Study area

The Milpa Alta area, with a surface of 28,375 ha (^{Diario Oficial de la Federación, 1997}) is located in the southeast sector of Mexico City between 19° 04’ and 19° 12’ N and 98° 57’ and 99° 08’ W. It has mountainous topography and altitudes of 2,230 to 3,200 m (^{Rodríguez and López, 2006}). The climate is sub-humid temperate with rains in summer [C(w2)(w)], mean annual temperature of 14.4 °C and annual rainfall of 878.9 mm (^{García, 1988}). Rainfall is heterogenic in its distribution, from 580.6 mm in the lower altitudes to 1,200 mm in the higher elevations (^{IMTA, 1996}).

Logarithmic diagrammatic scale

To evaluate the incidence and severity of nopal cacarizo, a logarithmic diagrammatic scale was constructed using the methodology proposed by Mora-Aguilera et al. (2003)^[2]. A digital camera of 5 mega pixels was used to randomly photograph 60 mature cladodes of various levels of severity of nopal cacarizo. Then, with the image editor of Adobe Photoshop (Adobe Sytem Incorporated, 2010), the area invaded by the nopal cacarizo was measured using the program ImageTool^® (^{Wilcox et al., 2002}) and the upper and lower limits were determined. Once the maximum limit of the scale was defined, this value was put in the program 2LOG ver. I (Osada-Velázquez and Mora-Aguilera, 1997^[3] to define the number of classes (^{Tovar-Soto et al., 2002}; Mora-Aguilera et al., 2003^[2]; ^{Gomes et al., 2004}).

The program 2LOG ver. 1 was used to obtain a logarithmic scale that was converted to a diagrammatic scale through the association of a photographic image whose severity best approximated the mean points of each class. Thus, the diagrammatic scale was defined with five classes (0-4) (Figure 1).

Figure 1 Logarithmic diagrammatic scale of severity of nopal cacarizo. Ranges represent the percentage of the average area invaded in the cladodes. 

The diagrammatic scale was validated before using it, for which the real values obtained with the program PhotoShop and Image Tool were used, and those obtained for the evaluator. These data were used to make an analysis of simple linear regression with the program Validar-PER ver. 1.1 (Mora-Aguilera, 2009^[4]) and the values of r² and b₁ close to 1.0 indicated maximum precision and exactness, respectively.

Incidence and severity of nopal cacarizo

Sampling

Simple stratified sampling was carried out based on altitude and cardinal points in Milpa Alta in the spring-summer of 2010. For the distribution of the crop in the study area, five sampling zones were defined by altitude (masl): 1 3070 to 2720, II) 2919 to 2770; III) 2769 to 2620; IV) 2619 to 2470 and V) 2469 to 2320, and in each zone four sampling sites were selected; 20 plots of 5 to 10 years of age and whose plants did not exceed 1.60 m height, where the principal management system of pests and diseases was by fertilization with organic manure and chemical control.

Incidence

Incidence was evaluated as the presence or absence of nopal cacarizo on O. ficus-indica. In each plot 10 randomly selected plants were examined. The response variables were as follows: number of cladodes per plant, and number of cladodes with at least one pustule of nopal cacarizo. Each sampling point was geo-referenced with the global positioning system (GPS 12 XL Garmin Olathe, KS, USA). Determination of incidence was carried out according to the proportion or percentage of plants with symptoms using the following formula (^{Tovar-Soto et al., 2002}):

Severity

To evaluate severity (area of the cladode affected by nopal cacarizo), 10 plants were randomly selected from each plot with the size indicated above. In each plant an estimation was made of the degree of damage in three strata using the logarithmic diagrammatic scale. The basal and second cladodes were considered as low stratum, third cladode as middle stratum and those that grow above the third cladode as upper stratum. Total number of cladodes per stratum was determined along with the number of cladodes per level of damage per stratum. Severity was estimated using the following formula (^{Tovar-Soto et al., 2002}):

The data obtained of incidence and severity were captured on a calculation sheet of the program Excel and were imported to the program SURFER 32 ver. 6.04 (^{Surface Mapping System, 1997}) to determine the distribution of the nopal cacarizo. In addition, the dimension of both was estimated graphically with the data previously recorded on the calculation sheet and later as a graph. Then the options “Classed post and Contour” were used to generate bi and tridimensional maps.

