Materials and methods

The research was conducted at UAEM’s University Center in Tenancingo, located at 18° 58’ 05.53’’ N and 99° 36’ 50.51’’ W, 2068 masl. In 2016, manzano pepper plants showing wilt symptoms were collected at the following locations of the southern zone of the State of Mexico: 5 in Ahuacatitlán, Ixtapan de la Sal; 5 in El Zarco, Tenancingo; 3 in Santa Ana, Tenancingo; 4 in Tepoxtepec, Tenancingo; 3 in Matlazinca, Villa Guerrero; 4 in San Miguel, Ixtapan de la Sal; 5 in El Potrero, Coatepec Harinas; 5 in Ixtlahuaca, Coatepec Harinas; 3 in San Nicolás, Tenancingo; and 4 in Las Cabañas, Tenancingo. The collection sites were classified according to their cropping intensity (IC) that was defined based on the cultivated area: low: up to 3 ha; intermediate: 3.1-8.0 ha; high: more than 8.1 ha. Samples wrapped with wet brown paper were placed in transparent plastic bags and transported in a cooler to the laboratory, where were kept at 4 ºC until the next day, when they were used.

Isolation of the associated organisms. Eight segments of root and eight segments of stem were taken from each of the 41 plants collected in the 10 sampled sites, with a total of 656 samples. The samples were disinfected; for fungi, the samples were placed in Petri dishes containing a potato-dextrose-agar (PDA) culture medium, according to the method of ^{López (1984}); oomycetes were cultivated in 20 g of maize flour, 18 g of agar-agar, 0.8 mL of pimaricin, 0.02 g of rifamycin and 0.25 g L-¹ of ampicillin diluted in distilled water, according to the method of López et al., (2009), modified from ^{Kannwischer and Mitchell (1978}). The cultures were incubated at 24 ^oC in darkness and monitored every 24 h to observe fungus growth and development. When fungi and oomycetes had grown, successive transfers were made to obtain pure cultures and then prepare monosporic and hyphal tip cultures. The colonies were kept in tubes containing a PDA culture medium covered with sterile mineral oil.

Morphological identification of the associated organisms. The associated organisms were identified using taxonomic keys for fungi and oomycetes. The keys of ^{Booth (1971}) and ^{Leslie and Summerell (2006}) were used to characterize fungi micronidia and macronidia size and shape, and to describe chlamydospores, which are an important structure for identification; and the keys of ^{Singlenton et al. (1992}) and ^{Watanabe (2002}) to characterize sclerotia, ramification angles and mycelium. The keys of ^{Erwin and Ribeiro (1996}) and ^{Gallegly and Hong (2008}) were used to identify oomycetes colony growth, type of mycelium, and sporangia and chlamydospores shape.

Molecular identification of the associated organisms. DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method and sodium acetate. Universal PCR reactions were performed for fungi and oomycetes using the ITS-1 5’-tccgtaggtgaacctgcgg-3’ and ITS-4 5’-tcctccgcttattgatatgc-3’ primers (^{White et al., 1990}) that amplify fragments of 500-900 base pairs (bp). The reaction mixture in a final volume of 25 μL was made from a mixture of 10 µL of ultrapure water, 12 µL of MyTaq Mix® (Bioline), 1 µL of F primer, 1 µL of R primer and 1 µL of DNA. For oomycetes, the thermal program was set at 94 °C for 2 min, followed by 35 cycles at 94-55-72 °C for 30-30-60 s, respectively, and one final extension at 72 °C for 5 min. For fungi, the thermal program was set at 94 °C for 2 min, followed by 35 cycles at 94-60-72 °C for 30-30-60 s, respectively, and one final extension at 72 °C for 5 min. The amplified products from the PCR reactions were separated by electrophoresis using tris-borate-EDTA (TBE) and 1.2% agarose gels.

Sequencing. The PCR amplified fragments were purified using the EZ-10 Spin Column Handbook kit (Bio Basic Canada, Inc.) and then sequenced in the Molecular Biology Laboratory of FES-Iztacala, Universidad Nacional Autónoma de México (UNAM). The sequences in FASTA format (Chromas 2.6.5 version) were aligned with the National Center for Biotechnology Information (NCBI) gene bank database for consensus and identity -BLAST (Basic Local Alignment Search Tool) (available at https://www.yeastgenome.org/blast-fungal).

