Infection of resistant and susceptible Eucalyptus grandis genotypes by urediniospores of Puccinia psidii

P u b l i s h i n g

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Volume 30, 2001© Australasian Plant Pathology Society 2001

AustralasianPlant Pathology

A journal for the publication oforiginal research in all branches of plant pathology

© Australasian Plant Pathology Society 2001 10.1071/AP01038 0815-3191/01/030277

Australasian Plant Pathology, 2001, 30, 277–281

Infection of resistant and susceptible Eucalyptus grandis genotypes by urediniospores of Puccinia psidii

A. A. XavierA, A. C. AlfenasAC, K. MatsuokaA and C. S. HodgesB

ADepartamento de Fitopatologia, Univesrsidade Federal de Viçosa, Minas Gerais, Brazil.BDepartment of Plant Pathology, NC State University, Box 7616, Raleigh, NC 27695, USA.

CCorresponding author; email: [email protected]

Abstract. Germination of urediniospores, appressorium formation and penetration by Puccinia psidii Winter werestudied 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 bothgenotypes. Direct penetration by P. psidii between the anticlinal walls of the epidermal cells occurred. In thesusceptible genotype, primary mycelia and haustoria were observed 12 and 18 h, respectively, and in the resistant18 and 24 h after inoculation. After the formation of the first haustoria, dead cells developed and were followed bya hypersensitive reaction in the resistant genotype.

Additional keywords: histopathology, pathogenesis, resistance.

IntroductionPuccinia psidi Winter, the cause of Eucalyptus rust,

infects species in more than ten genera and numerous speciesin the family Myrtaceae. It occurs in South and CentralAmerica, several islands in the Caribbean and in southFlorida in the United States (Coutinho et al. 1998). This rusthas been one of the most important diseases of Eucalyptus inBrazil since the 1970s when eucalypt cultivation began toexpand (Ferreira 1989). Rust is also considered a potentialthreat to Eucalyptus grandis Hill ex Maiden, one of thespecies most susceptible to the disease, in Australia where itis native, and in South Africa where it is planted over largeareas (Coutinho et al. 1998).

Eucalyptus rust primarily affects young plants, includingnursery seedlings, plants in clonal gardens, newly plantedtrees up to 2 years old, and coppice shoots which develop aftertree harvesting. The disease is characterised initially by theformation of small, light-green, slightly raised pustules thatdevelop on young leaves or shoots. The pustule colour changeslater to a deep yellow with the development of urediniospores,or more rarely, to reddish-brown with the development ofteliospores (Alfenas et al. 1989; Ferreira 1989).

Selection of resistant eucalypt genotypes has been one ofthe main disease control measures (Ferreira 1989). Thehost–pathogen interaction involving biotrophic pathogensgenerally follows the gene-for-gene relationship proposed byFlor (1955). In Eucalyptus spp. resistance to rust is controlledby 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 onlyof Hunt’s (1968) report on the germination of urediniospores

and type of penetration on leaves of Syzygium jambos (L.)Alston (= Eugenia jambos L.). Studies of pathogenesis enableobservation of changes in the host cells in the presence of thepathogen, allowing comparison of behaviour betweensusceptible and resistant hosts. Such studies also allow aninsight into disease development and supply information forcytochemical and immuno-cytochemical studies that arenecessary to explain resistance mechanisms (Yokoyama et al.1991). This study was carried out to determine the pre- andpost-penetration phases of the interaction between P. psidii andE. grandis, and the morphological changes in host tissues inresistant and susceptible genotypes of E. grandis.

MethodsCuttings from a rust susceptible (UFV-1) and a resistant (UFV-2)

clone of E. grandis were used. The plants were kept in the greenhouseand fertilised every 15 days with 20 g mL–1 Ouro Verde (Green Gold)fertiliser (N, P, K and micronutrients). Pruning to induce new shootgrowth was carried out whenever necessary.

An isolate of P. psidii obtained from a single pustule from E. grandisin Viçosa, MG, was multiplied on young S. jambos leaves using thetechnique of Ruiz et al. (1989). Spores were collected 12 days afterinoculation with a fine brush. A spore suspension at 105 urediniosporesmL–1, containing 0.05% of Tween 20 was used.