Identification of the insect

To confirm the species of red chinch bug, five specimens were collected from each sampling site and they were taken to the entomology laboratory of the Plant Health department of the Colegio de Postgraduados mounted on entomological needles. The insects were then identified with a dissection microscope and with the keys for the genus Hesperolabops, published by ^{Froeschner (1967)}. In addition, the species was confirmed by Dr. Harry U. Brailovsky of the Biology Institute of UNAM, specialist in the family Miridae. The specimens were kept in the taxonomy laboratory of the Agricultural Parasitology Department of the Universidad Autónoma Chapingo.

Statistical analysis

For the distribution of nopal cacarizo in the field, the calculations of aggregation and spatial dispersion were used, which were determined through the indices of Morisita and Lloyd (^{Campbell and Madden, 1990}) using the program MorLloyd version 1.0 (Rivera-Valencia and Mora-Aguilera, 2010^[5]. The aggregation and spatial dispersion index of H. nigriceps was estimated with the same program.

The experimental design was completely randomized with five treatments; each altitude was considered a treatment and the four plots within each altitude were the replicates. Given that the data did not have normal distribution, even when transformations were attempted (Shapiro-Wilk), they were processed using the non-parametric test of Friedman. Tests were also made of simple correlation between the presence of H. nigriceps and the incidence of nopal cacarizo. The variables were healthy cladode, cladode with nopal cacarizo and the presence of red chinch bug, and cladode with the presence of red chinch bug without nopal cacarizo.

Incidence and severity of nopal cacarizo

The validation of the logarithmic diagrammatic scale to determine severity of nopal cacarizo, estimated by regression analysis, presented a precision of r²=0.988 and an exactness of b₁=1.012, which indicated that the values of severity were adequate for this evaluation. The incidence and severity of the nopal cacarizo in Milpa Alta were 56.4±18.5 % and 10.5±6.4 %, respectively. According to the aggregation indices of Morisita (0.990) and of dispersion of Lloyd (0.980), the incidence of nopal cacarizo presented a uniform type distribution in the region (Figure 2A), whereas the severity was of the aggregated type Morisita (1.767) and Lloyd (2.535) Figure 2B). These data suggest a possible vegetative transmission of the causal agent influenced by agronomic management, as occurs with other phytosanitary problems such as chlorotic spots associated with O. ficus-indica (^{Alonso-Barrera et al., 2015}).

Figure 2 Spatial distribution of incidence (A: uniform type) and severity (B: aggregated type) of nopal cacarizo in Milpa Alta, Mexico City, 2010. 

Although the bidimensional maps and the indices of Morisita and Lloyd reveal certain aggregation of the severity of nopal cacarizo in the western zone of Milpa Alta, no statistical differences were found between altitudinal categories (F=0.57; p=0.943), nor between the cardinal points (F=1.0; p=0.464).

Individually, in each plant the highest concentration of nopal cacarizo was measured in the low and middle strata, where the mother, second and third cladodes are located (Table 1) and which correspond to the cladodes of highest age in a plantation and highest concentration of red chinch bug, an observation which coincided with that registered by ^{Palomares-Pérez et al. (2010)}.

Association of nopal cacarizo-Hesperolabops nigriceps

All of the specimens of chinch bugs collected in the plots corresponded to H. nigriceps, and there was a significant correlation between the presence of H. nigriceps and nopal cacarizo (r=0.765). Furthermore, the indices of Morisita (1.003) and Lloyd (1.006) indicated that the concentration of red chinch bug in Milpa Alta was aggregated and coincided with the areas (west) where the highest severity of nopal cacarizo was concentrated (Figure 3). It is possible that the western topographical zone presents the optimum conditions for the development of this problem or that the phytosanitary management, principally in the cutting and transport of the cladode to be planted, are the factors that favor the development of this damage, as occurs with the dissemination of other problems of nopal such as black spot or chlorotic spots (^{Quezada-Salinas et al., 2006}; ^{Alonso-Barrera et al., 2015}).