Pathogenicity in vitro. Pathogenicity was evaluated on C. pubescens M3 genotype, the most cultivated material in the study region. Seeds were disinfested and inoculated following the method of ^{Apodaca-Sánchez et al. (2001}), and then placed in Petri dishes containing an agar-water culture medium. Each strain obtained from the monosporic and hyphal tip cultures was considered as a treatment of 5 repetitions with 10 seeds each. Disinfested seed immersed in sterile distilled water was used as control. The pathogenicity was evaluated using the incidence and severity levels measured at day 10, according to ^{Herrera and Laurentin (2012}), and radicle and hypocotyl symptoms were recorded. The incidence was determined using the percentage of seedlings showing disease symptoms. The severity was measured using the lesion length along the seedling and expressed as percentage. The obtained values were averaged within each experiment unit. The ratio between incidence and severity was calculated proportionally using the severity/incidence quotient (^{Segura et al., 2009}; ^{Fernández et al., 2010}).

Interaction in multiple infections. Disinfested seeds of the M3 genotype were placed in a test tube containing an agar-water medium, kept in darkness at 25 °C, and, when germinated, at intervals of 12 h light and 12 h darkness at 25 °C. At day 7, the seeds were inoculated following the method of ^{Herrera and Laurentin (2012}). A 100 µL of 1 x 10⁶ spore suspension and 100 µL of monosporic and hyphal tip culture of the associated organisms, alone and combined, were applied to seven treatments with 10 replications each: P. capsici (P); P. capsici plus R. solani (P+R); F. oxysporum plus P. capsici (F+P); F. oxysporum plus P. capsici plus R. solani (F+P+R); F. oxysporum (F); and R. solani (R). The treatments were incubated at intervals of 12 h light and 12 h darkness at 25 °C and monitored every 24 h to evaluate the disease severity, which was determined by lesion length/seedling length x 100.

Pathogens-genotype interaction. C. pubescens M1-M16 genotypes collected at different sites of the southern zone of the State of Mexico were used because they are morphologically contrasting with different traits such as flower and fruit (^{Martínez, 2016}). Seedlings with four true leaves were inoculated with the identified pathogens, alone and combined, according to the following treatments (T): F. oxysporum (T1); P. capsici (T2); R. solani (T3); F. oxysporum + P. capsici (T4) and F. oxysporum + P. capsici + R. solani (T5). The inoculation method used was that described by ^{Martínez et al. (1996)}. The plants inoculated were transplanted to 1-liter polystyrene cups containing peat and expanded mineral perlite (agrolita) at a 2:1 ratio, respectively, with five replications per treatment each. The disease severity was measured by the percentage of the seedling’s hypocotyl and root using a 1-9 scale developed at CIAT (^{Abawi and Pastor-Corrales, 1990}). Data were logarithmically converted to obtain a general linear model using the InfoStat statistical software (^{Di Rienzo et al., 2016}).

Statistical analysis. The data obtained were subjected to an analysis of variance and the treatments were compared using Duncan’s test (p=0.05) and the InfoStat program.

Results and discussion

Isolation of the associated organisms. Fusarium oxysporum, Phytophthora capsici and Rhizoctonia solani (Table 1) were isolated from samples collected at the study sites. F. oxysporum was constantly found in all the sites (9/10), while P. capsici was found only in sites with intermediate and high cropping intensity (Table 1). The presence of R. solani in one sampled site suggests a limited participation in wilt damage. The three associated organisms were isolated at one site, and this indicates that the pathogen may individually or collectively affect the host. These results coincide with those reported by ^{Anaya-López et al. (2011}), who mentioned F. oxysporum as the most common pathogen, followed by R. solani in C. annuum crops. According to ^{Nelson et al. (1983}), the frequency variation of the associated organisms that cause wilt is affected by diverse factors, among which climate conditions are crucial. Compared to the other communities, the climate at El Potrero, the site where the three associated organisms were found, is warmer and rainy, and favors the development of the associated organisms. On the other hand, the crop systems, the use of fungicides and the genotype of the same crop itself, are decisive factors that favor the presence and survival of the organisms associated with the fungal complex (^{Guigón-López et al., 2001}; ^{Lozano et al., 2015}). The crop’s phenological stage at which samples are collected could influence the presence of the fungal complex, as has been observed in the case of C. annuum seedling stage (^{Vásquez et al., 2009}).

