P u b l i s h i n g
Australasian Plant Pathology Volume 30, 2001 © Australasian Plant Pathology Society 2001 A journal for the publication of original research in all branches of plant pathology
For editorial enquiries and manuscripts, please contact: Australasian Plant Pathology Editor-in-Chief Dr Eric Cother Orange Agricultural Institute NSW Agriculture, Forest Road Orange, NSW 2800, Australia Telephone: +61 3 6391 3886 Fax: +61 3 6391 3899 Email:
[email protected] For general enquiries and subscriptions, please contact: CSIRO Publishing PO Box 1139 (150 Oxford St) Collingwood, Vic. 3066, Australia Telephone: +61 3 9662 7626 Fax: +61 3 9662 7611 Email:
[email protected] Published by CSIRO Publishing for the Australasian Plant Pathology Society
w w w. p u b l i s h . c s i ro . a u / j o u r n a l s / a p p
Australasian Plant Pathology, 2001, 30, 277–281
A. A. Xavier et al.
Infection of resistant and susceptible Eucalyptus grandis genotypes by urediniospores of Puccinia psidii
AP01038
A. A. XavierA, A. C. AlfenasAC, K. MatsuokaA and C. S. HodgesB A
Departamento de Fitopatologia, Univesrsidade Federal de Viçosa, Minas Gerais, Brazil. Department of Plant Pathology, NC State University, Box 7616, Raleigh, NC 27695, USA. C Corresponding author; email:
[email protected]
B
Abstract. Germination of urediniospores, appressorium formation and penetration by Puccinia psidii Winter were studied on detached leaves of resistant and susceptible clones of Eucalyptus grandis Hill ex Maiden. More than 90% of germination and appressorium formation were observed 12 and 18 h, respectively, after inoculation for both genotypes. Direct penetration by P. psidii between the anticlinal walls of the epidermal cells occurred. In the susceptible genotype, primary mycelia and haustoria were observed 12 and 18 h, respectively, and in the resistant 18 and 24 h after inoculation. After the formation of the first haustoria, dead cells developed and were followed by a hypersensitive reaction in the resistant genotype. Additional keywords: histopathology, pathogenesis, resistance. Introduction Puccinia psidi Winter, the cause of Eucalyptus rust, infects species in more than ten genera and numerous species in the family Myrtaceae. It occurs in South and Central America, several islands in the Caribbean and in south Florida in the United States (Coutinho et al. 1998). This rust has been one of the most important diseases of Eucalyptus in Brazil since the 1970s when eucalypt cultivation began to expand (Ferreira 1989). Rust is also considered a potential threat to Eucalyptus grandis Hill ex Maiden, one of the species most susceptible to the disease, in Australia where it is native, and in South Africa where it is planted over large areas (Coutinho et al. 1998). Eucalyptus rust primarily affects young plants, including nursery seedlings, plants in clonal gardens, newly planted trees up to 2 years old, and coppice shoots which develop after tree harvesting. The disease is characterised initially by the formation of small, light-green, slightly raised pustules that develop on young leaves or shoots. The pustule colour changes later to a deep yellow with the development of urediniospores, or more rarely, to reddish-brown with the development of teliospores (Alfenas et al. 1989; Ferreira 1989). Selection of resistant eucalypt genotypes has been one of the main disease control measures (Ferreira 1989). The host–pathogen interaction involving biotrophic pathogens generally follows the gene-for-gene relationship proposed by Flor (1955). In Eucalyptus spp. resistance to rust is controlled by a major dominant gene (Junghans et al. 1999). However, little is known about the histological aspects of this interaction. The available literature on P. psidii pathogenesis consists only of Hunt’s (1968) report on the germination of urediniospores © Australasian Plant Pathology Society 2001
