The prickly pear (Opuntia spp.) is widely distributed throughout the American continent, mainly in the central area. It belongs to the Cactaceae family, in which the most important species due to its economic value is found: O. ficus-indica, which is planted for its fruit (tuna), as well as for its cladode (nopal) (^{Caruso et al., 2010}). Its diverse uses include production for the food, medical and chemical industries, and it has a high environmental and symbolic value (^{Márquez-Berber et al., 2012}). Mexico is the main producer of prickly pear (O. ficus-indica), with a surface of 12,799 ha planted with this crop. The main producing areas are Morelos, with 4,107 ha; Mexico City, with 2,482; State of Mexico, with 1,016 ha and Tamaulipas, with 957 ha (^{SIAP, 2019}). However, like many other crops, its productivity can be affected by several factors, including phytosanitary factors. In the municipality of San Juan Ixcaquixtla, Puebla, the cladodes have been reported with symptoms of dark brown to black spots, which only affect the surface (cuticle, epidermis and part of the parenchyma). At the end of the cycle of the disease, the affected areas can be detached, showing scars and deformities on the cladode. This disease has been called black scab, and it causes deformities and a reduction of the photosynthetic area of the prickly pear; its incidence was of approximately 60%. Similar symptoms, cause by tissue necrosis, are caused by different pathogens in the prickly pear, including Fusarium sporotrichioides, Lasiodiplodia theobromae, related to the necrosis of cladodes (^{Swart and Kriel, 2002}); Pseudocercospora opuntiae, causal agent of the black scab on prickly pear (^{Ayala-Escobar et al., 2006}; ^{Quezada-Salinas et al., 2006}); Fusarium lunatum (^{Flores-Flores et al., 2013}) and Pectobacterium spp. (^{Torres-Bojórquez et al., 2016}). The symptoms observed in San Juan Ixcaquixtla do not coincide with any reports, therefore the aim of this investigation was to identify the causal agent on black scab on prickly pear.

In the town of Santa Cecilia Clavijero, in the municipality of San Juan Ixcaquixtla (18° 26’ 25.80” N, 97° 47’ 35.63” W) Puebla, during 2018-2020, cladodes were collected with different degrees of progress of the disease called black scab. Pieces of tissue with symptoms, 1 cm² in size, were cut from the diseased-healthy tissue transition (cuticle-epidermis-collenchyma). The pieces were disinfested with a 1% sodium hypochlorite (NaOCl) solution for 1 min, washed twice with sterilized distilled water, dried on sterilized drying paper, and planted in a wet chamber in a potato-dextrose-agar (PDA) medium and later incubated at 25±2 °C for five days. The fungal cultures developed and the structures formed in the chamber were transferred to a potato-dextrose-agar medium (natural: potato 200 g, dextrose 14 g, agar 18 g, water 1 L) with added lactic acid (1.5 mL at 10%) and incubated under continuous white fluorescent light at 25±2 °C.

The fungi were purified with monoconidial cultures in water-agar at 2% (W/V). The induction of reproductive structures was carried out in PDA at 25±2 °C. The identification and morphological description of the isolations was carried out with temporary and permanent preparations which helped observe the morphology and the size of 100 spores of each isolation under a compound microscope at a magnification of 40X. To determine the species, taxonomic keys were followed and comparisons were made with reports from scientific literature (^{Boerema, 1993}; ^{Bensch et al., 2010}; ^{Bensch et al., 2012}; ^{Chen et al., 2015}; ^{Nam et al., 2015}; ^{Pan et al., 2018}; ^{Mapook et al., 2020}).