Pre-penetration and colonisation phases of P. psidii were studied ondetached new leaves (the first half-opened leaf from the apical branchof the plant) from the susceptible and resistant E. grandis genotypes.The petiole of each leaf was wrapped in moistened cotton wool andmaintained in a tray (36 × 22 × 6 cm) under saturated moistureconditions. The leaves were inoculated by placing six 2-µL drops ofspore suspension containing 105 urediniospores mL–1 on the abaxialleaf surface. The inoculated leaves were incubated at 20 ± 1°C in agermination chamber (FANEM 347 CDG model) under continuousdarkness during the first 24 h, followed by a 12 h photoperiod at

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approximately 27 µmol m–2 s–1. Samples were collected at 6 h intervalsup to 60 h after inoculation, bleached in chloral hydrate (Longo et al.1994) and observed under a Leica DM RBE microscope, using phasecontrast.

A randomised complete block design with three replications was usedfor each sample period. One replication consisted of a single leafcontaining six micro-drops of inoculum. Fifty well-spaced urediniosporeswere assessed for each replication for germination frequency, appressoriumformation, direct and stomatal penetration, and formation and developmentof primary and secondary hyphae and haustoria.

The colonisation phase was studied in young detached leaves (firsthalf-opened leaf on the apical branch of the plant) inoculated aspreviously described. The leaves were examined 3, 6, 9 and 12 daysafter inoculation. Leaf sections were fixed in 3% glutaraldehydesolution containing sodium cacodilate buffer (0.05 M HCl, pH 6.9) for24 h, and then dehydrated in butyric alcohol series (Sass 1958). Theinfiltration solution was a mixture of 50 mL glycol metacrylate (basicTechnovit 7100 resin) and 0.5 g dybenzoil peroxide, constantlyagitated. A catalysing agent (Hardener II) was added to the infiltrationsolution for blocking at 1:15 (v/v) (Martins et al. 1995). Thepolymerisation temperature was 37°C for 24 h. The samples werestored in a dryer at room temperature after blocking. Serial sections3-µm thick were made with a Reichert Jung 2045 rotary microtome.The sections were individually distended in drops of distilled waterplaced on slides, dried, fixed and subjected to the Johansen (1940)hydration and triple staining procedure. The sections were thenmounted in Canada Balsam. The inter- or intra-cellular patterns ofhyphal development in the leaf mesophyll and the formation ofreproductive structures were observed with a light microscope.

ResultsThere were no differences between susceptible and

resistant genotypes for urediniospore germination,appressorium formation and host penetration. Ninety percentof the urediniospores germinated 6 h after inoculation onleaves of both genotypes. After 18 h, more than 90% of thegerminated urediniospores had formed appressoria on bothgenotypes and, on average, these measured 13 × 16 µm(Fig. 1A). A thin hypha (infection peg) originated from theappressorium and penetrated, in most cases, between theanticlinal walls of the epidermal cells and then entered themesophyll. Twelve hours after inoculation, more than 75% ofthe urediniospores had penetrated the tissue, both in thesusceptible and resistant genotypes. Penetration throughstomata was low, 7 and 2% in the resistant and susceptiblegenotypes, respectively. No appressorium was formed in themajority of the cases where stomatal penetration occurred.

After penetration, the infection hypha increased in size,forming a sub-epidermal vesicle from which primary andsecondary hyphae developed. Primary hyphae were seen at12 and 18 h after inoculation in the susceptible and resistantgenotypes, respectively. The haustorium mother celldeveloped from a primary hypha in contact with themesophyll cell wall, and was separated from the infectionhypha by a septum. A hypha developing from the haustoriummother cell penetrated the mesophyll cell wall, but not theplasmalemma, and gave rise to a haustorium. The haustoriumwas at first globose, but later became lobed, was highlybranched and occupied a large part of the cell lumen

(Fig. 2A). Haustorium formation was observed 18 and 24 hafter inoculation in susceptible and resistant genotypes,respectively.

Histopathological differences between genotypes werenot observed in the pre-penetration phase. However, stainingagents accumulated in the cells containing haustoria and inadjacent cells of the resistant genotype, indicating cell death(Niks 1983b) (Fig. 2B). The hypersensitive reaction could beobserved macroscopically on leaves 48 h after inoculation.

In the susceptible genotype, sections made 3 days afterinoculation showed little intercellular development ofhyphae, but after this time the hyphae ramified and rapidlycolonised the leaf tissues. Hyphae were observed primarilyin the spongy mesophyll but extended as far as the palisadelayer. On the sixth day after inoculation, the colonised tissuesbegan to exhibit hypertrophy (Fig. 1B).

Uredinial primordia of the fungus could be seen as anaccumulation of hyphae beneath and perpendicular to theepidermis. Further development of these structures causedthem to break through the epidermis about 9 days afterinoculation (Fig. 1C). Basal cells, developed from thehymenial layer, gave rise to support cells. After, elongation,each support cell bore a single urediniospore delimited by aseptum. 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, urediniawere observed only on the abaxial surface (Fig. 1D), althoughunder natural conditions they can be amphigenous.

