Materiales y métodos

Study site and sampling. The study area was located in Isla, Veracruz, Mexico at 18° 8' N y 95° 35' O, 60 m above sea level, in warm-humid weather, average temperature of 24.9 °C and average annual rainfall of 2,316 mm. From September 2011 to March 2012, 40 plants with heart rot characteristic symptoms were collected and processed in the laboratory of post-harvest fruit diseases at the Colegio de Postgraduados, Campus Montecillo, Texcoco, Estado de Mexico.

Isolation. The sick pineapple plants were washed under a stream of clean water to eliminate soil excess. 5 mm² pieces between healthy and sick tissue were cut in basal leaves. They were disinfested through immersion in a solution of 3 % Sodium Hypochlorite for 4 min., rinsing 4 times with distilled water and dried with sterile paper towels. A total of 600 fragments were cultivated in three culture mediums: potato- dextrose- agar (PDA), nutrient agar, V8- agar (Natamycin 10 µg/L, ampicillin 292 µg/L, rifampicin 10 µg/L, pentachloronitrobenzene 0.10 g/L and hymexazol 0.25 µg/L), with a total of 150 cultivated boxes. All Petri dishes were incubated at 28 °C in white light as recommended by ^{Jeffers and Martin, 1986} and ^{Erwin and Ribeiro, 1996}. The purification of isolated samples* was done through hyphal head, once purified they were transferred to Petri dishes with v8-agar culture mediums (acidified with lactic acid at 25 %) for their characterization.

Additionally, 20 basal leaves were cut from the rot diseased plants, they were disinfested as it was described for the basal leaves and they were placed in distilled water at 28°C in white light. After the second day, white colored mycelium growth was observed, and a segment of mycelium was transferred from each leaf in acidified vegetable juice V8 culture medium.

Preparation of the inoculum. From the Phytophthora isolates in PDA culture medium, fragments were taken and placed in distilled water for the formation of sporangiums and the release of zoospores. A suspension of zoospores was prepared with the Neubahuer camera, with a concentration of 10⁸ zoospores ml^-1 (^{Rodríguez et al. 2002}).

Pathogenicity tests. In vitro pathogenicity tests were carried out in 60 crowns and 80 in vivo stems. Both materials were sun dried during 5 days and disinfected with 3 % sodium hypochlorite for 4 min. and were washed 4 times with distilled water. 4 lesions were inflicted in 60 crowns with a needle in the median stem of the crown, 30 of the crowns were placed in a recipient with 300 ml of the suspension of spores, 30 crowns were used as controls and were only added sterile water without zoospores. The treatment was placed under white light at 28°C until the appearance of symptoms. For the in vivo tests, 5 month old stems were used, cultivated in polyethylene bags in mixtures of soil and tezontle (3:2 v/v). In 40 stems, the soil was removed at about 8 cm around the heart of the pineapple, 4 lesions were inflicted on the median stem with the sterilized needle and 300 ml of inoculum were added along with mycelium fragments with sporangiums and was covered again with the removed soil. The same technique was applied to the 40 control samples, but only distilled water was used. Another polyethylene bag was placed on the other plants until the appearance of symptoms. For both treatments the damaged tissue was cultivated in acidified V8- agar culture.

Morphological characterization. It was characterized based on the mycelial growth, pigmentation and formation of reproduction structures of the colony growing in PDA and under perpetual white light at 28°C. The identification of the genus was done through the ^{Erwin and Ribeiro (1996)} key, and for the species with the ones described by ^{Gallegly and Hong (2008)}.

Molecular Identification. The extraction of DNA was done from a 6 day growth cultivar in a V8 liquid medium at 28 °C, using the CTAB protocol (^{Doyle and Doyle 1990}). The transcribed internal space of the ribosomal DNA was amplified using the ITS6 and ITS4 initiators (^{White et al. 1990}). The sequences obtained were compared with the database in the National Center for Biotechnology Information ^{NCBI (2012)}, USA (http://www.ncbi.nlm.nih.gov).

Results

Isolation of microorganisms. It was observed among the different mediums that in the V8 agar with antibiotic culture medium, Phytophthora sp. was isolated with more frequency, and less frequently in the nutrient and PDA agars. In the nutrient and PDA agars, Pythium was isolated with more frequency, the percentages are shown in Table 1.

Table 1 Percentage of isolated microorganisms in pineapple plants (Ananascomosus L. Merril) MD2 cultivar with signs and symptoms of heart rot. Isla, Veracruz, Summer, 2011. 

From the 20 leaves placed in flasks with distilled water, the growth of mycelium was observed after 2 days (Figure 1), and the fragments cultivated in PDA and acidified V8- agar developed colonies characteristic of Phytophthora sp.

Figure 1 A) and B) Phytophthora sp. Mycelium grown in distilled water for two days, in basal leaves collected from the field. 

