Biology and Histopathology of Ustilago Filiformis (= U. Longissima), a Causal Agent of Leaf Stripe Smut of Glyceria Multiflora
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JOURNAL OF PLANT PROTECTION RESEARCH Vol. 55, No. 4 (2015) DOI: 10.1515/jppr-2015-0059 Biology and histopathology of Ustilago filiformis (=U. longissima), a causal agent of leaf stripe smut of Glyceria multiflora Analía Perelló1*, Marta Mónica Astiz Gasso2, Marcelo Lovisolo3 1 Technologist Institute Santa Catalina (IFSC), 1836 Llavallol, Faculty of Agricultural and Forestry Sciences, National University of La Plata, Street 60 and 119, Buenos Aires, Argentina 2 Faculty of Agricultural and Forestry Sciences, National University of La Plata, Street 60 and 119, Buenos Aires, Argentina 3 Chair of Botany, Faculty of Agricultural Sciences, National University of Lomas de Zamora, 1832 Lomas de Zamora, Buenos Aires, Argentina Received: March 16, 2015 Accepted: November 12, 2015 Abstract: The aims of this study were to clarify the reproductive biology of the Ustilago filiformis Schrank, as causal agent of the stripe smut of Glyceria multiflora, determine the infection process of the pathogen and analyze the histological changes associated host- Glyceria any fungus attack. Moreover, the life cycle of the fungus was elucidated for the first time. Both teliospores and basidiospores were found to be equally efficient in producing the infection in Glyceria plants after the plants had been inoculated. These results constitute an important contribution for the understanding of the epidemiology of the disease. Key words: basidiospores, infection cycle, stripe smut, teliospores Introduction dia, the USSR (Siberia); Europe – Austria, Bulgaria, Czech, Smut fungi are Basidiomycota fungi belonging to the Denmark, Finland, France, Germany, Hungary, Italy, Po- order Ustilaginales. They received this name due to the land, Romania, Spain, Sweden, Switzerland, the United very conspicuous symptoms of black teliospore masses Kingdom, the former Yugoslavia; and North and South resembling smut, which they often produce on the host America. plants. There have been over 1,100 smut species recog- In Argentina, the disease was detected in the cities nized in over 93 genera. Smuts infect more than 4,000 of Jeppener and La Plata which are in the Buenos Aires plant species belonging to approximately 75 different Province. The pathogen is transmitted through telio- plant families. The biggest group of this fungi infects spores spreading by wind and water (Hirschhorn 1986; monocots belonging to the Graminea, which include ce- Mordue 1991; Astiz Gassó et al. 2006). real crops. The smuts encompass, among others, the gen- Glyceria multiflora Steudel is a native species. It is era Ustilago and Tilletia. The latter cause, the so-called, aquatic, glabrous, perennial, rhizomatous with long til- bunt diseases of various cereal crops. Between the spe- lers, and it has wide ligule, narrow panicle lax, 20–60 cm cies genus Ustilago, the name of Ustilago longissima (based long, and reddish–dark fruit cariopsis (Burkart 1969). In on Uredo longissima Sowerby 1799) should be replaced by Argentina, it inhabits marginal areas of low grasslands Ustilago filiformis (based on Lycoperdon filiformis Schrank, of Mesopotamia and “Cuenca del Salado”, which are in in Hoppe 1793), accepted by the 13th International Bo- the Buenos Aires Province. In these areas, it is of high tanical Congress in Sydney, Australia, 1981 (Vánky 1985; value as cattle forage (Vidal and Piergentili 1973). The MycoBanK 2015). high productivity and good palatability of this species Ustilago filiformis is the causal agent of the stripe smut make it an interesting alternative in marginal farming in many species of the genus Glyceria, such as G. fluitans, areas of the country (Rossi et al. 2006, 2009; Alonso et al. G. grandis, G. multiflora, G. nemoralis, G. obtusa, G. plicata, 2009). and G. striata (Mordue 1991). Stripe smut produces chlo- Other smut species are recognised in terms of their rosis and necrosis on the leaves. The infected plants are life cycle and histopathology e.g. Sporisorium reilianum slightly stunted, and pale or yellowish-green. They pro- f. sp. zeae on Zea mays and Sorghum bicolor (L.) Moench duce new tillers partially atrophied of a pale greenish yel- (Wilson and Frederiksen 1970; Matyac 1985; Martinez low color, and have a weak aspect. The plants usually fail et al. 2002). to reach the flowering stage due to fungus damage. The The first aim of this work was to explain the repro- geographical distribution of the disease is generalised to ductive biology of the fungus U. filiformis. The second temperate areas of the world such as: Asia – Northern In- aim was to determine the pathogen’s process of infection. *Corresponding address: [email protected] 430 Journal of Plant Protection Research 55 (4), 2015 The third aim was to analyse the histological changes of Laboratory inoculation assay the host-Glyceria associated with the fungus attack. Small bits of Glyceria leaves (2 cm) were inoculated with a suspension of (1) basidiospores (106–8 spores ∙ ml–1) and Materials and Methods (2) teliospores (106–8 spores ∙ ml–1) in sterile distilled wa- The methodology used in this research was adapted and ter obtained from the culture media (PB). The control modified based on previous techniques used for the study plants received the PB media alone without the fungus. of smut fungi as mentioned by Fernández et al. (1978), Inoculated leaf samples were placed in Petri dishes with Matyac (1985), Craig and Frederiksen (1992), Maurya et sterile filter paper moistened with sterile distilled wa- al. (2010) and Kosiada (2011). ter to maintain a relative humidity of 80–90%. Then, the dishes were kept in an oven (BINDER KBF LQC) at Isolation of Ustilago filiformis and in vitro assays 25±2°C photoperiod 12 : 12 (L : D) h. After 24 h, samples were mounted in cotton blue lactophenol for observa- Glyceria samples of healthy plants and plants infected tion and histopathological examination under light mi- with U. filiformis, were collected in 2009 at Jeppener croscope. (35°16´39.5”S; 58°11´53.7” O/–35.277639, –58.19825), Bue- nos Aires Province, Argentina. Subsequently, symptom- Field inoculation assay atic plants were taken to the laboratory for morpho- logical and biometrical observations and to identify the The inoculum was prepared in Erlermeyer flasks with 200 smut. In addition, isolates of the fungus on agar media cm3 of PC media, and sprayed at a concentration of 106–8 were performed to study the type of teliospore germina- basidiospores ∙ ml−1 on the Glyceria plants at the seedling tion and the colony development of the fungus. Fungal stage (shoot apical meristem with axillary buds). The in- spores (0.5 g) were treated for 3 min in a 2% (w/v) hy- oculation was repeated every time plants produced new pochlorite sodium solution (55 g Cl/l), and afterwards buds during the vegetative stage (approximately every washed in sterile distilled water. After that, teliospores 20 days). Untreated plants (the controls) received PB me- were seeded on two agar media: (1) 2% Water Agar (AA) dia alone. Inoculation was performed during spring 2009 for observing the germination type, and (2) 2% Potato (November-December). Two containers of six plants each Dextrose Agar (PDA) for the study of colony characteris- one (replications) of G. multiflora were selected for field in- tics and fungus morphology. Cultures in PDA were kept oculation assays. Another two containers were selected as in the refrigerator at 5±2°C to be used in the artificial the control. For both the inoculated plants and the controls, inoculations carried out on the host, Glyceria (Craig and the observations and sampling started 24 h after the inocu- Frederiksen 1992). lations were done. Macro- and microscopic observations were performed to check if any new tillers were infected. Inoculum preparation Histopathological study For this experiment, basidiospores axenic cultures were obtained using the methodology described above. In Er- Samples of G. multiflora that had been inoculated under lenmeyer flasks with a 200-ml Potato Broth medium 2% field conditions were collected for the histopathological (PB), the fungal cultures were seeded. They were sub- study and compared with the untreated controls. Sam- sequently placed in a shaker 200 rpp, and placed in an ples were fixed in a mix of formalin-acetic acid-alcohol oven (BINDER KBF LQC) at 25±2°C in the dark. Every (FAA) and later treated with 10% hydrofluoric acid for 24 h, a sample was taken to quantify the basidiospores 24 h to soften the tissue and remove the silica from the concentration. The quantification was done with the aid epidermis. Inclusion was performed in paraffin. Trans- of a Neubauer haemocytometer. When the basidiospore verse and longitudinal sections (10−12 µ thick) were concentration reached 106–8 basidiospores ∙ ml−1, the sam- obtained using a microtome type Minot (Senior Rotary ples were placed in the refrigerator (5°C) to be used for Microtome Arcane Brand Model RTM-30). Samples future inoculations (Wilson and Frederiksen 1970; Mar- were double stained with safranin-fast green. The tech- tinez et al. 2002). nique described by Bracegirdle and Miles (1975) and D´Ambrogio Argüeso (1986) was used for the histopatho- Glyceria plants logical determination. Two containers (2 m wide × 10 m long) coated with thick black plastic 200 μ were conditioned to transplant the Results and Discussion Glyceria plants in environmental conditions similar to Identification of the species and colony morphology those where this species develops. Twelve healthy Glyce- ria plants were obtained from the wetlands of Jeppener. Macro- and microscopic observations of infected Glyceria In spring (September−October), as soon as the emergence plants confirmed that the sori observed in the inoculated of tillers began, the samples were kept for laboratory as- diseased plants agree with the ones described for the species says. Four other containers, twelve plants in total, were U. filiformis. This species produces sori in leaves resulting in selected for being inoculated under field conditions.