DiscussionThis is the first histopathological investigation of the

interaction between P. psidii and E. grandis and describes theinfection process of the pathogen in a resistant and asusceptible genotype at various times after inoculation. Ahigh percentage of urediniospore germination was observedin both resistant and susceptible genotypes 6 h afterinoculation and by 18 h, more than 90% of the germinatedspores had produced appressoria on both genotypes. Similarresults were obtained when studying the interactions onresistant and susceptible genotypes of wheat and Pucciniagraminis f. sp. tritici Eriks & H.Henn. (Liu and Hardner1996) and Puccinia recondita Roberg ex Desmaz f. sp. tritici(Ortelli et al. 1996).

Infection pegs produced from the appressoria penetratedbetween the anticlinal walls of the leaf epidermis directlyinto the mesophyll of the leaf, similar to that reportedpreviously for P. psidii on S. jambos (Hunt 1968). Theoccasional penetration through stomata without theformation of appressoria was probably random, although thismode of penetration has been reported for P. striiformisWestend. in wheat (Swertz 1994). Non-stomatal penetrationfor dikaryotic phase rust spores is unusual, and has beenobserved 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) urediniacontaining urediniospores, (D) 9 days after inoculation. Bars = 10 µm.

280 A. A. Xavier et al.

(Bonde et al. 1982) and Ravenelia humphreyana P. Henn. inCaesalpinia pulcherrima (L.) Sw. (Hunt 1968). However, thepathway of penetration varied among these rusts. ForP. pachyrhizi, penetration occurred directly through the outerwall of the epidermal wall and into the cell lumen, or lesscommonly, the infection peg penetrated between twoadjacent epidermal cells and entered one of the epidermalcells through the anticlinal wall . For P. zeae, penetration wasalso between two adjacent epidermal cells and then throughthe anticlinal walls of one of the cells (Bonde et al. 1982).Penetration by R. humphreyana occurred directly, with theinfection peg penetrating directly into the epidermal cell andenlarging as a vesicular haustorium (Hunt 1968).

No differences between genotypes were detected forpenetration or vesicle formation, but the formation ofprimary infecting hyphae and haustoria was delayed by 6 hin the resistant genotype as compared to the susceptiblegenotype. Broes and Lopes-Atilano (1996) also observed adelay in the formation of primary hyphae in resistant wheatcultivars inoculated with P. striiformis f. sp. tritici.

Soon after the formation of the haustoria in the leafmesophyll cells of the resistant genotype, a rapid cell necrosiswas observed in adjacent cells. This type of reaction, with celldeath after the formation of the haustorium, is similar to thatfound by various authors in other pathosystems (Littlefieldand Heath 1979; Niks 1983a; Taylor and Mims 1991; Liu andHarder 1996; Ortelli et al. 1996). Hypersensitivity is thoughtto be one of the most important defence responses of the plantto pathogens. In cases of resistance involving biotrophicinteractions, which are generally controlled by gene-for-geneinteraction, it is believed that elicitors are direct products fromavirulent genes; these elicitors interact with the correspondingresistant gene product culminating in cell death (Heath 1997).According to Yamamoto (1995), the resistance involved in thistype of reaction may be controlled by a few dominant genes.Another mechanism may be involved in this resistanceresponse but would not be evident because the adoptedtechnique would not allow it. For example, the speed at whichpapillae are laid down has been related to resistance in theinteraction between Hemileia vastatrix Berk. & Br. and thecoffee leaf, as demonstrated by Matsuoka and Vanetti (1993)in resistant genotypes of coffee, where such a mechanismaccounts for the restriction of pathogen development.

The leaf tissue of the susceptible genotype was littlecolonised 3 days after inoculation. The first visual symptomsof disease were generally observed from 3 to 5 days afterinoculation, and were characterised by small protrusions onthe epidermis resulting from hypertrophy of the colonisedtissues. Although in vivo sporulation was observed 6 daysafter inoculation , sporogenous tissue was detected only insections made 9 days after inoculation and by the twelfth dayurediniospores were well developed.

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Fig. 2. Development of Puccinia psidii in (A) susceptible and (B) resistantEucalyptus grandis genotypes. Vesicle (V) and haustorium (H) in asusceptible genotype. Stain accumulation in cytoplasm (Aniline Blue +Trypan Blue) due to cell death in the resistant genotype after haustoriumformation (H). Bars = 10 µm.

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Received 12 September 2000, accepted 14 May 2001