and type of penetration on leaves of Syzygium jambos (L.) Alston (= Eugenia jambos L.). Studies of pathogenesis enable observation of changes in the host cells in the presence of the pathogen, allowing comparison of behaviour between susceptible and resistant hosts. Such studies also allow an insight into disease development and supply information for cytochemical and immuno-cytochemical studies that are necessary to explain resistance mechanisms (Yokoyama et al. 1991). This study was carried out to determine the pre- and post-penetration phases of the interaction between P. psidii and E. grandis, and the morphological changes in host tissues in resistant and susceptible genotypes of E. grandis. Methods Cuttings from a rust susceptible (UFV-1) and a resistant (UFV-2) clone of E. grandis were used. The plants were kept in the greenhouse and fertilised every 15 days with 20 g mL–1 Ouro Verde (Green Gold) fertiliser (N, P, K and micronutrients). Pruning to induce new shoot growth was carried out whenever necessary. An isolate of P. psidii obtained from a single pustule from E. grandis in Viçosa, MG, was multiplied on young S. jambos leaves using the technique of Ruiz et al. (1989). Spores were collected 12 days after inoculation with a fine brush. A spore suspension at 105 urediniospores mL–1, containing 0.05% of Tween 20 was used. Pre-penetration and colonisation phases of P. psidii were studied on detached new leaves (the first half-opened leaf from the apical branch of the plant) from the susceptible and resistant E. grandis genotypes. The petiole of each leaf was wrapped in moistened cotton wool and maintained in a tray (36 × 22 × 6 cm) under saturated moisture conditions. The leaves were inoculated by placing six 2-µL drops of spore suspension containing 105 urediniospores mL–1 on the abaxial leaf surface. The inoculated leaves were incubated at 20 ± 1°C in a germination chamber (FANEM 347 CDG model) under continuous darkness during the first 24 h, followed by a 12 h photoperiod at
10.1071/AP01038
0815-3191/01/030277
278
approximately 27 µmol m–2 s–1. Samples were collected at 6 h intervals up to 60 h after inoculation, bleached in chloral hydrate (Longo et al. 1994) and observed under a Leica DM RBE microscope, using phase contrast. A randomised complete block design with three replications was used for each sample period. One replication consisted of a single leaf containing six micro-drops of inoculum. Fifty well-spaced urediniospores were assessed for each replication for germination frequency, appressorium formation, direct and stomatal penetration, and formation and development of primary and secondary hyphae and haustoria. The colonisation phase was studied in young detached leaves (first half-opened leaf on the apical branch of the plant) inoculated as previously described. The leaves were examined 3, 6, 9 and 12 days after inoculation. Leaf sections were fixed in 3% glutaraldehyde solution containing sodium cacodilate buffer (0.05 M HCl, pH 6.9) for 24 h, and then dehydrated in butyric alcohol series (Sass 1958). The infiltration solution was a mixture of 50 mL glycol metacrylate (basic Technovit 7100 resin) and 0.5 g dybenzoil peroxide, constantly agitated. A catalysing agent (Hardener II) was added to the infiltration solution for blocking at 1:15 (v/v) (Martins et al. 1995). The polymerisation temperature was 37°C for 24 h. The samples were stored in a dryer at room temperature after blocking. Serial sections 3-µm thick were made with a Reichert Jung 2045 rotary microtome. The sections were individually distended in drops of distilled water placed on slides, dried, fixed and subjected to the Johansen (1940) hydration and triple staining procedure. The sections were then mounted in Canada Balsam. The inter- or intra-cellular patterns of hyphal development in the leaf mesophyll and the formation of reproductive structures were observed with a light microscope.
