Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] Sociedad Mexicana de Fitopatología, A.C. México
Hernández-Lauzardo, Ana Niurka; Bautista-Baños, Silvia; Velázquez-del Valle, Miguel Gerardo; Trejo- Espino, José Luis Identification of Rhizopus stolonifer (Ehrenb.: Fr.) Vuill., Causal Agent of Rhizopus Rot Disease of Fruits and Vegetables Revista Mexicana de Fitopatología, vol. 24, núm. 1, enero-junio, 2006, pp. 65-69 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México
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Identification of Rhizopus stolonifer (Ehrenb.: Fr.) Vuill., Causal Agent of Rhizopus Rot Disease of Fruits and Vegetables Ana Niurka Hernández-Lauzardo, Silvia Bautista-Baños, Miguel Gerardo Velázquez- del Valle, and José Luis Trejo-Espino, Instituto Politécnico Nacional, Centro de Desarrollo de Productos Bióticos, km 8.5 Carr. Yautepec-Jojutla, San Isidro, Yautepec, Morelos, México CP 62731. Correspondence to: [email protected] (Received: September 14, 2005 Accepted: December 26, 2005)
Hernández-Lauzardo, A.N., Bautista-Baños, S., Velázquez- definición del micelio, esporangios y esporangiosporas del Valle, M.G., and Trejo-Espino, J.L. 2006. Identification cuando los aislamientos se cultivaron en papa-dextrosa-agar of Rhizopus stolonifer (Ehrenb.: Fr.) Vuill., causal agent of (PDA). La velocidad de crecimiento y esporulación fue mayor Rhizopus rot disease of fruits and vegetables. Revista en este medio de cultivo. Las esporangiosporas fueron Mexicana de Fitopatología 24:65-69. principalmente globosas, seguidas de formas elipsoidales y Abstract. Rhizopus stolonifer commonly causes postharvest angulares. Los valores de longitud, diámetro y área de los diseases on many fruits and vegetables. The objectives of esporangióforos, esporangios y esporangiosporas variaron this investigation were to evaluate the growth rate and entre los aislamientos. La ornamentación de las sporulation of three strains of R. stolonifer on different media, esporangiosporas mostró estrías continuas a lo largo de la to quantify the sporangiophores, sporangia, and superficie de las esporas. El PDA fue el medio más adecuado sporangiospores of three strains by image analysis, para caracterizar a R. stolonifer. Basándonos en la morfología determining the relative frequencies of spore shapes, and by y en la caracterización cuantitativa, los tres aislamientos scanning electron microscopy, to confirm their shape and correspondieron a Rhizopus stolonifer. ornamentation. Results indicated a better definition of mycelia, sporangia, and sporangiospores when isolates were Palabras claves adicionales: Esporangióforos, esporangios, grown on potato-dextrose-agar (PDA). Growth rate and esporangiosporas, análisis de imágenes, microscopía sporulation was higher on this culture medium. electrónica de barrido. Sporangiospores were mainly globose, following the ellipsoidal and angular shapes. Values of length, diameter, Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. is the causal agent of and area of sporangiophores, sporangia, and sporangiospores rhizopus rot disease of various fruits and vegetables. R. varied among isolates. Ornamentation of sporangiospores stolonifer commonly causes postharvest diseases on many showed continuous ridges alongside the spore surface. PDA fruits and vegetables. It typically causes a soft, watery rot, was the most adequate medium to further characterize R. and is a fast growing fungus developing on a broad range of stolonifer. Based on the morphological and quantitative temperatures and relative humidities (Nishijima et al., 1990). characterization, the three isolates corresponded to R. Phytopathogenic fungi are usually identified taking into stolonifer. account morphological criteria. The most important characteristics to identify fungi morphologically are the spores Additional keywords: Sporangiophores, sporangia, (shape, size and colour) and fruiting bodies (Agrios, 2001), sporangiospores, image analysis, scanning electron and to a lesser extent mycelia; thus, it is highly microscopy. recommendable to follow keys to genera for an accurate identification. To identify R. stolonifer, Farr et al. (1989) Resumen. Rhizopus stolonifer comúnmente ocasiona recommend turning to the generic description published by enfermedades postcosecha en muchas frutas y hortalizas. Los Schipper (1984). It is important to identify and quantify the objetivos de esta investigación fueron evaluar la tasa