Introduction

Pepper (Capsicum annuum L.) is one of the main commodity crops worldwide. During 2013, Mexico was the second producer of fresh green peppers with 2.2×106t, behind China that produced 15.8×106t (FAOSTAT 2015).

Bell or sweet peppers are different from hot peppers because they contain low quantities of the pungent substance capsaicin in the fruit parts (Chávez-Mendoza et al., 2013; Sora et al., 2015). In Mexico, most of the bell pepper crop is exported, mainly to Canada and the US (Ayala-Tafoya et al., 2015). During 2012, over 75% of fresh bell pepper imports into the US came from Mexico. Pepper crops are commonly subjected to the attack of soil-borne pathogens, which are very difficult to control, and are responsible for high disease incidences and heavy economic losses worldwide (Kamoun et al., 2015). One of the main diseases seriously impacting bell pepper yields is root and crown rot caused by Phytophthora capsici. Around the world, the oomycete P. capsiciLeonian, is one of the most destructive soil-borne pathogens on peppers (Hwang and Kim, 1995; Ristaino and Johnston, 1999). On chili pepper, this pathogen can cause root rot and crown rot (Hausbeck and Lamour, 2004; Dunn and Smart, 2015), while other researchers have reported that this pathogen attacks the base of the stem (Ristaino and Johnston, 1999).

Management of P. capsici relies on integrated approaches such as cultural practices, chemical control, host resistance (Gevens et al., 2006) and grafting ( Morra and Bilotto, 2010). Chemical treatments to reduce the incidence of root and crown rot relied in the past on methyl bromide fumigation (Wang et al., 2014). An alternative to substitute this fumigant is metam sodium (sodium N-methyl dithiocarbamate; metam-Na; Amvac Chemical Corp., Newport Beach, CA), which has a broad spectrum in biocidal activity in soil (Kreutzer, 1963). This registered fumigant (Duniway, 2002) is widely used in agricultural production for controlling soil-borne pathogens (Klose et al., 2008). Soil fumigation is one of the most effective methods to control pathogens and consequently maintain good yields (Hamm et al., 2003) and prevent environmental pollution (Arbeli and Fuentes, 2007). Even though studies have shown that metam sodium significantly reduces the inoculum of soil-borne pathogens (Hamm et al., 2003; Gerik, 2005; Gerik et al., 2006), little has been investigated on the effectiveness of this fumigant to reduce the incidence P. capsici in Mexico. Another approach to manage this disease is the use of resistant pepper plant materials (Gilardi et al., 2013). In recent years, grafting of pepper commercial valued types on resistant pepper rootstocks has been used to reduce the root rot and to increase yields (Rouphael et al., 2010). This practice has been successfully applied for bell pepper in Korea (Jang et al., 2012) and in Italy ( Morra and Bilotto, 2010; Gilardi et al., 2013).

In several studies about root rot in pepper plantations in Mexico, the causal agent of this disease has been attributed to diverse pathogens such as Fusarium oxysporum, Rhizoctonia solani (Gonzalez et al., 2004; Mojica-Marín et al., 2009), Pythium spp., Sclerotium rolfsii (Gonzalez et al., 2004) and Phytophthora capsici (Romero-Cova, 1988; Zapata- Vázquez et al., 2012).

Most studies report .. capsicias the causal agent of root rot in the Mexican states of Durango (Mojica-Marín et al., 2009), Zacatecas, Guanajuato, San Luis Potosi (Anaya- López et al., 2011), Chihuahua (Guigón-López and Gonzalez- Gonzalez, 2004; Ávila-Quezada et al., 2005; Silva-Rojas et al., 2009) and Aguascalientes (Velásquez-Valle et al., 2003). In Chihuahua only .. capsici has been considered to cause root rot, and two compatibility types have been documented from naturally infested pepper fields (Silva-Rojas et al., 2009). Nevertheless, few studies have been conducted to identify the populations of this pathogen affecting different types of pepper in this state (Anaya- Lopez et al., 2011).