Healthy cladodes were planted in sterilized soil and kept for six months in a greenhouse in the Colegio de Postgraduados, Mexico, at a temperature of 24±3 °C and a relative humidity of 70%. Three fungi (Cladosporium cladosporioides, Aplosporella hesperidica and Didymella glomerata) were consistently isolated from the pieces planted in the culture medium and inoculation was carried out following two methods, by sprinkling and injection of conidia. With a Neubauer chamber, suspensions were prepared at a concentration of 2×10³, 3×10³ and 6×10³ conidia mL⁻¹ for each of the three fungi isolated. Manual sprinklers, previously disinfected with a 1.5% hypochlorite solution, were used to apply the suspension, along with a sterilized hypodermic syringe, on cladodes previously washed three times with sterilized distilled water. The inoculated treatments consisted of C. cladosporioides at 6×10³ conidia mL⁻¹; A. hesperidica, 6×10³ conidia mL⁻¹; D. glomerata, 6×10³ conidia mL⁻¹; C. cladosporioides, 3×10³ conidia mL⁻¹ + A. hesperidica, 3×10³ conidia mL⁻¹; C. cladosporioides, 3×10³ conidia mL⁻¹ + D. glomerata, 3×10³ conidia mL⁻¹; A. hesperidica, 3×10³ conidia mL⁻¹ + D. glomerata, 3×10³ conidia; C. cladosporioides, 2×10³ conidia mL⁻¹ + A. hesperidica, 2×10³ conidia mL⁻¹ + D. glomerata, 2×10³ conidia mL⁻¹; sterilized distilled water.

The injection method consisted in injecting 1 mL of each treatment subepidermally, whereas for sprinkling, 4 mL of inoculant were sprinkled on both sides of the cladode. Each treatment consisted of three repetitions (three cladodes) with a completely random design. The experiment was carried out twice. The inoculated cladodes were kept in a greenhouse in the Colegio de Postgraduados at a temperature of 24±3 °C and a relative humidity of 70% until symptoms developed. The organisms that induced the black scab symptoms were re-isolated to verify their identity and comply with Koch’s postulates. Pieces of 0.5cm in size were cut from the cladodes inoculated with symptoms similar to those observed on the field, they were washed three times with sterilized distilled water, dried, planted in Petri dishes with a natural PDA medium, and incubated under continuous white fluorescent light at 25±2 °C.

On the field, the symptoms caused by black scab were characterized by the development of spots, 1 to 4 cm in diameter, initially light brown in color and which turn dark brown to black in time, with a moist consistency and surrounded by dead light brown tissue. When the spots coalesced, large areas developed which, in some cases, covered up to 70% of the surface of the face of a cladode. The lesions only affected the cuticle and epidermis, but not any internal tissues (collenchyma, chlorenchyma, storage parenchyma cells, vascular system and medulla). In advanced stages, the affected part detaches and falls to the ground, and this causes deformities and damages in the superficial photosynthetic area.

Out of the total of isolations obtained from symptoms of the black scab, three fungus species were identified and morphologically characterized: 1) Cladosporium cladosporioides developed maroon-olive green cultures on the front, and almost black on the reverse; mainly immersed mycelia, but also aerial mycelia; lone conidiophora, cilyndrical, pale to maroon-olive green in color, micronematous, smooth, occasionally warty, arising terminally from ascending hyphae and not branched, or occasionally branched, one of the top septa slightly darkened, where ramoconidia are formed; conidia in long, branched chains that disarticulate easily, mostly unseptated, elliptical to lemon-shaped, pale to maroon-olive green and smooth, sized 3.0-5.5 × 2.5-3.0 µm, these characteristics were consistent with reports by ^{Bensch et al. (2010)}, ^{Bensch et al. (2012)} and ^{Nam et al. (2015}). 2) Aplosporella hesperidica showed culture with slightly elevated mycelia, with white aerial hyphae on the surface, extending from the center, obverse of the white culture. Conidiomata, sixed 398 × 516 µm, semi-immersed, eruptive, uniloculate, in groups, globose, dark maroon. Conidiophores reduced to conidiogenic cells. Conidiogenic hyalin cells, holoblastic, oblong or cylindrical to bulbous. Conidia sized 18×12 µm, maroon, septated, ellipsoidal to oval. Characteristics correspond to those reported for this species by ^{Mapook et al. (2020}). 3) In Didymella glomerata, cultures were initially white in color but later became olive green to dark maroon in color. Conidia, developed in ostiolate pycnidia, ellipsoid, unicellular, hyaline, sized 5.0×3.0 µm. Pycnidia, dark brown in color, sized 70×90 μm. These morphological characteristics coincided with reports by ^{Boerema (1993}), ^{Chen et al. (2015}) and ^{Pan et al. (2018}).