de possible sources of variability of R. stolonifer. For example, crecimiento y esporulación de tres aislamientos de R. nutrient status is known to affect growth of various fungi. To stolonifer en diferentes medios, cuantificar los carry out the morphological characterization, selection of the esporangióforos, esporangios y esporangiosporas de los tres best media will be important since nutrients containing the aislamientos mediante análisis de imágenes, determinando media might or might not facilitate the development of la frecuencia relativa de las formas de las esporas, y mediante structures such as sporangiophores, sporangia, and microscopía electrónica de barrido, confirmar su forma y sporangiospores. For R. stolonifer, it is reported different ornamentación. Los resultados mostraron una mejor growing media to evaluate in vitro development. Schipper 66 / Volumen 24, Número 1, 2006
(1984) mentions malt-agar as the best growth medium, while fruit to obtain pure cultures. To obtain monosporic cultures, in many other studies potato-dextrose-agar is used as the most serial dilutions were prepared from pure cultures and common, but it may not be the best medium to grow this individual spores were collected and grown on PDA. fungus (Mari et al., 2002; Nishijima et al., 1990; Northover Morphological and growing evaluations. A small portion and Zhou, 2002). Currently, image analysis is a fast (0.5 cm diameter) of each pure culture was placed in the center quantitative method commonly used to analyze image features of Petri plates containing PDA, malt-agar (MA) or Czapeck giving a more precise and accurate data. For example, in plant media (CZ). For morphological characterization, mycelia diseases, image analysis is also a reliable method to identify description was carried out taking into account particular fungus corresponding to the genus Penicillium, it determines features of mycelium and rhizoids (Schipper, 1984). To fungal biomass, variations on conidial morphology after determine the growth rate of each isolate, mycelial growth chitosan, and characterizes the morphological differences was measured every 6 h during an incubation period of 96 h broth cultures (Dorge et al., 2000; Hernández-López, 2002; or when mycelium reached the edge of the Petri plate. Lucatero et al., 2004; Morgan et al., 1991). Scanning electron Sporulation was evaluated after 48 and 72 h. Spore microscopy is another reliable method to integrate fungal suspensions were prepared by flooding colonies with distilled identification, allowing the observation of distinct shapes and water, agitating with a sterile glass rod and filtering through ornamental definition of spore surface (Schipper, 1984). The glass wool. Spore counting was done using a Neaubauer objectives of this investigation were to evaluate the growth haemocytometer and a microscope (Nikon, Alphaphot-2 YS2) rate and sporulation of three strains of R. stolonifer on at 40x magnification. The experiment was repeated three times different media; to quantify sporangiophores, using five Petri plates as an experimental unit. Treatments sporangiospores, and sporangia of the three strains by image were arranged in a completely randomized design. analysis, determining the relative frequencies of the various Microscopic studies. Sporangiophores, sporangia, and shapes of R. stolonifer spores, and to confirm shape and sporangiospores of each isolate were measured after ornamentation by scanning electron microscopy. incubation on PDA for 48 h. Images of sporangiophores and Microorganisms and culture conditions. Three isolates of sporangia were obtained using a stereoscopic (Nikon, R. stolonifer were obtained from naturally infected tomatoes SMZ1500) and images of sporangiospores using a microscope (Lycopersicon esculentum Mill.) harvested from three (Nikon, Alphaphot-2 YS2) with a charged video camera (DL different regions of the state of Morelos, Mexico: Cuautla 330 DAGE-MTI). Magnifications of the images were 4X, (R1), Oaxtepec (R2), and Yautepec (R3). Infected fruit was 8X, and 10X for sporangiophores, sporangia, and placed in moist chambers at 25°C until symptoms appeared sporangiospores, respectively. Images were analyzed using (ca. four days). Portions of the infected tissue were placed Meta Imaging series software, (version 4.0 for Microsoft on Petri plates containing PDA and re-inoculated on tomato Windows, Universal Imaging Corporation). The length (ìm)
A B C
D E F