Morphological description of the associated microorganisms identified. Fusarium oxysporum developed abundant aerial, hyaline and septate mycelium, with radial growth habit and orange pigmentation, oval microconidia of 8.6 x 4.3 µm average size with 0 or 1 septum, abundant in fake heads and in monophialides, a lower number of macroconidia with attenuated apical cell and foot-like basal cell with 3-4 septa of 23.8 x 4.2 µm average size, as well as abundant intercalary and terminal chlamydospores on hyphae (Figure 1).

The first four days, Rhizoctonia solani developed mycelium white in color, which turned brown at day five, and sclerotia. Microscopical observations showed young vegetative cells with a ramification next to the distal septum, a 90° angle, narrow hyphae and formation of septa at a short distance from the point of origin to the hyphal ramification, 5-8 μm wide hyphae and sclerotia 1-3 mm in diameter (Figure 1).

Phytophthora capsici developed mycelium white in color, rosette-like growth in PDA culture medium, and coenocytic, hyaline and torulose mycelium; ovoid, lemon-like and not fully papillated sporangia of 11 a 54 µm long and 9 a 36 µm wide, and intercalary chlamydospores of 9.5 µm in diameter (Figure 1).

Different authors (^{Lozano et al., 2015}) have reported the associated organisms as the causal agents of wilt in C. annum. These organisms have been found in northern and central Mexico (^{Anaya-López, 2011}). However, in the case of C. pubescens, even when the three associated organisms have been found, the presence of P. capsici is associated with intermediate and high cropping intensity in the production zone of the State of Mexico (Table 1).

Molecular identification of the associated organisms. The PCR-amplified DNA fragments of the three associated organisms confirmed the identity of the pathogen, which had been previously morphologically established. The amplified fragment of F. oxysporum had a molecular weight of 900 base pairs (bp), and P. capsici and R. solani had an approximate weight of 650 bp (Figure 2).

Figure 1 Associated organisms that caused wilt in C. pubescens. 1. F. oxysporum a) microconidia, b) hyaline mycelium, c) septate mycelium. 2. R. solani a) septa close to the point of origin to the hyphal ramification, b) thick and septate mycelium, c) 90° angle. 3. P. capsici a) sporangia, b) hyaline and torulose mycelium. 

The sequenced fragments of the F. oxysporum strain were aligned with the F. oxysporum f.sp. lycopersici sequence (accession CM000593.1), and the consensus showed 88% identity with fragments from the region 248-270. The sequenced fragments of the P. capsici strain were aligned with the P. capsici_ LT1534 sequence version 11 accession PcapLT1534_SC064, and the consensus showed 83% identity with fragments from the region 146-259. Similarly, the DNA sequenced fragments of the R. solani strain were aligned with sequences from the NCBI’s gene bank, and its highest level of identity (90%) coincided with that of R. solani, accession number JATN01000256.1 (Version JATN01000256.1 GI: 576995022) with fragments from the region 144-292.

Pathogenicity in vitro. The symptoms observed were necrotic root apex, hypocotyl and/or cotyledons. The F. oxysporum, P. capsici and R. solani isolates were pathogenic to manzano pepper seedlings but symptoms appeared at different time. The expression of symptoms when F. oxysporum was inoculated varied 8-12 days after being cultivated depending on the strain. In the case of seedlings inoculated with P. capsici and R. solani, symptoms appeared 10 days after inoculation.

Figure 2 Electrophoresis in 1.2% agarose gel in TBE for ITS DNA amplification of fragments of F. oxysporum (F), P. capsici (P) and R. solani (R); M lanes correspond to the 100 bp molecular marker. 