Results There were no differences between susceptible and resistant genotypes for urediniospore germination, appressorium formation and host penetration. Ninety percent of the urediniospores germinated 6 h after inoculation on leaves of both genotypes. After 18 h, more than 90% of the germinated urediniospores had formed appressoria on both genotypes and, on average, these measured 13 × 16 µm (Fig. 1A). A thin hypha (infection peg) originated from the appressorium and penetrated, in most cases, between the anticlinal walls of the epidermal cells and then entered the mesophyll. Twelve hours after inoculation, more than 75% of the urediniospores had penetrated the tissue, both in the susceptible and resistant genotypes. Penetration through stomata was low, 7 and 2% in the resistant and susceptible genotypes, respectively. No appressorium was formed in the majority of the cases where stomatal penetration occurred. After penetration, the infection hypha increased in size, forming a sub-epidermal vesicle from which primary and secondary hyphae developed. Primary hyphae were seen at 12 and 18 h after inoculation in the susceptible and resistant genotypes, respectively. The haustorium mother cell developed from a primary hypha in contact with the mesophyll cell wall, and was separated from the infection hypha by a septum. A hypha developing from the haustorium mother cell penetrated the mesophyll cell wall, but not the plasmalemma, and gave rise to a haustorium. The haustorium was at first globose, but later became lobed, was highly branched and occupied a large part of the cell lumen
A. A. Xavier et al.
(Fig. 2A). Haustorium formation was observed 18 and 24 h after inoculation in susceptible and resistant genotypes, respectively. Histopathological differences between genotypes were not observed in the pre-penetration phase. However, staining agents accumulated in the cells containing haustoria and in adjacent cells of the resistant genotype, indicating cell death (Niks 1983b) (Fig. 2B). The hypersensitive reaction could be observed macroscopically on leaves 48 h after inoculation. In the susceptible genotype, sections made 3 days after inoculation showed little intercellular development of hyphae, but after this time the hyphae ramified and rapidly colonised the leaf tissues. Hyphae were observed primarily in the spongy mesophyll but extended as far as the palisade layer. On the sixth day after inoculation, the colonised tissues began to exhibit hypertrophy (Fig. 1B). Uredinial primordia of the fungus could be seen as an accumulation of hyphae beneath and perpendicular to the epidermis. Further development of these structures caused them to break through the epidermis about 9 days after inoculation (Fig. 1C). Basal cells, developed from the hymenial layer, gave rise to support cells. After, elongation, each support cell bore a single urediniospore delimited by a septum. These support cells were of various sizes and shapes, but were generally short rectangular to bulbous or rectangular. Paraphyses were not observed. On inoculated leaves, uredinia were observed only on the abaxial surface (Fig. 1D), although under natural conditions they can be amphigenous. Discussion This is the first histopathological investigation of the interaction between P. psidii and E. grandis and describes the infection process of the pathogen in a resistant and a susceptible genotype at various times after inoculation. A high percentage of urediniospore germination was observed in both resistant and susceptible genotypes 6 h after inoculation and by 18 h, more than 90% of the germinated spores had produced appressoria on both genotypes. Similar results were obtained when studying the interactions on resistant and susceptible genotypes of wheat and Puccinia graminis f. sp. tritici Eriks & H.Henn. (Liu and Hardner 1996) and Puccinia recondita Roberg ex Desmaz f. sp. tritici (Ortelli et al. 1996). Infection pegs produced from the appressoria penetrated between the anticlinal walls of the leaf epidermis directly into the mesophyll of the leaf, similar to that reported previously for P. psidii on S. jambos (Hunt 1968). The occasional penetration through stomata without the formation of appressoria was probably random, although this mode of penetration has been reported for P. striiformis Westend. in wheat (Swertz 1994). Non-stomatal penetration for dikaryotic phase rust spores is unusual, and has been observed previously only for P. pachyrhizi Syd. (Bonde et al. 1976; Marchetti et al. 1975) in soybean, P. zeae in maize
Infection of Eucalyptus grandis by Puccinia psidii
279
Fig. 1. Pathogenesis of Puccinia psidii in Eucalyptus grandis. (A) Penetration between epidermal cells, Ap = appressorium and U = urediniospore. (B) Colonisation of the mesophyll 6 days after inoculation. (C) Support cell containing urediniospore; s = support cell and u = urediniospore. (D) uredinia containing urediniospores, (D) 9 days after inoculation. Bars = 10 µm.
