate, host species, and the stage of plant growth during which the infection occurs. The disease incidence resulting from PMMoV infection in commercial bell pepper fields in Florida, USA varied from <1% to 30% [79]. In another study in Grady County, Georgia, USA, an entire jalapeno pepper (Capsicum annuum L.) field was reported to have been devastated by PMMoV [80]. Fruits of infected plants were deformed, mottled, reduced in size, and had off-colored sunken parts.

Management of Pepper Mild Mottle Virus (PMMoV)
Diseases originating from PMMoV infections are exceptionally difficult to control when they occur. Once infected with PMMoV, it becomes extremely difficult to recover plants using chemical or physical treatments [81]. Therefore, to control infections with and the spread of PMMoV, growers must observe good cultural and sanitation practices in their production systems. Avoiding sources of infection by the disinfection of working tools and removal and destruction of infected plants can help to control the spread of the pathogen. Working tools or equipment and stakes must be disinfected to minimize possible transmission. The pathogen is seed-borne; therefore, clean seeds must be used to establish pepper production. Sanitary certification and cross-protection management are important factors to control PMMoV. Care must also be taken to avoid abrasions and induced wounds on plant parts, as this is ideal for viral entry into host tissues. It is also essential to gain an understanding of which weed species can act as hosts of the virus. In addition, the genetic relationships among existing strains of PMMoV must be studied to facilitate accurate diagnosis in research. Knowledge about causal agents and symptoms associated with the virus as well as regular inspection of plants for the early detection of PMMoV infection are required. Under less severe conditions, plants showing noticeable symptomsof viral infection and adjacent plants should be removed immediately, but care must be taken to avoid touching healthy plants with contaminated hands or tools. Rotation with resistant crops and sterilizing soils before planting for greenhouse cultivation are good practices to break the disease cycle via inocula from plant debris [82]. The planting of resistant pepper genotypes is also highly recommended.

Diversity of L Resistance Genes and PMMoV Pathotypes
Due to the rapid spread and damaging nature of PMMoV, it is necessary to incorporate resistance alleles from PMMoV-resistant cultivars into the genome of commonly cultivated peppers. The L genes (L1-L4) that functionally control resistance of peppers to tobamovirusesdiffers across different pepper species and cultivars. Based on differences in the L gene allele, Capsicum species have been divided into L1, L2, L3, and L4 classes [10]. On the other hand, tobamoviruses are classified into four pathotypes-P0, P1, P1.2, and P1.2.3-according to their ability to systemically infect Capsicum species carrying L gene alleles L1, L2, L3, and L4, respectively [10,83]. Following infection, the PMMoV CP induces expression of the L genes and thus induces a HR in the host plant [84]. Two L gene alleles, L3 and L4, confer high degrees of resistance to PMMoV. Pepper plants harboring the L3 gene show resistance to the P1.2 pathotype but are susceptible to P1.2.3. Plants that have the L4 geneshow resistance to these two pathotypes and have a broader scope of resistance against tobamoviruses. The crop germplasm represents a significant tool for identifying disease resistance genotypes for crop breeding. Extensive screening of diverse accessions to identify PMMoV-resistant resources has been conducted using various gene markers (Table 3). Although commercial pepper varieties have been developed that carry L genes that confer resistance to the pathogen, there are strains of PMMoV that can overcome some of these resistance genes. Moreover, previous studies revealed that many host plant species harbor specific genes encoding a protein known as the TOBAMOVIRUS MULTIPLICA-TION (TOM) susceptibility protein, which interacts with the viral replication protein [85]. The mechanism underlying the host plant-viral protein interaction favorably promotes certain Tobamovirus replication complexes and pathogen multiplication, and leads to effective infectivity on the host. Molecular studies usingthe CRISPR/Cas9-derived suppression of certain host TOM-related genes have been successfully conducted in different plant species, including Arabidopsis, tomato, and tobacco [85], but this technique has not been fully explored for resistance breeding against PMMoV.

Pepper Genetic Resources with Resistance against PMMoV
Pepper cultivation is widely distributed across the globe, with specific selections conducted over the past several years in different locations, resulting in differences in cultivar adaptation potential in diverse environments. Although pepper has a wide range of cultivars, many cultivated varieties display susceptibility to PMMoV [90]. Germplasm screening is an important means of identifying Capsicum accessions that may contain genes capable of conferring resistance to the virus. Nonetheless, the resistance of Capsicum species to PMMoV differs considerably across different genotypes (Figure 4), with some genotypes succumbing easily to specific strains but showing resistance to other strains (Table 4). This phenomenon can be attributed to the presence of variability in strains or isolates of the pathogen. A comprehensive characterization that combinesphenotyping and genotyping strategies isrequired to identify useful PMMoV-resistant accessions [81]. Thus, pepper breeders screened diverse pepper germplasm resources and identified some accessions with resistance against different PMMoVpathotypes that are useful for further breeding (Table 4).   germplasm resources and identified some accessions with resistance against different PMMoVpathotypes that are useful for further breeding (Table 4).

PMMoV as an Indicator of Water Quality
Pepper is the most important spicy vegetable crop, with worldwide consumption of its fresh fruits and processed dry powder, as wellas an ingredient in some industrial consumable products. Strategies to control PMMoV in pepper fields will help to increase fruit yield and reduce the likelihood of water contamination [7]. There are research studies that have confirmed the detection of PMMoV in domestic and aquatic water resources, as well as in the excreta of animals, including humans ( Table 5). The pathogen is known to have survival ability in the human gastrointestinal tract, and thus can be transmitted via human excreta [92]. Human immune response and clinical symptoms linked to PMMoV infection have also been investigated experimentally and include fever, abdominal pains, and pruritus (7). Though this finding may be possible, the detected symptoms may also result from other cofactors [93]. The key source of the pathogen's presence in human excreta has been attributed largely to the consumption of PMMoV-infected pepper and its processed products, while human excreta-derived pollution of water resources is the reason for the presence of the virus in water bodies [94][95][96]). Therefore, the presence of PMMoV in water is currently considered an indicator for water quality assessment [8,[97][98][99]. Moreover, the stable nature of PMMoV in water raises additional concerns in relation to their possible transmission via contaminated irrigation water resources [92,100,101].   Surface water samples from inlets, exposed to runoff and septic seepage, and coastal sites, exposed to ocean outfalls RT-qPCR 1. PMMoV detected more frequently than other microbial source tracking markers. [118]

Conclusions
PMMoV is an economically significant pathogen responsible for yield losses in pepper production, and poses a serious threat to agriculture and food sustainability. Detection of the virus in water resources, including aquatic, irrigation, pond, underground, and domestic water, as well as in the excreta of animals, including humans, raises additional concerns requiring extensive research to develop solutions that can circumvent potential risks associated with the pathogen in relation to human health. The application of molecular breeding techniques has prospects for the development of new cultivars that are resilient against PMMoV. Mutagenesis, including physical, chemical, and biological techniques, can be used in combination with next-generation sequencing to explore and exploit beneficial candidate genes for molecular breeding targeting the development of PMMoV-resistant genotypes.

Conflicts of Interest:
The authors declare no conflict of interest.