The aim of this work was to determine if soil fumigation and use of resistant rootstocks have the capability to reduce root and crown rot incidence in bell pepper plots.

Materials and Methods

A field experiment was carried out in Delicias, Chihuahua, Mexico, in a P. capsici naturally infested site at an elevation of 1180masl.

Soil physical-chemical characteristics

Four samples from superficial soil (0-30cm) were taken for physicachemical analysis in early April 2011. The soil of the experimental site had a sandy clay loam texture (29.84% clay, 12.08% silt and 58.08% sand), with pH of 7.72 and organic matter content of 1.68%. In addition, soil content (ppm) reached 50.17 inorganic N, 64.14 P, 912.51 Na, 1994.56 K, 4021.55 Ca, 408.42 Mg, 7.44 Fe, 5.47 Mn, 7.02 Zn and 2.17 Cu. Moreover, CIC values showed 32.5me/100g, and electrical conductivity was 0.84ds·m-1.

Metam sodium application

The advantage of soil applied pesticides is that air pollution is avoided (Arbeli and Fuentes, 2007). Metam sodium (MS) was applied into prformed beds under a plastic mulch via the drip irrigation system at a dose of 600 lit·ha-1. This application method enhances the chemical efficiency, as reported by Overman (1982) and Overman and Price (1983). The dose ‘Lyzania’ and ‘Camila’. The ‘Fascinato’ variety was also grafted onto ‘Robusto’ and ‘Terrano’ rootstocks. All these plant materials or genotypes were bred by Syngenta Seeds, Houston, TX, USA. Seedlings were transplanted at a 0.45m spacing along into raised double-row 27m long beds. The trials had 120 or 240 plants (Table I).

Fertilization and irrigation

Fertilizers and water were applied via a drip irrigation system. Irrigations were done three days per week. Each day included two events of 1h duration; the first one at 8:00 and the second 2h later. Irrigation was suspended on those days when rainfall occurred. The growing season lasted 220 days. The following dosages (g·m-2) of fertilizers were soilapplied: NH.NO. (50.4), UAN32 (37.7), 5-30-00 (N-P-K) (56), KNO. (44.8), Ca (NO.). (162.3), K.SO. (201.3), and MgSO. (107.5).

All the experimental area was covered with a fixed shade net, which was installed previous to the beginning of the experiment. It kept air temperature between 30ºC at day to 18ºC during the night.

Disease incidence and statistical analysis

To quantify changes in incidence over time, the disease was assessed visually. Assessments were made in intervals of 14 days during the harvest period, from July 12 to October 3, 2011. The variable recorded was disease incidence, represented by the proportion of plants expressing wilt or being dead in the area as a whole (Campbell and Madden, 1990).

Disease incidence was adjusted over time through the following general linear model by logistic regression analysis:

where Pi: probability of Yi=1, and 1-Pi: probability of Yi=0. The disease progression model was tested for each trial.

Pathogen isolation from soil

Seven soil samples were collected in July 2011 for pathogen isolation and characterization. Soil cores (500g each) were randomly sampled, at least 10m apart within the experimental area and taken from the superficial soil layer (3-20cm). Once collected, samples were transported to the laboratory.

A 10g subsample was then taken from each sample. The subsample was suspended in 90ml of sterile distilled water. One ml of the suspension (1:100) was placed into an empty Petri dish and PARPH selective media was poured and distributed throughout the dish (Erwin and Ribeiro, 1996), forcing the mycelium to grow from underneath the media towards the surface, free from bacteria ( Martin et al., 2012).

Petri dishes were incubated at 28ºC and examined daily for colony growth during one week. Growth samples were placed on slides and examined under a Zeiss microscope (Carl Zeiss, New York) at ×40 and ×100 magnification to verify presence of pathogen mycelia and structures.

Once colonies of Phytophthora developed sufficiently, isolates were prepared and then plated out onto corn meal agar media (CMA; Fluka, Sigma-Aldrich) for morphological and molecular characterization. Three replications were made for each soil sample.