Pathogenicity tests. In the in vitro tests, the symptoms presented a pale green color and necrosis on the tips of the leaves within 7 to 9 days. Furthermore, rotten middle leaves presented a light brown color in the middle of the rosette, and Phytophthora mycelium could also be observed (Figure 2). 100 % of the inoculated crowns got the disease. The control samples did not present any symptoms or signs and developed an abundant radical system. From the necrotic tissue, isolation was once again performed in acidified V8- Agar wherein Phytophthora nicotianae was isolated. For the in vivo tests, the symptoms and signs took 16 days to manifest, starting with a change in the color of the leaves to a lighter green, the tips of which were necrotic around the heart of the pineapple. Within 20 days, heart rot was evident, the center of the rosette was brown and mycelium was present, the base of the middle leaves had a putrid smell; 38 (95%) of the 40 inoculated plants showed symptoms of the sickness. The control samples did not present symptoms or signs of the disease. From the necrotic leaves isolations were performed in acidified V8- agar culture medium, where the presence of Phytophthora nicotianae was corroborated.

Figure 2 Síntomas y signos de la pudrición del cogollo de la piña. A) Pruebas in vitro de la pudrición del cogollo de la piña. B) Signos y síntomas en coronas inoculadas. C) Pudrición del cogollo. D) Hoja de corona con pudrición. E) Corona de piña 9 ddi. F) Signos del oomiceto en plantas inoculadas. G) Signos y síntomas en plantas inoculadas en invernadero. H) Cogollo podrido de la planta de piña. I) Cogollo y raíz de planta testigo de prueba in vivo. J) Cogollo y raíz de planta inoculada. K) Planta de piña 16 ddi. 

Morphological characterization. The growth of mycelial hyaline was carried out in a V8- agar culture medium, non-defunct, spherical and/or limoniform sporangiums with a prominent papilla, the average size of 100 sporangiums was 44.6 x 36.8 µm; spherical, non-papillate chlamydospores were present, terminal and intercalated hyalines of 30.2 µm (average of 100 chlamydospores) (Figure 3). The formation of sporangiums in distilled water appeared within 48 hrs. under perpetual white light. The characteristics coincide with those reported by ^{Erwin and Ribeiro (1996)} and ^{Gallegly and Hong (2008)} for Phytophthora nicotianae.

Figure 3 Asexual structures of Phytophthoranicotianae. A) Spherical Sporangium. B) Limoniform Sporangium. C) Ovoid Sporangium. D) Interspersed Chlamydospore. E) Terminal Chlamydospore. 

Molecular identification. The product of amplification that was obtained with the ITS4 and ITS6 initiators was of 850 bp (Fig. 3). The comparison of the nucleotide sequence of the isolate showed 100% similarity with the Phytophthora nicotianae sequence.

Discussion

The in vivo and in vitro heart rot results similar to those observed in the cultivation fields of Isla, Veracruz, that start with aqueous lesions at the base of the leaves (tissue without chlorophyll) and the heart of the fruit, the leaves turn a lighter green, the tips become necrotic and develop a characteristic putrid smell, all caused by Phytophthora nicotianae, coinciding with the description by ^{Espinosa and Adam (1972)} in Mexico and by ^{Joy and Sindhu (2012)} in India. These symptoms are probably present due to the physical obstruction of the vascular system caused by the growth of hyphae and the penetration of other secondary organisms (fungi and bacteria), increasing the formation of gums that originate due to oxidation and the accumulation of cellular degradation residues, as happens with ornamental plants (^{Arévalo-Galarza, 2012}; ^{Agrios, 2005}). Subsequent to inoculation and the formation of zoospores, these are attracted to the elongation zone and differentiation of the root, where they form cysts, and even invade the secondary roots and not the tips (^{Galiana et al., 2005}; ^{Attard et al., 2010}). Most cases of heart rot in pineapples in the sampling areas were observed during the rainy season, with temperatures that oscillated between 24-26 °C and a relative humidity of 90-100 %; the high relative humidity induces the development of the diseases caused by Phytophthora sp. in pineapple plantations (^{Duniway, 1983}).

Additionally, when cultivating in slope areas, cultural labors (fertilization, weed control, soil removal, etc.) and lesions caused by other organisms (nematodes and rodents) to the radical system, facilitate the dispersion and the infection risk by Phytophthora sp. as mentioned by ^{Erwin and Ribeiro, 1996}, ^{Elliot, 1989}, ^{Jung and Blaschke, 2004} and; ^{Galiana et al., 2005}.

Conclusion

Pathogenicity tests, as well as morphological identification and molecular characterization, confirmed that Phytophthora nicotianae is the causative agent of heart rot in pineapple in the municipality of Isla, Veracruz, Mexico.