Symptoms began appearing in the cacti 50 d after inoculation. The only cladodes with symptoms were those inoculated with the method of conidia injection and with the combination of all three fungi, namely C. cladosporioides, A. hesperidica and D. glomerata. Neither the cladodes inoculated with one and the combination of two fungi nor the control presented any symptoms. These began with the appearance of light brown lesions, 2 to 6 mm in diameter (Figure 1A). For the next two weeks, the lesions acquired a dark brown to black color, and were surrounded by dead, light colored tissue (Figure 1B and C); these symptoms corresponded to those observed on the field in the municipality of San Juan Ixcaquixtla, Puebla. The reisolations obtained from inoculated plants coincided with the cultural and morphological characteristics of the isolations of plants gathered from the field, thus showing that C. cladosporioides, A. hesperidica and D. glomerata are the causal agents of black scab in prickly pear cladodes.

Cladosporium cladosporioides is the causal agent of floral necrosis, rotting and loss of young fruits in papaya tree (Carica papaya) in Mexico (^{Vásquez et al., 2012}); sooty mold in tangerines (Citrus reticulata) in Japan (^{Tashiro et al., 2013}) and the rotting of fruits in zapote mante (Pouteria campechiana) in Mexico (^{Nabor-Romero et al., 2018}). On the other hand, A. hesperidica has been reported as a saprophyte in dead Citrus aurantium branches in India (^{Rao, 1969}) and dead Chromolaena odorata stalks (^{Mapook et al., 2020}), and as a pathogen causing the early rot of the stem of Citrus sinensis in Zimbabwe (^{Yang et al., 2017}). Finally, D. glomerat, has been reported as a pathogen, causing leaf smut in pistachio (Pistacia vera) in the United States (^{Moral et al., 2018}) and black stain on kiwifruit (Actinidia chinensis) in China (^{Pan et al., 2018}), showing the impact of these pathogens on different economically important species. No reports have yet been found on the interaction of these three fungi on the development of the disease on prickly pear or on any other host. Only C. cladosporioides was reportedly found to infect cladodes in Brazil (^{Souza et al., 2010}). Due to this, according to scientific literature, this is the first report on C. cladosporioides, A. hesperidica and D. glomerata as causal agents of black scab in cacti in Mexico.

The pathogens that coexist in one same host can affect each other through antagonism or benefit through synergy, and in the latter, different interactions have been reported. In Pisum sativum, the development of smut increases with the coinfection of Mycosphaerella pinodes and Phoma medicaginis var. pinodella (^{Le May et al., 2009}); the foliar stain of eucaliptus is caused by numerous species of fungi of the genus Teratosphaeria (^{Crous et al., 2009}), and in South Africa, two species -Teratosphaeria juvenalis and T. verrucosa- coexist on the same leaves, and even on a same stain, causing infection (^{Crous et al., 2009}). In this study, results show that the presence of all three pathogens in the same organ of the plant at the same time leads to the development of the disease and its reproduction. As mentioned earlier, the three fungi cause similar symptoms, that is, rot and necrosis, symptoms that are also typical of the black scab disease in prickly pears. In later works, we suggest an evaluation of the time intervals in inoculation, in order to determine is simultaneous inoculation is crucial or which are the time intervals in which the infectious feature of the fungi C. cladosporioides, A. hesperidica and D. glomerata are maintained. Due to the fact that black scab is a potential threat to the production of prickly pears and nopal, it is necessary to evaluate different control strategies that help develop management plans. In addition, it is worth highlighting the need for complementary molecular studies to corroborate the identity of the causal agents described in this investigation.

Figura 1 Pathogenicity tests for Cladosporium cladosporioides, Aplosporella hesperidica and Didymella glomerata on prickly pear (Opuntia ficus-indica), under greenhouse conditions. A) Initial symptoms, light brown lesions, B) Later development of symptoms, light brown to black spots, surrounded by dead light-colored tissue, C) Cladode with symptoms, each lesion represents a point of inoculation with the injection of conidia