Pathogen isolation from plants

Symptomatic entire bell pepper plants were randomly collected in the experimental site to isolate the pathogen. The plants were transported to the laboratory, washed with tap water and cut in half. Then, small pieces of tissue were cut from the margins of lesions located on root, crown and stem. These tissue pieces were surface disinfested as reported by Foster and Hausbeck (2010), and blotted dry with filter paper to avoid bacterial contamination (Martin et al., 2012). Tissues were then plated out on PARPH medium. Isolates were transferred to Petri dishes with CMA and incubated for was based on label application instructions for this fumigant in the field (Gan et al., 1999). Given MS biocidal effects its application took place 30 days before transplanting to prevent seedlings damage.

Transplantation

Four commercial bell pepper varieties and one grafted onto two rootstocks were used to study the incidence of root and crown rot. All the seedlings were transplanted on April of 2011. The four non-grafted varieties were ‘Fascinato’, ‘Janette’, 7-15 days at room temperature (21 ±2ºC). Pure cultures were obtained by hyphal tips.

Six trials of bell pepper were compared as a strategy for controlling P. capsici. Four self-rooted varieties (V) and two grafted plants (G) were established in a metam sodium pretreated soil.

N=120 or 240 bell pepper plants per trial, Yi: initial disease incidence, Yf: final disease incidence.

The two parameters of Hick paradigm were estimated: intercept (β0) and slope (β1). Pr > 2 significantly different at P>0.0001

Ha: β1 of the trials 1 and 2≠β1 of the trials 3-6; Ho: β1 of the trials 1 and 2=β1 of the trials 3-6. a: 90 days after transplanting, b: 174 days after transplanting.

Results

Disease temporal progress and statistical analysis

According to the graph exploration, the patterns of the disease epidemics differ between the group of varieties and the group of grafted plants with respect to epidemic onset and Yf (Table I).

Initially, disease incidence increased slowly in all trials. Disease occurrence on self-rooted bell pepper varieties was first detected 90 days after transplanting. In the two grafted plants trials the disease started 104 days after days after transplanting. By comparing the group of self-rooted and grafted plants, it is noticeable that in the latter the proportion of dis- eased plants remained low for a period of approximately 174 days.

Disease incidence rose significantly until day 132 for grafted plants and until 146 days for self-rooted plants. It reached an asymptote in root- stocks trials toward the end of the epidemics (Figure 1) where- as no asymptote was reached on the disease incidence in the case of the varieties.

The general linear model appropriately fits the disease progress data over time. The model effects were found to be statistically significant at P<0.0001. When ‘Robusto’ and ‘Terrano’ rootstocks were planted on the fumigated soil some control of P. capsiciwas achieved. In this case, only few plants showed symptoms, representing 16% incidence in ‘Terrano’, and 13.7% in ‘Robusto’. Disease incidence followed a sigmoid function.

In general, the four varieties had a high disease incidence. However, differences among varieties can also be appreciated. For instance, self-rooted Lyzania plants (trial 5) were the most tolerant variety with the lowest slope β.= 0.3248. In contrast, plants grafted onto ‘Robusto’ rootstock were the most tolerant in all evaluation dates with the lowest Yf (13.7%) and the lowest slope (β.= 0.2312) in log odds ratio. The general lineal model fit- ted well the measured data for the two rootstocks and the four self-rooted varieties (adjusted R2, P<0.0001).

Discussion

The results indicate that the use of grafted bell pepper varieties on resistant pepper rootstocks planted on a pre-fumigated soil could provide an effective management strategy to reduce Phytophthoraroot and crown rot incidence.

The linearization of disease progress curves was essential to determine the epidemic speed. We fit the epidemics with the general linear model, which is recognized as an appropriate model to describe soilborne diseases (Liu et al., 1995) such as Phytophthora root and crown rot (Ristaino, 1991).