Fig. 1. Growth of Rhizopus stolonifer on different media, after 24 h: A) potato-dextrose-agar (PDA), B) malt-agar (MA), C) Czapeck (CZ), and after 48h: D) PDA, E) MA, F) CZ. Revista Mexicana de FITOPATOLOGIA/ 67
Table 1. Growth rate of three isolates of Rhizopus stolonifer series for five min each, CO2 dried (SAMDRI-780B grown on potato-dextrose-agar (PDA), malt-agar (MA), and Tousimis), and sputter-coated with gold palladium in a Czapeck (CZ) media during an incubation period of 96 h at Nanotech sputter coated (BAL-TEC SDC 050). Samples were 25°C. kept in a desiccator until examination with a scanning electron Rate of growth (mm/h)z using a microscope Karl Zeiss DSM 940 operated at 30kV. Isolates PDA MA CZ Micrographs were taken on Polaroid type 52 film (Polaroid R1 2.29 a 1.84 b 1.93 c Corp.). R2 2.32 a 1.96 b 1.94 b Data and statistical analysis. Treatments were arranged in R3 2.17 a 1.87 b 1.92 b a completely randomized design. Data were analyzed through z Different letters among rows indicate significant differences ANOVA (Sigma Stat version 2.0). Median separation by (Tukey, p > 0.05). Kruskal-Wallis was carried out for sporulation, and mean separation by Tukeys’s multiple range test (P < 0.05) for of sporangiophores, diameter (ìm) and elliptical form factor growth rate. Means and standard deviations were carried out (EFF) of sporangia, and area (ìm2), length and EFF of from data of sporangiophores, sporangia, and sporangiospores were measured on 90 or 300 observations sporangiospores. Relative frecuencies among the observed per isolate. Ranges of EFF were given as follow: globose shapes of sporangiospores of each of the three isolates of (0.5-1.15), ellipsoidal (1.16-1.30), and angular (> 1.31). Rhizopus were also calculated. Scanning electron microscopy. Sporangiospores of a 72 h The three isolates showed a well defined ramified mycelium incubation period grown on PDA were fixed with 6% development on PDA after 24 h (Fig. 1A). Although less glutaraldehyde for 24 h at room temperature, rinsed three ramified, almost similar growth was observed when grown times with phosphate buffers 0.02 M, fixed with 2% osmium on MA (Fig. 1B). On CZ, mycelium growth was scarce (Fig. tetraoxide for 2 h at 20°C, dehydrated in a graded ethanol 1C). After 48 h, abundant cottony and aerial mycelium was accentuated in the three cultures grown on PDA, showing the distinctive characteristics of the genus Rhizopus such as 50 the formation of sporangiophores and sporangia (Fig. 1D), A R1 R2 R3 bbb 45 and characteristics of the stolonifer specie such as the 40 formation of complex rhizoids, well defined (image not 35 shown). When incubated on MA, R. stolonifer growth was 30 less defined with occasional formation of sporangiophores 25 and sporangia (Fig. 1E). After 72 h on this medium, there 20 Spores/mL was abundant production of fruiting bodies (data not shown). 15 The strains grown on CZ did not show fruiting bodies at any 10 incubation time (Fig. 1F). The highest growth rate of R. 5 a a a aaa stolonifer during the four days incubation period was shown x 1000 0 on PDA on the three isolates tested (R1 = 2.2, R2 = 2.3, and CZ EMA PDA R3 = 2.1 mm h-1, Table 1). Sporulation was markedly higher Culture media using PDA as growing medium for the three isolates tested 700 B R1 R2 R3 c 600 c 50 c R1 R2 R3 500 45 400 40 35 300 30 Spores/mL 200 25 20 100 bbb aaa 15 x 1000 0 10 Relative frequency (%) CZ EMA PDA 5 Culture media 0 Fig. 2. Sporulation of three isolates of Rhizopus stolonifer Angular Ellipsoidal Globose (R1, R2, R3) incubated on Czapeck (CZ), potato-dextrose- Shape agar (PDA), and malt-agar (MA) for 48 h (A) and 72 h (B). Fig. 3. Shape ocurrence of sporangiospores from three Different letters indicate significant differences among media isolates of Rhizopus stolonifer (R1, R2, R3) grown on potato- (Kruskal-Wallis, p = 0.05). dextrose-agar for 48 h (n = 100). 68 / Volumen 24, Número 1, 2006
Table 2. Quantitative observations by image analysis of sporangiophores, sporangia, and sporangiospores of three isolates of Rhizopus stolonifer grown on potato-dextrose-agar for 48h. Sporangiophoresy Sporangiay Sporangiosporesz Isolates Length Diameter EFF Area Length (ìm) (ìm) (dimensionless) (ìm2) (ìm) R1 2173.3 ± 277 364.0 ± 85 1.0 ± 0.06 110.9 ± 29 13.9 ± 1.9 R2 2230.1 ± 362 327.4 ± 77 1.1 ± 0.07 117.4 ± 28 14.4 ± 2.0 R3 2317.4 ± 418 332.1 ± 74 1.0 ± 0.06 98.9 ± 26 13.2 ± 1.8 Means and standard deviations of 90y and 300z observations.