Statistical differences in incidence and severity were observed on F. oxysporum (P≤ 0.01 colonies, with maximum and minimum values between 34.4 and 92.8% incidence, and 48.8 and 81.6% severity, respectively, and on P. capsici colonies (P≤ 0.05, with maximum and minimum values between 49.2 and 63.6% incidence, and 69.3 and 90.9% severity, respectively, that were associated with the collection site, a fact that may indicate intraspecific heterogeneity associated, among other factors, with pathogens natural spread, such as that reported for powdery mildew (Leveillula taurica) in tomato crops (Solanum lycopersicum) (^{Guzmán-Plazola et al., 2011}), and resistance development by the pathogens due to the selection pressure caused by fungicides (^{Silva-Rojas et al., 2009}). The pathogens incidence was 65% in F. oxysporum, 56. 4% in P. capsici, and 76% in R. solani. On the other hand, the severity values were 81.4% in P. capsici, 64.6% in F. oxyspoum, and 65. 5% in R. solani. These values indicate a more devastating effect caused by oomycetes, which come from a major manzano pepper production zone, where chemical products are used to control diseases, a fact that, according to ^{Silva-Rojas et al. (2009)}, causes P. capsici to become resistant.

Values higher than 1 in the severity/incidence quotient (Figure 3) indicate a higher level of pathogenicity given that although the pathogens showed a relatively similar incidence, the infection process was faster (severity) on the tissue area. The severity/incidence quotient of F. oxysporum was lower than 1.0 in five out of nine sites where F. oxysporum was found (Figure 3), which represented higher levels of incidence than those of severity, but the average value of the quotient of the nine pathogen colonies was very close to one, which suggests a proportional relation between the pathogen’s incidence and severity. In contrast, the severity/incidence quotient of P. capsici had values higher than 1.0 in all the evaluated colonies (Figure 3), which indicates that the rate of damage was faster even though the incidence was lower. In the case of R. solani, the quotient value was similar to that of F. oxysporum.

Figure 3 Severity/incidence quotient of F. oxysporum, P. capsici and R. solani in manzano pepper seedlings in 10 production sites of the southern zone of the State of Mexico. 

Interaction in multiple infections. The infectious process after inoculations, alone and combined, was faster and symptoms appeared three days after inoculation (dai) (Figure 4). At five dai, plants infected with P. capsici died (Figure 5). Similar results of Capsicum spp. indicate that this is the most lethal pathogen around the world (^{Lamour et al., 2012}). Additionally, reports by ^{Sanzón et al. (2012}) describe root necrosis 24 h after inoculation and plant death at day five, as well as evidence of plants being invaded by mycelium and sporangia. However, when P. capsici was combined with other pathogen(s), although no significant differences were found in some treatments (Figure 5), the level of damage was low, which could be due to pathogens competition for nutrients (^{Abdullah et al., 2017}). The average severity values from three-day measurements (Figure 5) showed a significant variation (p≤0.05) in the infection process rate between treatments, which also indicated that the P. capsici inoculum alone was the most severe because of the faster damages accumulation.

In the case of F. oxysporum, seedlings developed brown spots on the hypocotyl five ddi, which, according to ^{Sanzón et al. (2012}), are caused by a higher accumulation of polyphenols in the stem tissue that causes the cells in contact to die. Cell wall degradation could be due to activity of lytic enzymes that are produced by the pathogens (^{Feng et al., 2010}).

Figure 4 Damage caused in C. pubescens five dai by: A) F. oxysporum, B) P. capsici, C) R. solani, D) P. capsici + R. solani, E) F. oxysporum + R. solani, F) P. capsici + F. oxysporum and G) F. oxysporum+ P. capsici + R. solani. 

Figure 5 Accumulated and average severity of associated organisms alone and combined in C. pubescens plants, 5 days after inoculation. P= P. capsici, P+R= P. capsici + R. solani, F+P= F. oxysporum + P. capsici, F+P+R= F. oxysporum + P. capsici + R. solani, F= F. oxysporum and R= R. solani. The error bars correspond to the standard error. Columns with the same letter are not statistically different (p≤0.05). 

When F. oxysporum + P. capsici + R. solani were combined, a co-infection interaction occurred where the pathogenicity was lower compared to that of the infection produced by P. capsici alone (Figure 5). In this interaction complex, the response elicited by one pathogen may be modified in the presence of another pathogen. This complex interaction tends to alter the course of the disease because the co-existing pathogens compete for growth and nutrients in the same host, and suboptimal nutrition leads to a competition whereby some species may dominate (^{Abdullah et al., 2017}). However, the severity and type of competition are determined by nutrient consumption over time (^{Chesson, 2000}).