280
Fig. 2. Development of Puccinia psidii in (A) susceptible and (B) resistant Eucalyptus grandis genotypes. Vesicle (V) and haustorium (H) in a susceptible genotype. Stain accumulation in cytoplasm (Aniline Blue + Trypan Blue) due to cell death in the resistant genotype after haustorium formation (H). Bars = 10 µm.
(Bonde et al. 1982) and Ravenelia humphreyana P. Henn. in Caesalpinia pulcherrima (L.) Sw. (Hunt 1968). However, the pathway of penetration varied among these rusts. For P. pachyrhizi, penetration occurred directly through the outer wall of the epidermal wall and into the cell lumen, or less commonly, the infection peg penetrated between two adjacent epidermal cells and entered one of the epidermal cells through the anticlinal wall . For P. zeae, penetration was also between two adjacent epidermal cells and then through the anticlinal walls of one of the cells (Bonde et al. 1982). Penetration by R. humphreyana occurred directly, with the infection peg penetrating directly into the epidermal cell and enlarging as a vesicular haustorium (Hunt 1968).
A. A. Xavier et al.
No differences between genotypes were detected for penetration or vesicle formation, but the formation of primary infecting hyphae and haustoria was delayed by 6 h in the resistant genotype as compared to the susceptible genotype. Broes and Lopes-Atilano (1996) also observed a delay in the formation of primary hyphae in resistant wheat cultivars inoculated with P. striiformis f. sp. tritici. Soon after the formation of the haustoria in the leaf mesophyll cells of the resistant genotype, a rapid cell necrosis was observed in adjacent cells. This type of reaction, with cell death after the formation of the haustorium, is similar to that found by various authors in other pathosystems (Littlefield and Heath 1979; Niks 1983a; Taylor and Mims 1991; Liu and Harder 1996; Ortelli et al. 1996). Hypersensitivity is thought to be one of the most important defence responses of the plant to pathogens. In cases of resistance involving biotrophic interactions, which are generally controlled by gene-for-gene interaction, it is believed that elicitors are direct products from avirulent genes; these elicitors interact with the corresponding resistant gene product culminating in cell death (Heath 1997). According to Yamamoto (1995), the resistance involved in this type of reaction may be controlled by a few dominant genes. Another mechanism may be involved in this resistance response but would not be evident because the adopted technique would not allow it. For example, the speed at which papillae are laid down has been related to resistance in the interaction between Hemileia vastatrix Berk. & Br. and the coffee leaf, as demonstrated by Matsuoka and Vanetti (1993) in resistant genotypes of coffee, where such a mechanism accounts for the restriction of pathogen development. The leaf tissue of the susceptible genotype was little colonised 3 days after inoculation. The first visual symptoms of disease were generally observed from 3 to 5 days after inoculation, and were characterised by small protrusions on the epidermis resulting from hypertrophy of the colonised tissues. Although in vivo sporulation was observed 6 days after inoculation , sporogenous tissue was detected only in sections made 9 days after inoculation and by the twelfth day urediniospores were well developed. References Alfenas AC, Demuner NL, Barbosa MM (1989) O eucalipto: a ferrugem e as opções de controle. Correio Agrícola 1, 18–20. Bonde MR, Bromfield KR, Melching JS (1982) Morphological development of Physopella zeae on corn. Phytopathology 72, 1489–1491. Bonde MR, Melching JS, Bromfield KR (1976) Histology of the suscept–pathogen relationship between Glycine max and Phakopsora pachyrhizi, cause of soybean rust. Phytopathology 66, 1290–1294. Broes LHM, Lópes-Atilano RM (1996) Effect of quantitative resistance in wheat on development of Puccinia striiformis during early stages of infection. Plant Disease 80, 1265–1268. Coutinho TA, Wingfield MJ, Alfenas AC, Crous PW (1998) Eucalyptus rust: A disease with the potential for serious international implications. Plant Disease 82, 819–825.