(R1 = 6.7 x 105, R2 = 5 x 105, and R3 = 5.5 x 105 spores/mL) three isolates. Surface ornamentations of R. stolonifer were after 72 h incubation period (Fig. 2). No sporulation was continuous and distinct ridges alongside the spore (Fig. 4). observed on CZ for the three isolates after 48 or 72. The Results of morphological and quantitative characterization relative occurrence of sporangiospore shape (EFF) was mainly of this study, corroborate that R. stolonifer was the organism globose (R1 = 47, R2 = 47, and R3 = 45%) followed by the evaluated. Schipper (1984) mentions as distinctive feature ellipsoidal (R1 = 38, R2 = 35, and R3 = 39%), and angular of this fungus the aerial and cottony mycelium. On PDA, the shapes (R1 = 15, R2 = 18, and R3 = 16%) (Fig. 3). The three isolates had higher growth rate, an adequate sporulation, quantitative observations carried out by image analysis and the formation of particular structures of R. stolonifer after showed differences among the three isolates (Table 2). The 48 h incubation, representing then the most adequate medium length of sporangiophores ranged between 2173.3 to 2317.4 to evaluate other descriptive characteristics of this specie such ìm. Sporangia diameter varied from 327.4 to 364.0 ìm. EFF as the formation of complex and well developed rhizoids (ca. 1.0) indicates that sporangia were closer to the circular (Romero-Cova, 1988). However, our results on MA medium shape. Area of sporangiospores varied between 98.9 to 117.4 differ from previous report by Schipper (1984) who indicated ìm2, while their length ranged between 13.2 to 14.4 ìm. The that MA was the best growing medium to characterize representative images of the scanning electron microscopy morphological features of R. stolonifer. In this study, PDA confirmed those diverse forms (globose, ellipsoidal, and overcame the other two culture media tested with the angular) already obtained by the quantitative analysis of the possibility of carrying out measurements of relevant structures
A B
C D
Fig. 4. Scanning electron microscopy of sporangiospores of Rhizopus stolonifer grown on potato- dextrose-agar. 30 KV 2000X (A and B), 5000X (C), and 10000X (D). Revista Mexicana de FITOPATOLOGIA/ 69 in less than 48 h. Values of sporangiophores and sporangia digitatum y Fusarium oxysporum de la Papaya (Carica reported in this characterization occurred within the ranges papaya). MSc. Tesis. Centro de Desarrollo de Productos already reported by Schipper (1984). To the best of our Bióticos, Instituto Politécnico Nacional. Yautepec, knowledge, this is the first report about measurements of R. Morelos, México. 101 p. stolonifer sporangiospores such as area, length, and EFF Lucatero, S., Galindo, E., and Sarralde-Corona, P. 2004. together with the relative proportion (%) of each distinctive Quantitative characterization of the morphology of shape (globose, ellipsoidal, and angular). We think that in Trichoderma harzianum cultured in shake flasks and future studies, mainly those related to control of R. stolonifer, containing Tween 40. Biotechnology Letters 26:41-44. it might be important to include these evaluations to quantify Mari, M., Gregori, R., and Donati, I. 2002. Postharvest control possible changes on the relative proportions of of Monilinia laxa and Rhizopus stolonifer in stone fruit sporangiospores by effect of any kind of treatment. With the by peracetic acid. Postharvest Biology and Technology aid of the scanning electron microscopy, we confirmed spore 33:319-325. shapes and surface ornamentation of R. stolonifer mentioned Morgan, P., Cooper, C.J., Battersby, N.S., Lee, S.A., Lewis, by Schipper (1984). S.T., Machin, T.M., Graham, S.C., and Watkinson, R.J. 1991. Automated image analysis method to determine Acknowlegments. This work was supported by the National fungal biomass in soils and solid matrices. Soil Biology Polytechnic Institute, Mexico, through the grant CGPI: and Biochemistry 23:609-616. 20040452. Nishijima, W.T., Ebersole, S., and Fernandez, J.A. 1990. Factors influencing development of postharvest incidence LITERATURE CITED of Rhizopus soft rot of papaya. Acta Horticulturae 269:495- Agrios, G.N. 2001. Fitopatología. Second Edition. Limusa. 502. Mexico, D.F. 809 p. Northover, J., and Zhou, T. 2002. Control of rhizopus rot of Dorge, T., Cartensen, J.M., and Frisvad, C. 2000. Direct peaches with postharvest treatments of tebuconazole, identification of pure Penicillium species using image fludioxonil, and Pseudomonas syringae. Canadian Journal analysis. Journal of Microbiology Methods 41:121-133. of Plant Pathology 24:144-153. Farr, D.F., Bills, G.F., Chamuris, G.P., and Rossman, A.Y. Romero-Cova, S. 1988. Hongos Fitopatógenos. Universidad 1989. Fungi on Plants and Plant Products in the United Autónoma Chapingo. Chapingo, Estado de México, States. First Edition. APS Press. St. Paul, Minnesota, USA. México. 347 p. 1252 p. Schipper, M.A. 1984. A Revision of the Genus Rhizopus. Hernández-López, M. 2002. Uso Potencial del Quitosano y Studies in Mycology. Serie No. 25. Centraalbureau voor Extractos Vegetales en el Control de Colletotrichum Schimmelcultures. Baarn, The Netherlands. 34 p. gloeosporioides, Rhizopus stolonifer, Penicillium