In the interaction F. oxyporum + R. solani, the pathogenicity was similar to that of F. oxysporum alone, and slightly higher to that of R. solani (Figure 5), which in general terms suggests that when both pathogens are combined, the presence of one of them does not alter the presence of the other. To this regard, ^{Alizon et al. (2013}), pointed out that, when the pathogens are combined, there is a wide range of responses from a determined host that have to do with evolutive and ecological aspects from both parts.

Pathogens-genotype interaction. The symptoms in plants inoculated with pathogens, alone and combined, appeared 15 dai. Plants infected with P. capsici showed looseness on the aerial part, necrotic lesions on the internal and external part of the stem crown, and necrotic radicle. Seedlings infected with R. solani showed chlorosis on the aerial part and necrotic roots; when the damage was severe, there was radicle strangulation; ^{González (2008}) mentioned non-compact rot with epidermis detachment. Seedlings inoculated with F. oxysporum showed chlorosis and, sometimes, curly foliage and necrotic radicle. Seedlings inoculated with F. oxysporum + P.capsici showed foliar chlorosis and wilt, as well as necrotic stem and radicle.

Seedlings inoculated with F. oxysporum + P.capsici + R. solani showed chlorotic leaves and stem crown, and necrotic radicle. According to ^{Velásquez-Valle et al. (2001}), the earliest evident symptom was a slight foliar looseness that was more visible as the day went on, as well as a slight yellowing of foliage. These symptoms were associated with root rot, softening of roots tip, root lesions that varied in size and color (from reddish to brown and black) and short and rotten root (^{Hamon et al., 2011}).

In infections caused by F. oxysporum, there were highly significant differences (P≤0.01) between genotypes with 0-70% variation between the lowest and the highest value, respectively, where the M8 genotype showed resistance, and the M9 genotype showed tolerance to the pathogen (Table 2). In seedlings inoculated with R. solani there were highly significant differences (P≤0.01), with minimum values of 0 % for M8 and maximum values of 76 % for M15. When inoculated with P. capsica, significant differences (P≤0.05) were observed, with 81-95 % variation in susceptibility between M8 and M7, respectively. In pathogen combinations, the intervals between maximum and minimum values were lower. In combinations of F. oxysporum + P.capsici with significant differences (P≤0.05), the maximum and minimum values were 67 % for M8 and 93% for M2; and the combination F. oxysporum + P.capsici + R. solani with highly significant differences (P≤0.01), the minimum and maximum values were 41% for M8 and 95% for nine genotypes (Table 2). The M8 genotype had the lowest level of wilt susceptibility and also showed other distinctive phenotypic differences compared to the rest of the genotypes analyzed (non-reported data), such as dense ramification and small fruits that are characteristic of rustic materials.

The M8 genotype, and likely, the M9 genotype, were both the least susceptible to wilt damage and can be a potential alternative for introducing the observed resistance in this study to commercial genotypes, such as M3, the most cultivated in the region. Moreover, the differential response of F. oxysporum, P. capscici and R. solani to some of the C. pubescens genotypes analyzed suggests the existence of different resistance mechanisms (^{Anaya-López, 2011}).

Conclusions

Three organisms were detected that cause manzano pepper wilt in the southern zone of the State of Mexico, from which Fusarium oxysporum was the most frequently present, followed by Phytophthora capsici and Rhizoctonia solani.

The molecular identification showed 88% identity of the amplified fragments of F. oxysporum using F. oxysporum f.sp. lycopersici race 2. P. capsici showed 83% identity with P. capsici_ LT1534, version 11, and R. solani had 90% identity with the anastomic group 3 (AG-3).

From the three pathogens identified, Phytophthora capsici causes the highest level of wilt severity in the southern region of the State of Mexico, although Fusarium oxysporum and Rhizoctonia solani also cause plant damage.

The M8 genotype was resistant to F. oxysporum and R. solani, and the M9 genotype could be tolerant to F. oxysporum. The M8 genotype was tolerant to P. capsici and to inoculation with the three pathogens, alone and combined, so it could be a potential genotype for studies about tolerance genes that could be used in genetic improvement programs.