Figure 3 Spatial distribution of the red chinch bug, Hesperolabops nigriceps, in the production zone of vegetable nopal in Milpa Alta, Mexico City. 

Nopal cacarizo is a phytosanitary problem of the first order in Milpa Alta according to some technical reports. With this study the symptoms of nopal cacarizo can be considered important and establish that the visual estimations made in the technical studies are probably overestimated, as occurs with other symptoms that are manifested on leaves or stems in the plant of interest (^{Cadena-Hinajosa et al., 2003}; ^{Soltero-Díaz and Williams-Alanís, 2003}).

A fundamental factor in the level of damage or severity was the age of the cladodes. The dry pustules are practically limited to the mature cladodes of more than one year of age, while the young cladodes rarely presented this symptom. ^{Palomares-Pérez et al. (2010)} mention that the young cladodes are cut by the producers when they present any damage that affects their appearance in the market, and this is probably one of the reasons why the damage is observed only on the mature cladodes and is related to the places where the red chinch bug prefers to feed. Perhaps the concentration of nutrients in mature and immature cladodes, as is reported in studies of nutrition in nopal (^{Villareal et al., 1963}; ^{Saag et al., 1975}; ^{Bravo-Hollins, 1978}; ^{McGarvie and Parolis, 1979}, ¹⁹⁸¹), is important for defining the feeding locations of this species of insect. In addition to the preference for the feeding sites for H. nigriceps, the symptoms are cumulative, that is, the mature cladodes may have symptoms caused by the feeding of chinch bugs from previous years.

Although no differences were detected between incidence and severity of nopal cacarizo at different altitude and cardinal points, it was evident that using the spatial interpretation and the indices of Morisita and Lloyd, the symptoms presented certain tendency of aggregation in the western zone, where the populations of H. nigriceps were concentrated. This confirmed the relationship nopal cacarizo-H. nigriceps and the aggregation habit of this species, as occurs with other species with gregarious habits of the same family such as Lygus Hesperus Knight and Sahlbergella singularis Haglung (^{Schotzko and O’Keeffe, 1989}; ^{Bisseleus et al., 2011}).

The presence of microclimates due to altitudinal changes is a factor which affects the distribution of bees and other pollinating insects (^{Gonçalves and Stort, 1978}; ^{Salamanca-Grosso, 2009}). In the case of H. nigriceps the presence of microclimates in the region of Milpa Alta, related to altitude (^{García, 1988}; ^{IMTA, 1996}; ^{Rodríguez and López, 2006}) does not seem to have an important effect on the distribution of the insect and nopal cacarizo. This could be attributed, at least in part, to the capacity of this species of insect to exploit the nopal resource in the environment of Milpa Alta.

One factor that could influence the distribution of nopal cacarizo is crop management. Although it is the area with greatest surface destined to this crop in Mexico (^{SIAP, 2013}), in Milpa Alta agriculture is a secondary activity and the producers destine relatively less time to management tasks and pest control, including H. nigriceps, compared with producers whose principal activity is agriculture (^{Vanegas-Rico et al., 2010}). Furthermore, apparently not enough attention is given to the origin of cladodes that are used to establish new plantations. The cycle of H. nigriceps is one year and the sites of oviposition in the cladode are not evident (Palomares-Pérez, unpublished data), therefore it is probable that eggs of the insect are present in the material used to establish new plantations (^{Badii and Flores, 2001}), thus favoring the dispersion of the red chinch bug and nopal cacarizo. To date there is no information of the effect of nopal cacarizo on O. ficus-indica. Nevertheless, due to the distribution of these symptoms in Milpa Alta, and the severity that can be reached in some plots, it is convenient to study this phenomenon and its possible consequences on the productive life of the plants.