It is likely that the effect of metam sodium resulted in slow disease progress at the beginning of the epidemic. Symptoms of the disease appeared 104 and 90 days after trans- planting in grafted and nongrafted plants, respectively. It is likely that the application of metam sodium resulted in a slow epidemic onset. Later, the non-grafted varieties showed a sharp upward ‘inf lection’ of disease incidence, probably caused by reduction of metam sodium effects. The loss of metam sodium effectiveness has been reported previously due to fumigant degradation in soil. In a study by Triky-Dotan et al. (2009) metam sodium significantly reduced the incidence of Pythium rot in peanut after one application. However, fumigant effectiveness was greatly reduced after the second application. The same situation occurred when Verticillium was controlled with single and double applications of metam sodium; this fumigant was even less effective in the third application. Caution should be taken on the extensive use of a fumigant for disease management as it can can render P. capsici field populations resistant (Triky-Dotan et al., 2009).

Even though bell pepper production was obtained in this study (data not shown), the disease incidence increased with time ( Yf=13.7% for ‘Robusto’ rootstock). This percentage was reached 146 days after transplanting.

Since P. capsici is one of the most destructive pathogens to chili pepper and bell pepper (Hausbeck and Lamour, 2004) studies of soil fumigants (Linderman and Davis, 2008) and the use of resistant root- stocks (Louws et al., 2010) are essential to reduce disease incidence to assure crop yield. A considerable amount of research has been done on chemical control (Silvar et al., 2006; Keinath, 2007; Dunn et al., 2010;Foster and Hausbeck, 2010) and resistant pepper cultivars, although only a few have been conducted on combining resistant rootstocks planted on prefumigated soil (Morra and Bilotto, 2006). In the present study, results revealed that bell pepper grafted on resistant rootstocks could be an alternative to reduce the incidence of Phytophthora crown and root rot. Our findings confirm those of Gilardi et al. (2013), where ‘Terrano’ and ‘Robusto’ bell pepper root- stocks showed resistance to P. capsici.

The use of grafted pepper cultivars onto resistant pepper rootstocks in combination with metam sodium soil applications before transplanting could be an effective alternative to suppress disease progress caused by P. capsici. Rotation schemes are a cultural practice that somehow controls the disease. Nevertheless, management strategies that rely only on crop rotation may not provide an effective control of P. capsici, given the wide host range that this pathogen has, and the long time that propagules are cable to survive (Hwang and Kim, 1995). It should also be considered that the experimental site had a high propagule population, as given by the high incidence in the self-rooted control plants.

The phylogenetic analysis containing 14 sequenced P. capsici isolates of bell pepper, and other references showed that the dataset fitted into one clade. Previous research work on P. capsici found temperate isolates from the Solanaceae family to be grouped together in the same clade ( Bowers et al., 2007).

Our results provide compelling evidence that the P. capsici population showed no genetic differences within this experimental area. This supports the statement of Lamour and Hausbeck (2003) and Hu et al. (2013) who mentioned that P. capsici isolates from a single field experiment outcrossed within the population and, as a result, unique genotypes are present. Previous studies in Mexico (Silva-Rojas et al., 2009) have found both P. capsici compatibility types, which indicates that sexual reproduction is involved.

Since P. capsici is a destructive pathogen on bell pepper, avoiding the introduction of soil from another infested area is a required action to prevent genetic recombination (Gevens et al., 2006). Some future research priorities are identified that would be valuable in a better understanding of the epidemic. Large pathogen samples from different locations in northern Mexico would increase the robustness of these findings.

Studies on managing Phytophthora root and crown rot must continue to improve integrated management practices. This may include fumigants combination, fungicides, use of grafted plants, appropriate soil drainage and crop rotation.

Acknowledgements

The authors thank to the National Council for Science and Technology of Mexico (CONACyT) who support the Project 195770 PROINNOVA-CONACyT, and to the pepper production company Los Alamos of Delicias, Chihuahua, Mexico. We also thank Hilda Sáenz-Hidalgo, Ezequiel Muñoz- Márquez and Alexandro Guevara- Aguilar for disease incidence assessment and Juan Manuel Villa for agronomical practices in the experimental site.

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