Infection of Eucalyptus grandis by Puccinia psidii
Ferreira FA (1989) ‘Patologia florestal: principais doenças florestais no Brasil.’ (Viçosa, MG Sociedade de Investigações Florestais) Flor HH (1955) Host-parasite interactions in flax rust — its genetics and other implications. Phytopathology 45, 680–685. Heath M (1997) Signalling between pathogenic rust fungi and resistant or susceptible host plants. Annals of Botany 80, 713–720. Hunt P (1968) Cuticular penetration by germinating uredospores. Transactions of the British Mycological Society 51, 103–112. Johansen DA (1940) ‘Plant microtechnique.’ (MacGraw-Hill Book: New York) Junghans DT, Alfenas AC, Bromonschenkel SH, Valle LAC, Shitiro O, Mello EJ (1999) Inheritance on genetic mapping of rust (Puccinia psidii) resistance on Eucalyptus grandis. In ‘XXXII Congresso Brasileiro de Fitopatologia’. Fitopatologia Brasileira 24, 294. (Suplemento). Littlefield LJ, Heath MC (1979) ‘Ultrastructure of rust fungi.’ (Academic Press: New York) Liu JQ, Harder DE (1996) Components of resistance in barley to stem rust: pre- and post-penetration development in seedling and adult plant leaves. Canadian Journal of Botany 74, 1305–1312. Longo N, Naldini B, Drovandi F, Gonnelli T, Tani G (1994) Penetration and early colonization in basidiospore-derived infection of Melampsora pulcherrima (Bulb) Maire on Mercuriales annua L. Cariologia 47, 207–222. Marchetti MA, Uecker FA, Bromfield KR (1975) Uredial development of Phakopsora pachyrhizi in soybeans. Phytopathology 65, 822–823. Martins EOMI, Carvalho AA, Figueiredo MB (1995) Relação entre Capsicum frutescens e os micélios mono e dicarióticos de Puccinia pompeana. Fitopatologia Brasileira 20, 613–617. Matsuoka K, Vanetti CA (1993) Mudanças histopatológicas causadas por Hemileia vastatrix em cafeeiros apresentando diferentes níveis de resistência. Fitopatologia brasileira 18, 484–493.
281
Niks RE (1983a) Comparative histology of partial resistance and the nonhost reaction to leaf rust pathogens in barley and wheat seedlings. Phytopathology 73, 60–64. Niks RE (1983b) Haustorium formation by Puccinia hordei in leaves of hypersentive, partially resistant and nonhost plant genotypes. Phytopathology 73, 64–66. Ortelli S, Giezendanner U, Nosberger J (1996) Effect of the Lr9 resistance gene on pathogenesis of the wheat leaf rust fungus. Plant Disease 80, 14–18. Ruiz RAR, Alfenas AC, Ferreira FA, Vale FXR (1989) Influência da temperatura, do tempo de molhamento foliar, fotoperíodo e intensidade de luz sobre a infeção de Puccinia psidii em eucalipto. Fitopatologia Brasileira 14, 55–61. Sass JE (1958) ‘Botanical microtechnique.’ (The Iowa State University Press: Ames, Iowa) Swertz CA (1994) Morphology of germlings of urediniospores and its value for the identification and classification of grass rust fungi. MS Thesis, University of Wageningen, Netherlands, BION-NWO. Taylor J, Mims W (1991) Infection of and colony development within leaves of susceptible and resistant pearl millet and two nonhosts by the rust fungus Puccinia substriata var. indica. Mycologia 83, 565–577. Yamamoto H (1995) Pathogenesis and host-parasite specificity in rust. In ‘Pathogenesis and host specificity in plant disease’. pp. 203–215. (Eds K Kohmoto, US Singh and RP Singh) (Elsevier Science: UK) Yokoyama K, Aist JR, Bayles CJ (1991) A papilla-regulating extract that induces resistance to barley powdery mildew. Physiological Molecular Plant Pathology 39, 71–78.
Received 12 September 2000, accepted 14 May 2001
http://www.publish.csiro.au/journals/app
